Basic Veterinary Immunology - Callahan

Basic
Veterinary
Immunology
callahan text.indd 1 3/11/14 1:40 PM

Basic
Veterinary
Immunology
Gerald N. Callahan and Robin M. Yates
Veterinary Clinical Laboratory Immunology chapter by Amy L. Warren
University Press of Colorado

Boulder

© 2014 by University Press of Colorado
Published by University Press of Colorado

5589 Arapahoe Avenue, Suite 206C
Boulder, Colorado 80303
All rights reserved

Printed in Canada
The University Press of Colorado is a proud member of

the Association of American University Presses.
The University Press of Colorado is a cooperative publishing enterprise supported, in part, by Adams State University,
Colorado State University, Fort Lewis College, Metropolitan State University of Denver, Regis University, University of
Colorado, University of Northern Colorado, Utah State University, and Western State Colorado University.
∞ This paper meets the requirements of the ANSI/NISO Z39.48-1992 (Permanence of Paper).
Library of Congress Cataloging-in-Publication Data

Callahan, Gerald N., 1946– author.
Basic veterinary immunology / Gerald N. Callahan and Robin M. Yates ; Veterinary clinical laboratory immunology
chapter by Amy L. Warren.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-1-60732-218-4 (pbk. : alk. paper)
I. Yates, Robin M. author. II. Title.
[DNLM: 1. Animal Diseases—immunology. 2. Animal Population Groups—immunology. 3. Immune System
Diseases—veterinary. 4. Immune System Phenomena. 5. Veterinary Medicine—methods. SF 757.2]
SF757.2
636.089'6079—dc23
2013035025
Designed and typeset by Daniel Pratt
23 22 21 20 19 18 17 16 15 14 10 9 8 7 6 5 4 3 2 1
Cover illustrations, top to bottom: Thoroughbred horse © pirita/Shutterstock; Viruses, detailed 3D illustration
© nobeastsofierce/Shutterstock; © William Zaun; Chinese shar-pei dog © Waldemar Dabrowski / Shutterstock;
© William Zaun.

Contents
Chapter 1: Overview of Chapter 2: Overview of the

Mechanisms of Defense 1 Innate Immune System 17
Clinical Correlation: Simian Acquired Clinical Correlation: Cyclic
Immune Deficiencies 1 Neutropenia in Gray Collies 17
Learning Objectives 1 Learning Objectives 17
Essential Involvement of Animal Anatomical and Physiological Barriers
Immune Systems in All Aspects of to Microbial Invasion 18
Animal Health 1 Epithelial Barrier 18
Immune System 2 Secretions 18
Cells of Defense 3 Mucus and Mucociliary Clearance 20
Communication between Cells of the Commensal Organisms 21
Immune System 3 Cells of the Innate Immune System 21
Communication through cytokines 3 Hematopoiesis 21
Other cytokines 4 Coordinating Hematopoiesis 23
Communication through other signaling Monitoring Hematopoesis 23
molecules 4 Overview of the Cells of the Innate
Communication through cell–cell Immune System 25
contact 4 Myeloid Innate Immune Cells 25
Innate and Adaptive Immune Systems 5 Neutrophils 25
Innate immune system 5 Eosinophils 26
Adaptive immune system 9 Basophils 27
Clinical Correlation Follow-Up 16 Monocytes 28
Student Considerations 16 Macrophages 29
Possible Explanations 16 Myeloid dendritic cells 30
Mast cells 31

Lymphoid Innate Immune Cells 31 Chapter 4: The Complement
Natural killer cells 31 System 53
Other Innate Immune Cells 33
Clinical Correlation: C3 Deficiency in
Clinical Correlation: Follow-Up 33
Brittany Dogs 53
Student Considerations 33
Possible Explanations 34 Learning Objectives 53
Complement Activation 54
Classical Pathway 55
Chapter 3: Innate Immune Lectin Pathway 57
Recognition 37 Alternative Pathway 58
Terminal Membrane Attack Pathway 60
Clinical Correlation: Sepsis in
Neonatal Foals 37 Regulation of Complement Activation 60
Learning Objectives 38 Outcomes of Complement Activation 62
Inflammation and Chemotaxis 62
Introduction 38
Enhancement of Phagocytosis
Evolutionarily Conserved Molecular (Opsonization) 63
Patterns 40 Promoting Humoral Immunity 63
Pathogen-Associated Molecular Clearance of Immune Complexes 64
Patterns 40
Pattern-Recognition Receptors 40 Student Considerations 64
Signaling Pattern-Recognition Possible Explanations 64
Receptors 41
Toll-like receptors 42
NOD-like receptors and RIG-like Chapter 5: Acute Inflammation 67
receptors 44
Phagocytic Pattern-Recognition Clinical Correlation: Acute Bovine
Receptors 44 Mastitis 67
Other Pattern-Recognition Receptors 46 Learning Objectives 67
Innate Recognition of Damage- Purposes of Acute Inflammation 68
Associated Molecular Patterns 46 Signs of Acute Inflammation 69
Inflammasome 48
Initiators and Mediators of Acute
Consequences of Danger Signal Inflammation 70
Recognition by Sentinel Cells 50
Vascular Changes in Acute
Activation of Macrophages 50
Inflammation 72
Proinflammatory Mediators 50
Leukocyte Recruitment 75
Leukocyte Extravasation 75
Diversity and Timeline of Leukocyte
Possible Explanations 51
Recruitment 79
Systemic Effects of Acute
Inflammation 80
Clinical Correlation Follow-Up 85
vi Contents

Chapter 6: Innate Cellular Organs and Cells of the Adaptive
Effector Mechanisms 89 Immune System 109
Primary Lymphoid Organs and
Clinical Correlation: Rattles in Foals 89 Hematopoiesis 110
Learning Objectives 90 Bone marrow 110
Classification of Innate Cellular Thymus 112
Effector Mechanisms 90 Secondary Lymphoid Organs 114
Lymph nodes 115
Phagocytes and Phagocytosis 91
Spleen 117
Phagocytosis Part I: Recognition and
Mucosa-associated lymphoid tissue 118
Adhesion of Particles on the Plasma
Membrane of the Phagocyte 91 Clinical Correlation: Follow-Up 119
Phagocytosis Part II: Membrane and Student Considerations 120
Cytoskeletal Reorganization to Possible Explanations 120
Mediate Particle Engulfment and the
Creation of a Phagosome 94 Chapter 8: Antigens and
Phagocytosis Part III: Maturation of Antigen Processing 121
the Phagosome into a Microbicidal
and Degradative Phagolysosome 94 Clinical Correlation: Caprine Arthritis
Extracellular Innate Effector Encephalitis 121
Mechanisms 99 Learning Objectives 122
Degranulation by Neutrophils and Stimulators of Adaptive Immunity:
Eosinophils 99 Pathogens, Antigens, and Epitopes 122
Release of Neutrophil Extracellular Major Histocompatibility Complex 124
Traps 100 History of the Major
Histocompatibility Complex 124
Natural Killer Cell–Mediated
Major Histocompatibility Complex
Cytotoxicity 101
Genetics 125
Recognition of Target Cells 101
Structure and Distribution of Major
Activating stimulus 101
Histocompatibility Complex Class I
Inhibitory stimulus 102
and Class II Molecules 129
Targeted Degranulation by Natural
Antigen–Major Histocompatibility
Killer Cells 102
Complex Interactions 131
Antibody-Dependent Cell-Mediated
Cytotoxicity 105 Antigen Acquisition, Processing, and
Presentation 132
Antigen-Presenting Cells 132
Major Histocompatibility Complex
Possible Explanation 106
Class I Antigen Processing and
Presentation 135
Chapter 7: Cells and Organs Major Histocompatibility Complex
Class II Antigen Processing and
of the Adaptive Immune
Presentation 136
System 109
Cross-Presentation 137
Clinical Correlation: Feline Clinical Correlation: Follow-Up 139
Lymphadenopathy 109 Student Considerations 140
Learning Objectives 109 Possible Explanations 140
Contents vii

Chapter 9: T-Lymphocyte Third Signal and T-Helper Cell
Development 143 Differentiation 168
Effector T Cells 169
Clinical Correlation: Severe Combined
Effector T-Helper Cells: Th1, Th2,
Immunodeficiency Disease in
Tf h, and Th17 Cells 169
Arabian Foals 143
Effector Regulatory T Cells: Treg, Th3,
Learning Objectives 143 and Tr1 Cells 171
T-Cell Receptor Genes 144 Effector Cytotoxic T Cells 173
Structure of the T-Cell Receptor 144 Clinical Correlation Follow-Up 177
T-Cell Receptor Gene Families 145 Student Considerations 177
Gene Rearrangement in the Thymus 145 Possible Explanations 177
T-Cell Selection in the Thymus 150
Products of T-Cell Gene
Rearrangement 150 Chapter 11: B-Cell
Positive Selection of Functional Development 181
T Cells 153
Clinical Correlation: Equine
CD4, CD8, and Major
Agammaglobulinemia 181
Histocompatibility Complex
Recognition 155 Learning Objectives 181
Negative Selection of Self-Reactive B Cells and the Structure of the B-Cell
T Cells 155 Receptor 182
T-Helper, Cytotoxic T, and B-Cell Genetics, Development, and
T-Regulatory Cells 156 Circulation 183
γ/δ T Cells 156 B-Cell Receptor Gene Families,
Clinical Correlation: Follow-Up 158 Genetic Rearrangement, and
Student Considerations 158 Junctional Diversity 183
Possible Explanations 158 Order and Regulation of B-Cell
Development 186
Immunoglobulin Isotypes 189
Chapter 10: T-Cell Activation 161 Other Populations of B Cells 191
Clinical Correlation: Canine B-1 B Cells 191
X-Linked Severe Combined Marginal-Zone B Cells 194
Immunodeficiency 161 Clinical Correlation Follow-Up 195
Learning Objectives 162 Student Considerations 195
Antigen Recognition by T Cells 162
First Signal: T-Cell Receptors and
Major Histocompatibility Complex– Chapter 12: B-Cell Activation
Antigen Interactions 162
and Differentiation 197
Second Signal and Major
Histocompatibility Complex– Clinical Correlation: Selective Immuno-
CD4/8 Interactions 164 globulin A Deficiencies in Dogs 197
Signal Transduction and T-Cell Learning Objectives 197
Activation 166
T-Dependent B (B-2) Cells 199
viii Contents

Interaction of B Cells with Antigens 199 Memory B Cells 238
B Cells as Antigen-Presenting Cells 203 Memory T Cells 238
Activation of B Cells by T-Helper Clinical Correlation Follow-Up 239
Cells 204 Student Considerations 239
Isotype Switching, Somatic Possible Explanations 239
Hypermutation, and Terminal
Differentiation of B Cells into
Plasma Cells 206Chapter 14: Fetal and
Antibody Effector Functions 213 Neonatal Immunity 241
Pathogen and Toxin Neutralization 213
Clinical Correlation: Failure of Passive
Opsonization and Activation of
Transfer 241
Complement 213
Distribution and Function of Fc Learning Objectives 241
Receptors 214 Fetal Development of the Immune
Clinical Correlation Follow-Up 215 System 241
Student Considerations 215 Maternal Transfer of Immunity 243
Possible Explanations 217 Prenatal Transfer of Immunity 243
Postnatal Transfer of Immunity 245
Chapter 13: Adaptive Immune Neonatal Acquisition of Memory and
Responses to Infections and Immune Capacity 248
Immunological Memory 219 Vaccination of Neonates 249
Clinical Correlation: Canine
Parvovirus and the Wolves of Isle
Royale 219
Learning Objectives 220
Adaptive Immunity: The Big Picture— Chapter 15: Vaccination 253
From Insult to Recovery 221 Clinical Correlation: Animal Vaccines 253
Routes of Infection 223
Mechanisms of Pathogenesis 223
Innate Response Cells and Cytokines Vaccines 253
of Innate Responses Direct T-Cell Vaccine Characteristics 253
Differentiation 225 Live-Attenuated Vaccines 255
Effector T Cells Home to Sites of Killed Vaccines 259
Infection and Inflammation 226 Component, Toxoid, Conjugate,
Antibody Responses Develop in Recombinant, and Naked DNA
Secondary Lymphoid Tissues 229 Vaccines 259
Importance of Different Adaptive Adjuvants 263
Responses to Clearance of Different Administration 266
Pathogens 233 Frequency 266
Immune Responses in the Gut 233 Passive Immunotherapy 267
Immunological Memory 237 Clinical Correlation Follow-Up 268
Primary and Subsequent Immune Student Considerations 268
Reponses 237 Possible Explanations 268
Contents ix

Chapter 16: Immune Chapter 18: Veterinary Clinical
Deficiencies and Immune- Laboratory Immunology 295
Mediated Diseases 271 Clinical Correlation: Transfusion
Clinical Correlation: Feline Reactions 295
Immunodeficiency Virus 271 Learning Objectives 297
Learning Objectives 271 Principles of Antibody-Based
Immune Deficiencies 272 Techniques 298
Congenital Immune Deficiencies 272 Generating Antibodies: Polyclonal
Acquired Immune Deficiencies 273 and Monoclonal Antibodies 298
Immune-Mediated Diseases 274 Production of polyclonal antibodies 298
Hypersensitivities Type I, III, and IV 274 Production of monoclonal antibodies 299
Type I hypersensitivities 274 Detecting Antibody–Antigen
Type III hypersensitivities 279 Reactions 300
Type IV hypersensitivities 282 Specific Techniques 300
Type II Hypersensitivities and Enzyme-linked immunosorbent
Autoimmune Diseases 283 assays 300
Immunoblotting 302
Clinical Correlation Follow-Up 284 Immunofluorescent microscopy 303
Student Considerations 284 Precipitation assays 304
Possible Explanations 284 Flow cytometry 305
Immunohistochemistry 306
Chapter 17: The Immune Diagnosis of Blood Type
System and Cancer 287 Incompatibilities 307
Blood Typing 307
Clinical Correlation: Canine Cancer 287 Blood typing cards 308
Learning Objectives 287 Typing gel 308
Background 287 Membrane dipstick 309
Crossmatching 309
Evidence That Mammalian Immune
Systems Affect Tumors 289 Diagnosis of Immunodeficiencies 312
Serum Immunoglobulin
Tumor–Host Interactions 290
Quantification 312
Outcomes 292 Phenotypic Analysis of Lymphocytes 312
Clinical Correlation: Follow-Up 293 Other Immunodeficiency Tests 313
Student Considerations 293 Diagnosis of Autoimmunity 313
Possible Explanations 294 Antinuclear Antibody Test 313
Coombs Test 314
Antiplatelet Antibody Test 314
Antithyroglobulin Autoantibody Test 315
Glossary 319
Index 327
x Contents

Basic
Veterinary
Immunology

Gerald N. Callahan and Robin M. Yates
Overview of Mechanisms of Defense Chapter 1

10.5876_9781607322184.c001
was a retrovirus—simian T-lymphotropic virus

Clinical Correlation: Simian Acquired Immune
III—a retrovirus very similar to the human T-
Deficiencies 1
lymphotropic virus III, the apparent (and later
confirmed) cause of human AIDS. The monkey
Essential Involvement of Animal Immune
Systems in All Aspects of Animal Health 1 virus was later renamed simian immunodefi-
Immune System 2 ciency virus (SIV).
Cells of Defense 3 Studies of wild African monkeys revealed that
Communication between Cells of the Immune green monkeys had SIV infection rates as high
System 3 as 30 to 50 percent with no overt clinical syn-
Communication through cytokines 3 drome. As with rhesus macaques, however, SIV
Other cytokines 4 infection of pig-tailed and crab-eating macaques
Communication through other signaling almost invariably resulted in a chronic wasting
molecules 4 disease and severe opportunistic infections, such
Communication through cell–cell contact 4 as oroesophageal candidiasis. What was under-
Innate and Adaptive Immune Systems 5 lying this clinical syndrome in these monkeys?
Innate immune system 5
Adaptive immune system 9
Learning Objectives
Student Considerations 16 After reading this chapter, you should be able to
• describe the immune defense system;
• understand how cells of the immune sys-
Clinical Correlation: Simian tem communicate;
Acquired Immune Deficiencies • understand the basic elements of innate
The story probably began almost 32,000 years immune mechanisms;
ago, but the villain was not discovered until • describe the basic elements of adaptive
1985. In that year, M. D. Daniel and his collabo- immune mechanisms.
rators completed their work on four rhesus ma-
caque monkeys. Table 1.1 lists the symptoms of Essential Involvement of
those monkeys (Figure 1.1). Animal Immune Systems in All
Four monkeys seriously ill—at least three of Aspects of Animal Health
them from what appeared to be opportunis-
tic infections—for no apparent reason. Daniel Roughly 4 billion years ago, life on Earth became
and coworkers began searching for the cul- complex. From the primordial ooze, life co-
prit. What they found in the affected monkeys alesced into individual organisms—eubacteria,
11

Figure 1.1. Rhesus macaque (© gopause / Shutterstock)
archaebacteria, and likely others. The brown Table 1.1. Symptoms of macaque monkeys at the New
seas turned azure and the pink skies turned England Regional Primate Research Center (adapted from
M. D. Daniel et al., 1985, Science 228: 201)
blue. Soon after, the first parasites appeared.
Animal Symptoms
Parasitism offered an energy-efficient alterna-
tive to survival on one’s own. Hosts provided 251–79 Malignant lymphoma
protection and energy with minimal or no in- 239–82 Macrophage infiltrates in brain, oroesopha-
geal candidiasis, cryptosporidiosis, intestinal
vestment by the parasite.
trichomoniasis
By the time metazoan life appeared, almost
220–82 Macrophage infiltrates in brain, oroesopha-
3 billion years later, parasitism had become a geal candidiasis, cryptosporidiosis, intestinal
high art. That constant evolutionary pressure, trichomoniasis
those threats to the survival of individual or- 142–83 Diarrhea, facial rash, generalized lymphade-
ganisms, produced immune systems. Both nopathy, splenomegaly
simple and complex mechanisms for defense
against parasites had, almost from the outset,
become essential components of all life, and vidual animals vanish. Because of this critical
still are. Without means for self-defense, no in- involvement of immune systems in all animal
dividual plants or animals would exist on Earth. functions, understanding immunity is essential
The proof of that is apparent in both pri- to understanding animal medicine, including
mary and acquired immune deficiencies in animal behavior.
animals as disparate as sharks and horses. Im-
mune-deficient animals rapidly disappear, and Immune System
in their places communities of other living
things—bacteria, viruses, fungi, and parasites— Animals have evolved numerous strategies to
abruptly arise. Without immune systems, indi- defend themselves against infections. These
2 Ov e rv i e w o f M e c h a n i s m s o f D e f e n s e

mechanisms range from independently acting All other vertebrates have both white and red
single proteins to highly trained cellular assas- blood cells (RBCs). RBCs, or erythrocytes,
sins that can specifically target infected cells. transport oxygen to animal tissues and deliver
Over evolutionary time, these various mecha- carbon dioxide to animals’ lungs. White blood
nisms have coalesced into unified defense sys- cells, or leukocytes, are, among other things,
tems called immune systems. Collectively, an cells of vertebrate immunity, both innate and
immune system includes the tissues, cells, and adaptive. White blood cells include T and B
molecules that work together to effect an im- lymphocytes (often referred to simply as T and
mune response designed to protect the host B cells and involved primarily in adaptive im-
animal from a given threat. Immune responses mune responses), as well as neutrophils, baso-
do not only secure the immediate survival of phils, eosinophils, NK cells (involved primarily
the infected host, but in many cases also bet- in innate immunity), and monocytes, which be-
ter prepare the animal for later exposures to a come, among other things, macrophages and
particular threat. dendritic cells (DCs; involved in both innate
The immune system is divided into two func- and adaptive immune responses). Neutrophils
tional subsystems: the innate immune system make up about 70 percent of all leukocytes in
and the adaptive (or acquired) immune system. the blood, followed by lymphocytes at around
The innate immune system mediates rapid, 20 percent, but the percentages of the various
early-phase responses to threats. These responses cell types vary among species, as well as with
include molecular and cellular mechanisms that the animal’s age and health. T cells can further
kill a wide variety of potential pathogens. The differentiate into T-helper (Th) and cytotoxic
adaptive immune system provides a delayed but T (Tc) cells. Th cells may differentiate into a
customized, or specific, response to infectious variety of Th cell types, including Th1, Th2,
threats, resulting in the production of antibod- Th17, and regulatory T cells, and each of these
ies, T-cell responses, and immune memory. Th-cell lineages perform specialized functions
For decades, immunologists considered and in adaptive immunity (see chapter 10). B cells
studied the innate and adaptive immune sys- ultimately differentiate into plasma cells that
tems separately. More recently, it has become secrete antibodies.
clear that these subsystems work together in an
overlapping and reciprocating fashion. Gener-
ally speaking, without a functional innate im- Communication between Cells
mune system, an animal is incapable of gen- of the Immune System
erating effective adaptive immune responses. It takes billions of immune cells to achieve
Without a functional adaptive immune system, effective defensive responses. Coordination of
most innate immune mechanisms are less ef- such massive numbers of cells requires a so-
fective. In truth, animal immunity involves a phisticated communication system involving
single collection of cells and their products that multiple cytokines, a variety of other soluble
act synergistically in defense of their host. How mediators, and direct cell-to-cell communica-
is this synergy orchestrated? tion via intercellular contact.
Cells of Defense Communication through cytokines

The only known vertebrate animals without Essentially, all cells use cytokines (soluble
red blood cells (RBCs) are crocodile icefish, proteins or glycoproteins) to communicate
which live in oxygen-rich cold water and ab- with one another. As with hormones, cytokines
sorb gaseous oxygen directly into their blood. may act directly on the cells that produce them
Ov e rv i e w o f M e c h a n i s m s o f D e f e n s e 33

(autocrine stimulation); on other nearby cells eration of cells in the bone marrow is apparent
(paracrine stimulation); or, via the blood, on as new colonies of cells.
distant cells (endocrine stimulation). Immune
cytokines fall into distinct categories based on
structure, function, and origin. Other cytokines
Interleukins (ILs) are a diverse group of cy- Several other immunologically important cy-
tokines produced predominantly by leukocytes tokines do not fit neatly within the preceding
(white blood cells) and act on other leukocytes categories. These cytokines include transform-
(inter, “between”; leukins, “leukocytes”). Acti- ing growth factor–β (TGF-β; so called because
vated T lymphocytes secrete the majority of it stimulates [among other things] growth simi-
ILs, but many other leukocytes, particularly lar to that seen in tumor cells). TGF-β has roles
macrophages and DCs, also produce cytokines in the regulation of immune reactions and tis-
to communicate with other cells. The abbrevia- sue repair. Other significant cytokines include
tion IL is followed by a number designating the tumor necrosis factors TNF-α and TNF-β (so
specific IL; for example, IL-2 is an IL that stimu- called because under some conditions these fac-
lates T-cell division. ILs have a variety of targets tors can cause tumor cell necrosis), which have
and functions within the immune system. roles in mediating inflammation and its sys-
Interferons (IFNs) are a group of cytokines temic effects. Table 1.2 lists some of the more
that are particularly important in the coordina- common cytokines and their functions.
tion of immune responses to infections, par-
ticularly viral infections. Because they were
first identified as molecules that interfered with Communication through other
signaling molecules
viral infection and replication, scientists com-
bined the word “interfere” with the particle suf- Aside from cytokines, the immune system
fix “-on” to form interferon. They include IFN-α uses several other molecules as messengers.
and IFN-β (released by virus-infected cells) and These molecules include the monoamines his-
IFN-γ, a proinflammatory cytokine produced tamine and serotonin, synthesized and released
predominantly by Th lymphocytes. from mast cells and basophils in response to in-
Chemokines are a group of cytokines that flammatory stimuli or allergens, which mediate
direct movements of immune cells (chemo, vascular changes associated with inflammation.
“chemical”; kines, “kinos or movement”). The Other important groups of immune signal-
movement of leukocytes among various tis- ing molecules derive from cellular lipids. These
sues is essential to all immune responses. Che- signaling molecules include the leukotrienes
mokines released by other immune cells direct and prostaglandins. Although derived from a
the movements of these leukocytes. Some che- common lipid precursor, members of these
mokines can also change the physiology of im- two molecular families have diverse functions,
mune cells so that these cells can perform par- including vascular changes, immune activation,
ticular functions (a process commonly referred chemotaxis, and regulation of inflammation
to as activation). and immunity.
Colony-stimulating factors (CSFs) are cyto-
kines that promote the proliferation and dif-
ferentiation of leukocyte precursors. The ma- Communication through cell–cell contact
jority of CSFs act on bone marrow stem cells Much of the communication between im-
and leukocyte precursors in the bone marrow, mune cells in the adaptive immune system relies
although some CSFs also act in peripheral tis- on direct molecular interactions between pro-
sues. CSFs were so named because the prolif- teins, complexes, and receptors on cell surfaces.

Table 1.2. Some of the cytokines involved in innate immunity
Mass
(kDa) Assembly Source(s) Target(s)
IL-1 17 Monomer Macrophages, endo- Endothelia (h coagulation, h inflammation), hepatocytes
thelia, epithelia (h acute phase proteins), hypothalamus (h fever)
IL-18 17 Monomer Macrophages NK cells (h IFN-γ), T lymphocytes (h IFN-γ)
TNF 17 Homotrimer Macrophages, T Endothelia (h coagulation, h inflammation), hepatocytes
lymphocytes (h acute phase proteins), neutrophils (h activation), hypo-
thalamus (h fever)
IL-6 26 Homodimer Macrophages, endo- Hepatocytes (h acute phase proteins), B lymphocytes
thelia, T lymphocytes (h proliferation)
IL-15 13 Monomer Macrophages NK cells (h proliferation), T lymphocytes (h proliferation)
IL-12 35/40 Heterodimer Macrophages, den- Th1 lymphocytes (h differentiation), Tc lymphocytes
dritic cells (h IFN-γ), NK cells (h IFN-γ)
IL-23 19/40 Heterodimer Macrophages, den- T lymphocytes (h IL-17)
dritic cells
IL-27 28/13 Heterodimer Macrophages, den- Th1 lymphocytes (inhibition and/or differentiation), NK
dritic cells lymphocytes (h IFN-γ)
IL-10 18 Homodimer Macrophages, T Macrophages, dendritic cells (i IL-12)
lymphocytes
IFN-α 21 Homodimer Macrophages All cells (h viral immunity, h MHC class I), NK cells (h
activation)
IFN-β 25 Homodimer Fibroblasts All cells (h viral immunity, h MHC class I), NK cells (h
activation)
Chemo- 8–12 monomer Macrophages, endo- Phagocyte (h migration), B lymphocytes (h migration),
kines thelia, fibroblasts, T lymphocytes (h migration), h wound repair
epithelia
Note: MHC = major histocompatibility complex.
This type of communication requires intimate humans. In vertebrate animals, the innate im-
cell–cell contact. T lymphocytes in particular mune system represents the first line of defense
often require contact with other cells for acti- against invading pathogens, but in invertebrate
vation and to perform their effector functions. animals it represents the only line of defense.
Over millions of years of evolution, the innate
immune system has become amazingly com-
Innate and Adaptive Immune Systems plex and sophisticated. The key feature of this
Although the innate and adaptive immune immune system is its ability to distinguish be-
systems work together as a functional unit, tween foreign (often microbial) molecules and
there are evolutionary and functional differences molecules normally encountered in healthy
between these two systems (see Figure 1.2). host tissue.
Unlike adaptive immune systems, which have
to learn the differences between self and non-
Innate immune system self, the innate system has evolved to recognize
The innate immune system is an evolu- aspects of non-self immediately and to react
tionarily ancient system present in one form instantaneously, even before birth. This ability
or another in every animal, from mollusks to is the result of evolution of the genes encoding

Table 1.3. Some cytokines involved in adaptive immunity
Mass
(kDa) Assembly Source(s) Target(s)
Lympho- 21–24 Homotrimer T lymphocytes B lymphocytes (h development), T lymphocytes (h develop-
toxin ment), neutrophils (h migration, h activation)
IL-2 17 Monomer T lymphocytes T lymphocytes (h survival, h proliferation, h cytokines),
B lymphocytes (h proliferation, h antibody production),
NK cells (h proliferation, h activation)
IL-4 17 Monomer Th2 lymphocytes B lymphocytes (h isotope switch IgE), Th2 lymphocytes
(h proliferation, h differentiation), macrophages (i IFN-γ
response), mast cells (h proliferation)
IL-5 26 Homodimer Th2 lymphocytes B lymphocytes (h proliferation, h isotope switch IgA),
eosinophils (h proliferation, h activation)
IL-13 13 Monomer Th2 lymphocytes, B lymphocytes (h isotope switch IgE), macrophages (h col-
NK-T lymphocytes, lage), fibroblasts (h collage), epithelia (h mucus)
mast cells
IL-17 35/40 Dimer T lymphocytes Endothelia (h chemokines), macrophages (h cytokines/che-
mokines), epithelia (h G-CSF and GM-CSF)
IFN-γ 40 Homodimer Th1 lymphocytes, B lymphocytes (h isotope switch), Th1 lymphocytes (h dif-
Tc lymphocytes, ferentiation), macrophages (h activation), various cells (h
NK cells antigen processing and h MHC class I)
Note: MHC = major histocompatibility complex.
numerous, highly conserved receptors that rec- that sometimes results in the direct lysis of the
ognize molecular patterns specific to pathogens microbe and always activates numerous sec-
(pattern-recognition receptors [PRRs]) or other ondary innate and adaptive immune responses.
receptors specific to the products of host tissue Specialized groups of cells carry out other
damage and necrosis. Several types of cells ex- innate effector mechanisms. Phagocytes are
press both types of receptors, but they appear a group of cells that can, via a process called
in the highest concentrations on the surfaces of phagocytosis, engulf and degrade microbes.
the sentinel cells of the innate immune system, This ability, plus specific cell-surface receptors,
including the macrophages and DCs found in allows phagocytes to selectively ingest indi-
practically every animal tissue. Once these cell- vidual microbes and, within a cytoplasmic sack
surface receptors bind their ligands, the cells called a phagolysosome, eliminate those invad-
release cytokines and chemokines that engage ing organisms. In this way, phagocytes remove
the rest of the immune system, which leads to and kill microbes without harming host cells.
the activation of antimicrobial effector mecha- The phagocytes of the innate immune system
nisms that find and kill invading microbes and include macrophages, DCs, and neutrophils.
parasites as well as dispose of damaged cells. In some cases, invading pathogens may be
The simplest of the innate antimicrobial too large or too numerous for phagocytes to
mechanisms involve individual secreted pro- handle effectively. In these cases, neutrophils or
teins or groups of them. For example, the eosinophils can also release antimicrobial and
complement system includes a series of plasma antiparasitic products directly into extracellular
proteins, most of which come from the liver. spaces. Although these products can harm by-
Activation of the complement system by invad- stander cells, they also effectively kill microbes
ing microbes initiates a biochemical cascade and large parasites such as worms.

Figure 1.2. Major differentiating features of the innate and adaptive immune systems
Other innate immune cells, NK cells, defend nize and eliminate threats derives from highly
against intracellular pathogens such as viruses. conserved genes within the germline chromo-
NK cells do this by physically touching host somal DNA of animals. Because these systems
cells to determine whether they are healthy or have been refined over eons of evolution, they
stressed and abnormal (potentially indicating regularly and effectively distinguish microbes
that they may be infected by a virus or are neo- from normal host tissue. However, the innate
plastic). When NK cells detect suspicious cells, system cannot identify subtle differences be-
they induce apoptosis of those cells and limit tween strains of microbes and so may be cir-
viral spread or neoplasia (Figure 1.3). cumvented by evolving pathogens, particularly
The entire repertoire of mechanisms that viruses. Another limitation of the innate im-
allows the innate immune system to recog- mune response is that its efficiency does not

Figure 1.3. Major innate and adaptive defense mechanisms against extracellular and intracellular
pathogens
Different aspects of innate and adaptive immunity have evolved to deal with intracellular and ex-
tracellular pathogens. The complement system and phagocytes are especially important aspects of
innate immunity that deal with extracellular pathogens. NK cells of the innate response help to elimi-
nate infected cells. During adaptive immune responses (discussed in the Adaptive Immune System
section), antibodies and some Th cells act most directly in protecting against extracellular infections,
and other Th cells and Tc cells are most effective against intracellular infections.

improve with repeated exposure to a given mi- immune response. The first time an animal
crobe. Unlike the adaptive immune system, the encounters a pathogen, the adaptive immune
innate immune system has no memory. system generates a primary immune response.
As a result, the innate immune system re- During second and subsequent encounters
sponds in an identical and predictable manner with that same pathogen, adaptive immune re-
to microbes, foreign bodies, and trauma. Also, sponses become more specific, faster, stronger,
innate responses do not improve with repeated and longer lasting (see Figure 1.4).
exposures. To escape these limitations, ver- Those differences between primary and
tebrates have evolved another set of immune subsequent immune responses constitute im-
mechanisms that are highly specific and evolve munological memory—the adaptive immune
over time to deal with ever-changing patho- system’s ability to remember it has seen an anti-
gens: the adaptive immune system. gen before and respond differently on a second
encounter with that antigen. Specificity and
memory are the hallmarks of adaptive immune
Adaptive immune system responses.
Beyond innate defenses, vertebrate animals Adaptive immune responses rely on lympho-
can muster a series of additional protective cytes, especially T and B lymphocytes. These
responses. These responses take longer to de- cells are the only ones in an animal’s body that
velop than innate responses, but add several re- specifically recognize and respond to antigens.
markable defense options. Adaptive responses On lymphocytes, antigen recognition occurs
to infection are adaptive because they are not through specific molecules called antigen re-
immediate responses and develop only over ceptors. These molecules are B-cell receptors
time. The adaptive immune response is also re- (BCRs) and T-cell receptors (TCRs; see Figures
ferred to as the acquired immune response be- 1.5 and 1.6).
cause the animal does not directly inherit these BCRs and TCRs are exquisitely specific for
immune responses but acquires them within individual antigens—nearly one BCR or TCR
its lifetime. Molecules that induce adaptive for each potential antigen. Because animals can
immune responses are called antigens. In the respond to essentially any antigen inside of an
natural world, antigens are molecular bits of animal, BCRs and TCRs must exist in nearly
organisms, including pathogens. Some antigens limitless numbers of specificities. That chemi-
may also induce innate responses, but the term cal feat results from a remarkable series of ge-
antigen is reserved for those molecules that netic rearrangements.
stimulate adaptive immune responses (gener- The genes that encode BCRs and TCRs ap-
ally, macromolecules, especially proteins and pear in pieces spread along particular chromo-
glycoproteins, but also carbohydrates as well as somes. During the development of B cells and T
some lipids and nucleic acids). cells, those genetic bits shuffle randomly like a
Innate immune responses, although rapid deck of cards to generate a nearly infinite num-
and powerful, lack fine specificity. For example, ber of possible receptors, including self-reactive
the inflammatory responses (aspects of innate receptors. Subsequent processes identify and
immunity) to type 1 and type 2 parvoviruses destroy self-reactive lymphocytes, minimizing
are the same. Adaptive immune responses are the possibility of autoimmunity. Nonetheless,
much more specific and customized to deal some self-reactive B and T cells can survive se-
with specific pathogens. For example, no mat- lection and may initiate autoimmune disease
ter how many times a dog gets infected with later in the animal’s life.
a parvovirus, the innate immune response re- In spite of their plasticity, specificity, and
mains similar, which is not true of the adaptive memory, adaptive responses lack the speed of

Figure 1.4. Primary and secondary antibody responses
After injection of antigen A on day 0, an animal produces a primary antibody response to antigen A.
This response lasts for 30–40 days and is dominated by a type of antibody called IgM (pentamers).
When again injected with antigen A plus a new antigen B, the animal produces a robust secondary
antibody response to antigen A but a primary response to antigen B.
innate responses and can take days to weeks to tivated. Once these cells are activated by APCs,
reach measurable levels. This time lag is the re- they begin to divide rapidly and further differ-
sult of a phenomenon called clonal expansion. entiate. Ultimately, these waves of cell division
After a primary immunization or infection, a lead to a dramatic increase in the number of T
special group of cells, called antigen-presenting cells specific for the immunizing or infecting
cells (APCs), ingests and processes pathogens antigens. This process is clonal expansion—the
and then presents pathogen-derived antigens to expansion of a few cells into enormous clones
T cells. At the time of immunization or infec- of the originally activated T cell—resulting in
tion, an animal usually has only a few T cells thousands to millions of cells that all share the
specific for infecting or immunizing antigens. At same antigenic specificity. A similar process oc-
this stage, these T cells are referred to as “naive” curs among B cells. After antigen binding and
T cells because they have not yet encountered interaction with T cells, a few antigen-specific
their specific antigens—they have yet to be ac- B cells rapidly divide to create massive clones

Figure 1.5. The B-cell antigen-specific receptor Figure 1.6. The T-cell antigen-specific receptor
As B cells mature in the bone marrow, they The antigen-specific portion of the TCR con-
express a complex of molecules called the BCR. tains two chains, either α/β or γ/δ. Together
This complex contains two immunoglobulin they make up the single antigen-binding site
heavy chains (H) and two immunoglobulin light of the TCR. The TCR also includes γ-, δ-, ε-,
(L) chains (essentially an antibody molecule) and ζ-chain molecules that contribute to signal
that, together, make up the antigen-specific transduction during T-cell activation. Ag =
portions of the BCR. In addition, the BCR con- antigen.
tains two other molecules, Igα and Igβ, which
participate in signal transduction and B-cell
activation. Ag = antigen.
through antibodies. After binding antigen and
interacting with certain T cells, B cells differen-
of antigen-specific B cells. Ultimately, these B tiate into plasma cells. Plasma cells secrete an-
cells differentiate into plasma cells and secrete tibodies (essentially a soluble form of the BCR)
antigen-specific antibody. All of this takes time, into lymph and blood. Antibodies are especially
a week to ten days in a primary adaptive im- effective in eliminating extracellular pathogens
mune response. (see Figure 1.8).
Once generated in sufficient numbers, T cells As mentioned earlier, unlike the innate im-
exert their effects directly, especially through mune system, the adaptive immune system re-
destruction of infected host cells and enhance- members its first encounters with antigens. So,
ment of other immune responses (see Fig- in a secondary adaptive immune response, lag
ure 1.7). B cells, however, provide protection times are shorter than primary immune response,
11

Figure 1.7. Some of the actions of effector T cells
After activation by APCs, T cells differentiate into a variety of effector cells that drive the progres-
sion of several types of immune responses, including destruction of virus-infected cells (first panel),
activation of macrophages to kill ingested bacteria (second panel), activation of B cells to produce an-
tibodies (third panel), stimulation of inflammation (fourth panel), and maintenance of self-tolerance
(fifth panel). Treg = regulatory T cell; CTL = Tc cells.
tertiary responses are even faster, and so on. Also, although innate immune responses may
Such immune memory results from the cre- occur nearly anywhere, adaptive immune re-
ation of specialized subsets of long-lived B and sponses arise at specialized sites in animals’ bod-
T cells, called memory cells, which remain after ies. Those sites include lymph nodes (see Figure
primary adaptive immune responses. This pro- 1.9), the spleen, and mucosa-associated lym-
cess increases the number of resident antigen- phoid tissue (MALT).
specific cells. The second time an animal en- As mentioned at the beginning of this chap-
counters the same pathogen or antigen, these ter, separating immune responses and immune
memory cells dominate adaptive responses. systems into innate and adaptive components is
The result is that secondary and subsequent primarily a matter of convenience and history.
adaptive immune responses happen much No adaptive response ever occurs in the ab-
faster, are more specific, are stronger, and last sence of an accompanying innate response, and
longer (see Figure 1.4). most innate responses in vertebrate animals in-

Figure 1.8. Effector mechanisms of antibodies
As shown in the left column, when antibodies bind to toxins (or viruses), the toxins can no longer
bind to specific receptors on host cells and are neutralized. The center column shows how antibody
binding to bacteria (a process called opsonization) enhances phagocytosis of pathogens by macro-
phages. The right column shows a similar process. Once IgM or IgG antibodies have bound to a
pathogen, the complex activates complement. Because macrophages and other phagocytic cells also
have receptors for complement fragments, this process also leads to more efficient phagocytosis of
pathogens.
13

Figure 1.10. (facing page) Overview of a typical
immune response
Figure 1.10 is a concept map of a typical im-
mune response involving both the innate
(yellow) and adaptive (blue) immune systems.
These two systems intersect to work together
toward a common goal: control or eradication
of a threat. Following is a chronological descrip-
tion of the typical immune response depicted.
Numbers in parentheses correspond to the
circled numbers in the figure.
(1) After recognition of a threat, sentinel in-
nate immune cells initiate immune responses. (2)
Recognized threats include microbial products in
normally sterile tissues and the products of cellu-
lar injury—hallmarks of infection or potential in-
fection. (3) Even without knowing precisely what
the threat may be, innate sentinel cells can recruit
other innate cells, such as neutrophils, eosinophils,
and monocytes, as well as allow serum proteins,
such as complement, to permeate the affected
tissue through a process called inflammation. (4)
Once recruited to the site of infection, innate cells
Figure 1.9. Canine lymphatics and lymph nodes and serum proteins deploy powerful (albeit non-
Lymphatics (upper portion) are a system of specific) antimicrobial and antiparasitic mecha-
open-ended vessels that return extravascular nisms. (5) During the innate immune response,
fluid from the periphery to the heart and blood. APCs, such as macrophages and DCs, ingest
Distributed throughout the lymphatics are a and process proteins, move to lymph nodes, and
series of small filtering stations called lymph present these proteins to lymphocytes, initiating
nodes. Each node has both cortical (outer) and adaptive immune responses. (6) Adaptive immune
medullary (inner) regions in which different responses rely on the activation of key antigen-
aspects of the immune response occur. specific lymphocytes (around 1 in each 100,000
lymphocytes). Once activated, these cells undergo
rapid replication, expanding a few key lympho-
duce coincidental adaptive immune responses cytes to millions of identical cells (clones), a pro-
cess called clonal expansion. (7) The result is an
(see Figure 1.10).
army of lymphocytes that specifically recognize
Every day, every animal encounters liter-
parts of the invading microbe and effect a targeted
ally trillions of potential threats in the form attack by producing antibodies or by mobilizing
of transmissible pathogens—viruses, bacteria, and coordinating other immune processes. Not
fungi, and parasites. These agents of disease, only do these adaptive immune responses directly
if unchecked, would quickly destroy us all. Im- kill invading microbes or infected cells, but many
mune systems have evolved to help prevent that. of them at the same time enhance the efficiency
To do this, immune systems recognize specific of innate antimicrobial mechanisms already in
pieces of pathogens as foreign and, in response, play. (8) During clonal expansion, a portion of key
induce a series of protective mechanisms that antigen-specific lymphocytes differentiate into
involve both innate and adaptive reactions. The long-lived memory cells—allowing more efficient
ability to generate those reactions is why we are adaptive immune responses on repeat exposure
to the same threat (immunologic memory).
all here. The remainder of this book explores

15

animals’ remarkable immune systems and their tic infections apparent in rhesus macaques and
interactions with the infectious world. other monkeys infected with SIV.
Clinical Correlation Follow-Up

Possible Explanations
Student Considerations As shown in this chapter, T cells are essen-
Further investigations of the affected mon- tial components of almost all adaptive immune
keys showed that SIV specifically infected T responses against both intra- and extracellular
cells, particularly Th cells, a type of T cell nec- pathogens. As SIV destroys more and more T
essary for almost all types of adaptive immune cells, these monkeys’ immune systems grow
responses and for amplification of many innate weaker and weaker. Eventually, the total num-
immune responses. ber of T cells drops below the levels needed
SIV is a retrovirus, which means that during to generate effective immune responses. At
infection, viral RNA serves as the template for that point, the monkeys begin to show signs
new complementary DNA that, with the help of of an acquired immunodeficiency syndrome.
an integrase, inserts itself into a host’s chromo- Although the innate immune system is still
somal DNA. This SIV provirus may remain inac- in play, without help from T cells it is quickly
tive for years, but once the host T cell activates— overwhelmed by normally innocuous mi-
for example, during an immune response—viral crobes, such as Candida albicans, that become
DNA becomes active, and those host cells begin opportunistic pathogens and blossom across
to produce infectious SIV, which results in host- mucosal surfaces, causing oroesophageal can-
cell malfunction and often host-cell death. didiasis. These and other opportunistic infec-
After reading this chapter, you should be able tions quickly overwhelm and kill infected
to propose an explanation for the opportunis- monkeys.

Robin M. Yates
Overview of the Innate Immune System Chapter 2

10.5876_9781607322184.c002
susceptibility to bacterial infections. Within

Clinical Correlation: Cyclic Neutropenia in
Gray Collies 17 eight to twelve weeks of age, affected puppies
Learning Objectives 17 develop a broad range of symptoms, including
Anatomical and Physiological Barriers to fever, diarrhea, and joint pain as well as eye, re-
Microbial Invasion 18 spiratory, and skin infections. As a result, these
Epithelial Barrier 18 puppies often die before they reach six months
Secretions 18 of age. Monitoring the cell composition of
Mucus and Mucociliary Clearance 20 the collies’ blood over time by means of serial
Commensal Organisms 21 complete blood cell counts reveals an interest-
Cells of the Innate Immune System 21 ing phenomenon. Every twelve to fifteen days,
Hematopoiesis 21 these puppies develop a marked neutropenia
Coordinating Hematopoiesis 23 (lack of circulating immune cells called neu-
Monitoring Hematopoiesis 23 trophils), after which the number of circulating
Overview of the Cells of the Innate Immune neutrophils rebounds. During these periods of
System 25 neutropenia, the animals are extremely suscep-
Myeloid Innate Immune Cells 25 tible to bacterial infections that often result in
Neutrophils 25 illness and dramatically shortened lives. How
Eosinophils 26 does a genetic disorder affect neutrophil num-
Basophils 27 bers when other leukocytes seem unaffected?
Monocytes 28 And if the immune system is so complex, why
Macrophages 29 does the periodic loss of one cell type have such
Myeloid dendritic cells 30 devastating effects?
Mast cells 31
Lymphoid Innate Immune Cells 31
Learning Objectives
Natural killer cells 31
Other Innate Immune Cells 33 After reading this chapter, you should be able to
• describe the anatomical and physical barri-
ers that guard against infection;
• discuss how leukocytes are generated in the
bone marrow (hematopoiesis);
Clinical Correlation: Cyclic • list the major cells in the innate immune
Neutropenia in Gray Collies system;
An unusual genetic disorder of collies results • list the basic morphological features of the
in gray coat color (Figure 2.1) and increased major innate immune cells;
17
17

• outline the origin and anatomical location
of the major innate immune cells;
• describe the basic functions of the major
innate immune cells;
• list and understand the basic function of
the lymphoid innate immune cells.
Only vertebrates have adaptive immunity; in

Figure 2.1. Gray-coatedcollie pup (photograph
most species in the biome, host defense starts
provided by Michelle Tennis)
and stops with innate immunity. Starfish, for
example, have only innate immune systems, Affected pups are gray (often with a pinkish
yet they can live in some of the world’s most hue) with pale brown noses.
microbe-rich environments and, for the most
part, successfully avoid infection. It is interest- that coat the exterior surfaces of an animal,
ing that many of the components of the innate including the gastrointestinal, respiratory, and
immune system in starfish are strikingly simi- reproductive tracts. Several features of mucous
lar to those in more complex animals, such as membranes and skin make them ideal barriers
mice, dogs, cats, pigs, horses, and humans. (Figure 2.3A–C). First, the flattened epidermal
For most of the twentieth century, immunol- and epithelial cells adhere to one another, form-
ogists focused on the adaptive immune system; ing tight junctions, which create a continuous
however, the innate immune system is an intri- impermeable shield. Second, in the skin, the
cate and sophisticated system in and of itself. outer layer of the epidermis is made up of dead
In this chapter, we introduce the major cells of cells that are filled with keratin—a mechanically
the innate immune system of animals and dis- strong protein that also repels water. Third, the
cuss their origins. The following chapters focus epithelial cells of the skin and mucous mem-
on how these components function to imme- branes continually grow and shed, repeatedly
diately protect animals from harm and how re-creating a fresh, microorganism-free surface.
the innate and adaptive immune systems work This process is particularly important in the in-
together to form an integrated defense system. testine, where epithelial cells in the colon have
a turnover rate of approximately six days. Dur-
ing intestinal parasitic infections, the intestinal
Anatomical and Physiological
epithelial cell turnover rate dramatically accel-
Barriers to Microbial Invasion
erates to expel the parasites.
Apart from what one normally thinks of as an
immune system, animals have innate anatomi-
cal and physiological barriers that are very effec- Secretions
tive in preventing infection. These barriers are In addition to the barrier role played by epi-
not usually included as bona fide components dermal and epithelial cells, the secretions these
of the innate immune system, but because their cells produce also play a role in innate defense.
contribution to host defense is so immense, we In the skin, specialized sebaceous glands adja-
review them here (Figure 2.2). cent to hair follicles produce sebum. Sebum is
lipid based and water-repellent. Sebum is also
acidic (with a pH of approximately 3 to 5),
Epithelial Barrier which inhibits the growth of many microbes.
The most obvious barrier to microbial inva- Numerous other secretions contain anti
sion is the skin and the mucous membranes microbial proteins that specifically target un-
18 Ov e rv i e w o f t h e Inn a t e Imm u n e Sy s t e m

Figure 2.2. Anatomical and physiological barriers in animals
Figure 2.3A–C. Epithelial barriers

Skin: keratinized squamous epithelium (panel A); mucous membrane: nonkeratinized squamous epi-
thelium (panel B); epithelium of the large intestine: columnar epithelium (panel C)
wanted microbes at key sites around an animal’s peptides defensins and cathelicidins, produced
body. Lysozyme is one of these secretions. It is by epithelial cells, also appear in body secre-
found in tears, saliva, milk, and in the mucus in tions. These small, positively charged peptides
the gut. Lysozyme specifically degrades the pep- insert themselves into negatively charged micro-
tidoglycan cell walls of bacteria. The cationic bial membranes and punch holes in them (most
Ov e rv i e w o f t h e Inn a t e Imm u n e Sy s t e m 19
19

as hydrochloric acid, which acidifies the gastric
contents to a pH of 2 or less. As food moves
out of the stomach into the duodenum, the pH
is rapidly increased by the addition of bile. Bile
also contains numerous detergents that, along
with the pancreatic enzymes, act to disrupt mi-
crobial membrane integrity.
Aside from the specific antimicrobial actions
of many secretions, the physical flushing of
milk, tears, saliva, and urine removes many mi-
crobes from the mammary glands, eyes, mouth,
and urinary tract. The importance of simple
unidirectional flushing of surfaces is very evi-
dent in animals that cannot fully empty their
urinary bladders (as can happen, e.g., in dogs
with lower spinal cord injury). In the absence
of regular flushing of the lower urinary tract,
microbes can more easily colonize and migrate
up the tract, predisposing these animals to both
Figure 2.4. Proposed mechanism of action of lower and upper urinary tract infections.
defensins
These antimicrobial peptides have clusters of
positive charges alongside regions of hydro-
Mucus and Mucociliary Clearance
phobicity that allow them to integrate into Mucosal surfaces (including those lining the
negatively charged membranes. Integration of respiratory and reproductive tracts as well as
several defensins can destabilize the microbial the small and large intestines) secrete large
membrane, creating pores through which water amounts of mucus. This sticky, viscous sub-
and solutes can pass. In sufficient numbers, stance coats epithelial cell layers, providing an
these pores can substantially perturb osmo
additional mechanical barrier to microorgan-
regulation of microbes and cause them to lyse.
isms by preventing them from binding to the ep-
ithelial surfaces. It also immobilizes microbes,
membranes in animal cells are positively charged making them easier to expel.
and resistant to these peptides; Figure 2.4). Although mucus produced by the intesti-
Some leukocytes also synthesize and secrete de- nal mucosa is cleared along with the intestinal
fensins, as do intestinal crypt Paneth cells. Other contents via peristalsis, mucus in the respira-
antimicrobial proteins can be found in milk, tory tract (along with trapped microbes and
where they help to inhibit microbial growth in debris) is expelled by a process referred to as
the lactating mammary gland. These proteins mucociliary clearance. Cilia are minute, finger-
include lactoferrin, which sequesters iron, mak- like projections on the apical (lumenal) surface
ing it unavailable for microbial growth, and lac- of the respiratory epithelium that move in co-
toperoxidase, which generates reactive oxygen ordinated waves (Figure 2.5). These waves act
compounds that damage microbes. to push mucus out of the airway and into the
The gastrointestinal tract produces multiple esophagus, inducing swallowing. Inactivation
secretions that not only mediate digestion but of mucociliary clearance results in the accumu-
also kill many unwanted microbes. The gastric lation of microbe-laden mucus in the airways.
mucosa secretes degradative enzymes as well In a rare heritable disease in dogs called ciliary

dyskinesia, affected dogs have an abnormality commensal organisms but inhibits growth of
in the structure of cilia that renders them non- the majority of other microbes.
functional. Because of this abnormality, these A growing body of evidence supports the clin-
dogs are unable to clear mucus and are prone ical benefits of pro-biotic supplementation of
to upper and lower respiratory tract infections. “good” microbes, including protection from gas-
trointestinal disease, reducing the length of gas-
trointestinal infections, and reducing the length
Commensal Organisms of antibiotic-induced diarrhea (a side effect of
The skin and the reproductive, respiratory, antibiotic-mediated destruction of natural flora).
and intestinal tracts of healthy animals are Commensal intestinal bacteria also have a pro-
colonized by an array of commensal organ- found influence on the development of the in-
isms, particularly bacteria. These organisms testine itself and the adaptive immune system.
typically do not cause disease but, among other Overall, the barriers inherent in an animal’s
things, prevent the growth of pathogenic mi- anatomy and physiology effectively prevent
crobes. Prevention is achieved in part by the the invasion of most environmental microbes.
well-adapted commensal organisms’ simply These barriers, however, are not impenetrable.
outcompeting foreign microbes for space and Microbial breach is common. The second line
nutrients but also by creating a chemical mi- of defense faced by invading microbes is the in-
croenvironment that allows some microbes to nate immune system.
flourish while others perish. For example, com-
mensal lactobacilli within the vagina create an
Cells of the Innate
acidic environment that favors the growth of
Immune System
The innate immune system is made up of a
host of immune cells as well as soluble fac-
tors such as complement and acute-phase pro-
teins (APPs, which are discussed in subsequent
chapters). Unlike the adaptive immune system,
which includes recognizable tissues and organs
such as lymph nodes, spleen, and thymus, the
innate immune system is spread throughout
every tissue of every animal. In spite of this dif-
fuse anatomical distribution, innate immune
cells have a common origin in the bone marrow.
Hematopoiesis
The production of immune cells in the bone
marrow is termed hematopoiesis (literally the an-
cient Greek words for blood and to make). As the
Figure 2.5. Scanning electron micrograph of name suggests, hematopoiesis produces not only
ciliated epithelium lining the respiratory tract immune cells but also red blood cells (RBCs) as
These long projections move in coordinated well as platelets. Amazingly, all of these arise
waves that push mucus and ensnared microbes during a highly coordinated and complex pro-
up and out of the respiratory tract. Image by cess that produces as many as 1011–1012 cells per
Charles Daghlian.
day from common hematopoietic stem cells.
21

Figure 2.6. An overview of hematopoiesis showing major points of differentiation and anatomical
location in healthy animals
Hematopoiesis has three major lineages: the dedicated immune cells in animals (Figure
erythroid, myeloid, and lymphoid. The ery- 2.6). Most of the cells of the innate immune
throid lineage primarily produces RBCs (eryth- system arise from the myeloid lineage. These
rocytes) and platelets. The remaining two lin- cells include the granulocytes (neutrophils,
eages, myeloid and lymphoid, produce all of eosinophils, and basophils), mast cells, mono-

cytes, macrophages, and myeloid dendritic cells ets or niches in the bone marrow that favor the
(DCs). The lymphoid lineage predominantly differentiation of particular hematopoietic lin-
produces lymphocytes (T and B cells) that eages, allowing different areas of the bone mar-
function in adaptive immune responses. The row to intensively support different hematopoi-
lymphoid lineage also produces natural killer etic activities (Figure 2.7).
(NK) cells that, unlike most T and B cells, func- Because the majority of innate immune cells
tion in the innate immune system. Other lym- derive from the common myeloid precursor
phoid cells have innate immune function, but stem cell, the activity of each differentiation
they apparently play only minor roles in innate pathway is what determines the final compo-
immune defense, and we consider them only sition of cells released from the bone marrow.
briefly here. Levels of growth factors (regulated by feedback
loops), along with external signals, regulate
these pathways. For instance, IL-5, IL-3, and
Coordinating Hematopoiesis GM-CSF favor eosinophil production; G-CSF,
With so many different bone marrow lin- GM-CSF, and IL-3 favor neutrophil production;
eages, all with quite different roles, it is impor- M-CSF, GM-CSF, and IL-3 favor monocyte pro-
tant that the bone marrow be able to selectively duction; and IL-4 and IL-9, along with GM-CSF,
increase or decrease the production of each of favor basophil and mast-cell differentiation, re-
the lineages in response to the body’s demands. spectively. Of course, other cytokines play roles
It would be wasteful to increase erythrocyte in the differentiation of these cells, but it is easy
production during inflammation and, likewise, to see that by varying the levels of cytokines, it
neutrophil production during anemia. For is possible to achieve a fine balance of lineage
these reasons, animals have evolved hemato- expansion or contraction.
poietic growth factors for specific regulation of At each step in a cell lineage, precursor cells
bone marrow cell differentiation and division. undergo irreversible commitments to fewer
The hematopoietic growth factors include a possible cell types. Expression of cell-specific
family of proteins known as colony-stimulating receptors and effector molecules as well as
factors (CSFs), which include granulocyte– changes in cellular morphology reflect this pro-
monocyte CSF (GM-CSF), granulocyte CSF gressive differentiation.
(G-CSF), and macrophage CSF (M-CSF). In ad- Once mature, the newly formed leukocytes,
dition to mediating inflammatory processes (as erythrocytes, and platelets move from the bone
we discuss in later chapters), several interleukins marrow into the blood through highly fenes-
serve as hematopoietic growth factors (includ- trated capillary networks. Some of the mature
ing IL-1, IL-3, IL-4, IL-5, IL-6, and IL-7). These leukocytic cells (particularly neutrophils and, to
interleukins play important roles in regulating a lesser extent, eosinophils and monocytes) re-
levels of immune cell production to meet the main in the blood, circulating through the body
immediate needs of an animal’s defense system. and exiting into surrounding tissues as needed.
Hematopoiesis in the bone marrow occurs in Other cells, such as mast cells, DCs, and mac-
areas between stromal reticular cells and adipo- rophages, are mostly directed to other tissues.
cytes (fat cells). Stromal reticular cells form a
very fine meshwork of spindled cells that pro-
vide the structural framework of the bone mar- Monitoring Hematopoiesis
row. In addition, stromal reticular cells (along Normally, the bone marrow of healthy ani-
with osteoblasts) produce and bind certain mals releases balanced numbers of mature leu-
hematopoietic growth factors, increasing their kocytes into circulation. But during disease,
local concentration. This process creates pock- when demand is high, increased numbers of
23

Figure 2.7. Bone marrow
Representation of localized hematopoiesis in the bone marrow. Hematopoietic precursor cells are
enmeshed in a fine stromal network that creates small niches or pockets where growth factors are
differentially concentrated. This compartmentalization is thought to support localized growth and
differentiation of particular lineages.
certain leukocytes, including their immature

forms, appear in the circulation. Blood counts
and differentials (the identification and propor-
tion of cell types in blood smears) provide evi-
dence of such changes. Large increases in leu-
kocyte counts, particularly neutrophils (termed
neutrophilia), often indicate active inflamma-
tion. When immature neutrophils (band neu-
trophils) appear in large numbers in the blood
(a phenomenon called a left shift), it is usually
indicative of severe, acute inflammation.
In addition, bone marrow aspirates allow Figure 2.8. Monitoring changes to hematopoiesis
practitioners to finely gauge the marrow’s re- can be achieved indirectly through complete
sponse to disease. During inflammation, the blood cell counts or directly by observing
number of cells differentiating into leukocytes changes in bone marrow aspirates.
increases. For example, in allergies, increased
numbers of eosinophil precursors appear in the a cannula directly into the marrow cavity. Be-
bone marrow. Bone marrow aspiration, how- cause of that, it is typically used only when the
ever, requires anesthesia and the insertion of bone marrow is suspected of failing or to diag-

nose bone marrow neoplasia. More commonly, of producing a variety of antimicrobial toxins.
practitioners exploit changes in circulating Under normal conditions, the majority of neu-
blood leukocytes to diagnose certain diseases. trophils remain in the blood in a resting state
for the entirety of their short lives (a few days).
However, circulating neutrophils have the abil-
Overview of the Cells of ity to swarm in huge numbers into inflamed tis-
the Innate Immune System sues where they unleash a storm of antimicro-
As mentioned earlier, the highly complex he- bial effectors. In these instances, neutrophils die
matopoietic process produces different subsets even more rapidly, often victims of their own
of innate immune cells. Here we provide only effector molecules. The extent of neutrophil
a brief introduction to these innate immune recruitment and death is often apparent as pus,
cells. More details appear in later chapters. which is composed mostly of dead neutrophils.
In their unactivated state in the blood, neu-
trophils are uniformly spherical. On activation,
Myeloid Innate Immune Cells typically accompanied with extravasation (exit
Neutrophils from the bloodstream), the neutrophils as-
These cells make up the majority of circulat- sume amoeboid shapes, allowing the cells to
ing leukocytes in many species and are capable crawl (chemotax) in the interstitial spaces. The
Figure 2.9. Canine neutrophil (mature) in a blood smear stained with Wright’s Giemsa
Name: Neutrophils
Other names: Polymorphonucleocytes; Heterophils (in birds and reptiles)
Classifications: Granulocyte, phagocyte
Lineage: Myeloid
Appearance: Segmented nucleus, granular cytoplasm
Location in health: Blood
Life span in health: Forty-eight to seventy-two hours
Primary function: Antimicrobial effectors, particularly in acute bacterial infection
Mechanism of action: Phagocytosis; Degranulation; Neutrophil extracellular trap formation
25

cytoplasm of neutrophils is filled with granules. Second, activated neutrophils can kill extracellu-
These granules fail to stain in common hema- lar microbes by degranulation and secretion of
tology and histology stains, resulting in a neu- antimicrobial proteins and the release of toxic
tral appearance, hence the name neutrophil. metabolites. Third, neutrophils release meshlike
The segmented nuclei of mature neutrophils substances called neutrophil extracellular traps
are unique and hard to miss in a blood smear. (NETs). These NETs, consisting of DNA and
The degree of nuclear segmentation is an indi- certain proteins, can trap and kill extracellular
cator of neutrophil maturation, with the imma- microbes (literally as a fishing net does). In addi-
ture band neutrophils possessing unsegmented tion to killing microbes, neutrophils participate
nuclei and older neutrophils having highly seg- in the coordination of inflammation through
mented nuclei. the release of inflammatory mediators.
As stated earlier, the primary function of
neutrophils is to kill invading microbes. Neu-
trophils achieve this through several effector Eosinophils
mechanisms. First, neutrophils engulf (eat) Eosinophils are especially effective in killing
microbes via phagocytosis and destroy them multicellular parasites such as parasitic hel-
in intracellular vesicles called phagolysosomes. minths (worms). Eosinophils also have some
Figure 2.10. Canine eosinophil in a blood smear stained with Wright’s Giemsa
Name: Eosinophils
Other names: Eosinophil granulocytes
Classifications: Granulocyte
Lineage: Myeloid
Appearance: Characteristic eosinophilic granules (staining red with eosin)
Location in health: Blood and tissues lining gastrointestinal tract and airways
Life span in health: Days to weeks
Primary function: Antiparasitic effectors, particularly in helminthic infection; Some antiviral action; Roles
in allergy
Mechanism of action: Degranulation; Limited phagocytosis

antiviral capabilities and are active in certain al- Once in the tissue and activated by parasite or
lergic responses (particularly in cats and horses). leukocyte products, resident or newly recruited
Their role in tumor immunity remains unclear. eosinophils kill invading parasites. Because mul-
After release from the bone marrow, mature ticellular parasites are too big to be engulfed by
eosinophils circulate in the blood and make up phagocytosis, eosinophils release antiparasitic
approximately 0.1 percent to 3.0 percent of leu- proteins and toxic metabolites into extracellular
kocytes in most species. In blood smears, their spaces to harm or kill these multicellular invad-
granules stain bright red. The morphology ers. They are also active in certain severe allergic
of eosinophil nuclei can range from round to responses in cooperation with mast cells and ba-
multilobular. Unlike neutrophils, eosinophils sophils and have roles in antiviral responses. For
normally exit the blood after a brief period the most part, however, eosinophils are innate
of circulation and spend most of their lives in granulocytes and specialists in combating para-
healthy tissues, particularly in the connective sitic, particularly helminth, infections.
tissues underlying mucosal surfaces. It is here
that eosinophils are most likely to encounter
multicellular parasites. During parasitic infec- Basophils
tions, eosinophils still circulating in the blood In most domestic species, basophils typically
are specifically recruited to the affected tissue. make up less than 0.5 percent of leukocytes
Figure 2.11. Bovine basophil in a blood smear stained with Wright’s Giemsa
Name: Basophils
Other names: Basophil granulocytes
Classifications: Granulocyte
Lineage: Myeloid
Appearance: Characteristic blue-purple basophilic granules with basic dyes
Life span in health: Days
Primary function: Mediator of inflammation
Mechanism of action: Degranulation
27

Figure 2.12. Feline monocyte in a blood smear stained with Wright’s Giemsa
Name: Monocytes
Classifications: Mononuclear phagocytes
Lineage: Myeloid
Appearance: Large round to kidney-shaped nucleus, diffuse pale blue-gray staining cytoplasm com-
monly containing vacuoles
Life span in health: Days (in circulation)
Primary function: Precursors of macrophages and DCs
Mechanism of action: Limited antimicrobial function in blood
circulating in the blood. Although rare, their Monocytes

characteristic dark-staining large cytoplasmic Monocytes are circulating precursors of,
granules (when stained with basic dyes) make among other cell types, macrophages and DCs.
it easy to identify them on a blood smear. Dur- In most species, monocytes make up 2 percent
ing multicellular parasitic infections and late- to 10 percent of circulating leukocytes. In a
phase allergic reactions, basophils migrate into blood smear, these cells appear to be 12–20 µm
inflamed tissues. Here, basophils coordinate in- in diameter with diffusely stained cytoplasm
flammation (in a manner similar to that of mast and large, round, indented, or kidney-shaped
cells) and promote the adaptive immune reac- nuclei. Monocytes typically circulate for a few
tions best suited to combating parasites (called days before exiting the circulation to differen-
a Th-2 response, covered in later chapters). Un- tiate into tissue macrophages or DCs. Under
fortunately, these functions of basophils also normal circumstances, monocytes perpetually
amplify the unwanted effects associated with replenish tissue macrophages and DCs in all
allergies (type I hypersensitivities). Overall, ba- tissues. However, in inflamed tissues, mono-
sophils are mediators of inflammation associ- cytes rapidly exit the blood to quickly populate
ated with parasitic infections and allergies. the area with large numbers of macrophages.

Figure 2.13. Murine tissue macrophage grown in cell culture and stained with Diff-Quik
Name: Macrophages
Other names: Tissue macrophages; Kupffer cells (macrophages in liver); Splenic macrophages (mac-
rophages in spleen); Microglial cells (macrophage equivalents in the central nervous
system); Alveolar macrophages (macrophages in airways); Peritoneal macrophages
(macrophages in the peritoneal cavity)
Classifications: Mononuclear phagocyte; Sentinel cell; Antigen-presenting cell
Lineage: Myeloid
Appearance: Round nucleus, clear-vacuolated cytoplasm, irregular cell shape
Location in health: Peripheral tissue
Life span in health: Months
Primary function: Immune surveillance, moderate antimicrobial capacity, limited antigen presentation
Mechanism of action: Detection of threats and release of inflammatory mediators; Phagocytosis
Monocytes themselves are phagocytic and pro- As mentioned earlier, macrophages arise from
duce some cytokines but are generally not very monocytes that exit the circulation. Once mono-
proficient at these tasks until they fully differ- cytes are in a tissue, various tissue factors drive
entiate into macrophages or DCs after exiting the monocytes’ maturation into macrophages.
the blood. Because this process occurs in practically every
tissue and organ, different combinations of tis-
sue factors produce numerous organ- or tissue-
Macrophages specific macrophage types. Although different
Within the immune system, macrophages macrophage types may differ in location, ho-
are sentinel cells, antimicrobial effector cells, meostatic function, morphology, and name,
and antigen presenting cells (APCs). During most perform similar functions in the immune
health, macrophages also perform numerous system. Macrophages are generally larger cells
housekeeping functions, such as removal of with copious amounts of membrane. They are
dead and damaged cells and cellular debris, extremely pleomorphic (i.e., they vary greatly
tissue remodeling, and maintenance of iron in their size and shape), adopting spherical, spin-
homeostasis. dle, or amoeboid cellular forms.
29

Figure 2.14. Murine DC grown in culture and stained with Diff-Quik
Name: Myeloid dendritic cells
Other names: Inflammatory DCs; Conventional DCs; Langerhaan cells (DCs in skin)
Classifications: Mononuclear phagocyte; Sentinel cell; Antigen-presenting cell
Lineage: Myeloid
Appearance: Round nucleus, clear cytoplasm, irregular shape with long branched projections
(dendrites)
Location in health: Tissue
Life span in health: Months
Primary function: Immune surveillance, antigen processing and presentation
Mechanism of action: Detection of threats and release of inflammatory mediators; Endocytosis and
phagocytosis
Macrophages are very active sentinel cells, al- Although macrophages are not as microbi-
ways on the lookout for signs of danger. They cidal as neutrophils, they are reasonably adept
commonly crawl around in the extracellular at phagocytosis and killing microbes inside
spaces and have high rates of endocytosis and of phagolysosomes. Unlike most neutrophils,
pinocytosis (two methods of taking up extra- however, macrophages can also keep pieces of
cellular fluid). These activities allow them to protein from the phagocytosed bacteria and, in
constantly patrol the tissue and sample mol- a process called antigen presentation, present
ecules in the extracellular fluid. Detection of these pieces to T cells of the adaptive immune
a threat is achieved using a variety of recep- system (see chapter 8).
tors that recognize microbial products as well
as products of tissue damage. Once activated,
macrophages produce proinflammatory cyto- Myeloid dendritic cells
kines that engage other aspects of the immune The term dendritic cell (DC) has been ap-
system and promote the recruitment of other plied to a number of different cell types with
leukocytes to the area. different origins and functions (see discussion

of other innate cells). Thus, the term does not out the body (especially the connective tissues
always define a single cell type. However, my- surrounding blood vessels and nerves, as well
eloid DCs (DCs of myeloid origin) are typically as in the lamina propria of mucosal surfaces),
just referred to as DCs (as we do here). where they then differentiate into mature mast
DCs are innate sentinel cells that are highly cells. Because mast cells act as sentinel cells,
efficient at processing and presenting antigens they express a variety of receptors that can de-
to T cells. Because antigen presentation is es- tect innate danger signals. Mast cells also have
sential for T-cell adaptive immunity, DCs func- receptors that recognize IgE, which is impor-
tion at the interface between the innate and the tant in adaptive immune responses to parasites
adaptive immune systems. and allergens.
DCs are present in practically every tissue The cytoplasm of a mature mast cell contains
and organ of the body. Like macrophages, DCs densely packed granules full of proinflamma-
derive from circulating monocytes that have ex- tory mediators. On detection of a threat (either
ited the blood and migrated into tissues. If given directly or through products released by other
the right stimulus, monocytes mature into DCs leukocytes or by complement activation), mast
that take up residence by extending their long cells degranulate and release the proinflam-
branched cellular projections between cells and matory granule contents into the extracellular
connective tissues, which gives the DCs their space. These granule components, together with
intricate shape as well as their name (dendron the lipid mediators and cytokines synthesized
is Greek for “tree”). Within tissues, DCs act as by the mast cells, act on local cells and vascula-
sentinel cells. They express numerous receptors ture, resulting in inflammation (particularly by
that detect tissue damage and infection, and in increasing vascular dilation and permeability).
response they synthesize cytokines that activate This inflammation not only alerts the immune
other immune cells. Similarly to macrophages, system to the threat but also assists in the re-
DCs can also endocytose and phagocytose mi- cruitment of effector cells that can combat the
crobes, but unlike macrophages, DCs are not threat. Mast cells also play important roles in the
very effective in killing the microbes within the coordination of chronic inflammation and tissue
phagolysosome. However, DCs are especially repair. Overall, mast cells are specialized sentinel
efficient at processing protein antigens, display- cells strategically positioned in tissues and, when
ing bits of those antigens on their surfaces, and triggered, quickly promote inflammation.
presenting them to T cells. In fact, DCs are the
only APCs that can effectively initiate T-cell re- Lymphoid Innate Immune Cells
sponses to new antigens. For this reason, DCs
are often referred to as professional APCs. Natural killer cells
NK cells earn their name because of their
ability to innately recognize and kill abnormal
Mast cells host cells without previous sensitization. NK
Mast cells, like macrophages and DCs, are cells do not express either T- or B-cell receptors,
sentinel cells involved in immune surveillance. which distinguishes them from NK T lympho-
On detection of a threat, mast cells rapidly re- cytes, which are named for their similarity to
lease potent mediators of inflammation, which both NK cells and T cells but function in both
are usually protective. Mast cells, however, adaptive and innate immune responses.
also play a major role in allergies (type I hyper NK cells arise in the bone marrow from the
sensitivities). lymphoid lineage, but go through a second
Mast-cell precursor cells arise in the bone round of maturation (termed licensing) in sec-
marrow and quickly migrate into tissues through- ondary lymphoid tissues (lymph nodes, spleen).
31

Figure 2.15. Canine mast cell from a fine-needle aspirate stained with Wright’s Giemsa
Name: Mast cells

Classifications: Sentinel cells
Lineage: Myeloid
Appearance: Round nucleus, cytoplasm densely packed with granules (purple)
Location in health: Tissue, particularly connective tissue surrounding vasculature and nerves, and the lamina
propria of the mucosa
Life span in health: Weeks to months
Primary function: Immune surveillance, mediator and amplifier of inflammation and allergy
Mechanism of action: Detection of threats and release of inflammatory mediators via degranulation (vasoac-
tive amines) or synthesis of lipid mediators and cytokines
Mature, licensed NK cells then enter the circula- anisms are ineffective against them. Similarly,
tion, where they can account for as much as 2 many tumor cells differ very little from normal
percent to 15 percent of lymphocytes. NK cells host cells. However, both virus-infected and
are larger than most circulating lymphocytes and tumor cells often express abnormal molecules
have cytoplasms that contain azurophilic gran- on their surfaces or lose markers that normally
ules (hence, they are often described as “large identify the host cells as self. NK cells use host
granular lymphocytes”). These granules contain cell–surface proteins to identify normal and ab-
proteins, called granzymes and perforins, that normal cells. If a host cell appears abnormal,
are key to NK cells’ cytotoxic abilities. NK cells the NK cell forms a tight adhesion to the target
also appear in normal tissues, particularly under- cell and precisely delivers cytotoxic molecules
lying mucosal surfaces; however, their function from its granules to the abnormal cell, forcing
in these locations is less well understood. it into apoptosis (programmed cell death). An-
NK cells constitute the first line of defense tibodies, produced during an adaptive immune
against viral infections and possibly some types response, can also trigger NK cell cytotoxicity
of tumor cells. Because viruses replicate inside in a process called antibody-dependent cell-me-
host cells, the majority of innate immune mech- diated cytotoxicity.

Figure 2.16. Canine NK cell in blood smear stained with Wright’s Giemsa
Name: Natural killer cells

Other names: Null lymphocytes
Classifications: Lymphocyte
Lineage: Lymphoid
Appearance: Large lymphoid cell, round nucleus, azurophilic cytoplasmic granules
Location in health: Blood, spleen
Life span in health: Weeks to months
Primary function: Destruction of virally infected or abnormal host cells (including tumor cells)
Mechanism of action: Recognition of virally infected or abnormal host cells and targeted release of cytotoxic
granules
Other Innate Immune Cells good at presenting antigen, but during viral
infections, they can produce IFN-α, a powerful
In addition to NK cells, B-1 cells and γδ T cells antiviral agent.
are innate immune cells of lymphoid origin. FDCs appear in the B-cell regions of lymph
Because both of these cell types share much in nodes and probably derive from mesenchymal
common with their adaptive immune cousins cells. They function to retain unprocessed an-
B-2 cells and αβ T cells, the specifics of these tigen on their surfaces, which supports B-cell
innate lymphocytes are covered in chapters 10 maturation during a humoral immune re-
and 12. sponse (covered in chapter 12).
As mentioned, DCs also have different sub-
types. In addition to myeloid DCs, there are
plasmacytoid DCs (pDCs) and follicular den- Clinical Correlation: Follow-Up
dritic cells (FDCs).
pDCs, named for their resemblance to Student Considerations
plasma cells, are of lymphoid origin and cir- As described at the beginning of this chap-
culate in the blood. They are not particularly ter, collies with cyclic neutropenia go through
33

Figure 2.17A–B. Cyclic neutropenia
Two blood smears (stained with Wright’s Giemsa) taken from a gray collie during (panel A) and after
(panel B) a period of neutropenia. Note the difference in mature neutrophils between smears.
cycles of neutrophil depletion that coincide cause. The mutation disrupts the normal traf-
with increased susceptibility to many types of ficking of granular proteins (such as elastase)
infections, especially bacterial infections. After to cytoplasmic granules during certain stages
reading this chapter, you should be able to in hematopoiesis. This disruption results in a
propose an explanation for the coincidence of cyclic arrest of maturation of myeloid progeni-
neutropenia and susceptibility to opportunistic tor cells in the bone marrow followed by their
bacterial infections. apoptosis—probably the result of buildup of in-
correctly trafficked granule proteins in the cell.
The end result of this mutation is that these
Possible Explanations gray collies have compromised production of
Canine cyclic neutropenia is a genetic disor- early myeloid cells.
der that affects melanocytes (resulting in the The understanding behind the cyclic nature
gray coat color) and bone marrow stem cells. of the defect is less clear, with several conflict-
Affected puppies exhibit cyclic fluctuations in ing theories proposed. However, it is likely that
all their blood cells (RBCs, platelets, and leu- the cyclic nature of the neutropenia is related
kocytes), but because neutrophils have the to feedback loops involving different stages of
shortest life span of these cells, the most char- myeloid precursor cells, which result in oscillat-
acteristic finding is cyclic neutropenia (Figure ing levels of growth factors, apoptosis, or both.
2.17A–B). Nonetheless, because neutrophils are one of
A mutation in the gene encoding the protein the primary innate defenders against bacterial
AP3β1 has been identified as the underlying attack, their depletion in periods of neutrope-

nia most likely underpins the clinical presenta- bolstering myelopoiesis by increasing the con-
tion of recurrent bacterial infections. centration of the hematopoietic growth factor
An interesting finding is that although these G-CSF through injection of exogenous G-CSF
puppies usually die at a very young age, there or increasing the expression of G-CSF through
has been some success in the treatment of this gene therapy.
lifelong disease. Successful strategies involve
35

Robin M. Yates
Innate Immune Recognition Chapter 3

10.5876_9781607322184.c003
foal’s blood via wounds, such as the umbilical

Clinical Correlation: Sepsis in Neonatal Foals 37
stump, or after ingestion or inhalation of envi-
ronmental bacteria. The original infection can
Introduction 38
also originate in utero.
Evolutionarily Conserved Molecular Patterns 40
The most common type of bacteria isolated
Pathogen-Associated Molecular Patterns 40
from septic foals is Gram-negative Escherichia
Pattern-Recognition Receptors 40
coli (E. coli), which accounts for 30 percent to
Signaling Pattern-Recognition Receptors 41
60 percent of reported cases. Bacteremic foals
Toll-like receptors 42
typically present with fever, malaise, and gen-
NOD-like receptors and RIG-like receptors 44
eral ill thrift. These symptoms can rapidly
Phagocytic Pattern-Recognition Receptors 44
progress to life-threatening sepsis, also known
Other Pattern-Recognition Receptors 46
as bacteremia-induced systemic inflammatory
Innate Recognition of Damage-Associated
response syndrome (SIRS). SIRS results from
Molecular Patterns 46
a systemwide uncontrolled activation of mul-
Inflammasome 48
tiple proinflammatory reactions. If not aggres-
Consequences of Danger Signal Recognition
by Sentinel Cells 50 sively treated, prolonged SIRS and its associated
Activation of Macrophages 50 shock (severe hypotension) can lead to multior-
Proinflammatory Mediators 50 gan failure and death (Figure 3.1).
Clinical Correlation: Follow-Up 50 It is easy to rationalize why bacteria in the
Student Considerations 50 blood (particularly in an immunocompromised
Possible Explanations 50 neonate) could result in severe illness. It is in-
teresting, however, that although the bacteria
themselves may be the instigators, they are not
Clinical Correlation:
the pathogenic force behind the symptoms of
Sepsis in Neonatal Foals
SIRS.
The medical management of septic foals is a What is doing all this damage? SIRS resulting
major challenge for equine practitioners. Sepsis from septicemia is primarily propagated by the
is a systemic inflammatory response to infec- innate immune system that is overactivated by
tion that in foals is a common sequela of bacte- disseminated bacterial products (not necessar-
remia (infection of the blood by live bacteria). ily the bacteria themselves). In the case of sepsis
Neonatal foals are particularly susceptible to from Gram-negative bacteria such as E. coli, one
bacteremia, especially foals of mares that are of the most potent activators of the immune
sick or experience abnormal or difficult par- system is lipopolysaccharide (LPS). LPS is also
turition or foals that fail to suckle soon after commonly referred to as an endotoxin (“inside
birth. Bacteria can gain entry to the neonatal toxin”). In fact, it is not a toxin in the classic sense
37
37

Figure 3.1. A septic neonatal foal undergoing intensive life-supporting therapy (photograph courtesy
of Dr. Michel Levy, University of Calgary)
(such as cholera toxin or botulinum toxin, which • explain the concepts of evolutionarily con-
have evolved to purposefully cause dysfunction) served molecular patterns;
but a structural component of the outer mem- • define the terms PAMPs, DAMPs, and PRRs;
branes of Gram-negative bacteria. However, • give examples of PAMPs and DAMPs and
LPS elicits such a strong inflammatory reaction the PRRs that recognize them;
in animals that it is often viewed as toxic, and the
• list the general functions and cellular loca-
reaction is often referred to as endotoxic shock.
tions of the major PRR families;
Experimentally, SIRS can be induced by the in-
jection of purified LPS in the complete absence
• explain the function and formation of the
inflammasome;
of live bacteria. How is a chemically inert mol-
ecule made up of lipid and sugars able to cause • list the consequences of sentinel cell expo-
so much damage? Clearly, in the case of septice- sure to PAMPs and DAMPs.
mia in a neonatal foal, the immune reaction to
LPS is detrimental. What is the advantage to the
animal, if any, of reacting to LPS at all? Introduction
The immune system of an animal cannot be
Learning Objectives fully activated in all tissues at all times. Not only
would this lead to the depletion of immune
After reading this chapter, you should be able to components and energy, but it would also cause
• describe in general terms how the innate tissue and organ damage. Instead, the immune
immune system recognizes threats; system needs to be in a dormant but alert state
38 Inn a t e Imm u n e R e c o gn i t i o n

Figure 3.2. The structure of LPS
LPS is a macromolecule consisting of a lipid component (lipid A), a core oligosaccharide, and a vari-
able polysaccharide side chain (O antigen). It is an integral component of Gram-negative bacteria,
conferring structural stability and chemical resistance to the outer membrane. The scanning electron
image of E. coli was created in the Rocky Mountain Laboratories, National Institutes of Health.
and be selectively activated when and where it tween self and non-self antigens (how it does
is needed. So, one of the first and most impor- this has preoccupied the field of immunology
tant functions of an animal’s immune system is for decades). But when an animal is faced with
to differentiate between threatening and non- an entity it has never encountered, it needs to
threatening situations. You are probably aware be able to instantly and instinctively determine
by now that the intricate adaptive immune whether it is a threat and be able to act accord-
system can learn over time to differentiate be- ingly. The innate immune system has evolved
Inn a t e Imm u n e R e c o gn i t i o n 39
39

to innately determine whether something is those species continue to express these specific
potentially hazardous or not without having to molecules.
learn how to make that distinction. It can then
selectively use offensive strategies to fight this
new threat. However, the importance of the in- Pathogen-Associated
nate detection of a threat does not stop with Molecular Patterns
the deployment of innate immune effectors; The basic structures of microbes (bacteria,
the innate responses also direct subsequent fungi, protozoa, and viruses) and mammalian
learned or adaptive immune responses. cells have many key differences. For example,
Animals encounter countless new foreign most bacteria have a particular cell wall struc-
antigens every day but only mount an adaptive ture that is made up of a substance called pepti-
immune response to some. Moreover, the most doglycan (a network of alternating sugars cross-
effective type of adaptive immune response linked by amino acids). Because mammalian
mounted (e.g., humoral vs. cellular) appears cells do not have cell walls, this arrangement of
to be directed by the type of infecting agent. molecules is absent from normal mammalian
For decades, the adaptive immune components tissue, and thus it is a distinguishing molecular
were heralded to be the “brains” making these feature (or pattern) of many bacteria. Hence,
intelligent decisions, but it is now clear that the peptidoglycan is an example of a pathogen-
innate immune system directs most subsequent associated molecular pattern (PAMP). The term
immune responses. Moreover, the first real ad- PAMP is somewhat misleading because these
vances in this understanding were not achieved molecular patterns are present in nonpathogenic
by studying the more advanced mammalian microbes as well. The term microbe-associated
system but by looking at the humble fruit fly, molecular patterns (MAMPs) is more accurate
which does not have an adaptive immune sys- but has not been widely adopted.
tem at all. In addition to acting as cues to distinguish
microbial from host material, different combi-
nations of PAMPs can give an animal’s innate
Evolutionarily Conserved
immune system an indication as to what type
Molecular Patterns
of microbe is present (bacteria vs. virus vs. pro-
At the molecular level, viruses, bacteria, fungi, tozoa, etc.), and thus the subsequent immune
parasites, and animals are all made up of the reaction can be crafted to best combat the par-
same elements arranged into similar carbon- ticular type of microbe. Obviously, the ability
based protein, lipid, nucleic acid, and carbohy- of the innate immune system to do this relies
drate building blocks. At numerous points in on its ability to somehow detect these PAMPs.
speciation, however, divergent evolution gen-
erated new arrangements of these molecules
Pattern-Recognition Receptors
and left behind some of the older ones. The
result is that certain molecules are peculiar to In subsequent chapters, you will read much
certain species. These molecules are sometimes about the adaptive immune system’s ability to
so fundamental to the existence of a certain or- learn to differentiate between self and non-self.
ganism that, even if selective pressure against a This ability involves random and nonrandom
particular molecule existed, the species could recombination of key areas of the chromosome
not simply change or eliminate that molecule. of lymphocytes, together with an accelerated
Because of that, even over evolutionary time selection process within an individual animal.
and even in the face of the increasing efficiency In contrast, the innate immune system’s ability
of evolving immune systems, all members of to detect evolutionarily conserved molecular

Figure 3.3. PAMPs of Gram-positive bacteria
A diagram of a Gram-positive bacterium and associated PAMPs recognized by the innate immune
system of mammals.
patterns such as PAMPs relies on numerous of structure and cellular location, but they can
receptors that are hardwired into the genome also be categorized on the basis of function: sig-
of animals after millions of years of evolution. naling PRRs (usually stimulating proinflamma-
These receptors are termed pattern recognition tory signaling and cytokine release) and phago-
receptors (PRRs) and can be found in organ- cytic PRRs (stimulating uptake of microbes by
isms throughout the biome (they are present endocytosis or phagocytosis).
in animals, plants, and even some microbes).
In essence, PRRs can be considered sensory
receptors, but instead of sensing tastes, smells, Signaling Pattern-
heat, or pain, they detect PAMPs. Numerous Recognition Receptors
PRRs have been identified in mammals. These Signaling PRRs (often also referred to as ac-
PRRs can be divided into families on the basis tivating or proinflammatory PRRs) appear on
41

a range of cells within the immune system but
are particularly concentrated in and on the sen-
tinel cells such as macrophages and DCs. The
primary function of these PRRs, after binding
to PAMPs, is to initiate intracellular signaling
pathways that lead to the activation of the host
cell. Activation of sentinel cells by PAMPs usu-
ally results in the release of proinflammatory
cytokines that alert other immune cells to the
presence of microbial products (recall that cy-
tokines are proteins released by cells to commu-
nicate to other cells), which in turn can initiate Figure 3.4. Drosophila flies that are deficient
a range of immune defense mechanisms. The in the toll receptor are susceptible to fungal
three major families of particular note within infections.
this functional category are the toll-like recep- This image is a scanning electron micrograph
tors (TLRs), the NOD-like receptors (NLRs) of an adult Drosophila that has been killed by
Aspergillus (200× magnification). The hairlike
and the RIG-like receptors (RLRs).
structures over the thorax are actually the ger-
minating hyphae of Aspergillus. (Lemaitre et al.,
Toll-like receptors 1996, Cell 86: 973–83)
Mammalian TLRs were actually discovered

because of their similarity to the toll recep-
tors of Drosophila melanogaster (fruit fly). Flies expressed on the plasma membrane of cells
that were deficient in the toll receptor were where they can detect extracellular microbes,
extremely susceptible to fungal infections (par- but some (notably TLR3, TLR7, and TLR9)
ticularly Aspergillus infections) and often died as are expressed on the intracellular endosomal
a result (Figure 3.4). Researchers found that the membranes, where they can detect the PAMPs
toll receptor detected fungal PAMPs and stimu- associated with intracellular bacteria and vi-
lated the release of antimicrobial peptides (sim- ruses (Figure 3.5). Each individual TLR detects
ilar to defensins) that protected fruit flies from a different set of PAMPs. These PAMPs include
fungus. Later, researchers noted that mammals LPS, peptidoglycan, unmethylated DNA (nu-
had genes that were similar to the Drosophila clear DNA from mammals is methylated, but
toll gene, which also mapped to a region of the prokaryotic DNA is unmethylated), single- and
genome that was known to confer sensitivity to double-stranded RNA (some viral genomes are
LPS (later found to be the gene for TLR4). This made of these), flagellin (a protein in bacterial
family of related genes was named TLRs. flagella), profilin-like protein (a protein found
TLRs are a family of numbered, structurally in some protozoan parasites), and various mi-
similar transmembrane proteins that detect a crobial lipoproteins, to name a few. In addition
broad range of PAMPs derived from bacteria, to individual receptor ligands, some TLRs can
viruses, fungi, and protozoa. TLRs are ex- join forces to detect other PAMPs. For example,
pressed predominantly on the sentinel cells of TLR2 can dimerize (form a pair) with TLR1 or
the innate immune system but are also found TLR6 to form a receptor that recognizes micro-
on many other leukocytes, as well as on key bial diacylated lipopeptides. Some TLRs also
areas of the epithelium exposed to the exter- cooperate with non-TLR accessory proteins, al-
nal environment (such as those lining the in- lowing them to bind to PAMPs that they could
testinal and respiratory tracts). Most TLRs are not bind to autonomously. As a family, TLRs

Figure 3.5. The cellular location and examples of known PAMP ligands for the TLR family
GPI = Glycosylphosphatidylinositol; dsRNA = double-stranded RNA; ssRNA = single-stranded RNA.
collectively detect at least one PAMP from vir- ways initiated by TLRs are also well conserved.
tually any microbe. All TLRs have a cytosolic TIR domain that, after
Although the number of TLRs that have detection of a PAMP, recruits signaling adapter
been identified varies slightly between different molecules (such as MyD88 and TRIF). These
vertebrate species (most mammals have approx- adapter molecules initiate signaling cascades in-
imately thirteen; chickens have approximately volving a slew of kinases, which serve to prop-
ten), the major individual TLRs (e.g., TLR4) agate and amplify the signal. One of the final
are surprisingly well conserved, displaying little steps in the signaling pathway is the release of
variation between species. The signaling path- the rapid-acting transcription factor NFκB from
43

its negative regulator (IκB), allowing it to move The PAMPs detected by many of the newly
from the cytoplasm into the nucleus (Figure discovered NLR members are currently un-
3.6). Within the nucleus, NFκB binds specific known. The best characterized NLRs are
sequences of DNA (called response elements) NOD1 (which is expressed in many cell types)
and recruits other transcriptional activators as and NOD2 (which is expressed predominantly
well as RNA polymerase. This process results in the sentinel cells). Both NOD1 and NOD2
in the expression of genes that lead to the ac- detect different patterns within peptidoglycan
tivation of the cell as well as the expression (recall that peptidoglycan is a meshlike com-
of numerous proinflammatory cytokines and ponent of bacterial cell walls). Because certain
chemokines. Aside from NFκB, several other pathogenic bacteria actually invade the cytosol
signaling pathways can be initiated by TLRs, re- of cells (such as Listeria monocytogenes, a major
sulting in the upregulation of a variety of genes problem in dairy and beef cattle), NOD1 and
associated with immune activation. Without a NOD2 have evolved to detect intracellular bac-
doubt, the TLR family is one of the most im- teria that are otherwise hidden from the rest
portant groups of PRRs in vertebrates. But be- of the immune system. After binding pepti-
cause TLRs are all transmembrane receptors doglycan, NOD1 or NOD2 initiates signaling
with their sensing domain always facing out- cascades that result in the activation of NFκB,
ward (detecting extracellular and endosomal leading to cellular activation and the expres-
PAMPs), infectious invaders of the cytosol can sion of proinflammatory cytokines and chemo-
escape their detection. kines, similar to TLR activation.
RLRs are a family of three RNA helicases
that sense the presence of viral RNA within
NOD-like receptors and the cytosol and hence are important in the de-
RIG-like receptors tection of certain viruses. The best character-
Two main families of PRRs sense PAMPs ized is RIG-I, which recognizes long double-
within the cytosol of cells: the NLRs, which stranded RNA, as well as single-stranded RNA
sense a variety of bacterial PAMPs (as well as containing a 5´-triphosphate. Although RNA
damage signals, which we discuss later), and the strands are in normal mammalian cytosol, they
RLRs, which detect viral RNA. Although TLRs are usually single stranded with a 5´ cap (no
can sense similar PAMPs, these cytosolic PRRs 5´-triphosphate); hence, RIG-I can differenti-
constitute a second line of detection of bacteria ate between normal host RNA and RNA that
and viruses that make their way into the cyto- is produced during viral replication. On recog-
sol of the cell. nition of viral RNA, RIG-I initiates signaling
As with TLRs, the NLRs encompass an pathways that not only result in the activation
evolutionarily conserved family of PRRs with of NFκB but also lead to the expression of type
more than 20 to 30 members in mammals (sea I interferons (cytokines that specifically elicit
urchins are thought to contain upward of 203 antiviral responses).
different NLR members). NLRs have a com-
mon tripartite structure consisting of a leu-
cine-rich pattern-recognition domain, a NOD Phagocytic Pattern-
domain (that facilitates oligomerization after Recognition Receptors
PAMP detection), and a variable protein–pro- All of the phagocytic cells, such as neutro-
tein interaction domain. NLRs are not mem- phils, macrophages, and DCs, express a collec-
brane bound but float in the cytosol of immune tion of phagocytic PRRs on their surfaces that
cells (although some nonimmune cells can also allows them to autonomously detect microbes
express certain NLRs). and phagocytose them. As you will learn in

Figure 3.6A–C. Activation of NFκB after binding of PAMPs by PRRs
Panel A: After PRR activation by PAMPs, intracellular signaling
(involving several steps not shown) (1) eventually results in the libera-
tion of NFκB from its inhibitor protein IκB (2), allowing it to move
into the nucleus (3) and turn on proinflammatory genes (4). One
of the outcomes is the synthesis and release of proinflammatory
cytokines (5). Panels B, C: The images show NFκB (green) trapped
in the cytosol before exposure to PAMPs (panel B) and its transloca-
tion to the nucleus after PAMPs have been detected (panel C). NFκB
is green, actin cytoskeleton is red, and nuclear DNA is blue. (Panel B
images reproduced courtesy of Cell Signaling Technology, Inc.
[www.cellsignal.com].)
45

subsequent chapters, phagocytosis of micro and C-reactive protein (CRP). MBL (not to be
organisms can not only lead to their destruc- confused with the membrane-bound MR) is an
tion, but also allow for their protein antigens to acute phase protein synthesized primarily by
be processed and presented to T cells (a critical the liver and released into plasma. Here, MBL
step in the initiation of the adaptive immune specifically binds to mannose residues on the
response). Numerous phagocytic PRRs can surface of microbes and initiates complement
recognize a wide variety of PAMPs. One of the activation, resulting in amplification of several
best-characterized phagocytic PRRs is the man- innate immune mechanisms. We cover com-
nose receptor (MR; CD206). plement activation in greater detail in chapter
The MR is a transmembrane protein ex- 4. CRP (the major acute phase protein in pri-
pressed on the surface of the macrophages and mates, rabbits, dogs, and pigs) is another plasma
DCs of mammals. The extracellular domain protein that can also be considered a secreted
is a C-type lectin (a lectin is a carbohydrate- PRR. It selectively binds microbial polysaccha-
binding protein), which allows it to selectively rides and glycolipids as well as phosphocholine
bind terminal mannose, fucose, and N-acetyl- associated with bacteria or damaged host cells.
glucosamine residues of modified proteins. Once bound, CRP can facilitate complement
These proteins are common on the surface of activation as well as enhance phagocytic clear-
microbes but absent from normal healthy host ance of microbes and dead cells.
cells. As such, the detection of these PAMPs by
the MR provides signals to the cell that stimu-
Innate Recognition of Damage-
late phagocytosis (or endocytosis) of the mi-
Associated Molecular Patterns
crobes that come into contact with phagocytes,
leading to their destruction and the presenta- To this point, we have focused on the innate
tion of their antigens to T cells (Figure 3.7). recognition of microbial products (PAMPs).
Recent research, however, has added a second
set of molecular patterns recognized by the
Other Pattern-Recognition Receptors innate immune system that are not foreign
Several other PRRs do not fit neatly within but endogenously derived (made by the host
the signaling or phagocytic PRR categories. animal). These molecular patterns are associ-
The formyl peptide receptors (FPR) found on ated with cellular and tissue injury, indicating a
the surface of neutrophils and macrophages, al- threat to the animal that may or may not be as-
though somewhat implicated in cellular activa- sociated with microbial invasion. The term that
tion and phagocytosis, primarily direct chemo- has been adopted for these signals is damage-
taxis. The PAMPs that these receptors detect associated molecular patterns, or DAMPs, although
are N-formylmethionine–containing peptides they have also previously been referred to as
(the amino terminus of bacterial proteins com- alarmins.
monly starts with the modified amino acid for- Irrespective of whether microbes are involved
mylmethionine, which is generally absent from or not, the immune system needs to respond to
mammalian proteins). On recognition of these tissue or cellular trauma. Tissues can be physi-
bacterial peptides, these G protein–coupled re- cally damaged (torn, crushed, cut), damaged by
ceptors coordinate movement of the neutro- heat and cold (burns, frostbite), or chemically
phil or macrophage toward the microbes. In damaged (acids, bases, toxins, poisons). Dam-
this way, these phagocytes can detect, locate, aged tissues need to be protected from micro-
and destroy bacteria. bial invasion and tissue repair mechanisms de-
Another category of PRRs is the secreted ployed; thus, the immune system needs to be
PRRs, such as mannose-binding lectin (MBL) alerted. DAMPs are derived from the normal

Figure 3.7. MRs and phagocytosis
MRs can facilitate phagocytosis of mi-
crobes through recognition of mannose
residues displayed on the microbe’s sur-
face. In this way, phagocytes, such as the
macrophage, can recognize and phagocy-
tose microbes in an autonomous fashion.
proteins and metabolites found in the cyto- have been identified in animals, ranging from
plasm or nucleus of host cells that are released nuclear proteins (e.g., high-mobility group box
after cellular necrosis (Figure 3.8). The extracel- 1 protein [HMGB1]), cytosolic proteins (S100
lular presence of these molecules indicates cel- calcium-binding proteins and heat shock pro-
lular injury in the vicinity. Numerous DAMPs teins), DNA (particularly mitochondrial DNA),
47

PRRs that also recognize PAMPs (e.g., HMGB1
is also recognized by the PAMP receptors TLR2
and TLR4).
Inflammasome
The presence of PAMPs or DAMPs indicates
that either a microbial invasion or some sort of
tissue damage has occurred, and the immune
system is thus stimulated to give a measured
response. The presence of both PAMPs and
DAMPs indicates both a microbial invasion
(most likely pathogenic) and tissue damage
have occurred—a serious threat. In addition
to the additive effects of PAMPs and DAMPs,
they can synergize through the formation of
the inflammasome—a signaling cascade that
results in the massive production and secretion
of IL-1β and IL-18 (both potent proinflamma-
tory cytokines).
Figure 3.8. Endogenous production of DAMPs Although a variety of inflammasome types
As shown, after injury, host cell necrosis can exist, most require two separate signals from
release DAMPs, often leading to inflamma- PAMPs, DAMPs, or both. The best studied is
tion. Programmed cell death via apoptosis does the NALP3 inflammasome (Figure 3.9). This
not release proinflammatory DAMPs and will large protein complex is formed in macro-
not activate the innate immune system during phages after stimulation by a PAMP, followed
homeostatic cell turnover. by a second signal provided by ATP (a DAMP).
The first signal can originate through the acti-
and purine metabolites (e.g., adenosine triphos- vation of a variety of PRRs (TLRs, NLRs, or
phate [ATP], adenosine, and uric acid) (Table RLRs), resulting in the NFκB-mediated expres-
3.1). In addition to DAMP release from necrotic sion of inactive (pro-forms) of IL-1β and IL-18.
cells, macrophages, neutrophils, and NK cells These proteins accumulate in the cytosol in
can actively secrete DAMPs after activation, their inactive forms. If damage has occurred to
which can act to amplify danger signals in a tis- host cells in the vicinity, released ATP, detected
sue after trauma or microbial invasion. through a purinergic receptor (P2X7 recep-
Numerous PRRs specifically recognize tor), initiates the assembly of a large cytoplas-
DAMPs. For instance, the nuclear protein mic complex, activating the protease caspase 1,
HMGB1, when released from cells, is recog- which efficiently cleaves the inactive IL-1β and
nized by a PRR called the receptor for advanced IL-18 into their active forms. These potent pro-
glycation end products (RAGE), which is ex- inflammatory cytokines are secreted from the
pressed on macrophages and certain endothe- macrophage in large quantities, inciting wide-
lial cells (cells that line blood vessels and lym- spread leukocyte activation and inflammation.
phatics). Detection of HMGB1 by RAGE results Aside from ATP, other DAMPs, such as uric
in signaling pathways that lead to NFκB acti- acid, as well as alum (a common vaccine adju-
vation (similar to activation by many PAMPs). vant), can provide the second signal needed for
Interestingly, some DAMPs are recognized by full inflammasome function.

Figure 3.9. Intracellular activation of inflammasomes
Full activation of the inflammasome requires activation by a PAMP (e.g., LPS) as well as a DAMP
(e.g., uric acid or ATP). This activation indicates that a microbial invasion and cellular damage have
occurred. The sentinel cell responds by secreting large quantities of the proinflammatory cytokines
IL-1β and IL-18.
Table 3.1. Examples of known DAMPs

DAMP Type Origin PRR
HMGB1 DNA-interacting protein Nucleus and cytoplasm RAGE, TLR2, TLR4
S100 proteins Calcium-binding protein Cytoplasm RAGE, TLR2, TLR4
Serum amyloid A Acute-phase protein Hepatocytes FPR, RAGE, TLR2, TLR4
DNA DNA Nucleus and mitochondria TLR9
Spliceosome-associated Protein Nucleus Mincle
protein 130 (SAP130)
ATP Adenosine derivative Cytosol P2X7 receptor
Uric acid Adenosine metabolite Cytosol NALP3
49

Consequences of Danger Signal proinflammatory cytokines that engage several
Recognition by Sentinel Cells other aspects of the immune response (Figure
3.10).
As covered in chapter 2, every tissue within an
animal contains innate immune sentinel cells
that constitutively express numerous PRRs Proinflammatory Mediators
on their plasma membranes and within their
Activated macrophages have an extremely
cytoplasm. The majority of these cells are de-
high capacity to synthesize and release me-
rived from the monocytic lineage, consisting
diators of inflammation. These mediators are
of blood monocytes, tissue macrophages, DCs,
covered in greater detail in chapter 5. However,
and a variety of tissue-specific monocytic cells,
in brief, they generally include three types of
including microglial cells (central nervous sys-
proinflammatory molecules: cytokines, che-
tem), Kupffer cells (liver sinusoids), and Lang-
mokines, and lipid mediators. The major pro-
erhans cells (skin). These relatively long-lived
inflammatory cytokines produced by sentinel
cells patrol the extracellular spaces of the tissue
cells in response to PAMPs and DAMPs are
in which they reside, constantly on the lookout
IL-1β, IL-6, and TNF-α. These cytokines act
for indicators of danger. In healthy tissue, these
locally by activating nearby leukocytes and en-
sentinel cells exist in an unactivated (resting)
dothelial cells, but when released in large quan-
state. However, binding of PAMPs, DAMPs, or
tities they can also act systemically, producing
both (via PRRs) activates these sentinel cells,
fever, malaise, and ill thrift. Chemokines are
enhancing their abilities to phagocytose and
also released by activated macrophages so that
destroy bacteria, to present antigens to the
additional leukocytes can be recruited to the
adaptive immune system, and to stimulate the
site of danger (recall that chemokines are pro-
synthesis and release of proinflammatory cy-
teins that stimulate directed movement of leu-
tokines and other mediators of inflammation.
kocytes). In addition to proinflammatory pro-
As you will see in later chapters, this step is the
teins, the activation of macrophages induces
first in initiating both the innate and adaptive
enzymes that can synthesize lipid-based proin-
immune responses.
flammatory mediators such as prostaglandins,
leukotrienes, and platelet-activating factor.
Activation of Macrophages These molecules can also assist in the recruit-
ment of leukocytes as well as mediate changes
Detection of PAMPs and DAMPs activates
to the local vasculature, which are essential to
macrophages, which allows these cells to bet-
inflammation.
ter combat microbes. The repertoire of mol-
ecules displayed on the surface of macrophages
changes, including increased numbers of phago- Clinical Correlation: Follow-Up
cytic receptors, adhesion molecules, and molec-
ular complexes that are needed to present anti- Student Considerations
gen to T cells. Antimicrobial effectors (covered After reading this chapter, you should under-
in chapter 6), such as enzyme complexes that stand why LPS, an innocuous bacterial product
produce oxidative products or proteins that kill in and of itself, can cause SIRS in neonatal foals
microbes directly, are also upregulated during with (Gram-negative) septicemia. You should
macrophage activation. As a result, activated also be able to reason why animals have evolved
macrophages more quickly and efficiently find, to recognize and respond to LPS, even though
engulf, and kill microbes in the vicinity. In addi- in cases of septicemia the response itself can be
tion, activated macrophages secrete a variety of life threatening.

Figure 3.10. Inflammatorymediators produced by macrophages after activation by PAMPs, DAMPs,
or proinflammatory cytokines
Exposure of a sentinel cell to PAMPs, DAMPs, or proinflammatory cytokines activates the cell, lead-
ing to upregulation of cellular antimicrobial defenses as well as release of proinflammatory chemo-
kines, lipid mediators, and cytokines. Many of these proinflammatory products can in turn activate
nearby leukocytes.
Possible Explanations the accessory proteins MD-2 and CD14), which

Infection of the blood by live Gram-negative would then initiate intracellular signaling in
bacteria, such as E. coli, would result in mas- the sentinel cells, resulting in the expression
sive amounts of LPS being liberated and dis- of multiple proinflammatory cytokines, che-
seminated throughout the foal’s body. Sentinel mokines, and enzymes that synthesize other
cells (particularly macrophages) in virtually proinflammatory mediators. The simultane-
every tissue will be exposed to LPS in signifi- ous, widespread release of these proinflam-
cant quantities. This particular PAMP is de- matory products, which can in turn stimulate
tected through the PRR TLR4 (with help from other leukocytes to release the same or similar
51

to activate in a focused response to an area of
infection (covered in chapter 5). However, sys-
temwide uncontrolled activation of multiple
proinflammatory reactions—including vascu-
lar changes (see Figure 3.11), clotting and plate-
let activation, production of oxidative radicals,
and leukocyte degranulation—leads to massive
disruption to systemic homeostasis and ulti-
mately SIRS.
Although LPS is the most cited initiator of
SIRS in sepsis, any PAMP from many varieties
Figure 3.11. Oral mucous membranes of a septic
of microbes can trigger SIRS in a septic animal.
foal (photograph courtesy of Dr. Michel Levy,
In fact, DAMPs released after severe trauma to
University of Calgary)
an animal, even in the complete absence of in-
Note the marked hyperemia as a result of
fection, can also initiate SIRS. It is important to
disseminated PAMPs and proinflammatory
mediators.
bear in mind that SIRS is an extreme example of
unregulated pattern recognition and response.
In a normal, healthy animal and in the majority
mediators, can result in a “cytokine storm” (or of those that are diseased, pattern recognition
hypercytokinemia). Ordinarily, the innate im- is one of the first and one of the most essen-
mune response to inflammatory mediators in tial mechanisms by which the immune system,
small, localized doses is advantageous to the when faced with a threat, can be activated in an
animal, because it allows the immune system appropriate manner.

Robin M. Yates
The Complement System Chapter 4

10.5876_9781607322184.c004
when the dogs reached middle age (four to

Clinical Correlation: C3 Deficiency in
seven years old), they developed renal disease
Brittany Dogs 53
involving the glomeruli, which was diagnosed
as mesangio-proliferative glomerulonephritis.
Complement Activation 54
As a result, many of the affected dogs died from
Classical Pathway 55
renal failure–related illnesses. The dogs in this
Lectin Pathway 57
colony were later discovered to have a geneti-
Alternative Pathway 58
cally determined deficiency in a blood protein
Terminal Membrane Attack Pathway 60
called complement C3. How could a deficiency
Regulation of Complement Activation 60
in this one complement component cause the
Outcomes of Complement Activation 62
Brittany dogs to develop devastating bacterial
Inflammation and Chemotaxis 62
infections and renal disease? In this chapter, you
Enhancement of Phagocytosis (Opsonization) 63
will discover that complement forms a very im-
Promoting Humoral Immunity 63
portant part of the innate immune system and
Clearance of Immune Complexes 64
that C3 in particular is an important player in
the effectiveness of the complement system.
Learning Objectives
Clinical Correlation: C3 After reading this chapter, you should be able to
Deficiency in Brittany Dogs
• explain in general terms the arrangement of
During the 1980s, a colony of Brittany dogs, bred a biochemical cascade such as the comple-
for research into hereditary canine spinal mus- ment system;
cular atrophy, were noted to have an increased • name the major pathways of the complement
susceptibility to bacterial infections and kidney system in mammals;
disease (Figure 4.1). At a young age (younger • identify the major similarities and differences
than two years old, many only a few months in the classical, lectin, and alternative path-
old), the dogs developed serious bacterial infec- ways of complement activation;
tions, including sepsis, pyometra, pneumonia,
and bacterial myositis at a surgery site. These
• describe the initiation of the classical, lectin,
and alternative pathways of complement
infections were caused by a variety of differ-
activation;
ent bacteria, including Pseudomonas, Escherichia
coli, Clostridia, and Klebsiella. With veterinary • identify the major convertases in the classical,
lectin, and alternative pathways of comple-
intervention (principally prolonged courses of
ment activation;
antibiotics), most of the dogs recovered. Later,
53
53

Figure 4.1. A Brittany dog (also referred to as a Brittany spaniel). Photo by Pharaoh Hound.
• describe in general terms the assembly and the innate immune system, certain triggers and
function of the membrane attack complex outcomes of the complement system involve
(MAC); parts of the adaptive immune system (such as
• give an example of how complement acti- antibodies). However, the complement system
vation is regulated; is always present and can act independently as
• list the major outcomes of complement a ready-made, innate immune defense system.
activation and describe the mechanisms The complement system consists of around
leading to them. sixteen different plasma proteins and glycopro-
teins that make up around 10 percent of the
total serum protein of mammals—a significant
investment of energy and resources for an ani-
Complement Activation
mal. As with most proteins found in plasma,
The complement system is made up of a col- the majority of the complement proteins are
lection of plasma proteins that are individually synthesized by the liver, but monocytes, mac-
inert but can interact to create a powerful innate rophages, and certain epithelial cells also con-
immune response. Complement was originally tribute. Many of these complement proteins
discovered in the 1890s as a heat-labile serum circulate in the blood as (inactive) serine prote-
factor that complemented the bactericidal prop- ase proenzymes. Activation of the complement
erties of a heat-stable serum factor (later iden- proenzyme to an active serine protease (usually
tified as antibodies). Like most components of as a result of cleavage by an activated comple-
54 T h e C o m p l e m e n t Sy s t e m

ment protein immediately upstream of it in mon terminal pathway (also known as a mem-
the pathway) permits it to, in turn, cleave (and brane attack pathway).
activate) downstream complement proteins. The nomenclature used to describe each
This activation forms a biochemical cascade. complement component typically uses C (for
Activation of the complement cascade results complement) and a number (e.g., C3). To com-
in many outcomes, including release of inflam- plicate matters, the numbers are only loosely
matory and chemotactic mediators, coating of arranged in the order by which they are acti-
microbes with complement proteins that “tag” vated in the pathway because the specific action
them as foreign (a process called opsonization), of some was discovered after the implementa-
and also direct lysis of microbes. tion of the numbering system. Sometimes a
But why is this system so overly complex? letter indicating a subunit or cleavage product
Why not have a few proteins (such as defensins of a complement molecule follows the num-
and C-reactive protein) that can act indepen- ber. For instance, C1 is made up of the proteins
dently and directly on microbes? The answer C1q, C1s, and C1r. When complement proteins
lies in the amplification power of a biochemi- are cleaved into two fragments, the smaller
cal cascade. Innate proteins (such as defensins) cleavage product is typically (but not always)
that act on their own cannot amplify. However, indicated by the letter a and the larger by the
an activated complement protein (although it letter b (e.g., when C3 is cleaved by a C3 con-
may not be able to effect an immune response vertase, the smaller fragment is named C3a
by itself ) can activate several other comple- and the larger is named C3b). When several
ment protein molecules in the next step in the complement proteins or cleavage products
pathway. Each of these activated proteins can form a complex together, the complex is typi-
in turn activate several complement proteins cally named using the complement numbers in
that follow. Thus, a small inciting stimulus can the order in which they join the complex (e.g.,
amplify to a huge response involving billions of cleavage products C4b and C2a form a complex
complement molecules. C4b2a). Complexes can also be referred to by
The complement cascade can be activated their function (e.g., C4b2a is also referred to
through three different pathways that are trig- as the classical pathway C3 convertase—the
gered by different stimuli: (1) the classical path- complex formed in the classical pathway that
way, (2) the lectin pathway, and (3) the alterna- cleaves [converts] C3). Last, other proteins or
tive pathway. cofactors integral to the complement pathways
The classical pathway is initiated by anti- are not named according to this system (e.g.,
bodies (particularly IgM and IgG) complexed factor B). It is easy to get lost in the details of
to antigens and thus occurs as a consequence these pathways, so it is important to focus on
of a humoral immune response (the adaptive the salient features common to each of them,
immune response that produces antibodies to particularly the creation of both a C3 and a C5
antigens). The lectin and alternative pathways, convertase and the initiation of the terminal
however, are triggered directly by microbial membrane attack pathway (Figure 4.2).
products or surfaces and thus are purely innate
responses. Although each pathway is triggered
by different stimuli, they share common fea- Classical Pathway
tures: they all consist of a proteolytic cascade As mentioned earlier, the classical pathway
that allows for signal amplification; they all re- is triggered by the antibody–antigen com-
sult in the creation of a C3 and a C5 convertase plexes produced after an adaptive humoral im-
(which can cleave the complement proteins C3 mune response (covered in chapter 12). So, even
and C5, respectively); and they all end in a com- though all of the complement components that
T h e C o m p l e m e n t Sy s t e m 55
55

make up this pathway are always present, the
activation of this pathway relies on the produc-
tion of specific antibodies that may take several
days to appear after a new infection. It is also
important to note that not all antibody types
can activate the classical pathway (sometimes
referred to as an antibody’s ability to fix com-
plement). In most domestic species, IgM and
certain isotypes of IgG are the most potent ac-
tivators of the classical complement pathway.
When one IgM or several IgG molecules are
bound to an antigen, the Fc (carboxy-termini
of the H chains) portion of the antibodies can Figure 4.3. Structure of the C1 complex binding
be recognized by the large multimolecular to IgG antibodies
complex named C1 (or C1qr2s2; Figure 4.3). The
backbone of C1 is made up of C1q. C1q itself is
a massive protein complex that has six globu- two copies of C1r and two copies of C1s, both
lar head domains connected by linear tail do- serine proteases. For C1 to become activated,
mains. Associated with tail domains of C1q are at least two globular heads of C1q must rec-
ognize and bind to two adjacent Fc regions of
antibodies bound to an antigen. This recogni-
tion induces conformational change in the C1q
protein, which activates the protease activity of
C1r through autocatalysis (meaning C1r cleaves
itself ). The active C1r subunits then cleave the
adjacent C1s molecules in the complex, which
activates their serine protease activities.
Now activated, C1s of the C1 complex
cleaves the nearby complement protein C4
into C4a and C4b (Figure 4.4), revealing a thi-
olester group on C4b that allows it to cova-
lently attach (through reaction with hydroxyl
or amine groups) to a nearby surface. Because
thiolesters are extremely unstable, lasting only
milliseconds, C4b can only attach to surfaces
in the immediate vicinity of the activated C1
complex (with luck, onto the surface of the
microbe or antigen complex that is being rec-
ognized by the initiating antibodies). The cova-
lently bound C4b can present C2 to C1s, which
Figure 4.2. A
simplified diagram to illustrate the cleaves it into C2b and the larger fragment C2a
common points of complement activation
(note that for historical reasons the naming of
The classical, lectin, and alternative pathways the larger C2 cleavage product C2a goes against
all create a functional C3 convertase and C5 the complement nomenclatural convention of
convertase and initiate the terminal membrane
labeling the larger fragment b). The bound C4b
attack pathway.
can then bind with the C2a fragment, forming

cleavage of C3 reveals a thiolester group in C3b,
which allows the fragment to covalently attach
to the surfaces in the immediate vicinity. As we
discuss later in the chapter, the covalent attach-
ment of C3b to a microbe or other antigen is
an important objective of the complement
system as it tags (a process called opsoniza-
tion) the object for phagocytosis and removal.
Although the attachment of C3b achieves one
goal of the classical complement pathway, the
next step is to initiate the terminal membrane
attack pathway that can mediate direct lysis of
the microbe. For this, a proportion of C3b will
complex with the C3 convertase C4b2a to form
the classical pathway C5 convertase (C4b2a3b).
This complex now cleaves C5 into C5a and C5b.
C5b remains associated with the C4b2a3b com-
plex and serves as an anchor that coordinates
the assembly of the MAC in the terminal mem-
brane attack pathway (as detailed in the Termi-
nal Membrane Attack Pathway section).
Lectin Pathway
The lectin pathway of complement activa-
tion is not reliant on any products of the adap-
tive immune response; thus, it is an indepen-
dent innate immune response. Lectins are pro-
teins that bind particular carbohydrate (sugar)
residues. The lectin complement pathway is
driven by lectins found in plasma, such as the
mannose binding lectin (MBL) and ficolins that
can recognize microbial-specific carbohydrates
displayed on the surface of various microbes.
MBL is a tetrameric member of the C-type lec-
tin family and shares structural similarities with
C1q. Instead of recognizing bound antibodies
(as does C1q), however, MBL binds mannose,
Figure 4.4. The classical pathway of complement
activation fucose, and N-acetylglucosamine PAMPs that
are commonly present in the cell walls of bacte-
ria and fungi, as well as on some protozoa and
complex C4b2a on the surface of the microbe viruses. Essentially, as mentioned in chapter 3,
or antigen. MBL can be considered an extracellular PRR.
C4b2a is the classical pathway C3 convertase. Binding to a microbial cell wall induces a con-
Its job is to cleave C3 (the most abundant com- formational change in MBL, which promotes
plement protein) into C3a and C3b. As with C4, activation of MBL-associated serine proteases
57

(MASPs) via autocatalysis (Figure 4.5). Simi-
larly to C1s, MASPs can in turn cleave C4 and
C2 to create the C3 convertase C4b2a, which is
assembled on the surface of the microbe. From
here on, the lectin pathway resembles the clas-
sical pathway, with C4b2a cleaving C3, leading
to deposition of covalently bound C3b on the
surface of the microbe and the creation of the
C4b2a3b C5 convertase, which initiates the ter-
minal membrane attack pathway. Ficolins are a
family of lectins that, like MBL, bind microbe-
associated carbohydrates and activate MASPs.
In essence, the lectin pathway is homologous
to the classical pathway but uses the innate rec-
ognition of microbial products (PAMPs) and
surfaces using lectins instead of the recognition
of antibodies by C1.
Alternative Pathway
The alternative pathway of complement acti-
vation is unlike the classical and lectin pathways
because it does not require specific recognition
of a microbial surface by antibodies or lectins.
It relies on the nonspecific, low-level hydroly-
sis of C3 (remember, C3 is the most abundant
complement protein and a key player in all path-
ways). In plasma, C3 is inherently slightly un-
stable, with a small portion of it spontaneously
degrading into its C3a and C3b components. As
you recall, this cleavage reveals the reactive thi-
olester group on C3b, which allows it to cova-
lently bind to surfaces in the immediate vicinity.
If C3b binds to a healthy host cell membrane, it
is rapidly degraded to prevent any further com-
plement activation. This degradation occurs
because host membranes are rich in sialic acid
residues that promote binding of C3b to factor
H (a negative regulator of complement activa-
tion; Figure 4.6A). Factor H, along with factor I,
inactivates and degrades inappropriately bound
C3b. Hence, on those surfaces on which comple-
Figure 4.5. The lectin pathway of complement
ment activation is not wanted, spontaneously
activation
bound C3b is destroyed as fast as it is deposited.
However, microbial membranes and cell walls
are typically rich in polysaccharides that do not

Figure 4.6A–B. The alternative pathway of complement activation
Panel A: In the presence of sialic acid residues, which are abundant on the surface of
host cells, bound C3b preferentially binds factor H and is degraded by factor I. Panel
B: On activating surfaces, bound C3b can bind factor B, which can be cleaved by factor
D, resulting in the activation of the alternative C3 convertase C3bBb. C3bBb can now
catalyze C3 proteolysis into C3a and C3b, eventually leading to more C3bBb, thus
creating an amplification loop.
contain sialic acid. Should C3b happen to cova- complex C3bBb. C3bBb is the alternative C3
lently bind to one of these so-called activating convertase. Now, instead of relying on the low
surfaces, C3b does not bind the regulatory fac- level of spontaneous hydrolysis of C3, this C3
tor H and instead adopts a conformation that convertase efficiently catalyzes the cleavage of
allows it to preferentially bind factor B, forming plasma C3, resulting in the rapid deposition of
the complex C3bB (Figure 4.6B). C3b onto the activating surface. A portion of
Another serum factor, factor D, cleaves the this bound C3b will recruit factor B and, with
factor B component of the C3bB complex, re- the help of factor D, will create more of the
leasing Ba and creating the active proteolytic alternative C3 convertase C3bBb. As you can
59

pathways (C4b2a3b), the alternative C5 conver-
tase (C3bBb3b) cleaves C5, which initiates the
terminal membrane attack pathway.
Terminal Membrane Attack Pathway

All complement activation pathways can ini-
tiate the common terminal membrane attack
pathway (Figure 4.7). The goal of this particu-
lar pathway is to construct a protein complex
that essentially punches a hole in the target
membrane and thus can directly lyse and kill
the microbe (Figure 4.8). This protein complex
is aptly named the membrane attack complex
(MAC). The terminal membrane attack path-
way is initiated by the activating pathways’ C5
convertase (C4b2a3b, generated by the classical
and lectin pathways, and C3bBb3b, generated
by the alternative pathway). These C5 conver-
tases cleave C5 into C5a and C5b. C5b recruits
C6 and C7 to the target membrane, forming
the complex C5b67. This complex can bind and
partially unfold C8, exposing a hydrophobic
region of the protein that allows it to wedge
itself deep in the target membrane. With C8
now inserted into the membrane, the complex
can recruit and insert ten to sixteen copies of
Figure 4.7. Theclassical, lectin, and alternative C9 into the membrane to create a cylindrical
pathways of complement activation pore (using C9 much like the wooden staves
that make up a barrel). The completed MAC
breaches the cell membrane of the microbe, al-
see, this process initiates an amplification loop lowing water to rush into the cell. If sufficient
leading to the accelerated deposition of C3b on numbers of MAC form on the membrane, the
the activating surface. (As a side note, because microbe is destroyed by osmotic lysis.
C3b deposition is common to all pathways, the
alternative C3 convertase C3bBb and the am-
Regulation of
plification loop that results are also inherent
in the classical and lectin pathways.) The C3
convertase C3bBb is itself relatively unstable One downside of an amplifying biochemical
but can complex with a plasma protein called cascade is that it can easily get out of hand.
factor P (also known as properdin), which helps Thus, it is not surprising that the system of
stabilize the complex. The alternative C5 con- complement activation is highly regulated by
vertase is created when an additional C3b mol- an equally complex system of complement
ecule complexes with the C3 convertase C3bBb control proteins (see Table 4.1) that not only
to form C3bBb3b (Figure 4.7). As with the C5 increases the complement system’s selectivity
convertase formed in the classical and lectin for non-self but also limits activated comple-

C4 and C2. C1 inhibitor can also inhibit MASPs
that have been activated by MBL; hence, it plays
a role in the regulation of the early stages of
both the classical and lectin pathways.
Other complement control proteins found in
plasma include factor H and C4b-binding pro-
tein. As mentioned earlier, factor H enhances
the degradation of C3b bound to membranes
rich in sialic acid and can thus (along with fac-
tor I) directly influence the formation and sta-
bility of the alternative C3 convertase (C3bBb).
The C4b-binding protein, as its name suggests,
preferentially binds C4b. Binding results in the
accelerated degradation of the C3 convertase
C4b2a; hence, it is a regulator of the classical
and lectin activation pathways.
In addition to plasma proteins, many com-
plement control proteins are displayed on the
surfaces of host cells. Decay-accelerating factor
(CD55) is displayed on the surfaces of erythro-
cytes, circulating leukocytes, platelets, and en-
dothelia, where, as its name suggests, it acts to
accelerate the decay of the alternative C3 con-
vertase (C3bBb), thus providing an extra level
Figure 4.8. Assembly
of the MAC by the terminal of security against inappropriate activation
membrane attack pathway of the alternative pathway on host cells that
The final MAC consists mostly of ten to sixteen are continually exposed to plasma. Similarly,
molecules of C9 arranged into a barrel-shaped complement receptor 1 (CR1 or CD35) and
pore within the target membrane. The diameter membrane cofactor protein (CD46) are mem-
of the inner pore is around ten nanometers and brane proteins that can inhibit either the alter-
allows fluid to rush into the target cell. native or the classical and lectin C3 convertase
by interacting with C4b or C3b. Several other
ment cascades from continuing indefinitely. complement control proteins found on the cell
The classical, lectin, alternative, and terminal membranes of host cells also specifically act to
membrane attack pathways can all be stopped prevent formation of the MAC. One of these
or downregulated at key points by soluble or control proteins, protectin (CD59), acts to pre-
membrane-associated complement control vent MAC formation on host cells by binding
proteins. Many of these control proteins func- the C5b678 complex and preventing the bind-
tion by inhibiting the protease activities or fa- ing and oligomerization of C9. Several other
cilitating the degradation of activated comple- host membrane glycoproteins act in a similar
ment complexes or convertases. fashion.
An example of an inhibiting protein is the Together, the complement control proteins
acute phase protein C1 inhibitor. As the name act to prevent the complement innate defense
suggests, the C1 inhibitor inhibits the protease system from acting on inappropriate targets
function of C1 (specifically C1r and C1s), which and also from acting in perpetuity. The inabil-
prevents excessive or inappropriate cleavage of ity of the control proteins to halt complement
61

Table 4.1. A summary of known complement control proteins and their function
Complement Pathway
control protein Location regulated Action
C1 inhibitor Plasma, serum Classical and Inactivates C1r, C1s, and MASPs
lectin
C4b-binding Plasma, serum Classical and Binds C4b; accelerates decay of
protein lectin the classical and lectin C3 conver-
tase (C4b2a)
Factor H Plasma, serum Alternative Binds C3b and accelerates decay
Decay-accelerating Cell membrane of erythrocytes, neu- Alternative Accelerates decay of C3 conver-
factor (CD55) trophils, lymphocytes, monocytes, tase (C3bBb)
platelets, and endothelial cells
Complement recep- Cell membrane of erythrocytes Classical, lectin, Binds C3b and C4a and inhibits
tor I (CD35) (primates), neutrophils, lymphocytes, and alternative alternative and classical/lectin C3
monocytes, and macrophages convertases
Membrane cofactor Widely expressed on cell membrane Classical, lectin, Binds C3b and C4a and inhibits
protein (CD46) of host cells and alternative alternative and classical/lectin C3
convertases
Protectin (CD59) Predominantly expressed on cell Terminal mem- Binds to C5b678 and prevents C9
membrane of erythrocytes, leuko- brane attack recruitment and MAC formation
cytes, and vascular endothelium
cascades can have devastating consequences. Inflammation and Chemotaxis

An interesting fact is that venom from the cobra As you recall, two major points of all com-
snake contains a C3b analogue (called cobra plement activation pathways are the assembly
venom factor) that can complex with factor of C3 and C5 convertases. These convertases
Bb and factor P to form a stable C3 convertase mediate the cleavages of C3 and C5, respec-
(cobra venom factor Bb). This convertase is not tively, resulting in the attachment of the large
recognized by the regulatory proteins factor H fragments C3b and C5b to microbial surfaces.
and I, and thus it will continually hydrolyse C3 It is easy to discount the two smaller frag-
(and C5), leading to complement depletion and ments C3a and C5a as useless by-products,
severe local tissue damage. but they actually play important roles in their
own right. Because both C3a and C5a are small
(around 10 kDa), they readily diffuse away
Outcomes of
from the site of complement activation. Nu-
merous leukocytes detect local complement
Aside from the direct complement-mediated activation through the recognition of C3a and
lysis of cells by formation of a MAC, numer- C5a by surface complement receptors, which
ous products of complement activation act to alerts these cells to a potential threat in the vi-
enhance immune defense within an animal cinity. Mast cells react to both C3a and C5a by
(see Table 4.2). These products include those degranulating, thus dumping loads of vasoac-
promoting inflammation, chemotaxis of leu- tive amines into the extracellular space, which
kocytes, phagocytic uptake of targets, humoral in turn leads to dilation and increased perme-
immune responses, and the clearance of im- ability of the local vasculature (both hallmarks
mune complexes. of acute inflammation, covered in chapter 5).

Table 4.2. A summary of outcomes of complement activation
Complement
Outcome products Action
Direct target lysis MAC Osmodysregulation and lysis of target cells
Tissue C3a and C5a Activation of mast-cell degranulation leading to release of vasoactive
inflammation amines (histamine and serotonin)
Endothelial C3a and C5a Increased expression of adhesion molecules
activation
Chemotaxis C3a and C5a Promotes migration of neutrophils, eosinophils, and macrophages toward
site of complement activation
Leukocyte C5a (C3a and Upregulation of adhesion molecules, phagocytic receptors, and antimicro-
activation C4a) bial effectors by neutrophils and monocytes
Opsonization C3b and iC3b Enhancement of particle phagocytosis by macrophages and neutrophils
Promotion of C3dg Enhanced B-cell activation, retention of antigen complexes in B-cell follicles
humoral responses
Immune complex C3b (and iC3b) Blocking of growth and facilitation of dissociation of immune complexes;
clearance immobilization and clearance of immune complexes through interaction
with CR1 on erythrocytes
With a small localized response, complement- Enhancement of Phagocytosis

mediated vascular dilation and permeability (Opsonization)
act to enhance the delivery of leukocytes and A major outcome of complement activa-
immune proteins to the site of complement tion is the covalent attachment of C3b to target
activation. C3a and C5a can also help recruit surfaces, which essentially tags these surfaces
circulating leukocytes by prompting endothe- as foreign, hazardous, or simply abnormal.
lial cells lining the local vasculature to express Particles with C3b and its breakdown product
adhesion molecules. But when generated in iC3b on their surfaces are recognized by mac-
large amounts systemically, C3a and C5a can rophages and neutrophils through complement
cause systemic anaphylactic shock. For this rea- receptors (specifically CR1, CR3, and CR4),
son, C3a and C5a are sometimes referred to as which mediate phagocytosis of the particles.
anaphylotoxins. Although microbes can be recognized by mac-
In addition, C3a and C5a in particular stimu- rophages and neutrophils without complement
late the chemotaxis (movement) of many leu- (e.g., via the MR), the presence of C3b and iC3b
kocytes, including neutrophils, eosinophils, and on their surfaces dramatically enhances their
macrophages. Diffusion of C3a and C5a away phagocytosis. This binding of C3b and iC3b to
from the site of complement activation estab- microbial surfaces is termed opsonization (cov-
lishes a chemotactic gradient, up which leuko- ered in greater detail in chapter 6).
cytes can migrate toward the potential threat.
These complement products also activate leu-
kocytes such as neutrophils, allowing these cells Promoting Humoral Immunity
to upregulate their receptors, adhesion mol- Another breakdown product of bound C3b
ecules, and the antimicrobial arsenal needed to is C3dg, which can be recognized by the CR2
find and combat microbes and other threats in expressed on both B cells and follicular den-
the tissue. dritic cells (FDCs). When a B cell recognizes a
63

specific antigen through its B-cell receptor, it is infections. You should also be able to derive a
stimulated to activate and proliferate (covered possible explanation for the development of kid-
in chapter 12). Recognition of C3dg by CR2, ney disease in these Brittany dogs (Figure 4.9).
in association with the specific antigen, signifi-
cantly enhances B-cell activation, which in turn
enhances the generation of antibodies directed Possible Explanations
against that antigen. The presence of C3dg in C3 is the most abundant complement pro-
antigen complexes is also thought to help retain tein. It is central to the classical, lectin, and al-
the antigen within the B-cell follicles of lymph ternative complement pathways and is also a
nodes through interaction with CR2 on FDCs. prerequisite for the initiation of the common
This retention promotes prolonged antigen–B terminal membrane attack pathway. So, essen-
cell interaction, which further acts to enhance tially, a deficiency in the C3 component renders
the humoral response to the complement-as- the whole complement system defunct, which
sociated antigen—yet another example of an would result in deficiencies in direct bacterial
innate immune system outcome that enhances killing by MAC, reduced bacterial phagocytosis,
an adaptive immune response. compromised acute inflammatory reactions,
diminished activation and chemotaxis of leuko-
cytes, and perhaps even reductions in humoral
Clearance of Immune Complexes immune responses. All of these would directly
Immune complexes consisting of matrices of compromise the dog’s efficiency in mounting
antibodies and antigen can be generated during effective immune responses to invading bac-
an immune reaction to an abundant antigen. teria, and thus it is easy to understand why an
These complexes, if not cleared, can be depos- animal that is deficient in C3 would succumb to
ited in particular sites in the body of an animal bacterial infection.
(particularly in vessel walls, kidneys, joints, The renal disease that these Brittany dogs de-
and eyes), resulting in a type III hypersensitiv- velop later in life is harder to explain. It is inter-
ity (covered in chapter 16). Attachment of C3b esting to note that human patients with C3 de-
to these complexes (through classical comple- ficiency develop a similar glomerulonephritis.
ment cascade) interrupts the antigen–antibody The membrano-proliferative glomerulonephri-
interactions, preventing complex growth and tis that both dogs and humans with C3 defi-
facilitating their dissociation. Integration of ciency develop is thought to be the manifesta-
C3b into the complexes also allows them to be tion of an autoimmune-type disease. As you will
bound to the surface of erythrocytes through discover in chapter 16, glomerulonephritis (lit-
interaction with CR1. Once bound to erythro- erally, inflammation of the glomerulus) is one
cytes, the immune complexes are not inappro- of the more common manifestations of a type
priately deposited and can be safely removed III hypersensitivity, caused by antigen–antibody
by phagocytosis of the erythrocytes in the liver complexes that circulate through the blood and
and spleen. lodge in the maze of capillaries that form the
glomerulus. Once in the glomeruli, these an-
Clinical Correlation: Follow-Up tigen–antibody complexes trigger inflamma-
tory responses that damage the glomeruli. As
Student Considerations you learned in this chapter, deposition of C3b
After reading this chapter, you should be able on circulating immune complexes aids in their
to list the outcomes of complement activation dissociation, immobilization, and removal (via
and reason why a deficiency in C3 would inter- binding to erythrocytes and phagocytic clear-
fere with an animal’s ability to control bacterial ance in the liver and spleen). Without C3, im-

Figure 4.9. A Brittany dog. Photograph by Lori Branham.
mune complexes that develop during the mul- of a membrano-proliferative glomerulonephri-

titude of adaptive responses in the lives of C3- tis and, eventually, renal failure. Thankfully, and
deficient Brittany dogs lodge in glomeruli and presumably because of the immense selection
incite inflammation. Over a period of years, the pressure over eons of evolution, complete C3
inflammation in the glomeruli results in their deficiency in any animal species is considered
progressive dysfunction and the development to be extremely rare.
65

Robin M. Yates
Acute Inflammation Chapter 5

10.5876_9781607322184.c005
Acute mastitis is the sudden dysfunction of

Clinical Correlation: Acute Bovine Mastitis 67
the mammary gland, accompanied by swelling,
heat, and obvious pain and discomfort for the
Purposes of Acute Inflammation 68
cow. The milk expressed from the affected quar-
Signs of Acute Inflammation 69
ter often contains clots and debris and is unfit
Initiators and Mediators of Acute
for consumption. A hallmark diagnostic feature
Inflammation 70
of acute mastitis is the presence of leukocytes
Vascular Changes in Acute Inflammation 72
in the milk. Leukocytes, and indeed other so-
Leukocyte Recruitment 75
matic cells, can be detected using a cowside
Leukocyte Extravasation 75
test called the California Mastitis Test (CMT).
Diversity and Timeline of Leukocyte
Recruitment 79
This test uses the agglutination of DNA, which
Systemic Effects of Acute Inflammation 80
forms a gel in the milk, as an indicator of the
presence of leukocytes (see Figure 5.2). Nowa-
days, the CMT is gradually being replaced by
automated cell counting. Sudden onset of clini-
cal signs and a positive CMT or high leukocyte
counts in the milk from a quarter are indicative
of acute mastitis. They are handy diagnostic
Acute Bovine Mastitis
indicators, but what are the leukocytes doing
Everyone has had experience with acute in- there? How did they get there, and how did
flammation. It is characterized by hallmark fea- they know where to go?
tures and usually indicates that an infection is
present or that the tissue has had some physical Learning Objectives
or chemical damage. Dairy farmers deal with
inflammation on a twice-daily basis in the form After reading this chapter, you should be able to
of mastitis in their milking herds (Figure 5.1). • understand purposes of inflammation and
Mastitis is inflammation of the mammary its roles in immunity;
gland, which is almost always initiated by the
infection of these high-performing glands by
• describe the cardinal signs of acute inflam-
mation and explain the basic underlying
contagious or environmental bacteria. Eco-
pathophysiological processes;
nomically, mastitis is one of the most important
disease syndromes affecting the dairy industry. • list basic groups of mediators in acute
Clinical and subclinical mastitis results in lower inflammation and their origins and targets;
milk production and poor milk quality and ad- • describe the blood vessel changes that
versely affects the welfare of these animals. occur during acute inflammation;
67
67

Figure 5.1. Acute bovine mastitis (photograph courtesy of Dr. Gordon Atkins, University of Calgary)
This photograph is of a four-year-old, mid–second-lactation Jersey cow with severe mastitis of the
right hindquarter caused by Staphylococcus aureus.
• describe the steps of extravasation and urement, why is it inherent—in some form or
chemotaxis of leukocytes in the acute another—to all species in the animal kingdom?
inflammatory process; As we discussed in chapter 3, sentinel cells such
• appreciate the diversity of inflamma- as tissue macrophages and DCs are dispersed
tory processes and timelines of leukocyte throughout every tissue in the body. Although
recruitment; macrophages, by themselves, can phagocytose
• describe the systemic clinical findings that and destroy bacteria as well as clear cellular de-
may be associated with an acute inflamma- bris, in the face of major tissue damage or the
tory process; introduction of a large microbial insult, they
can be quickly overwhelmed. In these cases,
• describe the systemic laboratory find-
macrophages need to enlist the aid of large
ings that may be associated with an acute
numbers of other leukocytes. One of the most
inflammatory process.
well equipped of these leukocytes is the neu-
trophil, but monocytes, eosinophils, and even
Purposes of Acute Inflammation certain lymphocytes can also be recruited to
the affected tissue.
The acute inflammation of a tissue is com- Neutrophils are armed with numerous pow-
monly viewed as an undesirable symptom of erful antimicrobial effector mechanisms to de-
an injury or disease process. If this process fend against major threats. But as you learned
serves only to provide discomfort and disfig- in chapter 2, neutrophils spend most of their
68 Ac u t e In f l a mm a t i o n

the influx of leukocytes and plasma proteins to
the site of insult and the changes to the vascula-
ture needed to facilitate the movement of these
immune entities result in the cardinal signs as-
sociated with inflammation (Figure 5.3).
Signs of Acute Inflammation

The first known description of the signs as-
sociated with acute inflammation was found
in an Egyptian papyrus dating back to 3000
BC. However, it was during the period of the
Roman Empire when the encyclopedist Celsus
documented the cardinal signs of inflamma-
tion in the Latin terminology that is still in use
today:
1. rubor (redness);
2. tumor (swelling);
Figure 5.2. California Mastitis Test (CMT) 3. calor (heat);
The CMT pictured was performed on milk ex- 4. dolor (pain);
pressed from a normal quarter of an udder (left) 5. functio laesa (loss of function; added to
and a quarter of the same udder with severe Celsus’s list by the German pathologist
acute mastitis (right). CMT reagent was added Virchow in the late 1800s).
to both milk samples and mixed in a CMT
paddle. The gelatinous material formed in the Many of these signs are consequences of
positive sample is leukocyte DNA, which can be the local vascular changes initiated by inflam-
assessed when the mixture is swirled (top) or mation. Both rubor and calor arise from local
poured out of the paddle (bottom). vasodilation and increased blood flow to the
affected area. It is important to point out that
calor is due primarily to increased conduction
lives inside the circulatory system. To be effec- of core body temperature to the periphery by
tive in infected or damaged tissues, neutrophils increased blood flow to the area of inflamma-
need to rapidly and accurately find infected tion (as a result of vasodilation) rather than
and affected tissues, extravasate (get out of the local metabolic changes or pyrexia (fever).
circulatory system), and travel to the specific Tumor is primarily a result of increased per-
site of insult. In addition to cells, soluble com- meability or leakiness of the local vasculature,
ponents of the immune system (once again, leading to tissue edema. Dolor is attributed to
mostly found in blood), such as complement the release of inflammatory mediators such as
proteins, acute phase proteins, and antibodies, prostaglandins, neuropeptides, and cytokines
need to gain access to affected tissue. Delivery by damaged tissue and activated immune cells.
of cellular and soluble immune components These mediators can act on pain receptors, re-
to the site of injury significantly enhances the sulting in hyperalgesia, although the physical
local immune defenses, which act to eliminate and chemical trauma associated with tissue
microbes, trigger the adaptive immune system, edema and antimicrobial functions of immune
and initiate tissue repair. The culmination of cells undoubtedly contribute. Functio laesa re-
Ac u t e In f l a mm a t i o n 69
69

be debilitating. So when is it appropriate for an
inflammatory process to be initiated, and how
is it triggered?
Initiators and Mediators

of Acute Inflammation
The most common initiator of acute inflamma-
tion is the sentinel cell detection of microbial
or parasitic products within host tissues. Recall
from chapter 3 that the innate immune system
is equipped with numerous PRRs that can rec-
ognize the conserved molecular motifs associ-
ated with microbes—the so-called PAMPs. Sen-
tinel cells, such as macrophages, constitutively
express PRRs that enable them to continually
monitor the local tissue microenvironment
for signs of microbial invasion. The detection
of microbial products via PRRs leads to signal-
ing cascades within the macrophages, which
results in their activation. Once activated, the
macrophages synthesize and release a slew of
proinflammatory cytokines (such as IL-1β, IL-6,
and TNF-α) that act as signals to other inflam-
Figure 5.3. Inflammation of the fetlock of a matory cells and blood vessels in the area. Ac-
foal (photograph courtesy of Dr. Michel Levy, tivated macrophages can also release chemo-
University of Calgary) kines such as CXCL8 (also known as IL-8). As
This foal has a suppurative arthritis. There you have learned, chemokines are chemotactic
is diffuse, moderate swelling (tumor) of the cytokines consisting of small proteins that can
fetlock and reddening of the skin (rubor). If diffuse away from the secreting cell to set up
you were to palpate it, it would feel warmer
a concentration gradient. Using this gradient,
to the touch (calor) and would likely elicit a
leukocytes, such as neutrophils, can move to-
pain response (dolor). Clinically, the foal would
present with an abnormal gait caused by loss ward the particular site of insult by chemokine-
of full function of the limb (functio laesa). Also directed chemotaxis.
note that distal to the fetlock are two puncture Although microbial infections are the most
wounds oozing blood and suppurative material commonly considered triggers of inflammation,
(pus). many other tissue insults can initiate so-called
sterile inflammation. These insults include
trauma, physical and chemical injury, foreign
sults from a culmination of tumor, dolor, and bodies, or inappropriate products of adaptive
tissue damage, leading to general dysfunction immunity. Many of these insults result in the
and the instinctive behavior of the animal to release of DAMPs, which, although endog-
protect the inflamed region. enously derived, can also lead to the activation
Apart from dolor, which may provide a proof macrophages.
tective function for the inflamed area, the other Activation of macrophages with PAMPs,
cardinal signs have no obvious benefits and can along with activation by DAMPs, indicates in-

fection and tissue injury. Together, they com- thesize leukotrienes and prostaglandins from
bine to become a powerful stimulus to activate the normal cellular diacylglycerols or phospho-
assembly of the inflammasome within macro- lipids found in cellular or nuclear membranes.
phages, which dramatically enhances the release The first step is the liberation of a polyunsatu-
of IL-1β and IL-18, both potent proinflamma- rated 20-carbon fatty acid (called arachidonic
tory cytokines. The macrophage-derived pro acid) from diacylglycerols or phospholipids
inflammatory cytokines (IL-1β, IL-6, and TNF- (Figure 5.4), which is mediated by phospholi-
α) can act directly on endothelial cells in local pases (phospholipase C or phospholipase A2,
vasculature and can additionally act on neigh- respectively). Although leukotrienes and pros-
boring immune cells, often prompting them to taglandins derive from this common precursor
also release proinflammatory mediators. and have overlapping functions, it is important
One cell in particular that can rapidly amplify to differentiate between the two classes of mol-
proinflammatory signals in response to the cy- ecules because of their distinct contributions to
tokines produced by macrophages is the mast clinically important situations.
cell. As mentioned in chapter 2, mast cells are The enzyme lipoxygenase uses the freed
typically situated near blood vessels and nerves, arachidonic acid to synthesize leukotrienes.
and contain numerous cytoplasmic granules There are several immunologically important
rich in histamine and serotonin, both vasoactive leukotrienes, including leukotriene B4 and leu-
compounds. Mast cells and histamine are com- kotriene D4. These leukotrienes act locally as
monly associated with type I hypersensitivities powerful chemoattractants for neutrophils (B4)
(allergies) in response to an inappropriate hu- and effect changes in the vascular endothelium
moral response to allergens and are discussed (B4 and D4).
in depth in chapter 16. Aside from unwanted Prostaglandins are synthesized from the
degranulation in allergies, however, mast-cell arachidonic acid pool by cyclooxygenase (COX)
degranulation in response to proinflammatory enzymes. The two major COX isoforms, COX-1
cytokines such as IL-1 acts to rapidly facilitate and COX-2, are expressed at different levels in
the changes to local vasculature needed for in- different tissues. Although COX-1 is needed to
flammation. In addition to macrophage prod- synthesize prostaglandins with homeostatic
ucts, mast cells can be directly induced to de- roles, COX-2 is responsible primarily for pros-
granulate by surface-bound IgE (a specific anti- taglandin synthesis in inflammation. These en-
body isotype associated with allergies) after the zymes become critically important in the phar-
detection of specific antigens. Furthermore, macological management of inflammation
products of complement activation (C3a and because they are the target of the nonsteroidal
C5a), physical or chemical trauma, and even anti-inflammatory drugs (NSAIDs) commonly
extreme temperatures can induce mast-cell used in veterinary medicine (e.g., meloxicam
degranulation. [Metacam®]; phenylbutazone, or “bute”; and
Another group of important mediators of carprofen [Rimadyl®]). The major prostaglan-
acute inflammation is the eicosanoids—in par- din involved in inflammation is prostaglandin
ticular, leukotrienes and prostaglandins. Unlike E2. Prostaglandin E2 is synthesized by both
cytokines, which are proteins, the eicosanoids macrophages and mast cells and acts on local
are oxidized derivatives of fatty acids. These vasculature and endothelium.
compounds typically have a short half-life and Collectively, in response to local noxious or
thus act in an autocrine or paracrine capacity in microbial stimuli, macrophages, in concert
an inflamed tissue. Within minutes of detecting with other immune cells such as mast cells,
an inflammatory stimulus (e.g., cytokines or can rapidly release proinflammatory cytokines
trauma), both macrophages and mast cells syn- and other mediators such as histamine. These
71

Figure 5.5. Vascular changes in acute
inflammation
This figure shows the major changes to vas-
culature in response to inflammatory signals
detected within a tissue. In brief, indicators of
infection or trauma can be detected by sen-
Figure 5.4. Eicosanoid synthesis
tinel cells, such as macrophages, resulting in
Leukotrienes, and prostaglandins are synthe- the release of inflammatory mediators, which
sized from arachidonic acid released from is often amplified by neighboring mast cells.
membrane lipids. Pools of arachidonic acid can These inflammatory mediators act on local
be acted on by lipoxygenase or cycloxygenase vasculature to effect vasodilation, expansion of
enzymes to produce a variety of leukotrienes capillary beds, increased vascular permeability,
and prostaglandins, respectively. Cycloxygenases and decreased blood velocity in the postcapil-
can be inhibited by NSAIDs, which decrease lary venules.
prostaglandin synthesis and prostaglandin-
induced inflammatory sequelae.
1. vasodilation and local stasis of blood flow;
2. increased vascular permeability;
3. expression of cell-adhesion molecules on
mediators can act on local vasculature to initi-
vascular endothelium.
ate the vascular changes needed for the infiltra-
tion of cellular and plasma-derived immune Vasodilation occurs through the action of
mediators (see Table 5.1). inflammatory mediators on the arteriole and
the postcapillary venule smooth muscle. De-
creasing the tone of the periarteriolar muscu-
Vascular Changes in
lature acts to widen the lumen of the vessels,
Acute Inflammation
which in turn opens new capillary beds in the
Three main changes to local blood vessels occur affected tissue (Figure 5.5). As mentioned ear-
in response to inflammatory mediators: lier, these expanded capillary beds contribute

Table 5.1. A summary of the major mediators of inflammation
Inflammatory mediator Primary source Inflammatory action
Cytokines and chemokines
IL-1β, IL-6, and TNF-α Macrophages Leukocyte and endothelial activation; systemic
reactions
CXCL8 (IL-8) Leukocytes Leukocyte activation and chemotaxis
Plasma proteins
C3a Complement activation Mast-cell degranulation; smooth-muscle contraction
C5a Complement activation Leukocyte chemotaxis; mast-cell degranulation;
smooth-muscle contraction; vascular permeability
Bradykinin Kinin–kallikrein system Vasodilation; vascular permeability; pain
Vasoactive amines
Histamine Mast-cell degranulation Vascular permeability; smooth-muscle contraction
Serotonin (5-hydroxytryptamine) Mast-cell degranulation Vascular permeability; smooth-muscle contraction
Eicosanoids
Leukotriene B4 Leukocytes Leukocyte activation and chemotaxis; vascular
permeability
Leukotrienes B4, D4, and E4 Leukocytes (particularly Smooth-muscle contraction; vascular permeability
mast cells)
Prostaglandin E2 Leukocytes (particularly Vasodilation; vascular permeability; pain
mast cells)
to both rubor and calor by increasing the vol- ity also contributes to the local stasis of blood
ume of blood in the area. By widening the ve- flow; akin to the flow of water in a pipe with
nules, vasodilation also acts to slow the veloc- numerous holes in it, the more water that leaks
ity of the blood in these postcapillary vessels, out, the slower the flow of water in the pipe.
akin to water current in a river—the wider and In healthy tissue, endothelial cells lining the
deeper the river is, the slower and less turbu- microvasculature typically prevent the move-
lent the water flow. Coupled with the “leaki- ment of protein-rich fluid out of the blood
ness” of endothelium, vasodilation contributes vessels. They do this by creating a continuous
to the local stasis of blood flow. Slower velocity barrier on the inside of vessels through inter-
or stasis of blood flow is important to decrease cellular tight junctions, which is critical for the
the shear force on the leukocytes during the maintenance of fluid homeostasis. However,
process of extravasation, which we discuss in within minutes of the detection of infectious or
depth later. noxious stimuli by sentinel cells, inflammatory
Increased vascular permeability or leakiness mediators can act on endothelial cells (typically
is another important vascular change associ- in postcapillary venules) and trigger confor-
ated with inflammation. It permits the move- mational changes in these cells, leading to the
ment of fluid and plasma proteins—including breakdown of the endothelial barrier (Figure
complement, antibodies, and acute phase pro- 5.6A–D). Mediators such as histamine and IL-1
teins (discussed later in this chapter)—into the stimulate endothelial cells to contract or rear-
affected interstitial tissue. The protein-rich fluid range their cytoskeletons, which decreases
that escapes from vasculature is known as exu- their surface areas, creating gaps between en-
date. As mentioned earlier, vascular permeabil- dothelial cells and allowing protein-rich fluid
73

Figure 5.6A–D. Mechanisms that increase vascular permeability during inflammation
Four mechanisms are thought to contribute to the increase in vascular permeability during inflamma-
tion: endothelial contraction (panel A), active transcytosis (panel B), direct physical or chemical injury
to the vasculature (panel C), and injury to the vasculature after leukocyte extravasation or activation
(panel D).
(exudate) to leak out of these vessels and into cytosis is the active transport of fluid and mac-
the surrounding tissue. romolecules across the cytoplasm through a se-
Another mechanism by which increased vas- ries of separate or interconnected cytoplasmic
cular permeability is thought to be induced in vesicles. These vesicles effectively form tunnels
inflammation is through the upregulation of or channels through the cells, allowing protein-
transcytosis in venule endothelial cells. Trans- rich fluid to pass directly through the activated

endothelial barrier. Finally, aside from purpose- first to respond. Unlike macrophages, how-
ful changes to endothelial cells in postcapillary ever, before inflammation, neutrophils appear
venules induced by inflammatory mediators, almost exclusively in the blood. To be useful,
inadvertent injury to vasculature can also con- neutrophils need to find the general area of in-
tribute to vascular permeability after injury or flamed tissue, exit the circulation, and migrate
inflammation. Injury can include direct physi- within the tissue to the specific locale of insult.
cal or chemical injury to vascular beds by the They achieve this in four stages of extravasation
inciting insult (e.g., a heat or chemical burn) (Figure 5.7):
or collateral damage associated with leukocyte
1. tethering (margination) and rolling;
extravasation and the subsequent antimicrobial
effector function. 2. firm adhesion (pavementing) and crawling;
In addition to promoting vascular permeabil- 3. transmigration (diapedesis);
ity, the activation of vascular endothelium by 4. chemotaxis through tissue.
cytokines (such as IL-1 and TNF-α) induces the
Even though vascular changes help to slow
expression of cell-adhesion molecules. These
the blood flow in an inflamed region, normal
cell-adhesion molecules are displayed in high
neutrophil velocity in the circulation, coupled
numbers on the lumenal membranes of the
with the shear forces on stationary objects
endothelial cells that line postcapillary venules,
within blood vessels, makes the task of ex-
where they act to snag circulating leukocytes.
travasation difficult. Tethering and rolling of
This process serves to slow and tether use-
neutrophils on activated endothelium are nec-
ful leukocytes and also to signal to them that
essary to marginalize and slow down the oth-
a threat is in the vicinity and that they should
erwise fast-moving cells. As mentioned earlier,
extravasate.
IL-1 and TNF-α activate vascular endothelial
Overall, the changes to vasculature in the vi-
cells in venules to express and display cell-
cinity of a detected proinflammatory threat act
adhesion molecules. An important family of
to deliver more blood to the vasculature beds in
cell-adhesion molecules is the selectins, named
the area (vasodilation); slow the velocity of the
because they function as selective lectins (recall
blood flow (vasodilation and vascular perme-
that lectins are carbohydrate-binding proteins).
ability); allow protein-rich fluid into the tissue
Selectins are expressed on activated endothe-
(vascular permeability); and select, tether, and
lium, where they function to marginalize and
concentrate circulating leukocytes (cell-adhe-
slow down circulating leukocytes. E-selectin
sion molecule expression). Vascular changes are
binds to sialylated Lewis X–modified glyco-
important steps in the early process of inflam-
proteins that are displayed on the surface of
mation and account for many of the cardinal
circulating neutrophils and eosinophils. P-se-
signs (rubor, calor, and tumor), but they are just
lectin, which is also is displayed on activated
the beginning of the inflammatory process.
endothelium, binds platelet selectin glycopro-
tein ligand 1, which is a glycoprotein found
Leukocyte Recruitment on all circulating leukocytes. The interactions
between the selectins on activated endothelia
Leukocyte Extravasation and their glycoprotein ligands on leukocytes
As mentioned earlier, one of the major pur- are relatively low affinity. Through numerous
poses of inflammation is to recruit additional low-affinity selectin and ligand interactions,
leukocytes to the site of a detected threat. In however, leukocytes can loosely adhere to ac-
acute inflammation, neutrophils—well equipped tivated endothelium, causing them to be mar-
with an antimicrobial arsenal—are usually the ginalized within the vasculature and roll along
75

Figure 5.7. Leukocyte extravasation (video courtesy of Dr. Paul Kubes, University of Calgary)
Diagram (top) and video micrograph (bottom) showing the four stages of leukocyte extravasation.
The bottom panel is a sequential montage taken from a video micrograph of neutrophils extravasat-
ing from a postcapillary venule in the cremaster muscle of a mouse after experimental induction of
local acute inflammation. Endothelial cells and their tight junctions are highlighted in red. Neutro-
phils are green. From left to right, arrows indicate neutrophils undergoing (1) tethering and rolling,
(2) firm adhesion, (3) transmigration, and (4) chemotaxis. The video was taken using a mulitphoton
confocal microscope.
the endothelial surface. Through this process of chemokines, such as CXCL8, further slows
involving multiple weak interactions with pass- the neutrophils and induces conformational
ing leukocytes, the endothelium slows but does changes in a second group of neutrophil pro-
not stop the cells in the high-shear environment teins called integrins. These structural changes
of the blood vessel. switch the integrins from low- to high-affinity
Being tethered and rolled gives the leukocyte receptors. Lymphocyte function–associated an
time to detect local proinflammatory chemo- tigen 1 (LFA-1) is one such integrin found on
kines at the endothelial surface. The detection the surface of lymphocytes, monocytes, and

neutrophils. LFA-1 binds to intercellular adhe- typically occurs in the thin-walled postcapillary
sion molecule 1 (ICAM-1), which is another venules; however, transmigrating neutrophils
cell-adhesion molecule that endothelial cells must still penetrate the endothelial barrier and
express in response to proinflammatory media- the underlying basement membrane. Although
tors. In its low-affinity state, LFA-1 on neutro- neutrophils have been reported to exit the ves-
phils does not bind endothelial ICAM-1, but the sel by moving through intact endothelial cells,
conformational switch to its high-affinity state adhered neutrophils usually crawl to the junc-
quickly enables LFA-1 to bind ICAM-1 with tions between these cells to transmigrate. Here
great strength, allowing the rolling neutro- neutrophils, in cooperation with the endothe-
phil—if given the right chemokine stimulus— lial cells, break down the tight junctions of the
to slow its rolling down to a complete stop and endothelium, creating small gaps between the
to firmly adhere to the activated endothelium. endothelial cells. The neutrophils then attempt
Once stopped, the shear force on the adhered to squeeze through these gaps and digest the
neutrophil causes it to spread out from a spheri- underlying basement membrane through local-
cal shape to a flattened morphology. This stage ized secretion of collagenases. Using another
of firm adhesion is also referred to by patholo- series of cell-adhesion molecules, the leading
gists as pavementing, because the local endo- edge of each neutrophil adheres to the extra-
thelium starts to resemble a tiled pavement cellular matrix, allowing the neutrophils to pull
as more neutrophils adhere and flatten out. themselves out of the vasculature. It is not sur-
With the advent of sophisticated microscopic prising that by breaching the endothelial and
techniques, we now know that adhered neu- basement membrane barriers, the process of
trophils are not stationary on the endothelium transmigration additionally increases the per-
but crawl in their flattened state to try to find a meability (leakiness) of the vessel. Microscopic
suitable area to transmigrate. studies have also shown that the hole left by
The importance of the ability of neutrophils transmigrating neutrophils is commonly used
to firmly adhere to the endothelium is demon- by other transmigrating leukocytes, increasing
strated by the clinical manifestations of bovine the efficiency of extravasation. Now out of the
leukocyte adhesion deficiency (BLAD). BLAD circulation, the neutrophils must migrate to the
is an autosomal recessive disease that affects specific location of the potential threat.
Holstein cattle as a result of a mutation in the The recruitment of leukocytes to a spe-
gene that encodes β2-integrin (CD18). An ani- cific site of insult relies on the establishment
mal that is homozygous for this mutation has of chemical gradients of chemoattractants.
impaired expression of LFA-1 on its neutro- These chemoattractants can be endogenously
phils. Because LFA-1 is required to form a firm derived from activated macrophages and other
adhesion with activated endothelium (through immune processes or can be exogenously de-
interaction with ICAM-1), tethered and rolling rived microbial products diffusing away from a
neutrophils cannot stably adhere to the endo- site of infection. As mentioned earlier, sentinel
thelium and are thus unable to extravasate. cells such as macrophages, once activated by
Without the ability to mobilize its first respond- PAMPs, DAMPs, or both, produce proinflam-
ers to a site of inflammation, the animal suffers matory mediators. These mediators include
frequent and recurrent bacterial infections that chemoattractants, for example lipid-derived me-
result in premature death. diators such as leukotriene B4 and chemokines
Transmigration, commonly referred to as such as CXCL8. These endogenous chemoat-
diapedesis, is the movement of leukocytes tractants diffuse away from activated macro-
from the lumen of the blood vessel through phages at the site of insult, setting up a concen-
the vessel wall and into the interstitium. It tration gradient. Another powerful endogenous
77

Figure 5.8. Leukocyte chemotaxis (photograph courtesy of Dr. Paul Kubes, University of Calgary)
Diagram (left) and video micrograph (right) showing leukocyte chemotaxis up a chemoattractant gra-
dient. The micrograph is a sequential montage taken from a video of an activated neutrophil migrat-
ing up a concentration gradient of the chemoattractant of N-formylmethionine–containing peptide in
a petri dish. Note the neutrophil morphology, particularly the leading edge and trailing tail between
which the neutrophil can detect minute differences in concentration of the chemoattractant. The four
sequential photos are taken approximately 60 seconds apart using a phase-contrast microscope.
chemoattractant is a small protein by-product though, does the neutrophil detect a chemotac-
of complement activation, C5a. Recall from tic gradient and move in the right direction?
chapter 4 that following classical, alternative, When clinicians think of neutrophils, they
or lectin pathways of complement activation, usually picture a snowball-shaped cell with a
C5 is cleaved by the C5 convertases to produce segmented nucleus in blood smears. However,
a large C5b fragment (which is involved in ini- once out of the circulation, the neutrophil can
tiation of the MAC) and a small, often forgot- take on an amoeboid-like morphology with an
ten C5a fragment. C5a diffuses away from the extending pseudopod leading edge and a trail-
site of complement activation and establishes a ing tail (Figure 5.8). Using receptors for che-
chemoattractant gradient around complement- moattractants dispersed on its plasma mem-
activating surfaces. brane, the neutrophil extends its pseudopod in
Exogenously derived chemoattractants (such the region of the greatest chemotactic recep-
as microbial products—PAMPs) can also be tor activation and then pulls the rest of itself
used by neutrophils to navigate toward a nidus toward the stimulus. Clearly, these migrating
of infection. One particular chemotactic PAMP leukocytes can actually detect the very small
is N-formylmethionine–containing peptides. difference in concentration of a chemoattrac-
Because N-formylmethionine is common in tant between its leading edge and trailing tail
proteins of bacterial origin (but not common (some 8–12 µm apart). During the journey to
in host proteins), a neutrophil that migrates the site of injury or infection, stimulated by
up a gradient of N-formylmethionine pep- local proinflammatory mediators, the neutro-
tides should be moving toward its target. How, phil activates, making it much more efficient at

Figure 5.9. Eosinophilic inflammation
Histopathological section of the intestinal mucosa of a horse with a strongyle infection (arrow)
showing an acute inflammatory reaction with recruitment eosinophils. Note that a high proportion
of the cells have the bright red cytoplasmic granules characteristic of eosinophils.
locating, ingesting, and destroying pathogens sion molecules displayed on the endothelium,
(covered in chapter 6). and the types of adhesion molecules and che-
Once at the site of inflammation, the neu- mokine receptors on the surface of the circulat-
trophil is short-lived; thus, a constant stream of ing leukocytes. For instance, acute inflamma-
new neutrophils needs to be recruited. In many tion associated with parasitic infections (such as
bacterial infections, the neutrophils infiltrate strongyle infection in horses) is associated pri-
and die in such large numbers that they visibly marily with eosinophil recruitment (recall from
manifest as pus or suppuration. chapter 2 that eosinophils are best equipped to
fight helminthic infections; Figure 5.9). The re-
cruitment of eosinophils is mediated by release
Diversity and Timeline of of the chemokine CCL11 (also called eotaxin),
Leukocyte Recruitment which promotes the extravasation and che-
Although neutrophils are the most com- motaxis of eosinophils (as opposed to CXCL8,
mon early cells in many acute inflammatory which attracts primarily neutrophils).
conditions, other leukocytes (such as mono- Circulating monocytes are a common sec-
cytes, eosinophils, and lymphocytes) can also ond responder in acute inflammation, with the
be recruited to the site of inflammation. The majority being recruited one to two days after
mechanisms of extravasation and chemotaxis the initial insult, as the neutrophil recruitment
for these other leukocytes are similar to those wanes. As soon as they extravasate, these mono-
described earlier for neutrophils. Which leukocytes mature into macrophages, which further
cytes are recruited and the timing of their re- activate as they migrate toward the site of in-
cruitment are dependent on the type and extent flammation. As mentioned in chapter 2, macro-
of the tissue insult. Coordination of the type phages are moderately proficient at killing mi-
and timing of leukocyte recruitment to an in- crobes, but they also have the ability to phago-
flammatory site is achieved through a complex cytose and break down debris, as well as present
combination of chemotactic molecules and cy- antigen. Hence, these new macrophages start
tokines released in the area, the types of adhe- the task of cleaning up the dead neutrophils,
79

Figure 5.10. Phases of leukocyte recruitment in persisting inflammation
Although neutrophils are a common first responder in acutely inflamed tissue, mononuclear cells
and lymphocytes are often recruited in later stages of inflammation. This figure shows the phases of
leukocyte recruitment in a typical site of inflammation that persists over several weeks.
debris, and remaining microbes, which is not are dependent on the pathogen and tissue in-
only important in the fight against infection, but volved and the nature of the adaptive immune
a necessary step in the resolution of inflamma- response (e.g., Th1 vs. Th2 responses; covered
tion, wound healing, and tissue repair. in later chapters). Invariably, the neutrophil
In most instances, local acute inflammation plays a diminished role in chronic inflamma-
is self-limiting and quickly resolves without fur- tion, but macrophages, Th cells, Tc cells, and
ther sequelae. However, infection with a bona even B cells play major roles (Figure 5.10).
fide pathogen that is adapted to resist anti
microbial mechanisms of the innate immune
Systemic Effects of
system may cause persistent inflammation. In
Acute Inflammation
these instances, the adaptive immune system is
brought into play, and the acute inflammation Aside from the local effects of acute inflam-
may evolve into chronic inflammation. Chronic mation (rubor, tumor, calor, dolor, and functio
inflammatory processes are very diverse and laesa), significant localized inflammation or

Figure 5.11. Systemic effects of acute inflammation
Certain proinflammatory cytokines generated at the site of acute inflammation can be distributed
throughout the body of an animal, resulting in systemic inflammatory responses involving many
organs. ACTH = adrenocorticotropic hormone; CSF = colony-stimulating factors.
disseminated acute inflammation can have sys- 3. lethargy;

temic effects on the animal that may be clini- 4. muscular wasting (cachexia);
cally apparent. These effects become important 5. hemodynamic changes (shock);
indicators of an acute inflammatory process in
6. fever (pyrexia);
tissues or organs not immediately visible in a
7. leukocytosis (commonly neutrophilia);
clinical examination (e.g., pyothorax or prosta-
titis). In these situations, it is common for ani- 8. metabolic acidosis (lowered blood pH);
mals to be presented for these systemic symp- 9. alterations to acute phase proteins.
toms rather than for the inflamed tissue itself. The majority of the systemic effects of acute
Systemic effects of acute inflammation inflammation are mediated by the systemic re-
include lease of TNF-α, IL-1, and to a lesser extent IL-6
1. loss of appetite (anorexia); by activated leukocytes at the site of inflamma-
2. altered sleep patterns (increased slow-wave tion (Figure 5.11). These proinflammatory cyto-
sleep); kines can rapidly disseminate throughout the
81

body and mediate responses in many organs, extraordinarily conserved (even reptiles display
including the hypothalamus, liver, and bone fever in response to inflammation), and thus it
marrow. Generally speaking, many of these presumably offers some survival advantage to
systemic effects serve to conserve energy and the host.
mobilize resources (particularly protein) to aid Leukocytosis is the increased number of leu-
in the resource-expensive process of inflamma- kocytes circulating in the blood. Leukocytosis
tion. These systemic effects include anorexia, in response to acute inflammation is primarily
altered sleep patterns, lethargy, and cachexia a neutrophilia (increased numbers of circulat-
(which contributes to metabolic acidosis)— ing neutrophils), which is due to the increased
most of which are directed by TNF-α. Other release of neutrophils from the bone marrow
systemic effects, however, perform specific in response to IL-1 and TNF-α that provides
functions in the acute inflammatory process. greater numbers of these short-lived leuko-
Fever (pyrexia) is the elevation of the core cytes so that more can extravasate and contrib-
body temperature as a result of a higher-than- ute to the immune defense at the site of inflam-
normal thermoregulatory set point within the mation. Prolonged release of neutrophils from
anterior hypothalamus. This set point is raised the bone marrow will deplete its store of the
(typically between 1° and 5°C [~2° and 9°F]) mature cells, prompting increased synthesis
above the normal body temperature of the ani- of CSFs such as granulocyte CSF (products of
mal in response to pyrogens. Pyrogen is a term inflammation such as C3a also lead to eleva-
used for any substance that induces fever and tions in G-CSF). This increased synthesis in
can be exogenously or endogenously derived. turn enhances granulocyte differentiation from
Immunologically speaking, exogenous pyrogens myeloid precursor cells, and the release of the
(typically PAMPs or DAMPs such as LPS and ad- less mature band neutrophils. As mentioned in
enosine) can induce leukocytes to produce en- chapter 2, band neutrophils can be identified by
dogenous pyrogens, which are proinflammatory their unsegmented band nuclei. Clinically, this
cytokines such as TNF-α and IL-1 (Figure 5.12). release of less mature band neutrophils is re-
These endogenous pyrogens can be released ferred to as a neutrophilia with a left shift (more
into the circulation to act on the vascular en- circulating neutrophils that are on average less
dothelial and perivascular cells of the anterior mature) and is commonly associated with acute
hypothalamus. Here, they can stimulate the inflammation. It is one of the most important
synthesis of prostaglandins (particularly pros- clues a veterinarian has in gauging whether the
taglandin E2), which will then act on neurons animal’s clinical signs stem from an inflamma-
in the preoptic nucleus involved in thermoregu- tory process (especially if the site of inflamma-
lation. Nonsteroidal anti-inflammatory drugs tion is not immediately apparent). The degrees
(such as meloxicam) are often antipyretic as well of neutrophilia and left shift are also important
as anti-inflammatory because they block cyclox- diagnostic indicators of the severity of the in-
ygenase enzymes, thus reducing the production flammatory response occurring in the animal
of prostaglandins at the site of inflammation (Figure 5.14).
(anti-inflammatory) as well as in the anterior Another common laboratory finding in
hypothalamus (antipyretic). acute inflammation is the change to the acute
Fever in response to acute inflammation is phase protein (APP) profile of the serum.
well accepted, but what are the functions of APPs are serum proteins whose abundance is
fever? Surprisingly, it is poorly understood. altered in response to an acute inflammatory
There are many theories, but no one theory process. They can be divided into positive and
can explain the benefits of fever to everyone’s negative APPs, with serum levels of positive
satisfaction (Figure 5.13). Nevertheless, fever is APPs increasing, and those for negative APPs

Figure 5.12. Induction of fever
Exogenous and endogenous pyrogens stimulate the release of prostaglandins (prostaglandin E2) in the
anterior hypothalamus that act on the thermoregulatory neurons that control the body’s temperature
set point. Increasing the set point promotes metabolic and behavioral changes in the animal that serve
to increase heat production and minimize heat loss.
83

Figure 5.13. Proposed functions of fever
The true function of fever is nebulous. Over the years, numerous reports have shown immunological
benefits of fever to the host animal. This diagram attempts to summarize these possible benefits of
fever.
decreasing, in response to acute inflammation. onset of inflammation) and can reach levels
Positive and negative APPs in various species ten to one hundred times their normal serum
and their relative association with acute in- concentrations. Veterinarians exploit this fea-
flammation can be found in Table 5.2. APPs ture by using increased APP concentrations
are named as such because they are indicative as an indicator of inflammation, well before
of an inflammatory process taking place in the classic blood leukocyte concentration changes
animal, not necessarily because they have com- (such as neutrophilia). The use of APPs in di-
mon functions or are proinflammatory. In fact, agnostic medicine has received a great deal of
the reported actions of different positive APPs attention in recent years as an aid in the diag-
include a wide range of homeostatic, proin- nosis of a broad range of diseases, including
flammatory, and anti-inflammatory actions. bovine respiratory syncytial virus infections,
APP concentrations rise very quickly in the prostate cancer, bronchopneumonia, multiple
serum of animals with inflammation (peak- myeloma, mastitis, Streptococcus suis infection,
ing within twenty-four to forty-eight hours of starvation, and lymphoid neoplasia.

Figure 5.14. Neutrophilia left shift
As bone marrow stores of mature neutrophils diminish during the course of acute inflammation, less
mature neutrophils will be released into circulation, which is termed a left shift of the population of
circulating neutrophils. This figure shows the range of maturation in states of normal, left-shifted,
and severely left-shifted neutrophil populations.
Many of the positive APPs are produced by ment activation). Thus, CRP acts as a secreted
the liver in response to cytokines such as IL-6, PRR that enhances the clearance of microbes
although the gastrointestinal tract, kidney, and damaged host cells during periods of acute
heart, adipocytes, and even leukocytes them- inflammation. Numerous other pro- and anti-
selves have been shown to synthesize various inflammatory roles of CRP have been reported,
APPs. One of the more prominent positive including the induction of various cytokines,
APPs in dogs, rabbits, pigs, and primates is C-re- attenuation of neutrophil chemotaxis and de-
active protein (CRP). CRP is a pentameric pro- granulation, and promotion of tissue repair.
tein produced by hepatocytes and adipocytes in
response to IL-6. It contains domains that can
Clinical Correlation: Follow-Up
bind phosphocholine as well as certain poly-
saccharides and glycolipids that are commonly As we discussed at the start of this chapter,
found on the surface of bacteria, parasites, and acute mastitis is an acute inflammatory reac-
damaged cells. Other domains can bind cer- tion in the mammary gland, leading to its dys-
tain Fc (antibody) receptors on macrophages function (Figure 5.15). Lactating dairy herds
(enhancing the phagocytosis of microbes and are highly susceptible to mastitis, which is com-
dead cells) or can bind C1q (initiating comple- monly initiated by contagious or environmental
85

Table 5.2. Acute-phase proteins in domestic species
APP Immunologic function Species variability
C-reactive protein Binds to bacteria Major APP in dog and pig (increases 10 to
(CRP) Promotes complement binding and opsonization 100 times)
Induces cytokine production Not seen in cats
Modulates monocytes and macrophages
Serum amyloid A Chemotactically recruits leukocytes Major APP in dogs, cats, cattle, and pigs
Downregulates inflammatory response (increases 10–100 times)
Has an inhibitory effect on fever, oxidative burst
of neutrophils, and immune response
Involved in lipid metabolism and transport (e.g.,
cholesterol from dying cells)
Plays a pathological role in systemic (reactive)
amyloidosis
Haptoglobin Binds hemoglobin; reduces tissue toxicity and Major APP in pigs, cattle, and sheep
sequesters iron from microbe (increases 10–100 times)
Inhibits granulocyte chemotaxis and phagocytosis Moderate APP in dogs and cats (increases
2–3 times)
Alpha-1-acid Antineutrophil and anticomplement activity Major APP in cats (increases 10–100 times)
glycoprotein Increases macrophage IL-1R antagonist secretion Increases in feline infectious peritonitis and
inflammatory peritonitis in cats
Moderate APP in dogs (increases 2–3
times)
Minor APP in cattle
Ceruloplasmin Transports copper for wound healing, collagen Moderate APP in dogs (increases 2–3
formation, and maturation times)
Antioxidant Minor APP in cattle
Hepcidin Sequesters iron in anemia of chronic disease
Fibrinogen Coagulation Important indicator of acute inflammation
in horses and cattle
Albumin (negative) Decreases inflammation
Transferrin Iron binding, so reduced concentration sequesters
(negative) iron from bacterial use
bacteria. Hallmark characteristics of acute mas- Possible Explanations

titis include redness, heat, swelling and pain in High-performing mammary glands such as
the udder, and the presence of leukocytes in the those of a lactating dairy cow are highly suscep-
milk. tible to microbial infection with contagious or
environmental bacteria. These bacteria can pass
through the streak canal, particularly after milk-
Student Considerations ing, and proliferate in the gland. The proliferat-
After learning about the onset and developing microbes release PAMPs (such as endotoxin
ment of acute inflammation, you should be in the case of coliform mastitis), which are de-
able to describe the sequence of events that tected by sentinel cells (such as macrophages)
leads to acute mastitis and outline the key clini- resident in the gland’s tissue. These cells release
cal and laboratory findings that would aid a vet- proinflammatory cytokines, including TNF-α,
erinarian in his or her diagnosis. IL-1, and IL-6, and other mediators such as pros-

Figure 5.15. Acute bovine mastitis (photograph courtesy of Dr. Gordon Atkins, University of Calgary)
This photograph is of a five-year-old, mid–third-lactation Holstein cow with severe mastitis of the left
front quarter caused by Klebsiella bacteria.
taglandins and leukotrienes that, together with touch. When a milk sample is taken, increased
mediators from other cell types such as hista- numbers of infiltrated neutrophils along with
mine, result in changes to the local vasculature. sloughed epithelial cells can be detected by milk
These changes allow serum proteins containing smear, CMT, or automated cell counting. The
complement and antibodies into the tissue and cow may be febrile and show signs of lethargy,
also promote the tethering and adhesion of cir- anorexia, and altered behavior resulting from
culating neutrophils. Sensing chemokines ema- the systemic release of TNF-α and IL-1. Blood
nating from within the tissue, the tethered and samples would reveal a neutrophilia with left
rolling neutrophils extravasate and migrate up shift and, if measured, increased serum con-
a proinflammatory chemoattractant gradient. centrations of APPs, including haptoglobin and
Here, they can bolster the local macrophage serum amyloid A (the major APPs in cattle).
defense against the invading microbes by using To the dairy farmer, acute mastitis signifies a
their potent antimicrobial effector mechanisms. loss of income and considerable effort and time
The details of these mechanisms are discussed lost in both individual treatment (often intra
in the next chapter. gland antibiotic preparations) and herd preven-
Clinically, these changes manifest in a mam- tion. To the cow, acute mastitis is painful and
mary quarter that is swollen, red, and hot to the uncomfortable and is often accompanied by
87

systemic ill thrift. Nonetheless, as illustrated by tally important process of the innate and adap-
deficiencies such as bovine leukocyte adhesion tive immune systems and is essential for the
deficiency, acute inflammation is a fundamen- survival of animals in this microbe-rich world.

Robin M. Yates
Innate Cellular Effector Mechanisms Chapter 6

10.5876_9781607322184.c006
report as having “rattling breath” and “being

Clinical Correlation: Rattles in Foals 89
off feed.” Unfortunately, the filly dies before
the veterinarian arrives. On postmortem of
Classification of Innate Cellular Effector
the foal, the veterinarian discovers tuberculo-
Mechanisms 90
sis-like granulomas throughout the lungs and
Phagocytes and Phagocytosis 91
tentatively attributes the filly’s death to pneu-
Phagocytosis Part I: Recognition and
Adhesion of Particles on the Plasma
monia caused by a disease known as rattles
Membrane of the Phagocyte 91 (Figure 6.1).
Phagocytosis Part II: Membrane and Rattles, so named because of its presenting
Cytoskeletal Reorganization to Mediate sign of rattly breathing, is a severe form of
Particle Engulfment and the Creation of a bacterial pneumonia that commonly affects
Phagosome 94 foals. It is caused by the Gram-positive bacte-
Phagocytosis Part III: Maturation of the rium Rhodococcus equi, which is related to My-
Phagosome into a Microbicidal and
cobacterium tuberculosis (the etiological agent
Degradative Phagolysosome 94
of tuberculosis). R. equi is found in soil and is
Extracellular Innate Effector Mechanisms 99
endemic on certain horse farms. The majority
Degranulation by Neutrophils and
Eosinophils 99 of cases occur in hot, dry summers when con-
Release of Neutrophil Extracellular Traps 100 ditions are dusty. It is thought that foals inhale
Natural Killer Cell–Mediated Cytotoxicity 101 the bacteria in the dust, which are then depos-
Recognition of Target Cells 101 ited in terminal bronchi and alveoli. Here the R.
Activating stimulus 101 equi microbes are engulfed by resident alveolar
Inhibitory stimulus 102 macrophages. These specialized macrophages
Targeted Degranulation by Natural are the primary immune cells defending these
Killer Cells 102 respiratory surfaces. They are extremely profi-
Antibody-Dependent Cell-Mediated cient at recognizing and phagocytosing inhaled
Cytotoxicity 105 debris and microbes and very effective at killing
Clinical Correlation: Follow-Up 105 them. R. equi bacteria, however, are not typi-
Student Considerations 105 cally killed by alveolar macrophages in foals.
Possible Explanation 106 In fact, they can thrive within the macrophage.
Macrophages infected with R. equi quickly re-
Clinical Correlation: cruit other immune cells through the release of
Rattles in Foals proinflammatory cytokines, eventually form-
ing granulomas. How is the macrophage able
A veterinarian is called out to examine a four- to kill most microbes easily but unable to kill
month-old thoroughbred filly that the owners R. equi?
89
89

some that mediates the killing and diges-
tion of microbes;
• describe the process of degranulation of
neutrophils and eosinophils and list the
major products released by these cells;
• describe the formation and composition of
NETs and explain their proposed function;
• explain in general terms the recognition of
target cells by NK cells;
• explain how NK cells kill target cells;
• explain the augmentation of the innate effec-
tor mechanisms by the adaptive immune
system.
Classification of Innate
Cellular Effector Mechanisms
Thus far, you have learned that innate immune
cells can recognize microbes and parasites,
alert neighboring cells, and coordinate recruit-
ment of specific innate immune cells to a site
of infection. But what do these cells do when
Figure 6.1. Lung granulomas found at necropsy they get there, and how do they control infec-
in a five-month-old foal infected with tion? The mechanisms of microbial killing are
Rhodococcus equi (photograph courtesy of Dr. as diverse as the microbial threats to an animal.
Rachel Peters, Takeda Pharmaceuticals) These mechanisms depend on the type of in-
fection (particularly location and physical size
of the invader) and which innate immune cell
Learning Objectives responds to the infection.
Generally, the killing mechanisms can be cat-
After reading this chapter, you should be able to
egorized as
• explain in general terms the effector mecha-
nisms of the innate immune system; 1. phagocytosis by phagocytes and killing of
microbes within the phagosome;
• identify which innate immune effectors are
most efficient at controlling 2. release of antimicrobial products to kill
extracellular microbes and parasites;
»» small extracellular threats, such as bacteria,
3. targeted destruction of infected host cells
»» large extracellular threats, such as multicel-
by NK cell–mediated cytotoxicity.
lular parasites,
»» intracellular infections, such as viruses; Together, these mechanisms can defend
• list the major professional phagocytes and against pathogens large or small, intracellular
describe how they recognize, ingest, and or extracellular, often without any help from
kill target microbes; the adaptive immune system. The response to
• describe the process of phagosomal matu- a given infection may use effector mechanisms
ration and list attributes of the phagolyso- in just one, two, or all three of these categories.
90 Inn a t e C e l l u l a r E f f e c t o r M e c h a n i s m s

Although these cellular effector mechanisms ized PRRs and opsonic receptors on the surface
are considered part of the innate immune sys- of the phagocyte. As you recall from chapter 4,
tem (because they can be immediately deployed PRRs are immune receptors that can recognize
without previous exposure to the threat), these certain evolutionarily conserved molecular pat-
mechanisms also play an important role in the terns that are unique to foreign material such
elimination of infection after an adaptive im- as microbes (PAMPs) but are absent in normal
mune response has been mounted. In many healthy host tissue. In many circumstances, li-
cases, adaptive immune responses (such as the gation of these PRRs (e.g., by TLRs) leads to
production of antibodies) specifically act to en- proinflammatory signaling and activation of
hance the efficiency of these innate cellular ef- the cell. In addition to proinflammatory PRRs,
fector mechanisms. phagocytes express phagocytic PRRs. These re-
ceptors bind to PAMPs displayed on the surface
of microbes and instigate phagocytosis.
Phagocytes and Phagocytosis
Table 6.1 lists several known PRRs that can
As mentioned in chapter 2, both macrophages initiate phagocytosis. As covered in chapter 3,
and neutrophils (and to a lesser extent DCs) the best-characterized phagocytic PRR is the
have the ability to engulf small particles by a mannose receptor (MR, CD206). This recep-
process called phagocytosis. In fact, these cells tor is found on the surface of macrophages and
are so adept at “eating” particles that they are binds terminal sugar residues on proteins that
often collectively referred to as professional are common on the surface of microbes (but
phagocytes (i.e., professional eating cells). Once absent on healthy host tissues). When microbes
these phagocytes have phagocytosed microbes come into contact with the membrane of
and other material, they can kill and digest the phagocytes, the MR (along with other phago-
phagocytosed products within an intracellular cytic PRRs) binds to molecules on the microbe’s
vacuole called a phagolysosome, which allows the surface. The ligated receptors then initiate in-
cell to kill microbes, using high concentrations tracellular signaling pathways that coordinate
of toxic compounds and enzymes, while mini- phagocytosis of the microbe. Thus, through
mizing collateral damage to neighboring host phagocytic PRR, phagocytes can directly recog-
cells. nize and selectively engulf foreign particles but
The process of microbial killing via phago- leave normal healthy host cells alone. Similar
cytosis can be broken down into three stages: receptors, however, can recognize molecules
on the surface of host cells that have been dam-
1. recognition and adhesion of particles on
aged or have gone through apoptosis (such as
the plasma membrane of the phagocyte;
phosphatidyl serine). These receptors facilitate
2. membrane and cytoskeletal reorganization phagocytosis of dead and dying host cells (a
to mediate particle engulfment and the process often referred to as efferocytosis), which
creation of a phagosome; is necessary for homeostasis, wound repair, and
3. maturation of the phagosome to a microbi- removal of cells that have been killed by an in-
cidal and degradative phagolysosome. tracellular infection such as a virus. These recep-
tors, some of which are listed in Table 6.1, per-
mit professional phagocytes to recognize a wide
Phagocytosis Part I: Recognition range of particles from microbes to apoptotic
and Adhesion of Particles on the host cells and to stimulate their phagocytosis.
Plasma Membrane of the Phagocyte Although macrophages and neutrophils can
The recognition and adhesion of particles autonomously recognize and phagocytose for-
destined for phagocytosis involve both special- eign objects using their phagocytic PRRs, this
Inn a t e C e l l u l a r E f f e c t o r M e c h a n i s m s 91
91

Figure 6.2A–H.Phagocytosis of an IgG-opsonized particle (yellow arrow) by a tissue macrophage
(images acquired at the Live Cell Imaging Facility, University of Calgary)
This series of micrographs was taken over a period of 30 minutes and illustrate the three stages
of phagocytosis: recognition and adhesion of particles on the plasma membrane of the phagocyte
(panels A–B), membrane and cytoskeletal reorganization to mediate particle engulfment and creation
of a phagosome (panels C–E), and maturation of the early phagosome that will eventually become a
microbicidal and degradative phagolysosome (panels F–H).
process is dramatically enhanced by a process molecules essentially tag the microbe for phago-
known as opsonization. Opsonization is the cytosis (making it more tasty to phagocytes).
process whereby particles destined for phago- Phagocytes can strongly adhere to microbes
cytosis are coated with a protein-binding en- coated with the C3b and IgG opsonins through
hancer known as an opsonin (derived from the receptors for C3b (CR1 and CR3) and recep-
Greek opson, which is a condiment such as rel- tors that recognize the constant or Fc portion
ish that is added to food to make it more tasty, of IgG (Fcγ receptors) (Table 6.1). These recep-
hence opsonin). Mammals have two major op- tors also provide a powerful stimulus to initi-
sonins—complement (specifically C3b) and ate phagocytosis of the bound, opsonized mi-
antibodies (particularly certain IgG isotypes). crobes (Figure 6.2). Phagocytosis of opsonized
C-reactive protein (the major acute-phase pro- particles occurs with much greater efficiency
tein in many animal species) can also act as an than phagocytosis initiated through phagocytic
opsonin. These proteins are found in the serum PRRs alone (see Figure 6.3). Hence, although
and can bind to microbes. C3b covalently binds phagocytosis by macrophages and neutrophils
to microbes after initiation of the complement is considered an innate immune response,
cascade (recall from chapter 4). IgG noncova- the production of certain antibodies after the
lently, but very specifically, binds to antigens stimulation of an adaptive immune response
on the surface of microbes after a humoral im- acts to significantly enhance this innate effector
mune response (covered in chapter 12). These process.

Table 6.1. Examples of pattern recognition and opsonic phagocytic receptors that can be found on professional
phagocytes
Phagocytic receptor type Receptor example Ligand Major target
Direct PRR MR (CD206) Terminal mannose and Bacteria, fungi, viruses
fucose residues
Direct PRR Dectin-1 β-glucan residues Fungi
Direct PRR Scavenger receptor A I Polyanionic ligands (e.g., Diverse (e.g., foreign) particles,
LPS) microbes, and lipoproteins
Direct PRR Phosphatidyl serine receptor Phosphatidyl serine Apoptotic cells
Opsonic receptor CR 3 (CD11b–CD18) C3b Any surface labeled with C3b
Opsonic receptor Fcγ receptor 1 (CD64) Fc portion of IgG Any IgG-bound surface
Figure 6.3. A hypothetical representation of the rate of clearance of invading bacteria from an animal
in the presence or absence of opsonization
Bacteria alone could eventually be cleared from the body by phagocytosis via the direct recognition
by phagocytic PRRs (e.g., MR). However, the rate of clearance would be enhanced with the addition
of the complement- and Ig-based opsonins through more efficient bacterial recognition by phagocytic
cells.
93

results in tight adhesion of the particle to the
plasma membrane and also serves to cluster
the receptors together. This process of recep-
tor clustering is often enough to initiate re-
gional signals that orchestrate the cytoskeletal
rearrangement necessary for the engulfment of
the particle at the plasma membrane. Several
models of phagocytosis exist, depending on the
size of the particle and the type of phagocytic
receptors engaged, but this process essentially
results in involution of the plasma membrane
to which the particle is bound (Figure 6.5). This
inversion creates an inward-facing pouch of
plasma membrane that is eventually pinched
off, resealing the plasma membrane and creat-
ing a cytoplasmic vacuole (sac) containing the
Figure 6.4. Phagocytosis of bacteria by particle. This vacuole is called the phagosome,
neutrophils and for the first few seconds after being formed,
Neutrophil (yellow) in the process of engulfing it contains only the particle and the extracellu-
anthrax bacteria (orange) via phagocytosis. This lar fluid that was brought in with it—but all of
image was taken by Volker Brinkman using a this changes rapidly.
scanning electron microscope (reprinted from
Brinkman, 2005, PLoS Pathogens 1[3]: cover).
Phagocytosis Part III: Maturation of
the Phagosome into a Microbicidal
Phagocytosis Part II: Membrane and Degradative Phagolysosome
and Cytoskeletal Reorganization The interior of the phagosome—potentially
to Mediate Particle Engulfment containing living microbes—quickly evolves
and the Creation of a Phagosome from an innocuous environment with a neu-
The actual process of phagocytosis refers tral pH to an acidic, oxidative, degradative,
to the physical engulfment of particles bound antimicrobial chamber. This cellular process
to the plasma membrane, resulting in their is aptly named phagosomal maturation (Figure
being trapped in an intracellular vacuole called 6.6). For it to occur, the newly formed phago-
a phagosome. Macrophages and neutrophils some begins to fuse with endosomes and lyso-
can easily phagocytose irregularly shaped par- somes within the cytoplasm of the phagocyte.
ticles and particles as much as 5 µm in diameter Initially, the phagosome preferentially fuses
(about half the size of the phagocytes them- with the relatively immature early endosomes;
selves; Figure 6.4). Moreover, macrophages however, over a period of several minutes, the
and neutrophils can engulf numerous particles maturing phagosome starts to fuse with late
within minutes, and it is not uncommon to see endosomes and eventually with fully formed
these cells laden with upward of twenty mi- lysosomes. In neutrophils, the azurophilic gran-
crobes. Obviously, this level of phagocytosis re- ules—which are essentially modified lysosomes
quires significant alteration of the cytoskeleton that are densely packed with high concentra-
and membrane of the phagocyte. tions of antimicrobial proteins—also fuse with
Recognition of the particle by phagocytic the phagosome. This process of fusion be-
receptors on the surface of the phagocytes tween the phagosome and the endosomal and

Figure 6.5. Stepsof phagocytic
engulfment of a bacterium
Recognition of a bacterium by phago-
cytic receptors tightly binds the bacte-
rium to the plasma membrane and leads
to intracellular signaling that initiates
engulfment by phagocytosis. During the
process of phagocytic engulfment, the
cytoskeleton is remodeled to allow invo-
lution of the plasma membrane and the
creation of a new intracellular vacuole
called the phagosome.
lysosomal compartments results in the delivery actively pump protons into its interior, making
of hydrolytic enzymes and other antimicrobial it acidic (around pH 4–5). This acid not only
molecules (such as antimicrobial peptides) to serves to help kill any microbes directly but also
the phagosome. The phagosome also starts to activates many of the antimicrobial enzymes
accumulate complexes in its membrane that that have been delivered to the phagosome.
95

Figure 6.6. Maturation of the phagosome into a mature phagolysosome
After phagocytosis of a microbe, proton pumps are quickly assembled on the phagosomal membrane,
which serve to acidify its interior. In addition, endosomal and lysosomal compartments containing
hydrolytic enxymes and antimicrobial products progressively fuse with the nascent phagosome dur-
ing its maturation. These events rapidly remodel the newly formed vacuole into an acidic, digestive,
antimicrobial phagolysosome.

After extensive fusion with endosomes and ly- not generate an oxidative burst in phagocytes
sosomes, the mature phagosome is considered and thus suffer recurrent bacterial infections in
a hybrid vacuole called a phagolysosome. a condition called chronic granulomatous dis-
Another extremely effective strategy used ease. Although no naturally occurring NADPH
by the phagosome to kill ingested microbes oxidase deficiencies have been reported in vet-
is the generation of reactive oxygen species, erinary species, they undoubtedly exist but go
often referred to as the oxidative or respira- undiagnosed.
tory burst. This strategy is achieved by a mul- The phagosome can also produce another
tisubunit complex called NADPH oxidase oxidative radical group called the reactive ni-
that is assembled on the phagosomal mem- trogen intermediates (RNI). Using the amino
brane. This complex transfers unpaired elec- acid arginine as a substrate, inducible nitric
trons from NADPH to molecular oxygen (O2), oxide synthase (iNOS) produces the RNI nitric
generating the free radical superoxide (O2-) oxide (NO). Nitric oxide is produced by many
within the phagosome (Figure 6.7). Having cells as an important signaling molecule, but
an unpaired electron, superoxide is extremely at the high concentrations generated in the
reactive (and damaging) to molecules in its phagosomes of activated phagocytes (activa-
vicinity. If it does not react with an organic tion increases expression of iNOS—hence in-
compound, it typically reacts with hydrogen ducible nitric oxide synthase), the nitric oxide
ions and water to produce hydrogen peroxide free radical is antimicrobial, reacting with DNA
(H2O2) in a chemical reaction called dismuta- and microbial enzymes. Nitric oxide can also
tion. Although hydrogen peroxide is not a free combine with superoxide to produce a highly
radical per se, it is a powerful oxidant—par- unstable isomer of nitrate called peroxynitrite.
ticularly at a low pH, as in the phagosome. Peroxynitrite and its downstream products are
In the phagosome, hydrogen peroxide can be extremely damaging to the lipids, DNA, and
further converted into hydroxyl radicals (OH-), protein contained within the phagosome.
particularly in the presence of ferrous ions. Aside from a low pH and the production of
Similar to superoxide, these free radicals are oxidative compounds, fusion with lysosomes
extremely damaging to virtually all macromol- and granules delivers a variety of digestive
ecules within the phagosome. However, these enzymes and antimicrobial peptides to the
reactions do not necessarily stop here. phagosome. Because lysosomes function as the
Neutrophils make a heme-containing en- garbage disposal system of the cell, they con-
zyme called myeloperoxidase (MPO) in large tain numerous acid hydrolases that can break
amounts and store it in their granules. (An in- down a variety of macromolecules, includ-
teresting fact is that the heme group in MPO ing lipids, carbohydrates, proteins, and nucleic
has a green hue. It is this pigment that gives acids. Within their phagosomes, macrophages
pus, which is made up of dead neutrophils, its use these enzymes to digest and recycle com-
green tinge.) When these granules fuse with ponents of quiescent cells and debris in their
the phagosome, MPO can combine hydrogen normal homeostatic tasks. These enzymes,
peroxide with a chloride ion to make the hypo- though, also serve to help kill and digest phago-
chlorite ion (ClO-). (ClO- is the active ingredi-

cytosed microbes during infection. One ex-
ent in bleach, a powerful disinfectant.) tremely important set of hydrolases delivered
Hence, the oxidative burst in phagosomes to the phagosome is the proteases. As you will
generates numerous reactive oxygen species, learn in chapter 8, these enzymes function in
which serve to kill phagocytosed microbes the phagosomes of macrophages and DCs to
through oxidation. Humans and mice deficient break down protein antigens into small pieces
in parts of the NADPH oxidase complex can- that can be recognized by T cells. This process
97

Figure 6.7. Interaction of oxidative chemistries within the phagosome
Superoxide (O2-), hydrogen peroxide (H2O2), hydroxyl radicals (OH-), nitric oxide (NO), peroxynitrite
(ONOO-), and hypochlorite ion (ClO-), although not all free radicals, can all damage biologically
important molecules within the phagosome.

Extracellular Innate
Effector Mechanisms
Degranulation by Neutrophils
and Eosinophils
To defend against threats that are too big or
too numerous to phagocytose, neutrophils and
eosinophils can release their arsenals of gran-
ules into the extracellular space in a process
called degranulation. Unlike mast-cell degranu-
lation, which releases mostly vasoactive amines
such as histamine, degranulation by neutrophils
and eosinophils releases cytotoxic and degrada-
tive proteins and peptides. Although the aim is
to kill the foreign invader, a high amount of col-
Figure 6.8. The
environment within the lateral damage occurs to the host tissue.
phagolysosome In the face of significant proinflammatory
After minutes to hours, the newly formed stimulation and the presence of high levels of
phagosome is matured into an acidic, oxida- microbial products, neutrophils can induce de-
tive, digestive, antimicrobial compartment. granulation by fusing certain granules to the
Micrograph shows three tissue macrophages, cells’ plasma membranes. All of the granule
two of which have phagocytosed IgG-opsonized products are then released into the extracellular
particles (green). Nuclei are stained blue. space (many of these products are the same as
those delivered to the controlled environment
within the phagosome after phagocytosis).
These products include numerous proteases
is called antigen processing and is essential for (many of which degrade connective tissue),
the development of T-cell–mediated adaptive MPO, and antimicrobial peptides (such as de-
immunity. fensins and cathelicidins). In addition to the
Hence, professional phagocytes, such as release of these granule components, NADPH
macrophages and neutrophils, can recognize oxidase can form on the plasma membrane of
microbes (especially ones that have been opso- the neutrophil and generate superoxide in the
nized), engulf them by the process of phago- extracellular space. The very same dismutation
cytosis, entrap them in an intracellular vacuole reactions that occur in the phagosome can thus
called a phagosome, and then proceed to pump occur in the extracellular space. Hence, the out-
acid, toxic chemicals, and digestive enzymes wardly directed respiratory burst also generates
into the compartment (Figure 6.8). By carefully hydrogen peroxide, hydroxyl radicals, and (be-
selecting what to phagocytose and by contain- cause MPO is released by degranulation) hy-
ing the majority of the cytotoxic components pochlorite ion (ClO-). Although some of these
used to kill internalized microbes, phagocyto- products released by neutrophils are somewhat
sis is a relatively precise antimicrobial effector selective toward microbes (such as defensins),
mechanism that limits collateral damage to most (including proteinases, oxidative radicals,
neighboring host cells. But what happens if the etc.) are just as toxic to host cells as the targeted
invading threat is too big to phagocytose (such microbes. Excessive degranulation by neutro-
as a worm), or there are just too many threats phils can be life threatening to an animal and is
to handle? common in acute lung injury and septic shock.
99

plasma membrane and generate extracellular
superoxide and hydrogen peroxide during a re-
spiratory burst. Products released by activated
eosinophils, although antiparasitic, are also
extremely damaging to bystander host cells.
Eosinophil degranulation also triggers mast-
cell degranulation and is commonly associated
with allergies.
Release of Neutrophil
Extracellular Traps
More recently, researchers have discovered
that, when faced with high levels of proinflam-
matory stimulus, neutrophils can extrude a
sticky, meshlike substance into the extracellu-
lar space to which bacteria and yeast stick and
Figure 6.9. Acanine eosinophil in the process of
are killed. These substances are named neutro-
degranulation
phil extracellular traps (NETs). With the use
Note the release of the red granules from the
of NETs, neutrophils can efficiently trap and
eosinophil’s cytoplasm.
kill many microbes while minimizing collat-
eral damage to host tissue. NETs are formed
in the final stages of active neutrophil death,
Eosinophils, as mentioned in chapter 3, are in the presence of cytokines such as IL-8 and
specialist antiparasite leukocytes. Although PAMPs such as LPS in inflamed tissue. The
they often play roles in defending against cer- fibers of the NETs are actually strands of the
tain bacterial and viral threats, they are best neutrophils’ own DNA. Both nuclear and gran-
equipped to fight multicellular parasites—those ule membranes break down during the pro-
that are too big to phagocytose. As such, in cess of NET release, allowing antimicrobial
an acute inflammatory response to parasites, granule proteins (such as elastase, defensins,
the eosinophils are often the major recruited and MPO), along with nuclear histones (which
leukocyte. After activation by proinflamma- surprisingly also have antimicrobial proper-
tory stimuli, eosinophils can release their cy- ties), to stick to the negatively charged DNA
totoxic granule contents into the surrounding fibers. Decorated with antimicrobial proteins,
area via degranulation. Eosinophil granules these sticky networks of DNA fibers can not
contain several cytotoxic proteins, including only trap bacteria and yeast, but also kill them
major basic protein (a cytotoxin with antihel- directly (Figure 6.10). NET formation has also
minthic properties), eosinophil cationic protein been shown to occur in the blood, particularly
(a pore-forming protein that punches holes in in the capillaries of the lung and in liver sinu-
cell membranes; it is also reported to be a ribo- soids during periods of sepsis. Although these
nuclease that digests RNA), eosinophil-derived NETs may function to ensnare microbes in
neurotoxin (ribonuclease that has antiviral the blood, they may have detrimental conse-
properties), and eosinophil peroxidase (facili- quences to the animal. Not surprisingly, the
tates oxidative damage by generation of hydro- breakdown of the nucleus and the extrusion of
gen peroxide). As with neutrophils, eosinophils DNA during NET formation results in death of
can also assemble NADPH oxidase on their the short-lived neutrophil.

lies of conserved surface receptors that recog-
nize sick and stressed host cells, as well as those
cells that are not correctly displaying their self-
markers because they are most likely harboring
infectious agents (such as viruses) or are possi-
bly transformed (neoplastic) cells. In response,
NK cells can kill infected host cells, helping to
contain intracellular infectious agents while the
Tc lymphocyte arm of the adaptive immune
system prepares an antigen-specific response.
Recognition of Target Cells

The exact mechanisms by which NK cells
recognize potentially infected or transformed
host cells are largely unknown. Generally, the
killing of a particular host cell relies on the bal-
ance of the activating and inhibitory signals
from the surface receptors of the NK cells that
make contact with the host cell. These surface
receptors belong to two structural families: the
Figure 6.10. NETs
killer lectin-like receptors (KLRs; receptors that
This is a scanning electron microscope image of resemble the C-type lectins such as the MR)
a bacterium (Klebsiella pneumoniae) ensnared in a
and the killer cell immunoglobulin-like recep-
NET in an infected lung of a mouse. The image
tors (KIRs; receptors with domains that share
has been pseudocolored to differentiate the bac-
terium (pink) and the NET (green and white).
homology with parts of Igs). To make things
Image from “The tangled NETs of the immune more confusing, activating and inhibitory re-
system.” © Klaus Wilhelm, 2011. Originally ceptors belong to both families of receptors.
published in MaxPlanckReasearch 1:11, 72–78.
Activating stimulus
Receptors that provide activating stimulus
Natural Killer Cell–
in NK cells sense a variety of ligands that indi-
Mediated Cytotoxicity
cate abnormality on the surfaces of the target
Although phagocytes and granulocytes can cells. In some cases, NK cell–activating recep-
identify and destroy extracellular threats, threats tors can directly detect conserved viral proteins
that exist within host cells (such as virus and cer- on the surface of infected cells. Infection with
tain bacteria and parasites) are generally hidden certain members of the herpes virus family, for
from the majority of innate effectors. Identify- instance, can be directly detected by NK cells
ing potentially infected host cells, without prior through this type of receptor. Aside from these
exposure to the infective agent, is the specialty specific examples, most activating receptors are
of NK cells. As covered in chapter 2, NK cells thought to detect ill-defined ligands that indi-
are actually lymphocytes. Unlike most T and B cate an altered cellular state, which may arise
cells, however, they do not possess the receptor through infection with viruses, intracellular bac-
diversity generated by recombination of their teria or parasites, or a state of malignancy. These
chromosomal DNA. Instead, NK cells use fami- activating ligands are derived from alterations
101

to surface glycoproteins induced by metabolic unrecognizable MHC I, do not trigger inhibi-
stress or altered posttranslational modification tory signaling within the NK cell, and activating
of normal surface proteins. Metabolic stress receptors can fully activate the NK-cell killing
and altered protein processing are commonly mechanism (Figure 6.11).
found in virally infected cells (whose synthe-
sis machinery has been hijacked by viruses).
Transformed cells, which are replicating at an Targeted Degranulation by
abnormal rate, also show metabolic stress and Natural Killer Cells
altered protein expression. The major mechanism by which NK cells kill
their target cells is very similar to that used by
Tc cells (covered in chapter 10). Once an NK
Inhibitory stimulus cell detects a host cell that triggers its activat-
Even if host cells display surface ligands that ing receptors without triggering its inhibitory
indicate ill thrift, these cells are not killed by receptors, changes occur within the NK cell
NK cells if the cells are also displaying normal that allow it to kill the target cell with great
self-markers. This sort of inhibition is achieved precision. NK cells possess cytoplasmic gran-
through inhibitory receptors on the NK cell ules termed lytic granules. These lytic granules
that detect the normal expression of major contain cytotoxic molecules that can kill target
histocompatibility complex class I (MHC I) cells after degranulation. Unlike degranulation
molecules on host cells. MHC I molecules are by neutrophils and eosinophils, which results
usually expressed on every nucleated cell, and in widespread collateral damage, NK-cell de-
because they differ between individual animals, granulation is restricted to killing target cells,
they constitute an important marker to differ- but not neighboring normal cells. NK cells do
entiate self cells from non-self cells. They are this by forming a tight area of adhesion with
also a critical element in the adaptive immune the target cell through which degranulation can
response to intracellular infections because they occur without the cytotoxic components leak-
display samples of proteins that are present ing into the surrounding tissue. This adhesion
within the cell’s cytoplasm and “show” them complex, termed an NK immunological synapse,
to Tc cells. The details on MHC molecules are consists of a collection of adhesion molecules
covered in chapter 8. The presence of normal carefully arranged into a ring by the NK cyto-
levels of MHC I on the surface of target cells skeleton (Figure 6.12). This ring—almost like
indicates that, although they might be slightly a suction cup—acts to “dock” NK cells onto
altered, these cells are behaving relatively nor- target cells and form a tight seal within which
mally. This indication triggers inhibitory signals granule components may be released into the
that override the activating stimulus, thus pre- target cell while being fully contained between
venting activation of the NK-cell killing mecha- both cells. Figure 6.12 shows the NK immuno-
nism. However, altered levels or unrecogniz- logical synapse between an activated NK cell
able MHC I expression indicate that the host and a target cell and the beginning of the polar-
cell’s metabolism has significantly changed or ized degranulation.
that there is covert inhibition of MHC I expres- Degranulation within the NK immunologi-
sion. Purposeful downregulation of the surface cal synapse releases cytotoxic molecules that
expression of MHC I is a common strategy can act on the target cell. Two of these cyto-
used by many viruses to hide from Tc cells. toxic molecules are the proteins perforin and
Many transformed neoplastic cells also have de- granzyme. When released into the NK im-
creased levels of MHC I on their plasma mem- munological synapse, perforin inserts itself
branes. Hence, decreased levels of MHC I, or into the plasma membrane of the target cells

Figure 6.11. NK cell–mediated killing of target cells
Activation of the cytotoxic function of NK cells requires the presence of activating ligands and the
absence of inhibitory ligands on the target cell surface. The top panel depicts inhibition of NK killing
through recognition of normal MHC I on the surface of the target cell. Despite displaying activating
ligands, the host cell is spared by the NK cell. The lower panel depicts a target cell displaying both an
activating ligand and altered MHC I expression, which triggers the NK-cell killing mechanism, result-
ing in the death of the host cell via forced apoptosis.
103

Figure 6.12. NK cell degranulation
NK cells carefully degranulate into an NK immunological synapse (NKIS) formed with the target cell.
This series of micrographs depicts the NKIS between the NK cell (top) and the target cell (bottom).
The actin cytoskeleton (blue) and adhesion molecule CD2 (green) form a ring through which the
perforin (red) and granzyme can be delivered to the target cell. (From Orange et al., “The Mature
Natural Activating Killer Cell Immunologic Synapse Is Formed in Distinct Stages,” PNAS 100 (24): fig.
1, © 2003, National Academy of Sciences, USA)

Figure 6.13. Antibody-dependent, cell-mediated cytotoxicity
NK cells can recognize surface-bound antibodies (such as IgG) through the receptors that recognize
the Fc portion of the antibody (Fc receptors), thus providing a powerful activating stimulus to initiate
the NK cell’s cytotoxic function.
(perforating the membrane, as its name sug- tive immunity. Specifically, akin to opsoniza-
gests), forming pores. Granzymes are a family tion for phagocytosis, antibody-dependent cell
of serine proteases that can gain access to the cytotoxicity is a process through which the
target cell’s cytoplasm via the pores created by humoral immune system can augment NK cell–
perforin. Here granzymes can act on caspases, mediated cytotoxicity. After the production and
activating pathways that force the target cell to binding of antibodies to cell-associated viral or
go through apoptosis and die. The NK cell is re- neoplasm-associated antigens on the surface
sistant to its own perforin and granzymes and, of abnormal cells, NK cells can more readily
thus, after the directed apoptosis of the target identify these antibody-coated cells as targets.
cell, it can disengage and seek another abnor- Recognition of antibodies on the surface of a
mal cell for destruction. cell is achieved through the Fc receptors on the
NK cell, which serve to provide a very power-
Antibody-Dependent Cell- ful activating stimulus to initiate the NK cell’s
Mediated Cytotoxicity cytotoxic function (Figure 6.13).
As with many innate effector mechanisms,
NK cell–mediated cytotoxicity does not sim- Clinical Correlation: Follow-Up
ply serve to contain infection between expo-
sure and the more specific yet slow-reacting Student Considerations
adaptive immune response. It also acts in an After reading through this material, you
enhanced capacity after the induction of adap- should be able to list the ways in which macro-
105

Figure 6.14. R. equi and phagosome maturation
The R. equi bacterium actively arrests the maturation of the phagosome. The arrested phagosome
fails to fully acidify or fuse with the hydrolytic lysosomes, allowing the bacterium to survive within
the macrophage.
phages are able to kill ingested microbes. You ity of the bacterium to avoid being destroyed
should also be able to think of several points in by the macrophage after its phagocytosis in
that arsenal at which R. equi is able to hijack this the pulmonary alveolus. R. equi is thought to
machinery and avoid destruction. achieve this by blocking the maturation of the
phagosome that is created around these bacte-
ria. Without phagosomal maturation, the inte-
Possible Explanation rior does not acidify, and lysosomal products
R. equi is a well-adapted pathogen that has (such as digestive enzymes and antimicrobial
evolved several means for circumventing host products) are not delivered to the phagosome.
immune responses. One of these is the abil- The R. equi bacterium also has the ability to

prevent the assembly of the NADPH oxidase recruits other mononuclear cells, thus creat-
complex on the membrane of the phagosome, ing a local inflammatory reaction. If not given
which reduces the oxidative stress placed on the the correct stimulus, these monocytes-turned-
bacterium. It is thought that the phagosomes macrophages cannot kill the R. equi bacteria
containing R. equi bacteria maintain some fu- either but instead become infected themselves,
sion capability with early and recycling endo- further recruiting other immune cells, and so on,
somes, which can bring nutrients to the inhab- which eventually creates a chronic focal immune
iting microbe. Hence, the bacterium survives reaction and the formation of a granuloma.
and replicates within this intracellular niche and An interesting fact is that the macrophage
for the most part is protected from the majority does have the ability to kill R. equi within its
of immune effectors. The exact mechanism by phagosomes, but it cannot do it alone. It needs
which R. equi arrests phagosomal maturation is to be activated by the correct cytokines that
still debated, but certain lipid and protein prod- can be provided by the Th1 cells of the adaptive
ucts produced by the bacterium have been im- immune system (covered in subsequent chap-
plicated in this pathogenic feature. ters). Foals are particularly susceptible to R. equi
The infected macrophage, detecting the pres- because they do not generate a strong Th1 cell
ence of the microbe through PRRs such as the response. Adult horses, however, do generate a
TLRs, releases proinflammatory cytokines robust Th1-type adaptive immune response to
and chemokines into the surroundings, which R. equi and therefore rarely develop rattles.
107

Gerald N. Callahan
Cells and Organs of the

Chapter 7
Adaptive Immune System
10.5876_9781607322184.c007
lymph nodes swell during infections and auto-

Clinical Correlation: Feline Lymphadenopathy 109
immune reactions is essential to understanding
animals’ responses to and recovery from these
Organs and Cells of the Adaptive Immune
syndromes.
System 109
Primary Lymphoid Organs and Hematopoiesis 110
Bone marrow 110 Learning Objectives
Thymus 112
Secondary Lymphoid Organs 114
Lymph nodes 115 • understand the location, structure, and func-
Spleen 117 tion of the bone marrow, thymus, lymph
Mucosa-associated lymphoid tissue 118 nodes, spleen, and MALTs;
Clinical Correlation: Follow-Up 119 • know the origins, characteristics, and physi-
Student Considerations 120 cal properties of T lymphocytes, B lympho-
Possible Explanations 120 cytes, macrophages, and DCs;
• understand the organization and function of
Clinical Correlation: the lymphatics.
Feline Lymphadenopathy
A five-year-old intact male cat presents at a Organs and Cells of the
small-animal clinic. The owner states the cat is Adaptive Immune System
lethargic and will not eat or drink. A quick ex- In 1966, Roy Grist—a pathologist working with
amination of the cat reveals that the animal has a mouse colony in Glasgow, Scotland—discov-
a fever (>40°C, 104°F) and swollen superficial ered a very odd-looking mouse in one of his
cervical and mandibular lymph nodes. Lymph- cages. It was puny, hairless, and short-lived.
adenopathy (especially swollen lymph nodes) is After Dr. Grist made a brief report of his find-
a common presenting symptom for several dis- ing, an Edinburgh researcher asked Dr. Grist
eases of cats (see Table 7.1). if his Edinburgh group could try to establish
With cats, it seems obvious why neoplastic a line of these mice. Grist agreed and shipped
diseases of lymphocytes might lead to swol- a few mice off to the researcher. Because they
len nodes: uncontrolled proliferation of lym- were hairless, the mice became known as nude
phocytes. For the most common causes of mice (see Figure 7.2).
feline lymphadenopathy, however—all of the Beyond their lack of hair, closer examina-
infectious diseases and autoimmune diseases— tion revealed a much more remarkable finding:
the reasons are less clear. Understanding why these nude mice had no thymuses. In the early
109
109

Table 7.1. Causes of lymphadenopathy in cats
I. Proliferative and inflammatory lymphadenopathies
A. Infectious
1. Bacterial
a) Actinomyces app.
b) Corynebacterium
c) Mycobacteria
d) Nocardia app.
e) Streptococci (contagious streptococcal
lymphadenopathy)
f ) Yersinia pestis
g) Localized bacterial infection
2. Septicemia
3. Rickettsial
Figure 7.1. Domestic cat (© Pashin Georgiy /
a) Feline ehrlichiosis
Shutterstock)
b) Hemobartonellosis
4. Fungal
a) Blastomycosis
1970s, no one had any idea what thymuses did, b) Coccidioidomycosis
and these mice seemed to offer an avenue for c) Cryptococcosis
greater insight into the role of this organ. d) Histoplasmosis
One set of experiments involved transplant- e) Phaeohyphomycosis
ing skin from other species onto nude mice. f ) Phycomycosis
Normally, skin grafts elicit a powerful immune g) Sporotrichosis
response and do not survive longer than a few 5. Parasitic
days. Nude mice accepted skin grafts from a) Cytauxzoonosis
chickens, humans, cats, chameleons, fence liz- b) Demodicosis
c) Toxoplasmosis
ards, and frogs. Immunologically, nude mice
6. Viral
(unlike mice with thymuses) could not distin-
a) Feline immunodeficiency virus
guish between themselves and chickens, liz-
b) Feline infectious peritonitis
ards, humans, cats, or frogs (see Figure 7.3A–F). c) Feline leukemia virus
Clearly, the thymus was critical to develop- B. Noninfectious
ing any immunological sense of self. That dis-
1. Dermatopathic lymphadenopathy
covery added the final piece to the puzzle of
2. Idiopathic
the immune system. The thymus, the bone a) Distinctive peripheral lymph node hyperplasia
marrow, the lymph nodes, the spleen, and the b) Plexiform vascularization of lymph nodes
MALT somehow combine to produce the adap- 3. Immune-mediated disorders
tive immune response. a) Immune-mediated polyarthritides
b) Other immune-mediated disorders
4. Localized inflammation
Primary Lymphoid Organs
5. Postvaccinal
and Hematopoiesis
Bone marrow
By the forty-fifth day of gestation, the bone the circulating blood cells. Perinatally, nearly all,
marrow in fetal cats already provides 50 percent if not all, bones contain marrow, the jellylike ma-
of the blood cells. By birth, a cat’s bone marrow terial where all the cells of the blood originate
(as in most mammals) will supply 100 percent of in a process called hematopoiesis (see Figure 7.4).
110 C e l l s a nd Org a n s o f t h e Ad a p t i v e Imm u n e Sy s t e m

Figure 7.2. Nude mouse (photograph by Armin Kübelbeck)
This phenotype is the result of a gene mutation involving hair growth and thymus development.
Recall from chapter 2 that all white blood the remarkable specificity and memory aspects
cells, RBCs, and platelets arise from a single plu- of the adaptive immune response.
ripotential bone marrow stem cell. Early in he- The myeloid lineage produces the remaining
matopoiesis, two distinct cell lineages appear: cells of the blood, many of which participate
the lymphoid lineage and the myeloid lineage. in innate immune responses, as discussed ear-
The lymphoid lineage ultimately produces lier. After they migrate out of the blood vessels
two major types of lymphocytes: T cells and B and into the surrounding tissues, monocytes
cells (recall from chapter 2 that the lymphoid differentiate into macrophages—phagocytic
lineage also produces NK cells that contribute to cells found throughout the body—that are of
innate defense). The T lymphocytes play critical critical importance to innate defense. Other
roles in all adaptive immune responses through phagocytic cells, the DCs, are specialists in ac-
release of essential cytokines and through di- quiring and presenting antigens. Once a DC has
rect killing of pathogen-infected cells. The B acquired antigen, the cell moves through the
cells produce antibodies found throughout the lymphatics to the nearest lymph node and pres-
body. These antibodies provide a major part of ents that antigen to T lymphocytes.
mammalian defense against infections. The B Mast cells begin in the marrow and move
and T lymphocytes are also the only cells with into the circulation and from there into the sur-
antigen-specific receptors—the BCR (a special rounding tissues, where they play major roles
form of antibody) and the TCR (see Figure in innate responses, especially inflammation.
7.5A–B). Together, T and B cells also produce Granulocytes—so named because they contain
C e l l s a nd Org a n s o f t h e Ad a p t i v e Imm u n e Sy s t e m 111

111

Figure 7.3A–F. Skin grafts on nude mice
Panel A shows human (mammal) skin after 60 days. Panel B shows cat (mammal) skin at 51 days.
Panel C shows chicken (bird) feathers at 32 days; the feathers were already present when the graft
was made. Panel D shows chameleon (reptile) skin at 41 days. Panel E shows fence lizard (reptile) skin
at 28 days. Panel F shows tree frog (amphibian) skin at 40 days. © 1973 Rockefeller University Press.
(Originally published in Manning, Reed, and Shaffer, 1973, J. Exp. Med. 138:488–494.)
cytoplasmic granules (active in inflammatory neutrophils are clearest, but all play important
and other pathways)—include neutrophils, baso- roles in innate defense. (See chapters 2–6.)
phils, and eosinophils. Neutrophils (also known
as polymorphonuclear leukocytes because their
nuclei come in many shapes and sizes) are the Thymus
most numerous of all white blood cells in mam- Although all lymphocytes originate in the
mals. Of the three granulocytes, the functions of bone marrow, only B lymphocytes mature there

Figure 7.4. Bone marrow and hematopoiesis
A single pluripotential bone marrow stem cell gives rise to all of the cells of the blood. Among these
are the cells most important to the adaptive immune response, particularly B and T lymphocytes and
monocytes, which differentiate into macrophages and DCs.
or in other specialized organs such as the bursa the bone marrow to the thymus, where these
of Fabricius in fowl. In vertebrates, another set cells become T lymphocytes. This step is essen-
of lymphocytes migrates, via the blood, from tial in developing a functional immune system,

113

Figure 7.5A–B. Antigen-specific receptors on T and B cells
T (panel A) and B (panel B) lymphocytes—essential components of immune responses—are the only
antigen-specific cells in animals. Distinct antigen-specific receptors on each of these cell types allow
for direct interactions with pathogens and their antigens. The left panel shows the TCR embedded in
the membrane of a T cell. The right panel depicts the BCR embedded in the membrane of a B cell.
Ag = antigen.
and if it does not happen (as in nude mice), ani- Secondary Lymphoid Organs
mals lack any sense of immunological self or Because the thymus generates T lympho-
non-self. cytes and the bone marrow (and the bursa of
The mammalian thymus lies right above the Fabricius) generates B lymphocytes, these or-
heart and is largest shortly after birth; it then gans are sometimes referred to as the primary
decreases significantly in size and function after lymphoid organs. The secondary lymphoid
sexual maturity. However, the thymus func- organs are the sites where the cells of the im-
tions in all mammals throughout adult life. mune system and pathogens meet and immune
The outer or cortical region of the thymus responses occur. In animals’ bodies, infections
contains many lymphocytes called thymocytes may begin in three major places: the intersti-
(while they are in the thymus; see Figure 7.6). tial fluid between the cells, the blood, and mu-
The inner or medullary region of the thymus cosal surfaces. Specialized lymphoid tissues or
contains mostly epithelial cells. Somehow, in- organs have evolved to deal with each. Lymph
side this organ, T cells acquire the ability to nodes screen interstitial fluid (lymph) draining
react with non-self. from peripheral tissues, the spleen screens the

Figure 7.6. Structure of the mammalian thymus
The thymus contains several lobules, each of which can be divided into cortical (outer) and medullary
(central) regions. The cortex contains immature dividing cells, and the medulla contains more mature
cells. Many of these cells (thymocytes) are in the process of becoming T lymphocytes. In addition,
thymic epithelial cells populate both cortical and medullary regions. Finally, the medulla also contains
both macrophages and DCs that originated in the bone marrow.
blood, and the MALT monitors the mucosal beds—a lot of fluid leaks out into the tissues
tissues. that surround the blood vessels. This fluid—
which is a lot like plasma, minus some of the
big proteins—is called lymph. The total volume
Lymph nodes of lymph that forms every day in most animals
All mammals have two circulatory systems: is nearly equal to the total plasma volume.
cardiovascular and lymphatic. The cardiovas- Some means for collecting lymph and return-
cular system is a closed system of arteries and ing it to the circulation is essential, and that is
veins that moves blood through the body to the the function of the lymphatics (see Figure 7.7).
heart, to the lungs, to the heart, and back to the Each of the vessels of the lymphatics termi-
body. Along the way—especially in the capillary nates in a fingerlike projection with inward-

115

Figure 7.7. Canine lymphatics and lymph nodes
Lymphatics (upper portion) are a system of open-ended vessels that return extravascular fluid from
the periphery to the heart and blood. Distributed throughout the lymphatics are a series of small
filtering stations called lymph nodes. Each node has both cortical and medullary regions where dif-
ferent aspects of immune responses occur. The medullary regions contain B cells, DCs, T cells, and
macrophages. The cortical regions contain structures called lymphoid follicles. During an immune
response, germinal centers form inside of the lymphoid follicles. Germinal centers contain T cells,
FDCs, and other DCs and some resting as well as rapidly dividing B cells. Each lymph node connects
to the lymphatics via multiple afferent (inflowing) and one efferent (outflowing) lymphatic vessel as
well as to the circulatory system by an arteriole and a venule.

Figure 7.8. Canine spleen
The spleen (in green) is the site of most immune responses against blood-borne pathogens.
opening valves. Lymph flows out of the cap- paracortical areas (between the cortex and the
illaries, into the interstitial tissue spaces, and medulla), however, contain mostly T cells.
through the valves into the lymphatics. Once
inside the lymphatics, lymph (driven by the con-
traction of skeletal muscles) moves ultimately Spleen
to the thoracic duct and to the right subclavian Although spleen size varies considerably
vein and into the heart. Along the way, many among mammals, the basic structures of all
small nodes appear; these lymph nodes (see spleens are the same. Immunologically, the
Figure 7.7) are filtering stations. During infec- spleen does for blood-borne antigens what
tions, DCs from the periphery deliver antigens lymph nodes do for lymph-borne antigens (see
to the lymph nodes. Blood delivers T cells and Figure 7.8). In addition, with the help of resi-
B cells to lymph nodes. dent macrophages, the spleen recycles some se-
Inside of lymph nodes, T and B cells as well nescent RBCs.
as macrophages and DCs interact to produce Within the spleen, the white pulp contains
immune responses. The cortical regions of the lymphoid tissues. Here, T-cell–rich regions
lymph nodes contain lymphoid follicles and surround the splenic arterioles to form a struc-
germinal centers where B cells proliferate. The ture called the periarteriolar lymphoid sheath.

117

Figure 7.9. Structure of lymphoid tissue in the mammalian spleen
PALS = periarteriolar lymphoid sheath
Nearby are B-cell–rich areas that also contain lymph nodes, germinal centers develop in the
germinal centers (see Figure 7.9). spleen when immune responses occur.
The red pulp areas are sites of red blood cell
destruction; the white pulp areas contain the
lymphoid tissues in the periarteriolar lymphoid Mucosa-associated lymphoid tissues
sheaths. As in lymph nodes, the T and B cells The epithelial mucosa of all animals are,
are unevenly distributed between germinal throughout their lives, constantly exposed to
centers and the surrounding tissues. Also as in and colonized by potentially infectious agents,

especially bacteria. The largest of these surfaces
are the lungs and the gut. All mucosal tissues
contain specialized lymphoid aggregates called
MALT.
Gut-associated lymphoid tissues (GALT)—a
part of MALT—include Peyer’s patches in the
small intestine and the cecal tonsils. The lym-
phoid tissues of the Peyer’s patches are the
most organized and possibly most important
of these tissues (see Figure 7.10A–B). In the in-
testine, specialized epithelial cells called M cells
transport antigens from the gut lumen into the
lymphocyte-rich areas of the Peyer’s patches.
Approximately thirty to forty groups of Peyer’s
patches appear in an animal’s intestine as elon-
gated areas without villi.
These lymphoid tissues monitor the patho-
gens in the gut and deal with pathogenic organ-
isms, usually without harming the normal gut
flora. Mucosal immunology remains relatively
poorly understood, but it is unquestionably one
of the keys to understanding animals’ immune
systems and animal health.
Bronchus-associated lymphoid tissues—an-
other part of MALT—are not as structured as
GALT. However, the process by which they deal
with inhaled pathogens is very similar to what
occurs in GALT. Somewhat similar lymphoid
tissues also appear on other mucosal surfaces
Figure 7.10A–B. Lymphoid tissues in the small
such as the urethral and vaginal epithelia.
intestine
Despite their structural differences, the
Panel A is a diagram of a thin section of small
lymph nodes, spleen, and MALT—with few ex-
intestine. Within this section, the dark purple–
ceptions—all deal with antigens in very similar rimmed ovoid areas represent specialized
ways using the same types of cells. lymphoid tissue called Peyer’s patches—sites of
immune responses in the intestine. Panel B is
a diagrammatic representation of the interac-
Clinical Correlation: Follow-Up tion between the cells of a Peyer’s patch and an
During discussions, the cat’s owner shared that enteric pathogen. M cells carry antigens from
the gut lumen into the Peyer’s patch where DCs
about a week before the cat’s bout of lethargy
process and present these antigens to the im-
and fever, the cat had killed and partially eaten a mune system. As with splenic lymphoid tissues,
small feral rabbit. Wild rabbits are known reser- there are discrete T-cell–rich and B-cell–rich
voirs for tularemia, a sometimes fatal infectious areas where immune responses begin. From
disease in cats. The examining veterinarian re- these areas, cells enter the lymphatics and
quested a lymph node biopsy and a serum titer eventually the blood. From the blood, immune
for Francisella tularensis, the causative agent for B and T cells return to mucosal tissues and
tularemia. mediate protection.

119

The biopsy revealed areas of focal necrosis and into the lymph nodes. Much more com-
and lymphoid hyperplasia, whereas the serum monly, lymphadenopathy is the result of pro-
exhibited a titer of 1/80 for F. tularensis. Ap- tective immune responses against infectious
parently, the observed lymphadenopathy was agents. Once antigens (pathogens or pieces of
lymphadenitis, a result of the bacterial infection pathogens), T cells, and B cells arrive inside
as well as the cat’s immune response to the bac- of lymph nodes, immune responses develop.
terium. After antibiotic treatment, the lymph- Each of these immune responses involves mas-
adenopathy resolved, and the cat recovered. sive proliferation of lymphocytes or lymphoid
hyperplasia. These expanding cell popula-
tions cause lymph nodes to swell well beyond
Student Considerations their normal size, as with human mandibular
After having read this chapter and considered nodes during respiratory infections. This swell-
the nature, distribution, and function of lymph ing is a reflection of the essential role of the
nodes, you should be able to offer at least two lymph nodes in immunity and defense against
possible explanations for the symptoms ob- infections.
served in the patient described at the beginning Regardless of the cause, antibiotic therapy
of this chapter. would be the appropriate course of action for
the attending veterinarian. In this case, the na-
ture of tularemia and the fact that the mandib-
Possible Explanations ular and cervical nodes showed areas of focal
The association of lymphadenopathy with necrosis suggested that lymphadenitis was the
infectious diseases occurs for at least two rea- cause of the swollen lymph nodes. The pres-
sons. First, it is possible for the nodes them- ence of antibody against F. tularensis indicates
selves to become infected, resulting in inflam- the cat had eaten an infected tick while killing
mation (lymphadenitis). This lymphadenopa- or feeding on the rabbit, and the presence of
thy results from the inflammation and the this bacterium in the lymph nodes was the un-
movement of cells and fluid out of the blood derlying cause of the lymphadenitis.

Gerald N. Callahan
Antigens and Antigen Processing Chapter 8

10.5876_9781607322184.c008
infections, several nations will not accept goats

Clinical Correlation: Caprine Arthritis
from countries where CAEV is enzootic, such
Encephalitis 121
as the United States.
Genetic studies of symptomatic and asymp-
Stimulators of Adaptive Immunity: Pathogens,
Antigens, and Epitopes 122
tomatic goats have yielded some of the most
Major Histocompatibility Complex 124
significant insights into the pathogenesis of
History of the Major Histocompatibility
CAEV. Genetically, a major difference exists
Complex 124 between unaffected and affected goats. That
Major Histocompatibility Complex Genetics 125 difference maps onto a particular region of the
Structure and Distribution of Major caprine genome known as the major histocom-
Histocompatibility Complex Class I and patibility complex, or MHC.
Class II Molecules 129 Goats that develop arthritis, encephalitis, or
Antigen–Major Histocompatibility Complex both are more likely to carry one or both of
Interactions 131 two MHC alleles, Be1 and Be14. This finding
Antigen Acquisition, Processing, and suggests that the products of these MHC genes
Presentation 132
play major roles in the pathogenesis of CAEV.
Antigen-Presenting Cells 132
Associations between diseases and MHC al-
Major Histocompatibility Complex Class I
leles are not unique to goats. The first of these
Antigen Processing and Presentation 135
associations was identified in humans, and it
Major Histocompatibility Complex Class II
Antigen Processing and Presentation 136 showed a clear link between expression of a
Cross-Presentation 137 particular MHC class I allele, human leukocyte
Clinical Correlation: Follow-Up 139 antigen–Bw27, and ankylosing spondylitis, a
Student Considerations 140 debilitating autoimmune disease. Other stud-
Possible Explanations 140 ies soon uncovered similar associations in most
species of mammals. Table 8.1 shows MHC al-
leles associated with various diseases in cattle.
Clinical Correlation: Caprine
The MHC alleles of cattle are also called bovine
Arthritis Encephalitis
leukocyte antigen alleles.
When caprine arthritis encephalitis virus (CAEV) Today, it is clear that MHC genes play a role
infects a herd of goats (Figure 8.1), most animals in a variety of diseases in many mammalian
remain healthy even if infected. An unfortunate species. But how MHC genes affect disease
few, however, develop untreatable, debilitating processes remains murky. A more complete un-
diseases that manifest as polyarthritis and oc- derstanding of the relationship between MHC
casionally in kids as encephalomyelitis. Because and disease is likely to offer new insights into
CAEV infection is common and causes lifelong disease prevention and therapy.
121
121

ecules that bind to antibodies, MHC molecules,
or TCRs. The two antigen subcategories are im-
munogens—substances capable of stimulating
an immune response—and haptens—substances
that will not by themselves produce an immune
response but can do so when complexed with a
larger molecules, such as a host protein.
Despite these different ways of speaking
about molecules that interact with immune
systems, in practice most immunologists refer
to all immunogens and haptens simply as an-
Figure 8.1. SouthAfrican goat herd (© WOLF tigens. In the remainder of this book, we con-
AVNI/ Shutterstock)
sider antigens to be any molecules that can
stimulate adaptive immune responses.
Because of their roles in defense, immune
Learning Objectives
systems are generally thought to focus on non-
After reading this chapter, you should be able to self—things foreign to hosts. But not all foreign
things are antigenic, and sometimes parts of
• distinguish among pathogens, antigens, self may be antigenic. How do molecules that
immunogens, and epitopes;
are antigenic and those that are not differ?
• know the properties that make a molecule To be capable of stimulating an adaptive im-
antigenic;
mune response a substance must be
• know the general character of the mam-
malian MHC; • complex,
• distinguish between class I and class II MHC • organic,
genes and molecules; • degradable,
• understand how MHC molecules bind anti- • large.
gens and the differences between antigens
bound by class I or class II molecules; These properties are necessary for a mol-
• know the different classes of APCs and ecule to be antigenic, but not all complex,
their distributions; organic, degradable, large molecules are anti-
• understand how APCs process and present genic in any given animal. For example, bovine
antigens and the roles of MHC class I and
serum albumin fulfills all of these conditions
class II molecules in this process;
and is, thus, antigenic in mice, but under nor-
mal conditions bovine serum albumin is not
• explain why certain MHC genes may pre- antigenic in cattle. So, often (but not always),
dispose animals to some diseases.
an antigenic molecule must, in addition to all
of the preceding properties, be recognizably
foreign.
Stimulators of Adaptive
All living things carry unique chemical sig-
Immunity: Pathogens,
natures, which is what separates one animal or
Antigens, and Epitopes
even one bacterium from another. Among those
The word antigen apparently arose as a conden- chemical signatures, proteins are the most indi-
sation of the words antibody generator, but that vidually distinct. Among the biological macro-
is not exactly what all antigens are. More accu- molecules, no other group so finely differenti-
rately, antigens are molecules or pieces of mol- ates individual organisms.
122 An t i g e n s a nd An t i g e n Pr o c e s s i ng

Table 8.1. Cattle MHC alleles and disease susceptibility
Bovine major histo-
Disease Breed compatibility system Effect
Enzootic bovine leukosis
Seroconversion Holstein A14 Late
Holstein A15 Rapid
Guernsey A21 Late
Guernsey DA6.2, A12 Rapid
Peripheral lymphocytes and B-cell Shorthorn DA7 Resistance
numbers
Shorthorn DA12.3 Susceptibility
I. Shorthorn A6, EU28R Susceptibility
I. Shorthorn A8 Resistance
Holstein A12 and A15 Susceptibility
Holstein A14 and A13 Resistance
Holstein DRB22A Resistance
Holstein DRB21C Susceptibility
Holstein DRB3(ERmotif ) Resistance
Holstein DRB3(ERmotif ) Resistance
Mastitis
Clinical mastitis Norwegian Red A2 Resistance
Norwegian Red A16 Susceptibility
Swedish Red & White DQ1A Susceptibility
Holstein A11 Resistance
Holstein CA42 Susceptibility
Subclinical mastitis (cell count) Icelandic A19 Susceptibility
Simmental (S) or S × Red Holstein A15 High
Danish Black Pied A11, A30 Low
Danish Black Pied A21, A26 High
California Mastitis Test Holstein A14 Low
Helminths
Nematodes Belmont Red A7, CA36 Resistance
Africander × Hereford A9 Susceptibility
Protozoa
Theileria parva Bos indicus Class I Parasite entry
Ticks
Boophilus Microplus Brahman × Shorthorn A6, CA31 Susceptibility
Posterior spinal paresis Holstein A8 Susceptibility
Ketosis Norwegian Red A2, A13 Resistance
Retained placenta Dutch Friesian Compatibility Susceptibility
An t i g e n s a nd An t i g e n Pr o c e s s i ng 123
123

Proteins make all life possible. Proteins are, all pathogen parts are equally antigenic, not
of course, strings of amino acids linked by pep- even all the proteins. Beyond that, not all parts
tide bonds. But unlike carbohydrates or nucleic of pathogen-derived antigenic proteins are
acids, at every position in a protein there are equally antigenic (see Figure 8.2). Some pieces
twenty possible amino acids to choose from. of the pathogen’s proteins stimulate strong re-
That makes proteins unique identifiers—mark- sponses, whereas others go unrecognized. For
ers that set each animal apart from all the example, Figure 8.2 shows antigenic portions
rest. Adaptive immune systems exploit those of proteins that stimulate immune responses
differences. (red or yellow) and other portions that fail to
Proteins are the most potent of all antigens, stimulate immune responses (green).
and the antigenicity of most pathogens de- The parts of proteins that stimulate immune
pends on their protein components. Whether responses are called antigenic epitopes (or sim-
a protein stimulates an immune response, how- ply epitopes) and come in at least two forms—
ever, depends on the properties of that protein. continuous and discontinuous. A continuous
Artificial proteins constructed from a single epitope is the product of the contiguous amino
amino acid—for example, homoalanine—elicit acids in the primary sequence of the protein. A
no immune responses. Complexity appears to discontinuous epitope involves noncontiguous
be an absolute requirement for antigenicity. amino acids and results from the secondary, ter-
Similarly, a protein constructed from D-amino tiary, or quaternary structure of the protein, as
acids will not stimulate an immune response in shown in Figure 8.2. Because of the manner in
any animal. All naturally occurring amino acids which they acquire antigens, MHC molecules
are L-amino acids. Because of that, all naturally bind only continuous antigenic epitopes. Only
occurring proteases have evolved to focus on B-cell receptors and antibody molecules (see
L-amino acids and cannot split peptide bonds chapters 11 and 12) recognize and bind to dis-
formed between D-amino acids. To be antigenic, continuous antigenic epitopes.
a protein must be complex and degradable by
naturally occurring proteolytic enzymes.
Enzymatic degradation of proteins is essen- Major Histocompatibility
tial, because T cells—essential components of Complex
all immune responses—cannot recognize in-
History of the Major
tact proteins. Before an immune response can
Histocompatibility Complex
begin, antigenic proteins must be chopped into
pieces and presented by an MHC molecule, and Nearly as long as people have known about
MHC molecules only bind to peptide bits of the origins of cancers, scientists have been
protein antigens. interested in the role of immunity in defense
Small proteins (<2,500 molecular weight) against tumors. As discussed further in chapter
make poor antigens, and inorganic molecules— 17, in the early 1960s Sir MacFarlane Burnet and
unless coupled to proteins—generally do not Lewis Thomas even went so far as to propose
elicit immune responses. Inorganic molecules that a primary role of mammalian immune
are not usually major parts of the pathogenic systems was to detect and destroy tumors. This
world. Also, of course, immune systems have proposal came to be called the theory of im-
evolved to focus on pathogens—transmissible mune surveillance.
agents of disease: viruses, bacteria, fungi, and To investigate this theory, scientists began
parasites. studying mouse tumors. Initially, those studies
Almost all pathogens induce a wide range of focused on transplanting tumors from mouse
immune responses. Interestingly, though, not to mouse. Some transplanted tumors grew well

lografts depended on the similarity or dissimi-
larity of only a few genes. Because these genes
had a major effect on tissue (or histo-) compat-
ibility, they were called the major histocompat-
ibility complex, or MHC.
Each species has its own set of MHC genes
and, within each species, a unique name for the
MHC. Because these molecules were originally
found on white blood cells, almost every MHC
name contains the words leukocyte antigen. The
MHC in humans is also called the HLA (human
leukocyte antigen) complex, in monkeys it is
also called the RHA (rhesus leukocyte antigen)
complex, in cattle it is BLA (bovine leukocyte
antigen) complex, and so on. Only mice and
rats depart from this general rule. The MHC of
mice is called H-2 and the MHC of rats is called
RT1.
Although they were discovered during trans-
plantation experiments, these genes did not
evolve simply to frustrate transplant surgeons.
Instead, the products of MHC genes bind and
Figure 8.2. Antigenic
epitopes of an present antigens to T cells, and those T cells
immunogenic protein direct immune responses. Sometimes those im-
Not all portions of a protein activate the host’s mune responses cause graft rejection, but more
immune system. Those segments that do stimu- often they help to protect animals from the
late immunity are antigenic epitopes. They can pathogenic world.
be of two types: continuous epitopes involving
adjacent amino acids (shown in red) or discon-
tinuous epitopes formed by the secondary or Major Histocompatibility
tertiary structure of the protein (shown in yel- Complex Genetics
low). Here, each ball represents an amino acid.
The basic structure of the MHC is similar in
all mammals. Figure 8.3A–C shows the MHCs
in some mice, but not in others. For this reason, of horses, humans, and mice.
some investigators thought they had discov- Within each MHC are three distinct gene
ered tumor-specific factors that might be ma- types: MHC class I, MHC class II, and several
nipulated for cancer treatment. But in similar other genes called MHC class III. Also, the
experiments using normal tissues, scientists got products of DM, DN, and DO genes in humans
the same results. Instead of something unique and H-2M in mice, although structurally ho-
to tumors, what these scientists had discovered mologous to other MHC class II molecules, are
was a group of genes whose products deter- much less variable and function differently than
mined the immunological compatibility of tis- other MHC class II gene products.
sues (malignant and otherwise) transplanted From end to end, the MHC in mice and hu-
from one mouse strain to another. mans spans about 4 × 106 base pairs. Each MHC
Studies with other mammalian species yiel contains multiple genes encoding class I MHC
ded similar results: the success or failure of al- molecules and multiple genes encoding class
125

Figure 8.3A–C. Diagrammatic representation of the chromosomal organization of MHCs of horses,
humans, and mice
MHC class I genes are shown in red, and MHC class II genes are shown in yellow. All mammalian MHCs
contain additional genes (sometimes called class III genes) that are involved in some immune and non-
immune functions unrelated to the roles of MHC class I and II genes (shown in green and blue).
II MHC molecules. In the mouse MHC, three rate loci encode four distinct MHC class I mol-
separate loci code for three different MHC class ecules, and five separate regions produce five
I molecules, and two distinct regions code for separate MHC class II molecules. Expression of
MHC class II molecules. In horses, four sepa- MHC class I and II genes is codominant, mean-

Figure 8.4. MHC gene polymorphism
Except for the DRα locus, each MHC class I and MHC class II locus has many possible alleles, which
means that among populations of mammals enormous variety is present in the genetic characteristics
of the MHC.
ing that each animal expresses the alleles on animal’s immune system. Up to a point, the
both parental chromosomes. more MHC molecules an animal has, the more
MHC proteins, as mentioned earlier, bind pathogens it can effectively present to its im-
bits of protein antigens and present them to T mune system. Therefore, multiple loci help to
cells. If that does not happen—if some patho- protect each mammal from a diverse and evolv-
gen escapes MHC molecules—the host animal ing pathogenic world.
is more likely to succumb to that infection. To Beyond multiple loci, the genes of the MHC
deal with this potential problem, each MHC are the most polymorphic genes in mammals.
molecule has evolved to bind a variety of an- Thus, at every locus that encodes an MHC
tigens, but in spite of that evolutionary pres- molecule, there are many possible alleles. For
sure, no one MHC molecule can bind all po- example, humans have six loci involved in pro-
tential antigens. Instead, over time, the MHC ducing MHC class II molecules—DPα, DPβ,
of mammals has grown to include multiple loci DQα, DQβ, DRα, and DRβ. Every human has
encoding class I and II MHC molecules. Those all of these loci, but not all humans have the
additional loci add another level of protection. same allele at each locus. The human popula-
Multiple loci encoding MHC class I and class tion has at least 20 different alleles possible at
II molecules reduce the chance of an animal’s the DQα locus, 89 at the DPβ locus, 45 at the
encountering a pathogen that can escape the DQβ locus, 20 at the DQα locus, 323 at the
127

DRβ locus, and 2 at the DRα locus (see Figure
8.4).
As a result, in addition to multiple MHC loci
in each individual, there are multiple alleles at
each MHC locus in the population at large. So,
although every human being has a DRβ locus,
it is rare for two unrelated people to have the
same allele at that locus, meaning that among
humans, as with most other animal popula-
tions, finding two human beings with the same
set of genes in their MHCs is extremely im-
probable (except for identical twins). Because
the products of these genes also play a role in
graft rejection, MHC polymorphism is a major
reason for the difficulties in finding compat-
ible kidney, heart, or liver donors for identified
recipients. Much more important, MHC poly-
morphisms offer essential protection for both
individuals and species.
Usually, when an infectious disease strikes a
population of animals, some individuals will
die and others will survive. One significant dif-
ference between survivors and fatalities is often
the complement of MHC molecules present
in each individual. Some animals will have the Figure 8.5. African
cheetah
MHC molecule needed to present the critical (© Anna Omelchenko / Shutterstock)
antigenic epitope or epitopes to the immune African cheetahs are genetically very homoge-
system and neutralize the pathogen, and others neous and at risk for decimation by infectious
will not. pathogens.
Thus, species-wide MHC polymorphisms
dramatically reduce the likelihood that a single
infectious agent will destroy an entire popula- an infectious disease kills one animal, it is likely
tion of animals. Some notable exceptions to this that it will kill every animal.
include African cheetahs, Florida panthers, and For the lions of the Ngorongoro crater, geo-
the lions of Ngorongoro crater in Tanzania. graphical isolation led to genetic bottleneck-
Between 10,000 and 20,000 years ago, the ing. The result, however, was the same. In 1962,
cheetah population dwindled to one or two an infestation of biting flies and their parasites
mating pairs. All of the cheetahs alive today killed all but ten or so of these lions.
came from those mates. Because of this, chee- Much more recently, genetic bottlenecking
tahs are now genetically nearly identical. This happened in the Florida panther. All of the
sort of event is called genetic bottlenecking. Florida panthers alive in the twentieth century
Cheetahs are now so similar, they do not reappear to have come from a single female. Dis-
ject skin grafts from one another, their MHCs ease and human pressures nearly destroyed this
are nearly identical, and when their hides are population. In 1995, five Texas pumas were in-
stacked atop one another, all of the spots line troduced into panther habitat, and the result-
up perfectly. In a population such as this one, if ing hybrids appear to be doing well. However,

Figure 8.6A–C. MHC class I structure
Approximate representations of MHC class I molecules. Panel A shows a ribbon diagram of the MHC
class I α chain (MHC derived) and β2-microglobulin. The tertiary structure of the class I α chain
contains three separate structural domains: α1, α2, and α3. The α1 and α2 domains form a cuplike
groove near the N-terminal region of the molecules and form the peptide–antigen binding site. Flat
ribbons ending in arrows indicate regions of β-pleated sheet, and the helical ribbons indicate regions
of α-helix. Panel B shows a top view of the MHC class I peptide–antigen binding site. Note how the
α-helical regions frame the flat segments of β-pleated sheet to form the binding site. Panel C shows
a diagrammatic representation of an MHC class I molecule including the transmembrane domain of
the α chain. The purple band across the figure bottom represents the lipid-bilayer outer membrane of
a eukaryotic cell. (Panels A and B adapted from C. Janeway, P. Travers, M. Walport, and M. Shlom-
chik, 2006, Immunobiology, 122)
because of the MHC homogeneity in these spe- tide chains. MHC class I molecules contain an
cies, some scientists predict that none of these MHC-encoded α chain and a smaller molecule
populations will survive the next few decades. called β2-microglobulin. Both of the two chains
of MHC class II molecules, α and β, come from
genes within the MHC (see Figure 8.6A–C).
Structure and Distribution of Although MHC class I and II molecules have
Major Histocompatibility Complex similar tertiary and quaternary structures,
Class I and Class II Molecules these MHC molecules are chemically distinct.
Both MHC class I and II molecules are cell- Those chemical distinctions result in some im-
surface glycoproteins composed of two polypep- portant structural differences at the antigen-
129

Figure 8.7A–C. MHC class II structure
Approximate representations of MHC class II molecules. Panel A shows a ribbon diagram of the
MHC class II α chain and β chain. Each chain contains three separate structural domains: α1, α2, and
α3, as well as β1, β2, and β3. The α1 and α2 domains, along with the β1 and β2 domains, form a cuplike
groove near the N-terminal region of the molecules and form the peptide–antigen binding site. Panel
B shows a top view of the MHC class II peptide–antigen binding site. Panel C shows a diagrammatic
representation of an MHC class I molecule including the transmembrane domain of the α and β
chains. The purple band across the figure bottom represents the lipid-bilayer outer membrane of a
eukaryotic cell. (Panels A and B adapted from C. Janeway, P. Travers, M. Walport, and M. Shlomchik,
2006, Immunobiology, 123)
binding site. For this reason, MHC class I and binding grooves. Only the α chain spans the
class II molecules bind distinct sets of antigenic lipid bilayer. β2-microglobulin binds noncova-
peptides. In addition, the biosynthetic pathways lently to the MHC class I α chain and is mono-
of MHC class I and class II molecules differ (dis- morphic. Two short stretches of α-helix above
cussion follows). Because of that difference, a relatively flat plane of β-pleated sheet (all part
class I and II molecules bind antigenic peptides of the MHC class I α chain) frame the antigen-
in different intracellular compartments and binding groove of MHC class I proteins (see
present these antigens to distinct subsets of T Figure 8.6A–C).
cells. Both the α and β chains of MHC class II mol-
In MHC class I molecules, only the α chains ecules come from the MHC region, both span
are encoded in the MHC. The first and second the cell membrane, and both contribute to the
domains of these α chains—where all of the al- antigen-binding groove of the molecule. Again,
lelic polymorphisms reside—form the antigen- two coils of α-helix above a plane of β-pleated

sheet create the groove itself. In MHC class II
molecules, however, the ends of the α-helices
are further apart than in MHC class I mol-
ecules, leaving open ends to the groove. Be-
cause of this, MHC class II molecules can bind
slightly larger peptides than those bound by
MHC class I molecules. However, the peptides
bound by MHC class I or II molecules are not
large enough to have significant secondary or
tertiary structure. As a result, only linear, con-
tinuous antigenic epitopes bind to MHC mol-
ecules. Together, the binding cleft of MHC
molecules and their bound peptides create the
ligands recognizable to T lymphocytes. Figure 8.8. Antigen binding by MHC class I
Still, MHC class I and class II molecules play molecules
very different roles in developing immune re- This ribbon diagram shows an approximation
sponses, and the tissue distribution of MHC of an MHC class I molecule–peptide complex
class I and class II molecules reflects their from above. Two stretches of α-helix near the
unique roles. MHC class I antigens appear on N-terminal portion of the α chain form the
all or nearly all nucleated cells, whereas MHC boundaries of the binding cleft. The antigenic
class II molecules appear only on APCs. Recall peptide lies in the binding cleft of the MHC
from earlier chapters that macrophages and class I molecule. The diagram also shows the
noncovalent interactions (in red) between
DCs are APCs, as are B cells and other cells such
amino acid side chains of the MHC α chain and
as astrocytes in the central nervous system and
the peptide. (Adapted from C. Janeway,
thymic epithelial cells. P. Travers, M. Walport, and M. Shlomchik, 2006,
Immunobiology, 125)
Antigen–Major Histocompatibility
Complex Interactions
As mentioned, each MHC molecule will bind the MHC molecule, but the rest of the peptide
multiple (but not all) antigenic peptides, which is irrelevant to MHC binding. That degenerate
means the interactions between antigens and specificity allows MHC molecules to bind many
MHC molecules have some specificity. That different antigenic peptides. As a result—using
specificity comes from noncovalent interac- a relatively small amount of DNA—mammals
tions between certain amino acids in the bind- are capable of defending themselves against
ing cleft and amino acids in the antigenic pep- most pathogens.
tide (see Figure 8.8). MHC class II molecules bind peptides of
MHC class I molecules bind eight- to ten- varying lengths (typically from twelve to twenty-
amino-acid-long peptides (see Figure 8.8 and four amino acids). Because the binding cleft of
Figure 8.9), but not all of the amino acids MHC class II proteins is open at both ends, the
in each peptide are equally important to the absolute length of the peptide is less important
binding affinity. Instead, MHC class I–peptide than is the position of certain amino acids rela-
interactions depend on as few as two amino tive to one another (see Figures 8.10 and 8.11).
acids. The spacing of those two amino acids Again, not all of the amino acids in the pep-
is critical, because they must line up with cor- tide bind to the MHC class II molecule, and
responding amino acids in the binding cleft of as few as two amino acids may determine the
131

Figure 8.9. Molecular
properties of peptides
bound by MHC class I molecules Figure 8.10. Antigen binding by MHC class II
Upper and lower panels show the amino acid molecules
sequence of peptides that bind to two different This ribbon diagram shows an approximate
MHC class I alleles. Although all of the peptides representation of an MHC class II molecule–
differ, the green amino acids indicate the fea- peptide complex from above. Two stretches of
tures they share. In the first set of peptides, the α-helix—one from the N-terminal portion of
amino acid found at position 5 must be either the α chain and the other from the N-terminal
phenylalanine (F) or tyrosine (Y), and the amino portion of the β chain—form the boundaries of
acid found at the C-terminal position must be the binding cleft. The antigenic peptide lies in
leucine (L). Similarly, peptides that react with the binding cleft of the MHC class II molecule.
the second MHC class I allele must contain In addition, the diagram shows the noncovalent
tyrosine at position 2 and a C-terminal valine interactions between amino acid side chains of
(V), leucine, or isoleucine (I). In addition, the the MHC α and β chains and the peptide in red.
length of the peptides is critical. For the first (After C. Janeway, P. Travers, M. Walport, and
MHC class I allele, each peptide must be exactly M. Shlomchik, 2006, Immunobiology, 127)
eight amino acids long, whereas for the second
allele, each peptide must be exactly nine amino
acids long. The critical points of interaction for MHC class I and II molecules also routinely bind
the first allele are the amino group at the pep- and present self peptides. MHC molecules bind
tide’s N-terminus, amino acids 5 and 8, and the and present almost all intracellular and extracel-
hydroxyl group at the C-terminus. The amino lular proteins—self or non-self. For a variety of
acids at all of the other positions in these pep- reasons (discussed in subsequent chapters), this
tides are irrelevant to the peptide’s MHC class does not routinely result in autoimmune de-
I binding affinity. This specificity is relatively struction of self tissues. As you will see, though,
degenerate, meaning that any given MHC class in type II hypersensitivities and other disorders,
I allele will bind many peptide antigens, but not self-presentation and autoimmunity are at the
all. (Adapted from C. Janeway, P. Travers, M.
root of many animal diseases.
Walport, and M. Shlomchik, 2006, Immunobiol-
ogy, 126)
Antigen Acquisition,
Processing, and Presentation
capacity of a peptide to bind to an MHC class
II molecule. Antigen-Presenting Cells
It is important to recognize that, although this The function of MHC class I and class II mol-
discussion focuses on the antigens of pathogens, ecules is to present peptide antigens to T lym-

Figure 8.11. Antigen-binding motif of MHC class II molecules
Because the ends to the antigen-binding cleft of MHC class II molecules are open, the molecules bind
peptides of varying lengths (typically twelve to twenty-four amino acids). The gray regions represent
the portion of the peptide containing the anchor residues—the amino acids actually within the binding
site of the MHC class II molecule. The peptides that bind to this particular MHC class II molecule range
from thirteen to nineteen amino acids long. However, among all of these peptides, only amino acids 4
and 9 appear critical for binding to this MHC class II allele. Amino acid 4 must be aspartic acid (D) or
glutamic acid (E). Amino acid 9 must usually be a hydrophobic or uncharged amino acid—tyrosine (Y),
isoleucine (I), proline (P), threonine (T), phenylalanine (F), asparagine (N), or glutamic acid. As with
MHC class I molecules, this specificity is relatively degenerate (especially compared with the specificities
of B- and T-cell receptors). So, each MHC class II molecule can bind many different, but not all, peptides.
(Adapted from C. Janeway, P. Travers, M. Walport, and M. Shlomchik, 2001, Immunobiology, fig. 3.26)
Table 8.2. Distribution and properties of APCs

Macrophage DC B cell
MHC-II expression Low levels; induced by PAMPs, Always expressed Always expressed; inducible
DAMPs, and cytokines on activation
Antigen type and pre- Extracellular antigens: presen- Intracellular & extracellu- Extracellular antigen binds to
sentation by MHC tation via MHC II lar antigens: presentation specific Ig receptors: presenta-
via MHC I & MHC II tion via MHC II
Location Lymphoid tissue Lymphoid tissue Lymphoid tissue
Connective tissue Connective tissue Blood
Body cavities Epithelium
phocytes. The cells that use MHC class I and of antigens (see Table 8.2), and each APC has
class II molecules to perform this function are a unique distribution in peripheral tissues and
APCs. Outside the primary lymphoid organs secondary lymphoid organs. In addition, these
and the central nervous system, macrophages, cells differ in how they acquire antigen and
DCs, and B cells are the major APCs (see Fig- what they do with it (see Figures 8.12, 8.13, and
ures 8.12–8.14). 8.14).
All of these cells share certain characteristics, There are several important DC subsets, in-
but each excels at acquisition of certain types cluding lymphoid, plasmacytoid, myeloid, and
133

Figure 8.12. Macrophage photo and drawing
The large cell in the center is a macrophage with pseudopodia. Macrophages acquire antigens by
phagocytosis and endocytosis.
Figure 8.13. DC: photo and drawing

This cell is called a DC because of the long processes. These cells also acquire antigens through
phagocytosis or endocytosis.
follicular. Each differs in location and effective- Macrophages and DCs acquire antigens in
ness as an APC. In peripheral tissues, myeloid several ways—through fluid-phase macropino-
DCs seem to be most common. We focus pri- cytosis, endocytosis, and phagocytosis of whole
marily on myeloid DCs. pathogens. Endocytosis and phagocytosis can

Figure 8.14. B cells
Graphic representation of mammalian B cell (right panel) and micrograph of a B cell (labeled “lym-
phocyte” in left panel). This mononuclear cell is one of two types of white blood cells called lympho-
cytes, a B lymphocyte or B cell. It is the ultimate source of antibody molecules. B cells use BCRs to
specifically bind antigen, endocytose it, and process it.
occur via PRRs such as the MR and scavenger I molecules mostly present intracellular anti-
receptor (see chapters 3 and 6). Simple high- gens, and MHC class II molecules mostly pre
mannose glycoproteins are present in greatest sent antigens from outside of the cell (extracel-
quantities on prokaryotic organisms, because lular antigen).
these organisms do not have Golgi apparati A subset of all proteins produced in the cyto-
where most complex sugar modifications occur sol of eukaryotic cells includes misfolded, mu-
in eukaryotes. tant, damaged, and excess proteins. To maintain
B cells acquire antigens only through recep- cellular homeostasis, the cell needs to divest it-
tor-mediated endocytosis. For this purpose, B self of these proteins. For this purpose, eukary-
cells use a unique receptor—the BCR. The anti- otic cells have evolved specialized cellular struc-
gen-binding part of that receptor is, of course, tures called proteasomes—large barrel-shaped
the same as the antigen-binding region of an protein complexes in the cytosol of eukaryotic
antibody molecule. B cells are the only APCs cells. Proteolysis (the breakdown of proteins)
that acquire antigens in an antigen-specific occurs within the core of proteasomes. Each
manner. proteasome core contains one α ring at each
end and two β rings in between (see Figure
8.15).
Major Histocompatibility Beta rings have three enzymatically active
Complex Class I Antigen sites with three separate specificities. B1 cleaves
Processing and Presentation on the C-terminal side of hydrophobic resi-
Inside animals are two spaces where infec- dues; B2 cleaves on the C-terminal side of basic
tious agents may proliferate—inside of cells (in- amino acids; and B3 cleaves on the C-terminal
tracellular) and outside of cells in fluid spaces side of acidic residues. As a result, intact pro-
(extracellular). For an immune system to be teins enter one end of the proteasome, and
protective, it must have information about peptides of various lengths exit the other end
events in both of those two spaces. MHC class of the proteasome. Although they all perform
135

ER. As each MHC protein assembles, the prod-
uct extrudes into the lumen of the ER. Inside
the ER, nascent MHC class I molecules, as-
sociated with chaperone proteins, bind β2-
microglobulin, acquire peptide antigens, and
assume their final configurations. Because of
TAP transporters, the peptides available to
MHC class I molecules include not only those
present in the ER but also those created in the
cytosol (see Figures 8.16 and 8.17).
As MHC class I α-chain proteins extrude off
ribosomes and into the ER, they associate with
a series of molecular chaperones. These mol-
ecules, along with β2-microglobulin, force the
α-chain–β2-microglobulin complex into an an-
tigen-binding form. After antigen binding, the
MHC α-chain–β2-microglobulin complexes,
Figure 8.15. Mammalian proteasome along with the bound antigen, move from the
Proteasomes are cellular structures that digest
ER to the Golgi complex and finally to the cell
intracellular proteins into short stretches of surface. This process occurs inside most mam-
linear polypeptides and are essential to normal malian cells. For this reason, most cells present
antigen processing. an exterior image of internal events. As you will
see, a particular T-cell subset—CD8+ T cells—
takes advantage of this image to find and de-
similar functions, not all proteasomes are alike. stroy infected cells.
At least two proteasome subtypes have been
identified—immunoproteasomes and thymo-
proteasomes. How these subtypes affect im- Major Histocompatibility
mune responses remains unclear. Regardless, Complex Class II Antigen
one end of cellular proteasomes collects rep- Processing and Presentation
resentatives of all intracellular proteins (self What about extracellular infections, microbes
or non-self ) and digests them into smaller pep- that never reside in a cell’s cytosol, or organ-
tides. Mammalian immune systems use those isms such as leishmania that grow inside of in-
peptides and transporters associated with anti- tracellular vesicles? Monitoring for extracellular
gen presentation (TAPs) to deliver those pep- and intravesicular infections is the function of
tides into the endoplasmic reticulum (ER). MHC class II molecules. As with MHC class I
In the absence of infection, the proteins en- molecules, class II molecules are also synthe-
tering and the peptides leaving proteasomes are sized on and extruded into the lumen of the
all parts of self. As we discuss later, immune rough ER (see Figures 8.18 and 8.19).
systems—for the most part—ignore self. How- Unlike class I molecules, as soon as MHC
ever, if an intracellular virus or bacterium is class II α and β chains associate inside the ER,
actively assembling itself inside of a host cell, a protein called the invariant chain (Ii) binds to
the proteasomes will also be delivering peptides and blocks the antigen-binding cleft. Because
derived from that virus or bacterium. of this, no antigenic peptide inside of the ER
The first phase of MHC class I molecule as- can bind to an MHC class II molecule. Also, un-
sembly occurs on the ribosomes of the rough like MHC class I molecules, after MHC class II

Figure 8.16. Antigen processing and presentation by MHC class I molecules
As with all proteins destined for the cell surface, MHC class I molecules, as they are synthesized,
insert their N-termini into the ER but remain anchored in the ER membrane (A). Inside the ER, the
body of MHC class I α chains interact with a series of chaperone proteins (shown here in gray) that,
along with β2-microglobulin (purple), force the α chain into its final configuration (B). A subset of all
proteins made in the cytosol collects in proteasomes where enzymes cleave the proteins into small
peptide fragments. The TAP moves these peptides into the lumen of the ER (C). Once inside the ER,
the peptides encounter MHC class I molecules. Under the right conditions, the peptides bind into the
antigen-binding groove of MHC class I molecules. The chaperone proteins detach (D), and the MHC
class I–peptide complex continues on to the Golgi complex and then to the cell surface (E).
molecules pass through the Golgi complex, CLIP dissociates. Removal of CLIP leaves the
they move—via the trans-Golgi network—into antigen-binding groove empty so that exog-
acidic vesicles that fuse with lysosomes. enously acquired antigenic peptides can bind to
Pathogens and other antigens acquired by the MHC class II molecule. All of this ensures
macropinocytosis, phagocytosis, or receptor- that most MHC class II molecules bind antigens
mediated endocytosis end up in endosomes that came from outside of the APCs.
or phagosomes that eventually fuse with ly- From the endolysosome–phagolysosome
sosomes. So in the end, endosomes and lyso- compartment, MHC class II molecules, along
somes contain extracellular antigens and MHC with bound antigens, move to the surface of
class II molecules. the APCs, where they present an antigenic pic-
Recall from chapter 6 that lysosomes are ture of what is happening in the extracellular
acidic compartments filled with proteolytic en- space near the APC and within vesicles inside
zymes. As endosomes or phagosomes carrying the APC. A T-cell subset—CD4+ T cells—mon-
exogenously acquired antigens (which may in- itors MHC class II molecules and their con-
clude pathogens) fuse with lysosomes, protein tents and, when appropriate, initiates immune
antigens are quickly chopped into peptides. The responses.
enzymes in lysosomes also remove most of the
Ii from MHC class II molecules. The last piece
of Ii—the CLIP—remains bound to the bind- Cross-Presentation
ing groove until the very last, when an MHC As we have discussed, generally MHC class
class II surrogate molecule (human leukocyte II molecules present extracellular antigens, and
antigen-M in humans) alters the structure of MHC class I molecules present intracellular an-
MHC class II molecules in such a way that the tigens, which allows for presentation to specific
137

viruses). These cells recognize antigens bound
by MHC class I molecules and destroy infected
cells.
However, many effective immune responses
require antigen presentation to both Th and Tc
cells. For example, effective immune responses
to virus infections often require both antibody
and Tc cells. Antibody production usually in-
volves CD4+ Th cells that require antigen pre-
sentation via MHC class II molecules. CD8+ Tc
cell activation, however, requires antigen pre-
sentation in the context of MHC class I mole-
cules. Thus, the virus would have to both infect
the DC cell (entering the cytosol, a traditional
pathway for MHC class I presentation) and
enter the endosomal pathway through phago-
cytosis for MHC class II presentation. Phagocy-
tosis of viral particles by DC cells occurs regu-
larly and efficiently. However, virus infection of
DC cells is a relatively rare event.
To deal with this, DCs have evolved cross-
presentation, a process that allows extracellular
antigen to be processed and displayed in MHC
class I molecules (in addition to MHC class II
molecules). When a DC phagocytoses a dead
Figure 8.17. Overview
of antigen processing and cell containing viral particles, the DC can shut-
presentation by MHC class I molecules
tle viral antigens into both the MHC class II and
As antigens are synthesized inside of cells (A), a the MHC class I processing pathway and pre
portion of the antigenic proteins (B) enter cel-
sent to both CD4+ and CD8+ T cells.
lular proteasomes (C). Those organelles digest
The cellular mechanisms by which DCs
these antigenic proteins into peptides. TAPs (D)
deliver these peptides into the ER.
cross-present extracellular antigens to MHC
(E) There, antigenic peptides bind with MHC class I molecules are still obscure. Some anti-
class I molecules, and endosomes (F) transport gens appear to be simply exported from the
them to the cell surface (G), where they present phagolysosome to the cytoplasm where protea-
these peptides to T cells. somes process them. The TAP moves the result-
ing peptides into the ER lumen for loading onto
MHC class I molecules. Other antigens seem to
T-cell subtypes best equipped to respond to be cross-presented without the involvement of
antigens, based on whether a given antigen is the proteasome and TAP. In this case, it may
extracellular or intracellular. be that MHC class I molecules interact with
For instance (discussed in later chapters), antigens in specialized endosomes. Regardless,
CD4+ T (Th) cells are most effective against cross-presentation is an important process by
extracellular pathogens. These cells recognize which APCs can monitor antigens within the
antigens bound by MHC class II molecules. cytoplasm of other host cells and potentially
However, CD8+ T cells (Tc cells) are most ef- stimulate T-cell responses that most effectively
fective against intracellular pathogens (such as deal with intracellular infections.

Figure 8.18. Antigen presentation by MHC class II molecules
MHC class II molecules are also synthesized on ribosomes and extruded into the lumen of the ER.
However, as MHC class II proteins arrive inside the ER, another molecule—the invariant chain, or Ii
(shown here in orange)—binds to them (A). The Ii covers the antigen-binding site and prevents MHC
class II molecules from presenting the same set of peptides bound by MHC class I molecules. The
MHC class II–Ii complex then enters the endolysosomal pathway (B) and eventually ends up inside of
endolysosomes. There, a protease removes all but a small piece of Ii from MHC class II molecules.
That small remaining piece is called class II–associated Ii peptide (CLIP). As APCs acquire pathogens
from their surroundings via phagocytosis and endocytosis, the resultant phagosomes and endosomes
containing extracellular antigens eventually fuse with lysosomes to form phagolysosomes and endoly-
sosomes. Inside of these compartments, proteolytic enzymes cleave the pathogen’s proteins into pep-
tides (C). At the same time, the CLIP leaves the antigen-binding site of the MHC class II molecules,
which allows for binding of antigenic peptides in the endolysosome. Portions of the endolysosome
along with MHC–antigen complexes then return to the cell surface via recycling endosomes, which
fuse with the cell membrane (D).
The end result of antigen acquisition, pro- MHCs. Goats that develop arthritis or en-
cessing, and presentation within MHC mol- cephalitis often carry one or both of two MHC
ecules is a cell-surface representation of ev- genes—Be1 and Be14—and disease-free goats
erything occurring in and around host cells. have a higher incidence of Be7.
Mammalian immune systems rely on this infor- Among infectious diseases of goats, CAEV is
mation to detect and respond to infectious non- one of the most common. As with other ret-
self and sometimes to mutant self. roviruses, these infections are lifelong, and re-
crudescence or transmission of the virus may
occur at any time. Goats most commonly pass
the virus to kids in the colostrum and milk of
As we said at the beginning of this chapter, infected dams. Goats exposed to CAEV often
only a few goats infected with CAEV develop develop persistent infections, slowly developing
disease. Exactly why remains unclear. A major arthritis of their carpal joints and mastitis. Only
factor seems to be the makeup of the goats’ rarely do adult goats develop encephalitis.
139

sort of association is not unique to goats. So far,
it appears that MHC–disease associations occur
in all mammals.
Student Considerations
Why might a particular MHC allele make an
animal more or less susceptible to a certain dis-
ease? From what you have just learned about
the function of MHC molecules, you should
be able to offer one or two explanations for the
association between CAEV and MHC genes—
explanations that apply to any association be-
tween a disease and an MHC allele.
The exact reasons for these associations re-
main unknown, but at least two possibilities
seem obvious. Because of the roles of MHC
class I and class II molecules in antigen bind-
ing and presentation, a disease might arise in an
animal that was unable to present a critical anti-
gen and mount a protective immune response.
For example, a protective immune response
Figure 8.19. Overview
of antigen processing and against pathogen X occurs only if the host ani-
presentation by MHC class II molecules
mal recognizes antigenic epitope Y. If an animal
APCs ingest extracellular antigens (A) and expresses MHC alleles whose protein products
enclose them in endosomes or phagosomes.
do not bind and present antigen Y, then that ani-
These phagosomes and endosomes eventually
mal will succumb to the infection.
fuse with lysosomes (creating hybrid organ-
elles—phagolysosomes and endolysosomes—
Alternatively, the disease might result from
where proteases digest the antigens into an overreaction or cross-reaction of the im-
peptides. These phagolysosomes and endoly- mune system. Let’s say pathogen X also carries
sosomes then fuse with endosomes containing an antigenic epitope Z that looks a lot like a pro-
MHC class II molecules (E). At this point the tein on synovial cells. If an animal infected with
CLIP is removed from the MHC class II mol- pathogen X carries a certain MHC allele and
ecules and replaced with antigenic peptides. the product of that allele binds to the antigenic
MHC class II–antigen complexes then return epitope Z, then when this animal’s immune sys-
to the cell surface, where they present these tem reacts to epitope Z on pathogen X, it may
peptides to T cells. inadvertently also strike the synovial cells and
cause a progressive arthritis. Although one usu-
Genes Be1, Be14, and Be7 all encode MHC ally thinks of immune responses as being pro-
class I proteins. Goats carrying either Be1 or tective—the ultimate defense against infectious
Be14 genes are more likely to develop arthritis diseases—under certain circumstances, immune
after CAEV infection, and goats carrying the responses can harm host animals. Goats might
Be7 gene are less likely to develop disease. This also develop CAEV because their immune re-

bind and present the critical CAEV epitope are
Be1 and Be14. If that is true, then goats with ei-
ther or both of these alleles would mount inef-
fective immune responses, the infection would
go unchecked, and these goats would develop
arthritis and sometimes encephalitis.
One more observation about CAEV points,
however, toward an alternative explanation
for the observed differences in susceptibility.
In one study in which goats were intentionally
infected with CAEV, researchers found that im-
munosuppression of goats reduced the likeli-
hood that these goats would develop arthritis.
Although the pathogenesis of CAEV remains
largely unknown, this finding suggests that, in
addition to the virus, an inappropriate immune
Figure 8.20. Domesticgoats (© Mircea response plays a role in the pathogenesis of
Bezergheanu / Shutterstock)
CAEV.
Of the two possibilities discussed, the immu-
sponses against CAEV cross-react with and nosuppression studies have pointed toward over-
damage host tissues. active immune responses as the probable cause
MHC class I and class II gene products bind of CAEV. The association of this disease with
wide varieties of (but not all) antigenic epitopes MHC class I alleles Be1 and Be14 demonstrates
derived from pathogens. In goats, it could be the importance of these genes and their protein
that among the possible MHC class I alleles at a products in antigen presentation and protective
particular MHC locus, the only ones that fail to immunity or destructive autoimmunity.
141

Gerald N. Callahan
T-Lymphocyte Development Chapter 9

10.5876_9781607322184.c009
necropsy, severe atrophy of their thymuses and

Clinical Correlation: Severe Combined
lymph nodes.
Immunodeficiency Disease in Arabian
Foals 143
The only antibodies found in SCID foals
are remnants of those maternally transferred
T-Cell Receptor Genes 144
through colostrum. Breeding studies have
Structure of the T-Cell Receptor 144
shown that among Arabian foals, SCID is due
T-Cell Receptor Gene Families 145
to an autosomal recessive mutation, meaning
Gene Rearrangement in the Thymus 145
that the sire and the dam have both contributed
T-Cell Selection in the Thymus 150
one defective gene to an afflicted foal. More re-
Products of T-Cell Receptor Gene
cently, studies have identified the relevant gene
Rearrangement 150 as a DNA-dependent phosphokinase. How does
Positive Selection of Functional T Cells 153 such a mutation make these foals so susceptible
CD4, CD8, and Major Histocompatibility to infection?
Complex Recognition 155
Negative Selection of Self-Reactive T Cells 155
Learning Objectives
T-Helper, Cytotoxic T, and T-Regulatory Cells 156
γ/δ T Cells 156 After reading this chapter, you should be able to
• understand the structure and function of
the TCR and the CD3 complex;
• describe the unique arrangement of TCR
genes;
Clinical Correlation: Severe • understand the process and products
Combined Immunodeficiency of genetic rearrangement of TCR gene
Disease in Arabian Foals families;
Every year, about 3 percent of U.S.-born Ara- • describe the process and outcome of positive
bian foals (Figure 9.1) exhibit a devastating and and negative selection of T cells in the thymus;
uniformly fatal affliction. This disease—called • understand the origins of double-positive
severe combined immunodeficiency disease and single-positive T cells and the roles of
(SCID)—manifests as repeated and eventually CD4 and CD8 molecules on T cells;
fatal opportunistic respiratory infections, in- • describe the characteristics of naive T
cluding adenovirus, Pneumocystis carinii, and cells that emigrate from the mammalian
assorted bacterial infections, especially strepto- thymus;
cocci. Clinical analyses of these foals indicate • explain the nature of and evidence for cen-
dramatically low lymphocyte counts and, at tral and peripheral tolerance.
143
143

Figure 9.1. Arabian foal (© Pictureguy / Shutterstock)
T-Cell Receptor Genes termine the antigen specificity of the receptor.

Also, both α/β and γ/δ chain pairs associate
Structure of the T-Cell Receptor
with CD3—a complex of four different poly-
Immunity—the ability to detect and respond peptides (g, δ, ε, and ζ) that together form the
to potentially pathogenic microorganisms—de- signal-transduction machinery of TCRs. Be-
pends on an animal’s ability to distinguish self yond this, the similarities between α/β and γ/δ
from non-self—a critical distinction that keeps TCRs are few.
us all alive. The α/β TCRs recognize antigens only after
Among all the cells of the body, only lym- processing and presentation inside of MHC
phocytes have antigen-specific receptors. On T class I or II molecules. The γ/δ TCRs recognize
cells, these molecules are the TCRs (see Figure intact antigens in the absence of classical MHC
9.2). In their functional state, the N-termini of class I or II molecules. The structural variabil-
these molecules project into T cells’ surround- ity among α/β TCRs is much greater than that
ings, and the C-termini extend into the cytosol. among γ/δ TCRs. Also, γ/δ TCRs seem to have
These receptors contain two polypeptide evolved to recognize a relatively narrow range
chains—either α and β or γ and δ—that to- of specialized antigens, including the lipids of
gether form the antigen-binding site at their mycobacteria, some phosphorylated ligands,
membrane-distal N-termini. Both the α and β and heat-shock proteins. Here, we focus pri-
chains and the γ and δ chains span the plasma marily on α/β TCRs, the most common form
membrane with their C-termini extending into in most mammals, although species variations
the cytosol. Together, α and β or γ and δ de- can be substantial.
144 T - Lym p h o cy t e D e v e l o p m e n t

species can respond to essentially every poten-
tial pathogen. Considering the enormity of
the antigenic universe, that means that every
animal must be able to make a nearly unlimited
number of TCRs—literally trillions upon tril-
lions of TCRs. How is that possible?
T-Cell Receptor Gene Families

The essentially unlimited diversity among
mammalian TCRs is the result of a unique ge-
netic mechanism that operates only inside of
T and B lymphocytes: the random rearrange-
ment of germline gene segments. Most of
the original insights into gene rearrangement
came from studies of genes inside of B cells.
However, T cells use the same mechanisms for
generation of diversity. T-cell DNA-sequencing
studies revealed the remarkable finding that
Figure 9.2. T-cell α/β antigen-specific receptor TCR genes are not in contiguous segments of
The complete TCR includes an α and a β chain DNA. Rather, the DNA that eventually encodes
or a γ and a δ chain that together form the an- a TCR is in segments strung out along the DNA
tigen-binding complex. In addition, functional strand. Moreover, for each of the final pieces of
TCRs include a series of associated molecules
a TCR, there are multiple possible segments on
collectively called CD3. These include the γ, δ,
the chromosome (see Figure 9.3).
ε, and ζ chains that transduce activation signals
after antigen binding. Ag = antigen.
Gene Rearrangement in the Thymus
The N-terminal (antigen-binding) regions of The assembly of a TCR α chain begins with
α and β chains have highly variable amino acid a genetic rearrangement that randomly brings
sequences, whereas the C-terminal regions are together one Vα segment and one Jα segment.
invariant. These chemical differences reflect the The intervening DNA loops out and is excised.
functional differences in these two regions of Then ligases join the V and J segments. This VJ
TCRs—antigen binding versus membrane an- segment is then joined to the Cα constant re-
choring and signal transduction. gion segment (see Figure 9.4). After transcrip-
Amino acid sequence analyses of these re- tion, spliceosomes remove all of the excess
ceptors have revealed three clusters of variabil- RNA from the nuclear transcript, and the mes-
ity in the N-termini of α and β chains and γ and senger RNA encodes a single α chain from N-
δ chains. These regions are called hypervariable to C-terminus, including the constant region.
regions 1, 2, and 3, or—more commonly— The assembly of a β-chain gene is slightly
complementarity-determining regions (CDRs) more complicated. This process begins with a
1, 2, and 3 because these regions determine the genetic rearrangement that loops out interven-
complementarity of the fit between TCR and ing DNA and randomly brings together one Dβ
antigen, like a lock and a key. segment and one Jβ segment. The resulting DJ
TCRs are highly specific—nearly one TCR segment then randomly combines with one
for each antigen. In spite of that, most animal Vβ segment to generate a VDJ segment that
T - Lym p h o cy t e D e v e l o p m e n t 145
145

Figure 9.3. Arrangement of TCR genes on mammalian chromosomes
The genetic material that encodes TCR α and β chains is not contiguous. Instead, it exists in seg-
ments. These segments include variable (V), joining ( J), diversity (D), and constant (C) region seg-
ments. The numbers above each gene segment indicate the total number of distinct segments found
in each region of each animal. This figure shows human α (upper panel) and β (lower panel) gene
families. Each species has a unique number of each gene segment.
encodes the variable region of a TCR β chain, the mathematical power of random combina-
which is then joined to a Cβ gene segment to tion, it dramatically increases the number of
form a complete gene. Again, spliceosomes re- possible TCRs that a T cell can generate from
move intervening RNA and convert the nuclear a fixed amount of DNA. In addition, it appears
transcript into messenger RNA that encodes a that any TCR α chain can pair with any TCR β
complete β chain from its variable N-terminus chain, which creates even more diversity.
to its constant C-terminus. By themselves, though, genetic rearrange-
As with all molecules destined for the cell sur- ment and random pairing of α and β chains can-
face, the synthesis of TCR chains begins on the not account for the astronomical array of TCRs
surface of the rough ER. As they grow, these inside of every mammal. The real secret to
polypeptides extrude into the lumen of this or- TCR diversity lies in the ways in which the gene
ganelle. Inside the ER, disulfide bonds join the segments join during genetic rearrangement—
two chains. Later, inside the Golgi complex, en- a process called junctional diversification.
zymes add sugars to these glycoproteins, which Physically, rearrangement of TCR gene seg-
then move to the surface of T cells and join ments occurs after looping out and excision of
with the CD3 complex to form the TCR. intervening DNA to join randomly selected
This random association of V and J or V, gene segments (as with TCR α chain in Figure
D, and J gene segments allows for a variation, 9.5).
similar to shuffling a large deck of cards and The products of two genes—RAG1 and
selecting two or three cards at a time. Using RAG2—drive this process. The protein products

Figure 9.4. Assembly of an α/β TCR
Formation of an α/β TCR begins with rearrangement of β-chain genes (lower half of figure) with
the random joining of a Dβ and a Jβ gene segment. The gray areas between the lines indicate the
excised segments of DNA. Enzymes then randomly link this new DJβ segment with a Vβ segment to
form the variable region of a β chain. Spliceosomes convert this nuclear RNA transcript into messen-
ger RNA encoding a complete β chain. Once a complete β-chain polypeptide appears, rearrangement
of α-chain gene segment begins (upper half of figure). First, a randomly selected Vα and a Jα pair of
gene segments are joined to form complete α-chain variable region. Then, the VJ segment is joined
with a Cα constant region segment. After splicing, a complete α-chain message forms. After transla-
tion, the α-chain polypeptide joins the β-chain polypeptide and the α/β TCR appears on the cell
surface. (Note: For clarity, only a few gene segments are shown in this drawing.)
147

Table 9.1. TCR diversity (adapted from C. Janeway, P. Travers, M. Walport, and M. Shlomchik, 2006, Immunobiology, 151)
Immunoglobulin α:ß receptors

Element H L ß α
Variable segments (V) 65 70 52 ~70
Diversity segments (D) 27 0 2 0
D segments read in 3 frames rarely — often —
Joining segments ( J) 6 5(κ) 4(λ) 13 61
Joints with N- and P-nucleotides 2 50% of joints 2 1
Number of V gene pairs 3.4 × 10 6
5.8 × 10 6
Junctional diversity ~3 × 107 ~2 × 1011

Total diversity ~1014 ~1018
of these genes combine to form the lymphoid- cannot recognize and respond to. However,
specific components of the VDJ recombinase. that incredible diversity comes at a price. Also
This enzymatic complex—along with sev- shown in Figure 9.6, during joint assembly, en-
eral other enzymes, including essential DNA- zymes add or remove nucleotides randomly.
dependent protein kinases—accomplishes the At least two-thirds of the time, this results in a
rearrangement steps shown in Figure 9.5A–D. frameshift mutation and no TCR. Only when a
First, complexes of RAG1 and RAG2 bind near full codon appears or disappears will the rear-
relevant gene segments. The RAG proteins ranged gene produce a functional TCR.
then bind to one another, looping and excising As we show later, T cells that do not form
the intervening DNA. functional TCRs die. If instead the cell assem-
During joint formation, enzymes, including bles and expresses a functional TCR, TCR gene
DNA-dependent protein kinases, cut away the rearrangement ceases, which means that all the
hairpin loop and leave single-stranded DNA TCRs on a given T cell have exactly the same
ends behind. The enzyme, terminal deoxy- antigenic specificity. No T cell ever expresses
nucleotidyl transferase (TDT), then randomly more than one TCR or more than one antigenic
adds complementary nucleotides to both specificity.
strands, and DNA polymerases fill the resultant Similar to those of the gene families of α/β
gaps. During the process, at least one-third of TCRs, the gene families used to produce γ/δ re-
the time, TDT creates new codons that code ceptors appear in the genome as a series of frag-
for new amino acids at the VJ and VJD joints ments (see Figure 9.8). In fact the δ-chain gene
(see Figure 9.6A–D). In the final tertiary struc- family nests within the family of gene segments
ture of the TCR, the VJ and VDJ joints form that encode the α chain of the α/β TCR. All
CDR3, one of the most variable regions of the of the processes that operate in the generation
TCR and the region that directly contacts an- of α/β TCRs also operate in the generation of
tigenic peptides during antigen presentation to γ/δ TCRs. As mentioned, though, γ/δ T cells
T cells. Thus, as a result of junctional diversifi- are much less diverse and recognize antigens in
cation, the diversity of possible TCRs increases a distinct manner.
exponentially, and the number of TCRs with The human TCR γ locus resembles the TCR
unique antigenic specificities inside one animal β locus: it has two C genes, each with its own
appears to be effectively limitless (see Table 9.1). set of J gene segments. The mouse γ locus (not
Because of this, an animal will rarely encoun- shown) has a more complex organization and
ter a pathogen or an antigen that the animal three functional clusters of γ gene segments.

Figure 9.5A–D. DNA rearrangement during TCR α-chain formation
Alpha-chain gene family (panel A); attachment of recombination activating gene (RAG) protein enzy-
matic complexes (panel B); looping out intervening DNA (panel C); and excision of intervening DNA
and formation of DNA hairpin loops (panel D).
Panel A shows a representative α-chain gene family with V segments from 1 to n and a J segment.
Panel B shows the attachment of RAG complexes—the initiation of rearrangement. Panel C shows
how the RAG complexes bind to one another and loop out intervening DNA between randomly
selected V and J segments. Panel D shows how enzymes, including DNA-dependent protein kinase,
excise the intervening DNA. The space with the two dots indicates an indeterminate amount of DNA.
149

Figure 9.6A–D. Junctional diversification
Cleavage of DNA hairpins (panel A); random deletion of nucleotides and activation of terminal de-
oxynucleotidyl transferase (TDT) (panel B); junctional diversification (panel C); and DNA ligation and
formation of VJ joint (panel D).
During the final steps of gene rearrangement, a process called junctional diversification introduces
additional diversity into the TCR α-chain gene. Panel A shows the final excision of the loop of inter-
vening DNA, the formation of singled-stranded DNA ends with a final hairpin loop, and enzymatic
cleavage of the hairpin. In Panel B, the enzyme TDT binds into the remaining gap in the DNA and,
in Panel C, begins to randomly add nucleotides, completing the DNA gap between V and J. Finally, as
shown in Panel D, DNA ligases join the now double-stranded DNA ends and complete the VJ joint.
Each contains V and J gene segments and a C gene segments as well as at the V–D and D–J
gene. Rearrangement at the γ and δ loci oc- junctions.
curs in the same way as in the other TCR loci,
with the exception being that during TCRδ T-Cell Selection in the Thymus
rearrangement, both D segments can be used
in the same gene. The use of two D segments Products of T-Cell Receptor
greatly increases the variability of the δ chain, Gene Rearrangement
mainly because extra N-region nucleotides can Beyond the potential frameshift inefficiency,
be added at the junction between the two D random rearrangement and insertion and dele-

Figure 9.7. γ/δ gene segment families
The TCR γ and TCR δ loci—as with the TCR α and TCR β loci—have discrete V, D, and J gene seg-
ments and C genes. In humans, the locus encoding the δ chain is entirely within the α-chain locus.
tion of nucleotides has another downside: the leaves the bone marrow and migrates to the
specificity of the resultant TCR is unpredict- thymus (see Figure 9.8).
able. Because these processes are random, they When these lymphocytes arrive in the thy-
may generate T cells with no functional TCRs, mus, the thymic stroma delivers a signal that
T cells with self-reactive TCRs, or T cells with activates the Notch 1 gene, and TCR gene
non-self-reactive TCRs. The first group is use- rearrangement begins. Because these early-
less, the second dangerous, and only the third stage thymocytes lack CD4 and CD8, they are
can be safely expanded and exposed to the tis- double-negative cells. Three TCR gene fami-
sues of an animal’s body. lies begin rearranging simultaneously—the
In most mammals, T-cell development be- β-chain, the γ-chain, and the δ-chain families.
gins well before birth. During the last one-third If the cell successfully rearranges and synthe-
of gestation, a large population of lymphocytes sizes a complete β chain first, the cell goes on to
151

Figure 9.8. T-cell development
All lymphocytes originate in the bone marrow.
Some of these lymphocytes emigrate from the
bone marrow and travel—via the blood—to the
thymus. Whether these cells are identifiably dif-
ferent from other lymphocytes in the bone mar-
row is unclear. Once inside the thymus (prob-
ably through activation of the Notch 1 gene),
most of the newly arrived lymphocytes begin
to differentiate, which results in the activation
of gene rearrangement among the β, γ, and δ
gene families. Depending on the outcomes of
these rearrangements, the cells begin to express
α/β and CD4 and CD8 cell-surface proteins or
γ/δ TCRs. After positive selection, α/β T cells
no longer express either CD4 or CD8 and dif-
ferentiate along two independent lines as either
CD4+ Th cells or CD8+ Tc cells.
become an α/β T cell. If γ- and δ-chain genes

successfully rearrange first, the cell goes on to
become a γ/δ T cell.
Inside the thymus, gene rearrangement pro-
ceeds one allele at a time; that is, it begins on
only one of the two parental chromosomes. If
rearrangement at the first allele fails, only then
does gene rearrangement begin at the second
allele. Because of this, TCRs always represent
the products of only one parental allele. How-
ever, the opportunity to rearrange gene seg-
ments on both parental chromosomes means
that each lymphocyte has two opportunities to
produce a functional gene rearrangement of
each of the chains of the TCR.
Obviously, the success of this whole process
is essential to an animal’s survival—useless T ment event has to be detectable at the surface
cells have to be disposed of, self-reactive T cells of the T cell. With α/β T cells, rearrangement
have to be destroyed, and somehow non-self- begins with the β-chain gene family. Even if the
reactive T cells have to be identified and pre- T cell manages to successfully rearrange a β
served. To meet these needs, mammals have chain, TCR β chains by themselves cannot in-
evolved a means to monitor TCR gene rear- sert into the cell’s membrane. Only α/β pairs
rangement. Ultimately, the thymic stroma is arrive at the cell surface, but α-chain gene re-
responsible for this monitoring. arrangement cannot begin until it is clear that
For monitoring to be successful throughout a functional β chain exists. Somehow, the de-
T-cell development, each successive rearrange- veloping T cell has to signal the thymic stroma

that it has successfully rearranged a β-chain
gene.
To provide that signal, T cells use an α-chain
surrogate called pre-Tα. When pre-Tα joins
with an assembled β chain, the complex in-
serts into the developing cell’s membrane and,
along with the CD3 complex, forms the pre-
TCR. The appearance of the pre-TCR signals
the thymic stroma that the cell has created a
functional β-chain molecule. In turn, the thy-
mic stroma delivers a signal that ends β-chain
gene rearrangement and initiates α-chain gene
rearrangement. Once the cell has assembled a
functional α chain, the full TCR appears on the
cell surface along with two other molecules—
CD4 and CD8. CD4 and CD8 are cell-surface
proteins that interact with constant regions of
MHC class II and class I molecules during anti-
gen presentation (see Figure 9.9).
Figure 9.9. The fully assembled α/β TCR
C-termini of the α and β chains both span the
Positive Selection of cell membrane while the membrane-distal N-
Functional T Cells termini form the MHC–antigen (Ag) binding
site. In addition, all α/β T cells express CD4
At this point in thymocyte development, the
and CD8 before positive selection. These cell-
thymocyte has on its surface an α/β TCR (or
surface molecules bind to constant regions of
not, because many rearrangements go wrong), MHC molecules: CD4 binds MHC class II, and
the CD3 complex, and CD4 as well as CD8. Be- CD8 binds MHC class I. In the T-cell mem-
cause these thymocytes now express both of brane, the α/β heterodimer associates with
the markers of mature T cells (CD4 and CD8) another group of molecules collectively called
they are called double-positive cells. CD3. These include the γ, δ, ε, and ζ molecules,
Ultimately, this complex of molecules is des- either alone or in pairs.
tined to interact with the MHC–antigen com-
plex on the surface of APCs. To be useful, this
newly formed TCR must show signs that it is with the MHC–antigen complex on the surface
capable of detecting those complexes. Because of an APC (see Figure 9.10).
the processes of gene rearrangement and nu- Positive selection tests whether a newly cre-
cleotide deletion and insertion all occur ran- ated TCR can bind to the MHC class I or MHC
domly, this newly formed TCR might be use- class II molecules on thymic cortical epithelial
less, dangerous, or potentially protective. It is cells (these cells are also APCs). This binding is
the job of the thymus and its components to likely a low-affinity interaction between MHC
sort out the useless and dangerous T cells and and TCR. Most (maybe as much as 96 percent
destroy them. This sorting or selection process of ) thymocytes fail positive selection and un-
occurs in two steps—positive selection and neg- dergo apoptosis. Remember, genetic rearrange-
ative selection. ment and reassembly are not only random but
To be of use, the T cell must express a func- perhaps as much as two times more likely to
tional TCR: a TCR that can recognize and react generate no or nonfunctional TCRs.
153

Figure 9.10. T-cell selection in the thymus
Once formed, the α/β TCR must react with MHC molecules present on APCs (including thymic epi-
thelial cells) in the thymic stroma. The first of these tests, positive selection, determines whether the
new TCR can bind with low affinity to the MHC portion of an MHC–antigen complex on thymic epi-
thelial cells. If a T cell fails positive selection, it dies by apoptosis. If it survives positive selection, the
T cell must pass a second test—negative selection—a test for self-reactivity. If the new TCR strongly
binds to an MHC–antigen complex presented on bone marrow–derived macrophages or DCs in the
thymus, those T cells also undergo apoptosis. Only non-self-reactive cells emigrate from the thymus.

Figure 9.11. The roles of CD4 and CD8 molecules
By binding to a constant region found on all MHC class II proteins, CD4 molecules on Th cells
increase the TCR’s avidity for the MHC class II–antigen complex on the APC (A). By binding to a con-
stant region found on all MHC class I proteins, CD8 molecules on Tc cells increase the TCR’s avidity
for the MHC class I–antigen complex on the APC (B). Once a TCR has bound to an MHC class I
molecule, that T cell ceases to express CD4. Likewise, once a TCR has bound to an MHC class II mol-
ecule, that T cell ceases to express CD8. Neither CD4 nor CD8 contributes to the antigenic specificity
of the TCR, but the combination of TCR and CD4 or CD8 binding leads to activation of the T cell via
signals transduced through the CD3 complex of molecules.
CD4, CD8, and Major only CD4. CD4+ T cells recognize antigens
Histocompatibility only in the context of MHC class II molecules.
Complex Recognition CD4 is a cell-surface protein that binds to the
Also during positive selection, thymocytes constant region of MHC class II molecules (see
transform from double-positive cells to single- Figure 9.11). CD4+ T cells are also known as Th
positive cells. This transformation occurs dur- cells, because they assist several other cell types
ing the interaction of the newly formed TCR during immune responses.
with an APC. If the TCR has an affinity for an
MHC class I molecule, the thymocyte turns off
the CD4 gene and begins to express solely CD8. Negative Selection of Self-
That cell then commits for life to MHC class Reactive T Cells
I molecules and their antigens. CD8 is a cell- Through the preceding process, positive se-
surface protein dimer that binds to constant lection eliminates thymocytes that either have
regions of MHC class I molecules (see Figure failed to assemble a TCR or have assembled a
9.11). For reasons that will become apparent useless TCR and seals a T cell’s fate, commit-
later, CD8+ T cells are also called Tc cells. ting it to a CD4+ or a CD8+ lineage. However,
Conversely, if the newly formed TCR has af- even after positive selection, the remaining thy-
finity for MHC class II molecules, that lympho- mocytes are as likely to have self-reactive TCRs
cyte switches off the CD8 gene and expresses as they are to have non-self-reactive TCRs.
155

Because of their potential danger, the process lytic anemia in dogs and cats. The reasons for
of negative selection has evolved to eliminate this are not clear.
these self-reactive T cells.
Inside the thymus, if a T cell’s TCR binds
with high affinity to an MHC-antigen com- T-Helper, Cytotoxic T, and
plex present on an APC, the T cell usually dies T-Regulatory Cells
through apoptosis (see Figure 9.11). Because a Those T cells that survive positive and nega-
blood–thymus barrier keeps non-self out of the tive selection leave the thymus via the blood.
thymus, any reactive T cell in the thymus is a Along with NKT-lymphocyte cells (an innate
self-reactive T cell. The process of negative se- subgroup of T cells), these T cells include at
lection eliminates most thymocytes that react least three populations of cells: α/β Tc, α/β
with self as represented in the thymus. Th, and α/β Treg cells, as well as γ/δ T cells.
On the surface, although this process seems Tc cells express CD8 molecules on their sur-
to be an effective means for eliminating useless faces and react with antigens in the context of
and dangerous T cells, it also seems as though MHC class I. These cells are largely responsible
it might eliminate only those cells reactive with for the destruction of pathogen-infected cells.
self presented in the thymus, which might rep- After leaving the thymus, and reaction with
resent only a small portion of the total variety their specific antigens, CD4+ Th cells differenti-
of self molecules. ate into several distinct subsets, including Th1,
To deal with this, the thymus has evolved Th2, Th17, and probably some types of regula-
a genetic mechanism that allows for a much tory cells (see chapter 10). Th cells are active in
broader representation of self on APCs in the almost all aspects of every immune response.
thymus. A gene known as the autoimmune Th1 cells activate macrophages, sometimes as-
regulator, or AIRE, gene allows thymic epithe- sist Tc cells, and activate some NK cells and B
lial cells to express many of the proteins usually cells. Th2 cells drive the proliferation and dif-
found on other tissues of the body (see Figure ferentiation of B cells into antibody-secreting
9.12). The result is that most cells leaving the plasma cells. Th17 cells promote inflammation,
thymus are tolerant of (will not react to) self and Th3 cells probably help suppress auto
antigens. This form of tolerance is called cen- immunity. Some T-regulatory cells differentiate
tral tolerance, and it results from the process of in the thymus but operate outside the thymus
negative selection in the thymus. to help suppress self-reactive T cells that leave
Although negative selection on APCs ex- the thymus.
pressing the AIRE gene eliminates most self-re-
active T cells, a subset of these cells (T-regula-
tory [Treg] cells) survives and differentiates into γ/δ T Cells
a group of cells expressing FoxP3 molecules. The γ/δ T cells make up only a small fraction
Ultimately these cells, and probably at least a of the T-cell population in mice and humans.
few other cell types, help maintain some as- In ruminants and chickens, however, the major-
pects of self-tolerance beyond the thymus. Tol- ity of T cells are γ/δ T cells. In some species,
erance maintained outside the thymus is called γ/δ T cells line the epithelia of the gut and the
peripheral tolerance. reproductive system, whereas in other species,
Despite central and peripheral tolerance, it most γ/δ T cells are at the same sites as α/β
is obvious—because of autoimmune disease— T cells. The majority of γ/δ T cells express
that some self-reactive cells do escape and, neither CD4 nor CD8 molecules. In addition,
under the right circumstance, can attack self most γ/δ T cells use very few V gene segments
and create diseases such as autoimmune hemo- during assemblies of TCRs and may, therefore,

Figure 9.12. Expression of self in the mammalian thymus and the role of the AIRE gene
Because of the AIRE gene, APCs in the thymus express and present many peptides derived from non-
thymic tissues.
have a significantly narrower diversity than that cal MHC class I molecules but are monomor-
seen in α/β T cells. However, because of fre- phic in animal populations, are not encoded
quent and substantial nucleotide deletion and by genes within the MHC, and do not seem to
addition during gene rearrangement, the CDR3 be involved in classical antigen presentation.
region of the δ chain is the most diverse of all These MHC class Ib proteins present some
the antigen-specific receptors in mammals. unprocessed antigens, such as certain phos-
The γ/δ T cells recognize unprocessed anti- phoproteins present on cell surfaces. After in-
gens, including intact pathogens, and classical teraction with their target antigens, γ/δ T cells
MHC class I and II molecules are not involved respond in the same ways as CD8+ α/β cells.
in presentation of antigens to γ/δ T cells. Only That is, both cell types secrete cytokines and in-
a few of the antigens that γ/δ T cells recognize duce apoptosis in the cells bearing their target
have been identified, and the role of these an- antigens. Because γ/δ T cells are widespread
tigens in disease processes is not always clear. in the animal kingdom, and because—in some
For example, one subset of γ/δ T cells recog- species—these T cells make up a significant
nizes a group of molecules called MHC class part of the T-cell population, γ/δ T cells must
Ib molecules. These molecules resemble classi- be important to immune-mediated protection,
157

Figure 9.13A–D. The role of DNA-dependent kinases in gene rearrangement
Cleavage of DNA hairpins (panel A); random deletion of nucleotides and activation of TDT (panel B);
junctional diversification (panel C); and DNA ligation and formation of VJ joint (panel D). Note the
essential role the DNA-dependent protein kinase plays in excision of intervening DNA and formation
of the new DJ joint. (For a complete description of these events, see Figure 9.6.)
but the exact nature of that importance is only should be able to offer a complete explanation
beginning to appear. for the clinical laboratory and necropsy findings
with SCID foals and the reason they all die so
Clinical Correlation: Follow-Up young.
SCID in Arabian foals manifests as severe re- Possible Explanations
peated infections, often respiratory infections. During genetic rearrangement of TCR V, D,
These foals invariably die from these infections and J segments, a DNA-dependent phospho
by age five months. SCID results from an auto- kinase aids in cleaving the intervening DNA
somal recessive mutation in a gene encoding a and ligating the new joint between the rear-
DNA-dependent phosphokinase. Knowing this, ranged segments (see Figure 9.13A–B). Without
and the material covered in this chapter, you that enzymatic event, rearrangement is impos-

sible, and functional TCRs cannot be assembled penia and atrophy of both thymus and lymph
(see Figure 9.13A–D). nodes.
During maturation in the thymus, thymo- The reason that some of these foals survive
cytes without functional TCRs die during posi- for as long as five months is that antibody-con-
tive selection, which means that in affected Ara- taining colostrum—transferred from mare to
bian foals, no functional T cells ever emigrate foal shortly after birth—provides temporary
from the thymus. Also (as you will see in chap- protection against infection. Because these
ter 11), the same enzymes drive the rearrange- antibodies have a limited half-life in the foals’
ment of BCRs. In the absence of functional blood, protection wanes by five months. Left
receptors, B cells die. The inability to produce defenseless, these foals rapidly succumb to
either B or T cells explains the marked lympho- infections.
159

Gerald N. Callahan
T-Cell Activation Chapter 10

10.5876_9781607322184.c010
worsened. About three weeks later, one of

Clinical Correlation: Canine X-Linked Severe
these pups developed signs of a systemic infec-
Combined Immunodeficiency 161
tion, and the owner took the dog to the hospi-
tal. The animal was small and underweight and
Antigen Recognition by T Cells 162
exhibited a large papular–pustular dermatitis,
First Signal: T-Cell Receptors and Major
Histocompatibility Complex–Antigen
and the examining veterinarian could find no
Interactions 162 peripheral lymph nodes. More antibiotics were
Second Signal and Major Histocompatibility prescribed as well as adjunctive therapy, but at
Complex–CD4/8 Interactions 164 eight weeks old, the pup died, apparently from
Signal Transduction and T-Cell Activation 166 pneumonia. At about the same time, the other
Third Signal and T-Helper Cell Differentiation 168 pup that had early symptoms of an infection
Effector T Cells 169 worsened and began to show the same symp-
Effector T-Helper Cells: Th1, Th2, Tfh, and toms as the first pup. Antibiotic therapy had no
Th17 Cells 169 effect, and a suppurative otitis developed. After
Effector Regulatory T Cells: Treg, Th3, and vaccination with a canine distemper–hepatitis
Tr1 Cells 171 live-virus vaccine, this pup developed symp-
Effector Cytotoxic T Cells 173 toms of hepatitis, became hypothermic and
Clinical Correlation Follow-Up 177 icteric, eventually slipped into a coma, and was
Student Considerations 177 euthanized.
Possible Explanations 177 Later, the same breeding pair of hounds
produced a second litter of eight pups—seven
males and one female. All appeared normal at
Clinical Correlation: Canine
birth. By age two weeks, one male pup exhib-
X-Linked Severe Combined
ited skin lesions, and two more male pups de-
Immunodeficiency
veloped similar symptoms at age three weeks.
In 1978, a local breeder brought a litter of basset All the lesions resolved after antibiotic treat-
hound pups (Figure 10.1) to the Genetics-Pedi- ment. None of these pups had palpable lymph
atrics Clinic of the University of Pennsylvania nodes or a detectable thymus gland. Between
Veterinary Teaching Hospital. The litter in- twelve and sixteen weeks, all three of the af-
cluded five males and five females. At about age fected pups developed canine distemper and
three weeks, two of the male pups developed were euthanized. Even though all were unvac-
superficial pyoderma—a bacterial infection of cinated, none of these pups’ littermates devel-
the skin common in dogs—over their hind- oped canine distemper.
quarters. The attending veterinarians began At necropsy, all the affected pups showed
antibiotic therapy, but over time the infections signs of extensive infections, including canine
161
161

Table 10.1. B and T cells in dogs with X-linked SCID (adapted from Felsberg, Somber, Harnett, Henthorn, and Carding,
1988, Immunologic Research, 17: 63)
<4 Week >8 Week
Cells Normal XLSCID Normal XLSCID
B Cells 14.3 ± 15.9a 66.3 ± 4.6 12.9 ± 2.5 49.7 ± 15.1
T Cells 58.2 ± 4.6 1.4 ± 1.9 68.1 ± 5.8 23.7 ± 21.4
CD45RA*+ T cellsb 92.5 ± 2.6 88.1 ± 4.6 90.6 ± 3.2 8.0 ± 0.6
Notes:
a Percentage ± SD.
b Percentage of T cells that are CD45RA+.
* CD45RA+ represents naive T cells.
distemper, hepatitis, and pneumonia. The thy- Learning Objectives

muses of all affected animals were severely at-
rophied and mostly acellular. (See thymocyte
cell counts from affected animals in Table 10.1.) • describe the nature of the first, second, and
Overall, T-cell counts were dramatically third signals necessary for T-cell activation;
lower in affected littermates. An interesting • explain the consequences of each of these
finding was that affected animals had higher- signals on the process and direction of
than-normal B-cell counts. On the basis of T-cell differentiation;
these and other data, it appeared that the af- • describe the role of the CDR1, CDR2, and
fected pups’ thymocytes had some sort of de- CDR3 regions of the α/β TCR in binding
fect that prevented their normal proliferation to MHC–antigen complexes;
during thymic development.
Further investigations have confirmed that
• list differences and similarities in the activa-
tion signals for activation of Th and Tc
this, in fact, is the case. Because the relevant
cells;
gene is on the X chromosome of these dogs,
and the mutation results in a broad-spectrum • describe the differences in the requirements
immunodeficiency, this disease—which has for naive T-cell and effector and memory
counterparts in other species—is known as T-cell activation;
X-linked severe combined immunodeficiency • explain the origins and functions of the
(XLSCID). major classes of effector Th cells, including
More recent studies have shown that the af- Th1, Th2, Th17, Tf h, and Treg cells;
fected gene encodes a protein known as the cy- • explain the actions of Tc cells on infected
tokine receptor common γ chain, or simply γc. target cells.
Receptors for several distinct IL receptors, in-
cluding IL-2R, IL-4R, IL-7R, IL-9R, IL-15R, and Antigen Recognition by T Cells
IL-2R, contain γc (or CD132). Furthermore, γc is
also among the family of molecules called type First Signal: T-Cell Receptors
I cytokine receptors and is expressed on most and Major Histocompatibility
lymphocytes. The gene for γc is on the X chro- Complex–Antigen Interactions
mosome of mammals. Somehow, the effects of Inside of the thymus, T cells acquire a TCR,
the XLSCID mutation in dogs result in a pro- a CD4 or CD8 molecule, and the CD3 complex
found immunodeficiency as well as dramatic (see Figure 10.2). The TCR contains an α and β
atrophy of the thymus and near to complete chain or a γ and δ chain that together form the
absence of lymph nodes. antigen-binding site. Once the TCR has bound
162 T - C e l l Ac t i v a t i o n

Figure 10.1. Basset hound (© caimacanul / Figure 10.2. T-cell antigen-specific receptor
Shutterstock)
cific antigen–MHC target. It now appears that

antigen, signal transduction occurs via the CD3 both of these populations of naive T cells can
complex, which includes γ, δ, ε, and ζ chains. only be activated by antigen–MHC complexes
Mature T cells also express either CD4 or CD8 on DC APCs (most commonly in secondary
(not shown here), molecules that are not part lymphoid tissues). Once activated, however,
of the TCR but do help determine whether a both Th and Tc cells respond to other types of
T cell will react with antigen in the context of APCs, including B cells, macrophages, and in-
MHC I or II molecules. fected self cells. Also, activated T cells, includ-
Each of these molecules has an important ing effector and memory T cells, react more
function, but only the α and β chains (or γ and rapidly during antigen presentation, require
δ chains) directly interact with both antigen and fewer antigen–MHC complexes, and require
MHC molecules in MHC–antigen complexes. less or no costimulation. For now, we focus on
Thus, the α and β chains (or γ and δ chains) activation of mature naive T cells.
determine all of the antigenic specificity of a As mentioned in chapter 9, the N-terminal
TCR. (membrane-distal) regions of the TCR α and
After differentiation in the thymus, two pop- β chains contain most of the chemical varia-
ulations of mature, naive T cells emigrate to tion seen between TCRs of differing antigenic
the thymus—CD4+ Th cells and CD8+ Tc cells. specificity. Within those N-terminal regions,
Naive T cells are those T cells that have left the the greatest variation lies in the so-called CDRs.
thymus but have not yet encountered their spe- Each TCR chain has three CDRs, designated
T - C e l l Ac t i v a t i o n 163
163

CDR1α, CDR2α, and CDR3α for the α chain
and CDR1β, CDR2β, and CDR3β for the β
chain. Together, these CDRs form the antigen–
MHC contact face of the TCR (see Figure 10.3).
Crystallographic studies of TCR and MHC–
antigen complexes revealed first that the TCR
interacts simultaneously with both the MHC
molecule and the antigenic peptide. Further-
more, during this interaction each of the CDRs
of the α and β chains plays a different role.
During binding, CDR1α, CDR2α, CDR1β, and
CDR2β bind to the α-helical portions of the
antigen-binding cleft of either MHC class I or
II molecules. Remember that positive selection
of T cells inside the thymus relies on the TCRs’
Figure 10.3. Interaction of TCR and antigen–MHC
ability to bind to MHC molecules. Different
complex
T cells have affinities for different MHC mol-
The N-terminal (membrane-distal) regions of
ecules. These affinities result from the interac-
both the α and β chains of the TCR directly
tions of CDR1α, CDR2α, CDR1β, and CDR2β interact with the antigen–MHC complex.
with either MHC class I or II molecules. From crystallographic studies, it is apparent
However, CDR3α and CDR3β bind di- that CDR1α and CDR2α as well as CDR1β and
rectly to the antigenic peptides in MHC mol- CDR2β interact most directly with the MHC
ecules’ binding clefts. As described in chapter molecule, whereas the CDR3 regions of both
9, CDR3α and CDR3β are the most variable chains interact most directly with the antigenic
portions of TCR α and β chains. An animal peptide in the MHC binding cleft.
must generate TCRs reactive with relatively
few MHC molecules. Those MHC molecules,
though, may hold a nearly infinite number of least two other interactions must occur be-
antigenic peptides. So it makes sense that the tween T-cell and APC cell-surface molecules.
most variable parts of TCRs would be those The first of these involves two molecules dis-
that bind directly to antigens. cussed in chapter 9: CD4 and CD8.
The binding of TCRs to antigen–MHC com- As T cells undergo positive selection in the
plexes is as specific as antigen–antibody inter- thymus, they switch from being double positive
actions but of lower affinity, which means that (expressing both CD4 and CD8) cells to express-
nearly one TCR exists for every possible anti- ing only CD4 (MHC class II–specific) or CD8
gen. The lower affinity means that TCRs by (MHC class I–specific) molecules. CD4 and
themselves are not enough to anchor a T cell CD8 molecules strengthen the bond between
to its target. TCRs and MHC molecules by binding to a con-
stant region (the same regardless of encoding
allele) of an MHC molecule. Overall, this bond
Second Signal and Major with the constant region of the MHC molecule
Histocompatibility Complex– increases the avidity of the TCR for the APC
CD4/8 Interactions but has no effect on the antigenic specificity
Interactions between a TCR and an antigen– of the TCR (see Figure 10.4A–B). All Th cells
MHC complex are essential but not sufficient express CD4 molecules that bind to antigen–
(by themselves) to activate a naive T cell. At MHC class II complexes. All Tc cells express

Figure 10.5. T-cell costimulation
The initial phase of T-cell activation requires
two signals; a primary signal CD3 delivers after
the TCR has bound to the MHC–antigen com-
plex on an APC and a secondary signal CD28
delivers after it has bound to B7 on the APC.
CD28 on the T-cell surface and B7 molecules

on the APC surface. Although we focus only on
T-cell activation, it is also possible for APCs to
deliver negative or inhibitory signals to T cells,
which may be important in both downregula-
tion of immune responses and self-tolerance.
Complete activation of T cells is a two-step
process. In the first step, after TCR binding of
Figure 10.4A–B. Interactions between CD4 and
antigen–MHC complex, CD3 delivers a pri-
CD8 molecules with APCs
mary signal to the T-cell nucleus. In the second
The CD4 molecule on the surface of Th cells
step, B7 (CD80 and 86) on the APC delivers a
binds MHC class II molecules and strengthens
secondary signal through CD28 on the T cell
the interaction between Th cells and DC APCs
(upper panel). The CD8 molecule on the surface
(see Figure 10.5). Only after both interactions
of Tc cells binds MHC class I molecules and occur successfully will a T cell respond.
strengthens the interaction between DC APCs Because inappropriate activation of T cells
or target cells and Tc cells (lower panel). may pose a threat to self, as with MHC class II
molecules, B7 molecules appear only on APCs.
Moreover, until pathogens, cytokines, or T cells
CD8 molecules that bind to antigen–MHC class activate them, most APCs express fairly low lev-
I complexes. els of B7.
Beyond CD4 and CD8, T-cell activation re- If a T cell receives only signal 2, nothing hap-
quires one more interaction between molecules pens. But when a T cell receives signal 1 alone,
on T cells and APCs. This interaction involves that T cell becomes anergic (unreactive) to
165

further antigenic stimulation (see Figure 10.6A–
B). This requirement further guards against T-
cell activation by non-APCs or APCs that have
not been previously activated by danger signals
(PAMPs, DAMPS, or both), because these cells
are likely presenting only self antigen.
Signal Transduction and

T-Cell Activation
The first lymphocytes activated during most
adaptive immune responses are Th cells. Once
a Th cell receives both signals from an APC, a
series of events follows.
Through a series of phosphorylations and
dephosphorylations inside the T cell, as well as
the activation of enzymes and nuclear factors
such as NFAT and NF-κB, nuclear gene expres-
sion changes. The pivotal role of these steps
is most apparent after treatment with cortico-
steroids, which inhibit the activation of NF-κB
and result in marked immunosuppression.
Under normal circumstances, though, T-cell
activation induces several Th-cell genes to cease
or begin producing proteins. One particularly
important change is induction of expression of
the high-affinity IL-2 receptor and the cytokine
IL-2 (see Figure 10.7A–D).
Naive T cells express an IL-2 receptor (IL-2R)
composed of only β and γ chains (a γ chain is
common to many IL receptors). This form of
the IL-2R has only moderate affinity for IL-2,
and the resultant interaction between receptor
and ligand is too weak to push the naive T cells
into cell division. After a specific interaction
with its cognate antigen (the antigen–MHC
complex that specifically reacts with the cell’s
TCR) and costimulation, a T cell begins to ex-
Figure 10.6A–B. T-cell
responses to only primary
press the third (or α) chain of the IL-2R. Once
or only secondary signal
the α chain joins with the β and γ chains, the
By itself, interaction of B7 and CD28 (second
complete IL-2R develops high affinity for IL-2.
signal) elicits no T-cell response (upper panel).
At the same time, the newly activated T cell Interaction of TCR with antigen–MHC com-
begins to express IL-2. The interaction of IL-2 plex (primary signal) without the second signal,
with the high-affinity IL-2R causes the T cell to however, renders the T cell unresponsive to this
begin dividing rapidly, which results in a process antigen (lower panel).
called clonal expansion (the rapid expansion of

Figure 10.7A–D. T-cell activation and the appearance of the high-affinity IL-2 receptor
After antigen presentation and costimulation (A), T cells begin to express both the α chain of the IL-2
receptor and IL-2 (B). When the completed receptor binds IL-2, a signal is transmitted to the nucleus
(C) and the activated T cells begin to proliferate and eventually differentiate into either effector or
memory T cells (D).
167

a single antigen-specific T cell into a clone of
millions of identical T cells, all specific for the
same antigen). This process is ultimately re-
sponsible for the exquisite specificity of adap-
tive immune responses.
Third Signal and T-Helper

Cell Differentiation
After activation, Th cells may follow any one
of several paths of differentiation. Understand-
ing this process is essential to understanding
animals’ immune responses and how to ma-
nipulate those responses for maximum ben-
efit—either through vaccination or immune
regulation.
Ultimately, DC APCs determine the final di-
rections of naive Th-cell differentiation through
selective cytokine secretion (third signal). Dur-
ing interactions with Th cells, APCs secrete spe-
cific cytokines that react with specific receptors
on Th cells, altering gene expression inside the
Th cells and pushing the Th cells down one dif-
ferentiation pathway or another.
When, during antigen presentation, DCs se-
crete IL-12, Th0 cells become Th1 cells. When
APCs induce Notch-receptor signaling, Th0
cells develop into Th2 cells. When APCs pro-
duce IL-6 and TGF-β, Th0 cells become Th17
cells. Some regulatory T cells (in particular,
Treg cells) develop in the thymus and become
Figure 10.8A–B. Directactivation of Tc cells by
active in the periphery, but other Treg cells de-
highly effective APCs
velop from Th0 cells outside the thymus, in-
APCs (like DCs) that constitutively express high
cluding Th3 and TR1 cells. The formation of
levels of B7 can provide both first (MHC–anti-
these cells requires DC-derived IL-10, TGF-β, or
gen) and second (B7) signals to Tc cells (panel
both. No one knows exactly what induces the A). In response, the T cells begin to express the
APC or the Th cell to produce a particular set high-affinity IL-2R and to produce IL-2 (panel
of cytokines, but it depends, at least in part, on B), which leads to activation.
the nature of the innate response that precedes
T-cell activation. Different innate responses can
alter APCs in several ways, and they direct the II) molecules. Second, APCs can activate CD8+
Th-cell response down different pathways. cells in more than one way. Highly effective
The pathways to Tc-cell activation differ APCs, in particular DCs, that express abundant
from those leading to Th-cell activation. First, MHC–antigen complexes and B7 molecules can
interactions between APCs and CD8+ T cells deliver first and second signals and directly acti-
involve antigens bound by MHC class I (not vate Tc cells (see Figure 10.8A–B).

Activation of naive CD8+ cells by other APCs
(e.g., macrophages) expressing lower amounts
of B7, however, often requires the participation
of a Th cell. Constitutively, macrophages ex-
press lower levels of B7. After antigen-specific
interactions between macrophages and Th
cells, macrophage APCs express much higher
levels of B7, allowing for effective delivery of
the second signal and activation of Tc cells (see
Figure 10.9A–B). Regardless of the activation
pathway, in the end, the activated Tc cells begin
to express the high-affinity IL-2R and IL-2, re-
sulting in rapid expansion of antigen-reactive
clones of Tc cells.
The third signal for Tc-cell activation comes
from the APC in the form of IL-12. This cyto-
kine strongly directs immune responses toward
CD8+–Tc-cell responses, also known as adaptive
cellular immune responses.
As you may have noticed, throughout this
Figure 10.9A–B. Th cell–assisted activation of Tc
description—from antigen processing and pre-
cells
sentation to final activation of T cells—none
of the processes distinguish between self and APCs expressing lower levels of B7 may first
have to interact with Th cells. That interaction
non-self. APCs are, in fact, just as effective at
induces activation of the APC and higher levels
presenting self antigens as they are at present-
of expression of B7 and MHC (panel A), which
ing non-self antigens. Clearly, two factors that increases the efficiency of the APCs, provides
help to reduce the likelihood of self-reactivity additional IL-2, and leads to activation of the Tc
are central and peripheral tolerance. But even cell (panel B).
beyond that, antigen presentation—self or non-
self—fails to activate T cells in the absence of
danger signals. Such signals arise when host mentioned earlier, effector T cells have signifi-
cells bind to PAMPs and DAMPs from patho- cantly different minimal requirements for acti-
gens and the ensuing inflammation. In the vation, including lower or no requirement for
absence of danger signals and the resulting ex- costimulation via B7.
pression of costimulatory ligands on the APCs, All Th cells begin their lives as naive Th cells
antigen presentation not only may fail to acti- (sometimes called Thn cells); after activation,
vate T cells but may even render them tolerant they become Th0 cells. These Th0 cells have
of the presented antigen. the potential to become many other types of
Th cells (see Figure 10.10).
Effector T Cells As shown in the first set of arrows in Figure
10.10, APC- and Th-cell–derived cytokines di-
Effector T-Helper Cells: Th1, rect Th0 differentiation toward one of many
Th2, Tfh, and Th17 Cells possible subsets of Th cells. Despite all the pos-
After activation, naive T cells become effec- sible outcomes of Th-cell differentiation, all Th
tor T cells capable of mediating their effects in cells continue to express CD4 and α/β TCRs
immune responses and other host defenses. As and recognize only antigen in the context of
169

Figure 10.10. Th-cell activation and differentiation
After interaction with a DC APC, Th cells may differentiate into several different cell types, including
Th1, Th2, Tf h, Th17, Treg, Th3, and Tr1 cells. The pathway of Th-cell differentiation depends on the
cytokines produced during antigen presentation (arrows). Each Th-cell type secretes a characteristic
set of cytokines (far right) and participates in a unique way in the immune response.
MHC class II molecules. But the similarities most is known about Th1, Th2, and Th17 cells.
end there. The Th1 cells participate in inflammatory re-
Most immunological studies generally recog- sponses and in the production of certain types
nize four products of Th0-cell differentiation: of antibodies, and the Th2 cells mostly direct
Th1, Th2, Th17, and Treg cells. Of these, the B-cell differentiation and antibody responses.

Th17 cells play their largest roles in inflamma- and IL-25. IL-4 and IL-5 stimulate Th2-cell di-
tory responses. Differences in the cytokines the vision, as well as B-cell activation and division,
cell types produce make this possible. and direct B cells toward the production of spe-
The largest numbers of Th1 cells develop cific antibody types, including IgGs and IgE.
after infections with intracellular bacteria and IL-4 and IL-5 also activate eosinophils (along
viruses. Activated Th1 cells produce a series of with IgE, also important in some parasitic in-
proinflammatory cytokines, including γ-IFN, fections) and mast cells (see Figure 10.12). Also,
IL-2, TNF-α, TNF-β, Fas ligand (FasL), and IL-4 and IL-13 activate an alternative set of mac-
others (see Figures 10.11A–D and 10.12). These rophages important for immune response to
cytokines affect many different aspects of pro- parasites and tissue repair.
tective responses, especially innate effector re- IL-5 stimulates production of granulocytes in
sponses. Together, Th1 cytokines (particularly the bone marrow, and IL-13 stimulates produc-
IFN-γ and IL-2) activate macrophages, increase tion of TGF-β—a cytokine that stimulates cel-
inflammation, stimulate B-cell proliferation lular proliferation and differentiation—whereas
and differentiation, and accelerate T-cell pro- IL-25 is another proinflammatory cytokine. At
liferation. The Th1-cell–activated macrophages sites of inflammation, IL-5 also serves to acti-
express higher levels of B7 and MHC class II vate eosinophils, allowing them to more effec-
molecules, more effectively destroy ingested tively degranulate and mount antihelminthic
bacteria (particularly through upregulation of responses.
oxidative radical-generating machinery), more Beyond Th1 and Th2 cells, several additional
efficiently activate additional T cells (especially types of CD4+ T cells play various roles in stim-
Tc cells), and secrete additional TNF-α—a pow- ulating or inhibiting immune responses. Other
erful stimulator of inflammation. The Th1-cell– products of Th0 cells, Th17 cells, produce sev-
activated macrophages and inflammation are eral cytokines (including IL-17, the source of the
major players in protective responses against designation Th17) that favor inflammation. The
bacteria. After secretion, IL-3 induces increased cytokine IL-17 has multiple effects on events re-
production of macrophages in the bone mar- lated to inflammation (see Figure 10.13).
row. TNF-β further contributes to inflamma- In immune reactions to several types of
tion and emigration of monocytes from the pathogens, this inflammatory enhancement
blood and, along with FasL, helps to destroy is essential for control and elimination of the
chronically infected cells and release their con- pathogen. The cytokines IL-6 and IL-23, from
tents to fresh macrophages. In addition, Th1 APCs, favor development of Th17 from Th0
cells secrete macrophage chemotactic protein cells (see Figure 10.10).
1. This cytokine brings more macrophages to
sites of inflammation. Th1 cells and their cyto-
kines also favor production of specific antibody Effector Regulatory T Cells:
types and suppress activation of Th2 cells and Treg, Th3, and Tr1 Cells
some B cells. The last generally recognized group of CD4+
In addition, Th1 cells are effective activators T cells is the regulatory T cells. Some regula-
of B cells and induce isotype switches to partic- tory T cells, specifically Treg cells, arise in the
ular Ig isotypes (especially those isotype types thymus from populations of self-reactive CD4+
that are the best opsonins; see chapter 11) and T cells that survive negative selection and emi-
suppress activation of Th2 cells. grate to the periphery (see chapter 9). Other
Activated Th2 cells participate most di- regulatory T cells appear to arise after induc-
rectly in antibody-mediated adaptive immune tion outside of the thymus; these cells may be
responses. These cells secrete IL-4, IL-5, IL-13, descendants of Th0 cells. The Treg cells play a
171

Figure 10.11A–D. Actions of
effector Th1 cells
Activated Th1 cells secrete
unique sets of cytokines.
Panel A depicts the original
activation that leads to the
production of IFN-γ, and the
other cytokines represented
in panels B, C, and D activate
macrophages, enhance the
killing of bacteria, and induce
B-cell differentiation (panel
A), kill chronically infected
cells (panel B), induce T-cell
proliferation (panel C), and
activate vascular endothelium
to enhance exit of monocytes
from blood into inflamed and
infected tissues (panel D).

major role in the suppression of autoimmunity Three additional regulatory T-cell categories
through peripheral tolerance. (Th3, Tr1, and Th9) may also be important in
To maintain self-tolerance, Treg cells inter- prevention of autoimmunity. But recent evi-
fere with the normal activation of effector cells dence has indicated that all three cell types may
or destroy them, but exactly how Treg cells do arise from one of the four major types of Th
this is unclear. It now appears that many auto- cells, depending on which cytokines are present.
immune diseases result from dysregulation of, In summary, Th cells act at many points
or imbalances among, Treg-cell populations. while innate and adaptive responses are devel-
Beyond the four generally recognized Th-cell oping. In addition, Th cells help prevent some
subtypes, other CD4+ T cells seemingly par- destructive autoimmune and inflammatory
ticipate in certain types of immune responses responses. But despite their importance in im-
or immune suppression. The relationships munity, the exact number of T-cell lineages and
of these cells to commonly recognized Th their mechanisms of action are not known. It is
cells are unclear. Among them are T-follicular clear that Th cells come in many forms and par-
helper cells (Tf h). These T cells reside in the ticipate in essential ways in all aspects of adap-
follicles inside of lymph nodes and appear to tive immune responses.
play a role in directing Th-cell differentiation.
Because much of Th-cell differentiation occurs
after antigen presentation in the lymph nodes, Effector Cytotoxic T Cells
Tf h cells probably play important roles in the In the final steps of CD8+-cell activation,
lives of Th cells. Whether Tf h cells represent APCs provide first signal in the form of anti-
a T-cell lineage distinct from Th1 and Th2 cells, gen presentation in the context of MHC class I
however, is not clear. molecules and second signal through the inter-
173

Figure 10.12. Actions of activated Th2 cells
Th2 cells secrete several cytokines, including IL-4 and IL-5. These cytokines stimulate proliferation
and differentiation, activate mast cells and eosinophils, and stimulate proliferation and differentiation
of B cells.
action of B7 and CD28. Once activated, Tc cells They can extravasate at sites of inflammation
no longer require costimulation for further and look for infected cells exhibiting the same
activation. Activated Tc cells migrate from the MHC class I–antigen complex the APCs first
lymph nodes and circulate in blood and lymph. presented to these Tc cells.

Figure 10.13. Effects of Th17-derived IL-17
During responses against bacteria, fungi, and parasites, IL-17 attracts neutrophils and macrophages
(derived from monocytes) to sites of inflammation. Once there, this cytokine stimulates mucin secre-
tion and interacts with several other proinflammatory cytokines to enhance and maintain the inflam-
matory state.
The interaction between effector Tc cells and antigen-1 (LFA-1) and I-CAM-1. Only after these
potential target cells does not begin with MHC– adhesion molecules anchor a Tc cell is there an
antigen and TCR. Instead, effector Tc cells opportunity for TCR interactions with specific
browse through potential target cells using intra- MHC–antigen complexes. If no TCR binding
cellular adhesion molecules (I-CAMs or simply occurs, Tc cells detach from this potential target
CAMs) such as leukocyte function-associated and resume their searches for infected cells.
175

Figure 10.14A–C. Effector Tc cell function
Tc cells first attach to target cells using CAM (panel A). Then TCR interacts with antigen–MHC com-
plexes, and a shift occurs in the cellular architecture. The Golgi apparatus (GA) aligns between Tc and
target cells, the microtubule organizing center (MTOC) realigns the cellular architecture, and lytic
granules (LG) appear between effector and target cells (panel B). The release of the lytic granules as
well as the interaction between FasL on the Tc cell and Fas on the target cell induce apoptosis in the
target cell (panel C).
When a specific interaction occurs between infectious contents. Macrophages then safely
the T cell’s TCR and MHC–antigen complexes clean up the remains of the apoptotic cells, and
on the target cell, though, several changes occur no inflammatory response follows.
inside of the Tc cell (see Figure 10.14A–C). The By these means, Tc cells effectively rid an ani-
Tc cell’s Golgi apparatus aligns at the point of mal’s body of infected cells and bring infectious
cellular contact, and lytic granules accumulate diseases to a healthy conclusion. In summary (see
in the Tc cell near contact points with the tar- Figure 10.15), APCs activate Tc cells in secondary
get cell. The cellular architecture shifts as the lymphoid tissues. Activated Tc cells may become
Tc cell focuses its weapons on the target cell. At effector Tc cells or memory Tc cells. Effector T
this point, the Tc cell releases both perforins and cells migrate to areas of inflammation and kill
granzymes (a series of serine proteases) and ex- infected cells. Memory Tc cells are long-lived and
presses FasL. Perforins open small pores in the are the first to respond in secondary and subse-
target cells’ surfaces. When entering through quent immune responses. After infection, T cells
those pores, the granzymes initiate apoptosis develop into a formidable array of weaponry
in the target cells. When FasL reacts with Fas (see Figure 10.16). Each of these T cells plays a
on the target cell, it also induces apoptosis, and vital role in the developing immune response as
infected cells die without releasing all of their well as in the prevention of autoimmunity.

Figure 10.15. Life cycle of a Tc cell
APCs activate Tc cells in lymphoid tissues. Activated cells differentiate into memory or effector Tc cells.
Clinical Correlation Follow-Up this and previous chapters you should know
why the absence of IL-2 receptors might result
As we said at the beginning of this chapter,
in thymic atrophy. In addition, you should be
XLSCID in basset hounds (Figure 10.17) re-
able to offer a plausible explanation for the dra-
sults in severe atrophy of the thymus and the
matically reduced numbers of T cells.
lymph nodes as well as SCID. Affected animals
have dramatically fewer T cells but a normal
to above-normal number of B cells. The muta- Possible Explanations
tion responsible for XLSCID occurs in the gene There is no simple explanation for all of the
that encodes the common γ-chain receptor—a symptoms of XLSCID in basset hounds, but
molecule that forms a requisite part of many some of the contributing factors seem obvious.
different IL receptors, including IL-2R, IL-4R, The γc chain is an essential component of re-
and IL-7R. How could alterations in the γc gene ceptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.
account for the XLSCID? IL-2 and IL-4 play essential roles in lympho-
cyte proliferation. Both inside and outside of
the thymus, T-cell division depends absolutely
Student Considerations on IL-2. Outside the thymus, naive Th cells re-
On the basis of the material presented in quire IL-2 for proliferation and differentiation.
this chapter, you should be able to propose a In addition, IL-4 stimulates proliferation and
reason why the absence of IL-2 and IL-4 recep- differentiation of Th2 cells and B cells.
tors would result in severely atrophied lymph A complete lack of IL-2R and IL-4R would
nodes. Also, on the basis of information from result in no lymphocyte proliferation in either
177

Figure 10.16. Effector T cells
After activation by MHC–antigen complexes on APCs, T cells differentiate into a variety of effector
cells that drive the progression of a variety of types of immune responses.
the thymus or the lymph nodes and a complete with the limited number of T cells, explains the
absence of mature B cells. These factors would absence of mature B cells.
result in severe atrophy of both primary and Beyond IL-2 and IL-4, another factor is at
secondary lymphoid organs, as seen in these play here: IL-7. This cytokine plays a major
dogs. However, these factors would not explain role in the development of lymphoid progeni-
the continued presence of roughly 30 percent tor cells in the bone marrow. When pluripotent
of normal T-cell numbers by age eight weeks lymphoid stem cells lack receptors for IL-7, few
old. or no lymphocytes develop in the bone mar-
In addition, there is the problem of the row. As a consequence, none go to the thymus,
above-normal B-cell numbers in dogs with and none leave the thymus to populate the
XLSCID. Interestingly, all of the B cells in dogs secondary lymphoid tissues. This, too, could
with XLSCID are immature B cells (they only explain the absence of palpable lymph nodes
express IgM). Maturation of B cells (see chapter and the thymic atrophy in dogs with XLSCID.
11) requires interaction with Th cells and cyto- However, a complete lack of receptors for
kines produced by Th cells, such as IL-4. The IL-7 seems inconsistent with the numbers of
absence of functional receptors for IL-4, along lymphocytes remaining in basset hounds with

Figure 10.17. Tan and white basset hound (© Ewa Studio / Shutterstock)
XLSCID—especially B cells. A complete expla- molecules; reduced affinity of these molecules;

nation for this disorder will probably involve or both, but it seems likely that other factors
reduced numbers of IL-2R, IL-4R, and IL-7R contribute as well.
179

Gerald N. Callahan
B-Cell Development Chapter 11

10.5876_9781607322184.c011
tology testing of these animals shows normal

Clinical Correlation: Equine
numbers of lymphocytes, but no B cells, no
Agammaglobulinemia 181
IgM or IgA antibodies, very low concentrations
of IgG and Ig(T) (an isotype peculiar to horses)
B Cells and the Structure of the B-Cell
Receptor 182
antibodies, and normal T-cell responses in vitro
B-Cell Genetics, Development, and
and in vivo. Because shortly after birth most
Circulation 183 foals receive large amounts of Ig in their dams’
B-Cell Receptor Gene Families, Genetic colostrum, the immunodeficiencies of the foals
Rearrangement, and Junctional Diversity 183 become apparent only as maternal Ig disap-
Order and Regulation of B-Cell Development 186 pears from their blood. At this point, these foals
Immunoglobulin Isotypes 189 often succumb to persistent bacterial infections
Other Populations of B Cells 191 and die.
B-1 B Cells 191 Because of the similarities of this disease to
Marginal-Zone B Cells 194 X-linked agammaglobulinemia in humans, the
Clinical Correlation Follow-Up 195 underlying cause is likely a defect in a molecule
Student Considerations 196 called Bruton’s tyrosine kinase (BTK), an en-
Possible Explanations 196 zyme essentially involved in the function of the
BCR and other surface receptors. These foals’
illness is apparent, but what is less apparent is
Clinical Correlation: Equine how a protein-kinase defect could lead to a se-
Agammaglobulinemia vere immunodeficiency.
Antibody deficiencies, which may be selective
or complete, are among the most frequently Learning Objectives
diagnosed immune deficiencies in mammals.
One of these, equine X-linked agammaglobu- After reading this chapter, you should be able to
linemia, provides a useful insight into the na- • describe the structure of the BCR;
ture of B-cell development in horses and prob-
ably all other mammals. Among thoroughbred,
• explain the genetic mechanisms responsible
for the essentially unlimited diversity of
standard bred, and quarter horses, some af-
BCRs, including genetic rearrangement and
flicted males begin to develop severe bacterial
junctional diversification;
infections at about two to six months of age.
These infections include pneumonia, enteritis, • describe the mechanisms and the order of
B-cell development, including B-cell selection;
and dermatitis. Antibiotics and, interestingly,
plasma transfusions provide temporary relief • explain the differences between Ig isotypes
from symptoms and further infections. Hema- and their importance in defense;
181
181

B cells are the ultimate source of antibod-
ies, the end products of adaptive humoral im-
mune responses—humoral because physicians,
such as Galen in the second century BC, once
believed that all maladies arose as a result of
an imbalance in the humors (black bile, yel-
low bile, phlegm, and blood) of the circulating
fluids. Because of that, factors that appeared
in blood were called humoral factors. Because
scientists first discovered antibodies in serum,
this type of response came to be known as the
humoral immune response. B cells come in two
forms, B-1 and B-2. In mammals, B-2 B cells are
the most numerous and produce the largest va-
riety of antibodies.
Like TCRs, antibodies come in almost limit-
less forms, each with a unique antigenic speci-
ficity, but BCRs contain four polypeptide chains
and react with native antigen—that is, with
unprocessed antigens and no involvement with
MHC molecules (Figure 11.2)
The most common form of mammalian
Figure 11.1. Americanquarter horse (© BCRs contains four Ig chains—two H chains
Lenkadan / Shutterstock) and two L chains—all held together by disul-
fide bonds. This part of the BCR is essentially
an antibody molecule with an added mem-
• describe the nature of B-1 and marginal- brane anchor. Each of the four chains contains
zone (MZ) B cells and discuss the relevance a C-terminal constant region and an N-terminal
of these cells to defense against infections. variable region. Within these variable regions
of both H and L chains (just as with TCR α and
β chains) are three regions of hypervariability
B Cells and the Structure
called CDR1, CDR2, and CDR3. Together these
of the B-Cell Receptor
regions of one H chain and one L chain form an
In mammals, B cells—like all lymphocytes— antigen-binding site—two per BCR.
originate in the bone marrow. Unlike T cells, Unlike TCRs, antibodies exist in two forms,
though, all of the maturation of naive mam- as the cell membrane–bound BCR and as se-
malian B cells occurs in the bone marrow; no creted antibodies (see the Order and Regulation
other organ contributes to the first phases of of B-Cell Development section) that circulate
B-cell development. In birds, however, B-cell in blood, extravascular fluids, and lymph. These
development (along with some hematopoiesis) secreted forms were discovered first and named
occurs in the bursa of Fabricius, an organ on immunoglobulins or antibodies.
the dorsal wall of the cloaca. In the teleost fish, In addition, BCRs associate with two other
the kidneys are the major sites of B-cell devel- membrane-bound proteins—Igα and Igβ. To-
opment, and in amphibians B cells develop at gether, these extra two molecules function in
several sites, including spleen, bone marrow, signal transduction much as the CD3 complex
and kidney. on T cells. Also like CD3, Igα and Igβ have
182 B-Cell Development

Unlike TCRs, during its lifetime a B cell may
express several different forms of the BCR, all
with the same antigenic specificity. Different
forms of BCRs and secreted antibodies are
called Ig isotypes (see Immunoglobulin Iso-
types section). The differences between iso-
types are all found in the constant regions of
the H chains. The first BCR expressed by devel-
oping mammalian B cells is always IgM.
B-Cell Genetics, Development,

and Circulation
B-Cell Receptor Gene Families,
Genetic Rearrangement,
and Junctional Diversity
Once Igs were discovered, it was soon obvi-
ous that the chemical diversity among Igs out-
stripped that of any other animal protein. But
only after many years of study was the genetic
basis of this structural diversity unraveled.
These discoveries actually predated understand-
ing of the genetics of TCRs by a few decades.
Figure 11.2. The common mammalian BCR
The essentially unlimited diversity among
The BCR contains four polypeptide chains—
the common mammalian BCRs within one ani-
two identical H chains and two identical L
chains. The L chains are bound to the H chains
mal arises much as does the diversity of mam-
by disulfide bonds, as the H chains are bound malian TCRs. Inside the genome of mammals,
to one another. The C-terminal (membrane- sequences that encode BCR H and L chains ap-
proximal) portions of the H chains span the pear in segments or families of genes. H chains
cell membrane and are identical for each Ig have one gene family, and L chains have one or
isotype, and the N-terminal (membrane-distal) more. As with the TCR gene families, one BCR
portions of the H chains are highly variable. gene family (H chain) contains variable (V), di-
Similarly, the C-terminal regions of the L chains versity (D), and joining ( J) segments, and the
are constant within a given type of L chains, other gene family (L chain) contains only V and
and the N-terminal portions are highly variable. J segments (Figure 11.3). Camelids (camels and
Together, the N-termini of the H and L chains
llamas) are the one known exception to this. Ca-
make up the two membrane-distal antigen-
melid antibodies (not shown) have no L chains.
binding sites. Igα and Igβ molecules associate
with the antigen-binding portion of the recep-
The H chains by themselves form the antigen-
tor and are involved with signal transduction. binding sites of these antibodies. The Ig gene
Ag = antigen. families of camels and llamas, therefore, do not
include L-chain gene segments.
In mammals, bony fish, and amphibians, BCR
nothing to do with the antigenic specificity of gene rearrangement begins when the bone
the BCR. That specificity resides completely marrow stroma signals a developing B cell to
within the N-termini of the H and L chains. start rearranging H-chain genes, which results
B-Cell Development 183

183

Figure 11.3. BCR gene families
The BCR H-chain gene family resides on a single chromosome and contains multiple variable (VH),
diversity (DH), and joining ( JH) segments. The number of these segments and their arrangements
varies between species. The lowercase n indicates varying numbers. The Greek letter ψ indicates
pseudogenes (incomplete genes), and the numbers under brackets indicate the total number of germ-
line V, D, J, and C complexes. IgW and Ig new antigen receptor (IGNAR) are Ig isotypes unique to
cartilaginous fish. BCR L-chain gene families are on separate chromosomes and contain only V, J, and
C segments.
in looping out of intervening DNA and juxta- chain, L-chain rearrangement begins when a
position of randomly selected DH and JH seg- randomly selected VL gene segment associates
ments. Just as with TCR gene rearrangements, with a randomly selected JL segment. Then, fol-
enzymes excise the looped-out DNA and, as the lowing the processes of excision of intervening
DH and JH segments join, additional diversity DNA and junctional diversification, an L-chain
arises from the process of junctional diversifica- variable gene complex appears. Enzymes then
tion—the random addition and subtraction of transcribe these L-chain and H-chain genes,
nucleotides as the DH–JH joint forms (see Figure along with their C segments, into nuclear, or
9.6). Next, this DJ segment, through a similar, primary, RNA. Then spliceosomes convert
random process, joins with a VH segment to cre- these transcripts into messenger RNA (mRNA),
ate an H-chain variable-region gene complex which are translated into proteins that join to
(Figure 11.4). form a BCR (Figure 11.4).
After a successful H-chain gene family rear- A final L chain requires one VL, one JL, and one
rangement and production of a functional H CL segment. The gene rearrangement process

Figure 11.4. Assembly of L chains and H chains

185

first randomly couples one VL and one JL. The result of somatic hypermutation driven by for-
newly formed VJ segment, along with interven- eign antigen. But regardless of the underlying
ing DNA and a CL segment, are transcribed into mechanisms, the end result is enormous diver-
a nuclear RNA transcript. Spliceosomes con- sity among BCRs and the ability to react with a
vert this nuclear transcript in mRNA, and ribo- nearly unlimited variety of antigens.
somes create the final protein. Similarly, a sin-
gle H-chain gene begins when rearrangement
randomly couples a single DH segment with a Order and Regulation of
JH segment. This complex then randomly rear- B-Cell Development
ranges to join a VH segment. The newly created Just as with T cells, the development of BCRs
VDJ complex, along with intervening DNA and is a complex and random process with an equal
a CH segment, produce a nuclear RNA tran- or greater potential for generating useless or
script that is converted to mRNA and translated self-reactive molecules over protective BCRs.
into an H chain. Two identical H and two iden- So the question remains, how is all of this regu-
tical L chains join to form a BCR. lated and controlled during B-cell development?
The final products are mammalian BCRs, In most mammals, bony fish, and amphib-
each with a unique antigen-binding specific- ians, B-cell development begins in the fetal liver,
ity. The nearly unlimited diversity and anti- spleen, and bone marrow. In chickens, rabbits,
genic specificity of these BCRs is the result of and sheep, it begins in the GALT. However, the
random gene rearrangement and junctional steps in the developmental processes are similar.
diversification. First, the bone marrow or other stroma deliv-
Other species use very different mechanisms ers a signal that initiates genetic rearrangement
to generate BCR diversity. For example, in chick- in the pre–B cells. These cells begin rearrang-
ens, BCR diversity arises from an entirely differing H-chain genes, which starts (as mentioned
ent process called gene conversion (Figure 11.5). earlier and shown in Figure 11.4) with random
Chickens have only one VH, DH, and Cm (con- association of DH and a JH segment. This DJ
stant region for the H chain) gene segment. In- complex then randomly associates with a VH
side the bursa of Fabricius, genetic rearrange- segment, enzymes transcribe the VDJ complex
ment results in the appearance of the same along with a CH segment—in most cases, a Cυ
BCR on all B cells. In addition to the functional segment—into nuclear RNA, spliceosomes re-
gene segments in chicken Ig gene families, how- form this transcript into mRNA, and ribosomes
ever, are multiple pseudogenes—inactive partial make H chains from the message.
genes. During further development in chicken Pre–B cells that fail to successfully rearrange
B cells, additional rearrangement randomly H-chain genes and express a functional H chain
inserts pieces of these pseudogenes into the V are of no further use, so animals have evolved a
segments of both H- and L-chain genes, which means to test for a functional H-chain polypep-
dramatically increases the variety of BCRs and tide. Just as with T cells, B cells have evolved
the capacity for antigen recognition in chickens. a special gene to deal with this, a gene called
Cartilaginous fish have their H and L genes Vλ5 that produces a surrogate L chain. If the
already connected in the germline genetic con- newly formed H chain is functional, it will bind
figuration (Figure 11.3) and use still another to Vλ5, and the complex will move to the cell
means for diversification. Although structurally surface where stromal receptors test for the
unique (no L chains), camelid Ig genes diversify pre-BCR (pBCR). On one hand, if the receptor
via genetic recombination just as do Ig genes does not appear, then further rearrangement
in most other mammals. On the other hand, in on that particular chromosome is shut down,
sheep and cattle, antibody diversity arises as a and H-chain gene rearrangement begins on the

Figure 11.5. Diversification of chicken BCRs
Initially, all chicken B cells express the same BCR—the product of only one VH, one DH, and one JH as
well as one VL and one JL segment. After these cells enter the bursa of Fabricius, they begin to prolif-
erate, and during cell division, pieces of adjacent pseudogenes (normally inactive genes) are inserted
into the active H-chain and L-chain genes, adding to the diversity of expressed BCRs.

187

Figure 11.6. B-cell development
Gene rearrangement begins as B cells divide in the bone marrow. Cells that fail to produce a func-
tional H chain along with a surrogate L chain die. B cells that produce self-reactive BCRs undergo
further rearrangement, called receptor editing. If, after a second gene rearrangement, these B cells
still react with self, these cells die.
other chromosome of the parental pair. At the pears, rearrangement of L-chain genes begins
end of that process, the stroma again tests the on the other parental chromosome. If again no
pre–B cell for the appearance of a pBCR. If at BCR appears, the cell dies. But if a functional
this point the cell expresses no pBCR, the cell BCR does appear, the cell is immediately tested
dies (see Figure 11.6). for self-reactivity. Once identified, self-reactive
On the other hand, if the receptor appears, B cells are stimulated to undergo further rear-
the stroma stimulates the initiation of L-chain rangement, a process called receptor editing. If
gene rearrangement. VL and JL segments ran- these B cells either fail further productive rear-
domly recombine, and a nuclear transcript of rangement or again produce self-reactive BCRs,
this VJ segment, along with a CL segment, ap- these cells also die.
pears. Spliceosomes convert this transcript into Beyond this level of selection, one other pro-
mRNA, and ribosomes begin to make L chains. cess helps to ensure that self-reactive B cells do
If this rearrangement is successful and a func- not attack self outside of the bone marrow. For
tional L chain appears, it associates with the H a B cell to become activated, more than one
chain and moves to the cell surface as a fully BCR must bind to an antigen at the same time,
formed antigen receptor (BCR). The stroma and these BCRs must become cross-linked to
monitors this process as well. If no BCR ap- one another to generate the appropriate intra-

cellular signals. That process requires particu- ten antigen-binding sites (Figure 11.9). Secreted
late antigen with a repetitive antigenic epitope, IgA antibodies may appear as monomers or as
so that two or more BCRs can bind to the same dimers (Figure 11.8). The dimeric form of IgA
antigen at the same time and induce cross-link- appears predominantly on mucosal epithelia,
ing. So, all B cells that react with soluble pro- so it is sometimes also called secretory IgA.
teins (which lack repetitive antigenic epitopes), The feature that distinguishes one Ig isotype
such as serum albumin, become anergic, or un- from another is the amino acid sequence of
reactive to any further stimulation. the constant region of the H chain. Because of
As a result, in a process somewhat similar to that, different Ig isotypes expressed by single B
T-cell selection, most self-reactive B cells die or cells will all have the same antigenic specificity.
are switched off before they can do any dam- How is it possible for one B cell to produce
age. B cells with functional BCRs are called several different isotypes with the same antigenic
naive or immature B cells—naive until they en- specificity? The answer is through a combination
counter their particular antigens. These B cells of selective RNA splicing and genetic rearrange-
exit the bone marrow or bursa of Fabricius and ment. In addition to V, D, and J segments, every
enter the blood. B-cell differentiation does not, H-chain gene family has multiple C segments—
however, end in the bone marrow. For all B Cm, Cδ, Cγ (one or more), Cε, and Cα (one or
cells, many steps remain on the way to becom- more)—corresponding to the H chains for IgM,
ing antibody-secreting plasma cells. After enter- IgD, IgG, IgE, and IgA (Figure 11.9).
ing the blood, most B cells will eventually make Two distinct genetic mechanisms result in
their way into secondary lymphoid tissues, and expression of distinct Ig isotypes by developing
it is here, especially within the lymphoid fol- B cells. All B cells first express IgM, which re-
licles, that much of the remainder of B-cell de- sults from a simple read-through from the VDJ
velopment occurs (see chapter 12). complex to the first two C gene segments, Cm
and Cδ. During production of mRNA, splice
osomes remove the RNA encoding the IgD H-
Immunoglobulin Isotypes chain C region. Thus, the newly developed B
As mentioned earlier, antibodies come in cells express only IgM.
two physical forms—a membrane-bound form At a later stage in their development, most
as part of the BCR and a soluble form known as B cells briefly express both IgD and IgM. This
antibodies. In addition, both membrane-bound expression results from an alternative mRNA
forms and soluble forms of antibodies come splice (one splice removes Cm and the other re-
in chemically distinct forms known as isotypes moves Cδ), resulting in the simultaneous pro-
(Figure 11.7). duction of both isotypes and both types of
The numbers and characters of Ig isotypes BCRs. As you will see in chapter 12, at a still
vary markedly between species. Placental mam- later stage in B-cell development, B cells will
mals express IgM, IgD, IgG, IgA, and IgE. Also, often switch from expressing IgM and IgD to
most species that express IgG express multiple expressing another isotype—IgG, IgE, or IgA.
forms, or subtypes, of IgG. The same is often This process is called isotype switching. It is im-
true for IgA subtypes. In addition, both IgM portant to note that isotype switching to IgG,
and IgA appear in multiple structurally distinct IgE, and IgA occurs only after B-cell activation
forms (Figure 11.8). As BCRs, both IgM and IgA and expansion, which occurs only if the BCR
appear in their monomeric forms (shown in recognizes an antigen and, usually, receives fur-
Figure 11.7). When secreted, however, IgM ap- ther signals from Th cells. Exactly how a cell
pears as pentamer with ten H chains and ten chooses one isotype over another is not clear,
L chains creating five antibody monomers and but Th cells and their products appear to direct

189

Figure 11.7. Ig isotypes
Variations in the constant regions of Ig H chains result in multiple forms of antibodies called Ig iso-
types. The number of isotypes, their form, and their designations vary among animal species.
the process. The process of Ig–isotype switch Occasionally, B cells will make more than
beyond IgM and IgD results not from alterna- one class switch, but these secondary and later
tive RNA splicing, but from further genetic switches must involve downstream constant re-
rearrangement. For example, inside a cell des- gion genes because all of those upstream of the
tined to express IgG, the DNA that encodes Cm first switch were excised.
and Cδ loops out, and enzymes excise it. From The advantage of multiple Ig isotypes is
this DNA, these B cells then begin to make IgG. that each antibody isotype appears at varying
Because this rearrangement results in changes concentration and distributes differently inside
to the H-chain constant region only, no change animals’ bodies, and each can perform distinct
occurs in the antigenic specificity of the BCR. functions after binding to antigen (Figure 11.10
The same holds for switches to IgA or IgE. and 11.11).

terial and other infections (see chapter 4). Only
IgE binds to mast cells, a critical aspect of aller-
gies and defense against parasites. In addition,
each isotype has a unique distribution inside an
animal’s body (Figure 11.11).
Other Populations of B Cells

B-1 B Cells
In all mammals so far investigated, there
exists a distinct population of B cells called
B-1 B cells. The origins and function of these
cells are distinct from the B cells discussed so
far (sometimes called B-2 B cells or follicular B
cells because they are most commonly found
in lymphoid follicles). These novel B-1 B cells
arise early during fetal development (which is
the reason for the B-1 designation) in fetal liver,
whereas B-2 B cells arise later, mostly in fetal
bone marrow.
Often, but not always, B-1 B cells also express
a novel cell-surface molecule called CD5, so B-1
B cells are also called CD5+ B cells. The percent-
age of the total B-cell population accounted for
by B-1 B cells varies among species, from about
5 percent in primates and rodents to the major-
Figure 11.8. Polymeric forms of IgM and IgA
ity of B cells in rabbits and ruminants.
As a soluble antibody, IgM always appears as B-1 B-cell Ig gene segments do rearrange dur-
a pentamer with ten H and ten L chains and
ing development, but because these cells lack
ten antigen-binding sites. This process may or
TDT, there is no junctional diversification. In
may not require the participation of another
polypeptide called the J chain (unrelated to addition, B-1 B cells use only a few DJ and VDJ
JH segments). Also, as a soluble antibody, IgA combinations. Together, these two factors re-
appears in two forms, monomeric and dimeric, sult in a much lower BCR diversity among B-1
also with a coupling J chain. B cells than among regular, or B-2, B cells. Also,
after maturation in fetal liver, most B-1 B cells
migrate to the peritoneal and pleural cavities,
Each Ig isotype appears at different concen- not to lymphoid follicles. In the peritoneal and
trations in the serum, with IgG at the highest pleural cavities, it appears that further develop-
concentrations. The exact plasma concentra- ment of B-1 B cells is driven by encounter with
tions of each isotype vary among species. For a narrow range of specific antigens. The exact
example, numbers for horse Ig isotypes are ap- nature of these antigens is not always clear, but
proximately 1.96 mg/ml for IgA, 27 mg/ml for the most common appear to be bacterial poly-
IgG, 4.19 mg/ml for Ig(T), and 0.7 mg/ml for saccharides and lipids, as well as some auto
IgM. antigens (self molecules).
Only IgM and IgG activate complement, an After activation, B-1 B cells establish self-
essential element in innate defense against bac- renewing populations of active B cells, and

191

Figure 11.9. Ig H-chain constant genes
H-chain gene families contain V, D, and J gene segments that encode the variable region of the H
chain. In addition, each H-chain gene family contains multiple C-region gene segments corresponding
to multiple Ig isotypes.
Figure 11.10. Properties of primate Ig isotypes

Each mammalian Ig isotype has slightly different properties. These structural differences allow for
differences in distribution and function. The data shown in the upper half of this chart were derived
from humans and vary among species. IgG crosses the placenta in significant amounts only in mam-
mals with hemochorial (e.g., primates and rodents) or endotheliochorial placentation (e.g., dogs and
cats).
they constitute the majority, if not all, of the mals generate B-1 B cells in fetal liver. These B
B-1 B cells present in adult animals. That is, cells then migrate to the peritoneal and pleu-
early during fetal development, most mam- ral cavities, where they establish self-renewing

Figure 11.11. Distribution of Ig isotypes in animals’ bodies
Both IgG and IgM appear in the blood, but extracellular fluid contains mostly IgG and monomeric
IgA. Dimeric or secretory IgA is the major isotype on mucosal secretions of epithelia and appears
in breast milk. IgE is mostly found bound to mast cells, and mast cells appear in greatest numbers
beneath the epithelial surfaces of the respiratory tract, the gut, and the skin. The blood–brain barrier
normally partitions Ig from the brain.
populations of B-1 B cells that persist through- that some B-1 B cells can, even in the absence
out adult life; these are the only B-1 B cells in of antigen, differentiate into antibody-secreting
adults. plasma cells and are responsible for so-called
B-1 B cells do not require interactions with natural antibodies that appear even in germ-
Th cells to become fully active and differenti- free animals. Most B-1 B cells express only IgM
ate into antibody-producing plasma cells (as do and never switch to another isotype, nor do
B-2 B cells; see chapter 12). So the antigens that they further diversify their BCRs in spleen or
activate B-1 B cells are called thymus-indepen- lymph nodes through the process of somatic
dent antigens (see Figure 11.12). It also appears hypermutation (as do B-2 B cells; chapter 12).

193

Figure 11.12. B-1 B cells and thymus-independent antigens
In comparison with traditional or B-2 B cells, B-1 B cells and MZ B cells recognize distinct sets of
antigens called thymus-independent antigens because no T cells are involved in activation of B cells in
response to these antigens.
From all of this, it appears that B-1 B cells have sinuses and returning to the circulation. Obvi-
evolved to recognize and respond to commonly ously, then, the MZ B cells are well positioned
encountered antigens, such as the components to deal with antigens circulating in the blood
of bacteria. Because bacteria are among the and, as it turns out, especially well suited to
most numerous of all pathogens, it seems rea- deal with circulating encapsulated bacteria.
sonable that a rapid response element—such MZ B cells have a greater BCR diversity than
as B-1 B cells—would provide a considerable B-1 B cells, but not as great as that of B-2 B cells.
evolutionary advantage to animals that at birth MZ B cells produce mostly IgM, are longer-
must unavoidably and immediately deal with a lived than B-2 B cells, do not require T-cell help,
host of bacterial pathogens. The role of B-1 B and appear to have evolved to most effectively
cells in autoimmunity is also evident, but the deal with circulating pathogens, especially bac-
mechanisms and evolutionary significance of teria (see Figure 11.12). From a defense perspec-
this aspect of B-1 B cell function are much less tive, this is another relatively rapid response
clear. mechanism targeted toward bacteria, particu-
larly bacteria in the blood.
To summarize, three distinct populations of
Marginal-Zone B Cells B cells have evolved in most mammals to deal
Inside the marginal sinus of the white pulp with particular infectious threats. B-1 and MZ
of the spleen is yet another population of B B cells have relatively limited antigenic reper-
cells, the MZ B cells. These cells are physically toires and appear to have arisen to protect ani-
and functionally distinct from either B-1 or B-2 mals from commonly encountered pathogens,
B cells. The marginal sinus is the site where especially bacteria. B-2 B cells, however, have
blood exits the circulation, then traverses a re- an almost limitless capacity for BCR diversity
gion called the MZ before entering the venous and take somewhat longer to respond. But B-2

Figure 11.13. Thoroughbred horse (© pirita / Shutterstock)
B cells can do something that B-1 B and MZ B ment. The transduction of that signal depends
cells cannot, and that is, deal effectively with on active functional BTK.
constantly evolving pathogens of all sorts. As mentioned, the bone marrow stroma ini-
tiates and directs rearrangement of BCR genes.
Tyrosine kinases are important components of Student Considerations
many signal transduction pathways, and BTK On the basis of the material presented in this
happens to play an especially important role in chapter, you should be able to offer an explana-
signal transduction in B cells (see Figure 11.14). tion for the observation that horses with defec-
All B cells arise from a common hemato- tive BTK enzymes might exhibit a pronounced
poietic stem cell. Early on, the B-cell lineage agammaglobulinemia, have severely atrophied
diverges from the T- and NK-cell lineages. The lymph nodes, and, by age six months, uniformly
first recognizable B-cell precursor is the pre–B succumb to bacterial infections.
cell. Gene rearrangement begins inside of this
cell. The first outward sign of successful gene
rearrangement is the appearance of the new H Possible Explanations
chain along with Vλ5 in the form of the pBCR— As mentioned earlier, B cells (even B-1 B cells)
the pre–B-cell. For further rearrangement to that express the pBCR must receive and deliver
occur, the bone marrow stroma must deliver a another message before L-chain gene rearrange-
signal through the pBCR to the nucleus of the ment can begin. That signal depends on BTK. In
pre–B-cell to initiate L-chain gene rearrange- the absence of a second rearrangement signal,

195

Figure 11.14. The role of BTK in equine X-linked agammaglobulinemia (EXLA)
To ensure that each step proceeds normally, B cells have evolved means for signaling the stroma at
each major step of development. Rearrangement and production of H-chain genes occurs first, but H
chains by themselves cannot assume proper configurations and insert themselves into the cell mem-
brane. Because the cell membrane is the only surface accessible to the bone marrow stroma, B cells
have evolved a means to express isolated H chains using a surrogate L chain called Vλ5. Together, H
chain and Vλ5 form the pBCR at the surface of developing B cells. Normally, the pBCR interacts with
stromal receptors and, in return, the stroma delivers a signal through the BCR to pre–B cells’ nuclei.
The delivery of this signal depends on a complex set of second-messenger molecules. One of these
is BTK. In the absence of BTK, further Ig gene rearrangement ceases. These B cells never produce L
chains and eventually die via apoptosis.
B cells fail to initiate L-chain gene rearrange- no B cells and no antibodies. Without B cells,
ment. As a result, these cells never express a lymph nodes atrophy, and no lymphoid follicles
functional BCR that will deliver a second signal or germinal centers appear. As a result, as soon
to the bone marrow stroma and allow the pre–B as the maternally transferred antibodies disap-
cells to survive a second round of selection. As a pear from the foal’s blood, massive infections
result, animals without functional BTK produce ensue and the animal dies.

Gerald N. Callahan
B-Cell Activation and Differentiation Chapter 12

10.5876_9781607322184.c012
normal levels of IgG and IgM but depressed lev-

Clinical Correlation: Selective Immuno-
els of IgA. The degree of the suppression varies
globulin A Deficiencies in Dogs 197
from no IgA to a less-than-normal level of IgA.
T-Dependent B (B-2) Cells 199
Interaction of B Cells with Antigens 199 Learning Objectives
B Cells as Antigen-Presenting Cells 203
Activation of B Cells by T-Helper Cells 204
Isotype Switching, Somatic Hypermutation, • describe the manner in which B cells interact
and Terminal Differentiation of B Cells into with antigens and pathogens;
Plasma Cells 206
Antibody Effector Functions 213
• understand the role of B cells in antigen
presentation to Th cells;
Pathogen and Toxin Neutralization 213
Opsonization and Activation of Complement 213
• describe the signals necessary for B-cell
activation and their consequences;
Distribution and Function of Fc Receptors 214
Clinical Correlation Follow-Up 215 • describe the driving forces for Ig isotype
Student Considerations 215 switch and the roles of the various Ig
Possible Explanations 217 isotypes;
• understand the mechanisms, consequences,
and importance of somatic hypermutation;
Selective Immunoglobulin
• explain the final differentiation of B cells
into plasma cells and the secretion of
A Deficiencies in Dogs
antibodies;
As many as 80 percent of Chinese shar peis • explain the process of affinity maturation;
(see Figure 12.1) develop recurrent dermatitises • describe the differences between primary
(staphylococcal), demodectic mange (see Fig- and secondary humoral immune response
ure 12.2), thyroid disease, otitis externa, flea and the reasons for these differences;
allergies, cystitis, food intolerance, bronchi- • explain the effector actions of antibodies and
tis, and atopy. Similar disorders also appear in their importance in defense;
(among others) German shepherds, beagles,
Irish wolf hounds, and cocker spaniels.
• explain the various roles of Fc receptors in
immune responses and protection.
Most of the symptoms apparent in Chinese
shar peis share two features: they involve body Two of the most striking features of the im-
surfaces and appear to result from some sort of mune system and immune responses are anti-
inherited immune deficiency. Clinical labora- gen specificity and immunologic memory (see
tory analyses of serum from affected dogs reveal Figure 12.3).
197
197

Figure 12.1. Chinese shar pei dog (© Waldemar Dabrowski / Shutterstock)
After a primary immunization of an animal, levels of antibodies persist for very long peri-
no detectable change occurs in blood levels of ods of time—often years or even decades. The
antibodies for the first seven to ten days. This immune response to antigen B, injected at the
phase is called the lag phase, and it occurs be- same time as antigen A, exhibits features char-
cause it takes time to develop an adaptive im- acteristic of a primary immune response.
mune response. After the lag phase, IgM anti- Clearly, at day 28 some aspect of the im-
bodies begin to appear in the animal’s serum. mune system has changed, and it now carries
The amount of antigen-specific serum IgM a memory of the previously seen antigen A.
continues to rise for about another seven days Because of this memory, the animal’s immune
and then gradually falls off over the next two system now does something very different: it
weeks to near 0 by day 28. After a secondary produces more antibody, it produces a differ-
immunization with antigen A and antigen B, ent type of antibody, it produces it faster, and
the lag phase drops to about three days for an- the antibody persists in the serum for a much
tibodies to antigen A, followed by a dramatic longer time. Also, the antibodies produced dur-
rise of antibodies to a much higher level than ing secondary immune responses have a much
seen in the primary response. In most immune higher affinity for antigen and that affinity con-
responses, nearly all of these antibodies are tinues to increase as the secondary response
IgG (although they could also include IgE and continues. This progressive increase in affinity
IgA). Also, after secondary immunization, high is known as affinity maturation, and it occurs in
198 B - C e l l Ac t i v a t i o n a nd D i f f e r e n t i a t i o n

Figure 12.2. Dog with severe demodectic mange (© Robert Adrian Hillman / Shutterstock)
Demodectic mange is also known as demodicosis or red mange. A persistent infestation with Demodex
canis, a mite, causes this mange. It comes in two varieties: localized and generalized. Localized mani-
fests at four or fewer spots of mange. Most normal dogs are not susceptible to demodectic mange.
most immune responses and generates highly quence variability among BCRs lies within the
antigen-specific antibody. Memory and speci- CDRs of the H and L chains (Figure 12.5), the
ficity are the hallmarks of adaptive immunity part of the antibody molecule in direct contact
and result from the characteristics of T and B with antigen.
lymphocytes. As the H and L chains fold into their final
conformations, CDR1, CDR2, and CDR3 of
T-Dependent B (B-2) Cells both chains come to reside in the antigen-bind-
ing site—that is, the portion of the BCR that
Interaction of B Cells with Antigens interacts most directly with antigen.
The BCR contains not only two H and two Unlike T cells, B cells do not require antigen
L chains, but signal-transduction molecules as processing before antigen binding, nor is there
well. Igα and Igβ are the same in all BCRs and any involvement of MHC class I or II molecules
do not contribute to an antibody’s antigen-bind- in antigen binding by BCRs. BCRs bind intact
ing properties. The only parts of the BCR that antigens, whether that antigen is a whole bacte-
interact directly with antigens are the variable rium or virus or clumps of denatured proteins,
regions of the L and H chains (see Figure 12.4). such as the toxoid in tetanus vaccines.
As discussed in chapter 11, within the N- Also, because the regions between the mem-
terminus, variable regions of H and L chains brane-distal and membrane-proximal regions
are three hypervariable regions, CDR1, CDR2, of the H chains (sometimes called the hinge
and CDR3. Essentially, all of the amino acid se- regions) are very flexible, antibody and BCR
B - C e l l Ac t i v a t i o n a nd D i f f e r e n t i a t i o n 199
199

Figure 12.3. Primary and secondary humoral immune responses
At day 0, an animal received an injection of antigen A. On day 28, the same animal received an injec-
tion of both antigen A and antigen B. From day 0 on, the serum levels of antibodies to antigens A and
B were monitored and appear in arbitrary units on the y-axis.
molecules can open wide to reach and bind multiple bonds. The advantage to multiple
identical antigenic epitopes. That is, antibodies interactions between antigens and antibodies
can go from being Y shaped to being T shaped is that it makes the interaction highly specific.
to accommodate the span between epitopes. Hydrogen bonds, electrostatic interactions, Van
The chemistry of antigen–antibody interac- der Wahl’s forces, and hydrophobic interactions
tions is complex and involves multiple types of act over only very short distances, which means
bonds, including hydrogen bonds, electrostatic that the fit between an antibody and an anti-
interactions, Van der Wahl’s forces, and hydrogen must be exact or these bonds simply will
phobic interactions. Two things all of these not form. When the fit is right, though, despite
types of bonds share is that they are noncova- low affinity of individual interactions, the sum
lent and individually weak interactions. of these bonds results in very high overall af-
The most important of these chemical inter- finities between antibodies and their antigens.
actions varies between antibodies and their anti- When the fit is imperfect, the affinity decreases
gens, but all antigen–antibody binding involves (see Figure 12.6)

Figure 12.4. The common mammalian BCR
This form of the BCR contains four signal-
transduction molecules—two Igα chains and
Figure 12.5. Therole of the CDRs in antigen (Ag)
two Igβ chains. However, all of the interactions
binding by the BCR
between BCR and antigen (Ag) occur at the
N-termini (membrane-distal) portions of the H The N-terminus regions (membrane-distal
and L chains. portions) of H and L chains contain all of the
variability found among BCRs of different
specificity. Within these variable regions of the
H and L chains, the greatest variability lies in
As a result, a given antibody may bind (with three discrete regions called CDR1, CDR2, and
CDR3. In the final quaternary confirmation
varying affinities) to more than one antigen.
of the BCR, CDR1, CDR2, and CDR3 regions
For example, a rabbit immunized with bovine
of the H and L chains make up the bulk of the
serum albumin may produce antibodies with antigen-binding sites and make direct contact
high affinity for bovine serum albumin, but with antigens.
these same antibodies may also bind (with a
much lower affinity) to equine serum albu-
min—an antibody cross-reaction. In spite of
their remarkable specificity, essentially all an- Regardless of affinity—high or low—all anti-
tibodies have some potential for cross-reactiv- gen–antibody interactions are noncovalent and
ity. This consideration is especially important reversible, meaning that antigens are always
when using antibodies for immunotherapy or moving on and off antibody-binding sites at a
in clinical analyses (see chapter 17). given rate—simple equlibria determined by
201

Figure 12.7. The B-cell coreceptor
Besides BCRs, B cells express another com-
plex of molecules called the B-cell coreceptor.
Together, these molecules bind to fragments of
complement proteins and deliver an additional
signal to the B-cell nucleus.
As with CD4 and CD8 on T cells, B cells ex-

press another group of molecules besides the
BCR that are important to B-cell activation—
the B-cell coreceptor (see Figure 12.7). The B-
cell coreceptor is a complex of molecules that
bind a complement protein fragment and trans-
duce a second singal from the B-cell surface
to the nucleus to initiate the process of B-cell
activation.
Figure 12.6. The affinity of antibodies for antigens
As discussed in chapter 4, several pathogens
results from the complementarity of fit
activate complement directly, which results in
When antigens fit properly within an antibody- opsonization of pathogens with complement-
binding site, the maximum number of interac-
derived proteins, especially C3b. IgG or IgM op-
tions can occur between the two, generating the
sonization of pathogens also activates comple-
highest affinity possible (upper panel). If the fit
is less good, fewer specific bonds form (indi-
ment and leads to deposition of complement
cated in different colors), and the interaction is fragments on the pathogen. Binding of the
of lower affinity (lower panel). coreceptor to these complement fragments on
the antigen, along with the binding of the BCR
the Keq of the reaction. This fact is important to antigen, significantly enhances the strength
to remember in the later discussion of affinity of the binding between B cell and pathogen,
maturation. leading to more efficient B-cell activation. After

Figure 12.8. B cells as APCs
B cells acquire antigen through the Ig portion of their BCRs. After binding antigen and BCR cross-
linking (not shown), B cells internalize the antigen–antibody (Ag–Ab) complexes, and process both
antigen and antibody into peptides. MHC class II molecules bind these peptides and present them on
B-cell surfaces.
binding of the BCR to antigen and of the B-cell cess, the antigen–antibody complexes enter the
coreceptor to complement fragments, B cells endosomal pathway, are chopped into pieces,
begin to express high levels of MHC class II and are bound by MHC class II molecules as de-
molecules and B7, making them much more ef- scribed in chapter 8. After this processing step,
ficient APCs. the MHC class II–antigen complexes move to
So, BCRs can directly bind to native antigen, the surface of B cells, where the complexes be-
sometimes with very high affinities, but antigen come accessible to the TCRs of Th cells (see
binding alone (not even antigen binding by the Figure 12.8).
B-cell coreceptor along with BCR binding to The same processing and presentation clearly
antigen) is not enough to activate a B cell and occur with BCR fragments. Because most self-
push it into proliferation and differentiation. All reactive T cells die in the thymus, though, there
B cells, except for B-1 B cells and possibly some are usually no Th cells in the periphery that
MZ B cells, require other signals beyond BCR react with self Ig. In fish and amphibians, B
and coreceptor binding to antigen before acti- cells (as do macrophages and DCs) also directly
vation to proliferation and differentiation. Most phagocytose antigens for presentation to Th
of those signals come from Th cells activated cells.
by B cells acting as APCs. After antigen and coreceptor binding, B cells
begin to express higher levels of B7 and MHC
class II molecules. As a consequence, these B
B Cells as Antigen-Presenting Cells cells transform into highly effective APCs. After
After a B cell’s BCRs bind to antigen and antigen-induced activation, B cells also begin
cross-link to one another, some of the antigens, to express another molecule called CD40. Ulti-
along with some of the BCRs themselves, are mately, this molecule will also make B cells bet-
taken into the B cells in a process known as re- ter APCs through interaction with CD40 ligand
ceptor-mediated endocytosis. During this pro- (CD40L) on Th cells. CD40 molecules, in fact,
203

appear on all APCs, but CD40 plays an espe-
cially important role in B-cell activation. Dur-
ing interactions between macrophages or DCs,
CD40 binding to CD40L causes these APCs to
secrete cytokines. In the interaction between B-
cell APCs and Th cells, CD40 binding to CD40L
provides a costimulatory signal for the B cells
and promotes growth, differentiation, and iso-
type switching (further described in the Isotype
Switching section).
B cells may encounter antigen in the pe-
riphery or in secondary lymphoid tissues. But
regardless of where the antigen-induced activa-
tion begins, B-cell development finishes in sec-
ondary lymphoid tissues. In our discussion, we
focus on lymph nodes.
Activation of B Cells by T-Helper Cells

The final stage of differentiation for most B
cells requires the participation of a Th cell that
has undergone clonal expansion and further
differentiation after activation by an APC other
than a B cell, usually a DC. Although the BCR
on the B cell binds an antigenic epitope distinct
from the one bound by the TCR on the Th
cell, the antigen–MHC complex on the B cell
must bind to the Th cell’s TCR. For all of this
to occur properly, T cells, B cells, antigens, and
other APCs must all somehow come together
inside of lymph nodes.
After antigen binding or ingestion, DCs and
some macrophages arrive, along with antigen,
in lymph nodes via the lymphatics. Th cells
leave the thymus, and B cells leave the bone
marrow via the blood. The arterial circulation
and the lymphatics have no direct connection,
but inside of lymph nodes (and other second- Figure 12.9. B cells and Th cells enter lymph
ary lymphoid tissues), the endothelia of some nodes through HEVs and interact to effect
of the venules have a unique high-columnar B-cell activation
structure and are called high endothelial ve- Specialized venules inside of lymph nodes and
nules (HEVs). Because of specific interactions Peyer’s patches called HEVs have receptors
between lymphocytes and HEVs, lympho- for cell-surface proteins on T and B cells. This
cytes can move through HEVs from blood into interaction allows the T and B cells to exit the
blood and enter lymph nodes. Inside, Th cells
lymph nodes, allowing for the direct interaction
encounter antigen-presenting DCs.
among B cells, T cells, and antigen (see Figure

Figure 12.10. B-cell migration
Naïve B cells leave the bone marrow via the blood. From there, these B cells may encounter antigen
in the blood, lymph, or lymph node. If the B cells fail to bind antigen or fail to find the appropriate Th
cell inside a lymph node, they leave nodes via the efferent lymphatics and move on to the next node.
12.9). If, after the antigen-activated B cell enters vides the next signal needed to stimulate B-cell
the lymph node, it does not encounter a T cell division and differentiation (see Figure 12.11).
with the appropriate TCR, the B cell leaves the After interactions with specific antigens pre-
node via the efferent lymphatic and moves on sented on APCs, T cells divide, differentiate,
to the next node (see Figure 12.10). If, instead, and provide necessary, additional signals to B
a B cell binds antigen and encounters a Th cell cells that ultimately trigger B-cell division and
reactive with that same antigen, the Th cell pro- differentiation. These additional signals come
205

of eosinophils. Similarly, IL-13 stimulates B-cell
division and affects Ig class switch, and IL-25 in-
duces production of more IL-4, IL-5, and IL-13
and appears to help protect against certain par-
asitic worms.
Th1 cells produce IFN-γ, TNF, IL-2, and IL-
10. In some species, at least, IFN-γ stimulates
B-cell growth and Ig class switching. TNF stim-
ulates inflammation and B-cell differentiation.
IL-2 stimulates further T-cell division and also
affects class switching. IL-10 has multiple effects
on both inflammation and B-cell differentiation.
During primary immune responses, almost
all of the available B cells express IgM (as do all
naive B cells). After interaction with Th cells,
antigen-activated B cells move to the medul-
lary cords of the lymph nodes, differentiate in
plasma cells, and begin to secrete IgM—the
predominant antibody of primary immune re-
sponses (see Figure 12.12). As B cells differenti-
ate into plasma cells, the switch from the mem-
Figure 12.11. Antigen processing and brane-bound form of Ig (the BCR) to the secre-
presentation by B cells inside of a lymph node tion of soluble Ig is the result of an alternative
Mammalian B-cell BCRs bind to antigen and RNA splice that removes the membrane anchor
then, via receptor-mediated endocytosis, from the Ig H chains and creates a soluble form
internalize the antigen–antibody complexes
of the Ig molecule.
(upper portion of figure). The newly formed
Late in the primary response to a persistent
endosomes fuse with lysosomes that contain
both proteolytic enzymes and MHC class II
antigen and in all secondary immune responses,
molecules. The antigenic peptides bind to the most B cells will switch to another antibody
MHC molecules and the MHC–antigen com- isotype.
plexes return to the surface for presentation to
Th cells.
Isotype Switching, Somatic
Hypermutation, and Terminal
in the form of cytokines secreted by Th2 or Th1 Differentiation of B Cells
cells (see Figure 12.11). into Plasma Cells
Perhaps most important among the Th2- Some Th cell–activated B cells do not go to
derived cytokines is IL-4, which (among other the medullary cords. Instead, this population
things) stimulates B-cell division and induces of B cells moves into the lymphoid follicles.
clonal expansion of B cells. As a result, in a rela- There, these B cells interact with additional
tively short period of time after exposure, the Th cells and begin to divide, forming germi-
number of pathogen-specific B cells increases nal centers (see Figure 12.13A–C). As the B cells
to protective levels. IL-4 also plays a role in Ig divide in the germinal centers, Th-cell–derived
class switch (see Isotype Switching section). cytokines induce isotype switching from IgM
In addition, Th2-derived IL-5 stimulates B-cell to IgG or IgA or, occasionally, IgE (see Figure
division, antibody production, and the growth 12.14).

Figure 12.12. The primary immune response
After the initial interaction between antigen-activated B cells and Th cells, the B cells begin to divide,
migrate to the medullary cords, and differentiate into IgM-secreting plasma cells.
After switching isotype, B cells must continue the mutated BCR may be the same, better, or
to interact with Th cells that stimulate further worse than that of the original BCR.
divisions, or the B cells will die. During these To deal with this, animals have evolved a pro-
divisions, mutations begin to accumulate in the cess of selection to eliminate those B cells with
B cells’ Ig genes at a very high rate—hypermu- unchanged or lower antigen affinity to ensure
tation. Because hypermutation occurs beyond that the end result of somatic hypermutation
the B cells developing in the bone marrow, it is antibody with greater affinity for the immu-
is called somatic hypermutation. Protein-se- nizing antigen. Overall, somatic hypermutation
quencing studies have shown that these muta- and selection result in the affinity maturation
tions do not occur randomly throughout the seen during secondary and subsequent im-
sequence of the Ig molecule. Instead, they are mune responses.
concentrated into the CDR1, CDR2, and CDR3 As mentioned earlier, germinal centers also
regions of the L and H chains (Figure 12.15). contain cells called FDCs. These cells play a
CDR1, CDR2, and CDR3 regions of the L crucial role in affinity maturation. On the sur-
and H chains are, of course, the segments that face of FDCs are receptors that bind to antigen–
make up the antigen-binding sites and directly antibody complexes. As antigen–antibody com-
contact antigen. So mutations that arise as a re- plexes begin to accumulate during the primary
sult of somatic hypermutation have dramatic immune responses, some are trapped on the
effects on the antigen-binding affinity of a B surface of FDCs in the germinal centers. The
cell’s BCR. Because somatic hypermutation is a antigens in these complexes serve as a reservoir
random process, the antigen-binding affinity of for B-cell selection.
207

Figure 12.13A–C. B cells proliferate in lymph node
germinal centers
After activation in the medulla, a significant
population of B cells moves to the lymphoid
follicles (panel A), proliferates, and forms
germinal centers (panel B). Dividing B cells are
called centroblasts, and resting B cells are called
centrocytes. Germinal centers also contain Th
cells and FDCs (cells distinct from the antigen-
presenting DCs). In the germinal centers, B
cells undergo isotype switching and somatic
hypermutation, then emigrate the lymph node
and differentiate into plasma cells that migrate
to the bone marrow (panel C).
Each time a B cell interacts with a Th cell, division. If it fails to reacquire antigen, the B
the B cell is induced to divide. As that B cell cell will die by apoptosis.
comes out of the division cycle—goes from The only significant source of antigen in the
being a centroblast to being a centrocyte—it germinal center is the antigen–antibody com-
must again acquire antigen for presentation to plexes on the FDCs. As we have mentioned, all
another Th cell and enter another round of cell bonds between antigen and antibody are non-

Figure 12.14. Cytokine control of isotype switching in germinal centers
In the upper left corner, a B cell first encounters antigen. Inside of a germinal center in secondary
lymphoid tissue, this B cell interacts with a Th cell (upper right). Depending on the cytokines pro-
duced by the Th cell, the B cell switches from expressing IgM to expressing IgG, IgE, or IgA.
covalent and reversible. If, as the B cell comes once again, present that antigen to a Th cell and
out of cycle, its BCR has a higher affinity for enter a new round of division. If, however, the
antigen than the antibody on the FDC, the new affinity of new, hypermutated BCR is no better
hypermutated BCR can acquire antigen from or worse than the affinity of the antibody in the
the complexes on the FDCs and process and, antigen–antibody complexes on the FDCs, the
209

Figure 12.15. Somatic hypermutation
Late in primary immune responses and throughout secondary and subsequent immune responses
against a given antigen, mutations (shown here as colored vertical bars) rapidly arise in B-cell genes
encoding Ig molecules. These mutations fall mostly within the CDR1, CDR2, and CDR3 regions of
the L and H chains.
B cell will not be able to reacquire antigen, and or booster immunization, quickly differentiate
it will die (see Figure 12.16). Because most muta- into plasma cells producing isotypes other than
tions would result in a BCR that has lower or the M and very high-affinity antibody. These are the
same affinity for the antigen, it is not surprising cells that dominate secondary and subsequent
that many B cells die in this process of negative immune responses and, along with memory
selection. Macrophages in the germinal centers Th and Tc cells, are responsible for immuno-
remove dead B cells by phagocytosis. These logical memory. Figure 12.18 shows a summary
macrophages are often referred to as tingible- of these processes and how they relate to the
body (stainable-body) macrophages because differences between primary and secondary re-
their cytoplasm contains B-cell chromatin frag- sponses that we discussed at the beginning of
ments undergoing degradation and is character- this chapter.
istic of active germinal centers in a lymph node. After primary immunization (or infection),
Some of these high-affinity B cells emigrate naive B cells must acquire antigen, migrate to
from the lymph nodes via the efferent lymphat- the secondary lymph nodes, and find T-cell
ics, enter the blood, and home back to the bone help. Then, the first of these cells to be acti-
marrow. Along the way, these B cells differen- vated remains in the secondary lymphoid tis-
tiate into long-lived plasma cells and secrete sue and secretes IgM antibodies. These cells are
higher-affinity antibody from the bone marrow usually short-lived. This process takes seven to
(or sometimes from sites of inflammation) for ten days, and during that time antibody levels
months to years (see Figure 12.17). begin to rise in the serum. Late in the primary
Also in the germinal center, other B cells be- response, another group of B cells moves into
come long-lived memory B cells and remain in the lymphoid follicles and, after interactions
secondary lymphoid tissues. These cells persist with Th cells and FDCs, undergoes isotype
for a long time and, after secondary infection switch, somatic hypermutation, and selection.

Figure 12.16. Affinity maturation through somatic hypermutation and selection
Inside of lymph nodes, as they undergo the process of somatic hypermutation, B cells must repeat-
edly bind antigen through their BCRs to survive. The major source of antigen in germinal centers is
in the form of antigen–antibody complexes bound directly or indirectly (through C3b and C3d recep-
tors, CR1 and CR2) to FDCs. To bind that antigen, hypermutated BCRs must have greater affinity for
antigen than the antibody on the FDCs. When this happens, B cells survive and continue as high-
affinity B cells.
211

Figure 12.17. Life cycle of B cells
In most animals, B cells begin life in the bone marrow. They migrate from there, via the blood, to
secondary lymphoid tissues, where they interact with Th cells. Some B cells immediately become
plasma cells and secrete Ab from the medullary cords of the lymph nodes. Other B cells move to the
lymphoid follicles and, after interactions with Th cells and FDCs, switch isotypes, undergo somatic
hypermutation and selection, and become either long-lived memory B cells or antibody-secreting,
long-lived plasma cells at sites of inflammation, in the bone marrow, or in secondary lymphoid tissue.
Some of these high-affinity B cells migrate to process, another group of long-lived memory B
bone marrow. Along the way, they differenti- cells arises and remains in secondary lymphoid
ate into long-lived plasma cells secreting high- tissues awaiting another antigenic insult.
affinity antibodies specific for the immunizing In species such as chickens, rabbits, and sheep,
antigen. In the germinal centers of the lymph in which most B-cell development occurs in the
nodes, another group of high-affinity, isotype- GALT, the process is a little different (see Figure
switched B cells become long-lived memory B 12.19). Also, B-cell maturation does not involve
cells. After secondary immunization (or infec- germinal centers in all species, nor is it entirely
tion), memory B cells dominate the immune clear whether all species are capable of somatic
response. These B cells, again after interactions hypermutation (see Figure 12.20).
with Th and FDCs, generate mostly IgG or IgA On the surface, these species’ variations
of even higher affinity. From these B cells come might suggest that some species would be at
more long-lived plasma cells secreting high-af- greater risk of infection because of lower po-
finity antibody in the bone marrow. During the tential antibody diversity. In fact, some scien-

Figure 12.18. Primary versus secondary immune responses
tists have argued that the apparent low diversity host-cell molecules co-opted by bacteria and
among bony and cartilaginous fish might re- viruses) on host cells and either destroy those
flect the greater homogeneity of their environ- host cells or pathologically alter host-cell func-
ment compared with land species—a reduced tions. Antibodies bound either to the surfaces
need for highly diverse BCRs. No hard evidence of viruses or to bacterial toxins prevent interac-
exists for either fewer pathogenic threats or tion with host-cell receptors (see Figure 12.21).
greater susceptibility to infection among these
species. Regardless, it is apparent that at consid-
erable energetic and genetic expense, all mam- Opsonization and Activation
mals and several other species have evolved very of Complement
sophisticated means for generating astounding Antibodies can also bind to the surfaces of
amounts of antibody diversity. bacteria and coat them with antibody molecules,
all with the C-termini of their H chains facing
Antibody Effector Functions out. This process is opsonization, and the end
result is more efficient destruction of bacterial
Pathogen and Toxin Neutralization pathogens (see Figure 12.21). Recall from chap-
During pathogenesis, viruses and bacterial ter 6 that macrophages, neutrophils, and other
toxins both bind to specific receptors (normal cells have receptors, called Fc receptors, that
213

Figure 12.19. Acomparison of B-cell development in species in which that development takes place
in bone marrow (and often fetal liver) or GALT
In the so-called GALT species, such as rabbits, chickens, and sheep, Ig gene variability arises differ-
ently. Although the non-GALT species rely largely on genetic rearrangement and junctional diversi-
fication for generation of BCR diversity, GALT species use mechanisms such as gene conversion and
somatic hypermutation before B-cell contact with antigen to generate Ig diversity. In both GALT and
non-GALT species, final B-cell maturation occurs in secondary lymphoid tissues and involves somatic
hypermutation, selection, and affinity maturation. GALT species may also use further gene conver-
sion events and selection to enhance antibody affinity.
bind to the C-termini of Ig H chains. After bind- in more efficient interactions between phago-
ing antigens, the conformation of the C-termini cytic cell surfaces and pathogens and in turn
of Ig H chains changes slightly. Aggregation of leads to more rapid and more effective destruc-
Ig adds to these changes and to the antibodies tive of pathogens, especially bacteria. Activa-
reactive with Fc receptors. As a result, once bac- tion of the terminal membrane attack pathway
teria have been opsonized, macrophages, via Fc also leads to direct lysis of the pathogen.
receptors, bind, phagocytose, and destroy these
bacteria much more efficiently.
Similarly, after antigen binding, changes in Distribution and Function
the C-termini of the H chains of IgG and IgM of Fc Receptors
molecules activate complement via the classical Fc receptors actually appear on a variety of
complement cascade (discussed in chapter 4). cells and perform several functions beyond sim-
Complement activation results in the deposi- ply enhancing phagocytosis. For example, Fc
tion of complement fragments, especially C3b, receptors on FDCs (FcγRIIB) capture antigen–
onto pathogen surfaces. Macrophages, neutro- antibody complexes for selection after somatic
phils, and other phagocytic cells also have re- hypermutation. Also, mast cells, cells that play
ceptors for C3b. Thus, bound C3b also results critical roles in inflammation and allergies, have

Figure 12.20.The forms of antibody and the use of hypermutation, Ig class switch, and germinal
center formation in various classes of chordates
Fc receptors for IgE (Fcε). When the IgE bound Clinical Correlation Follow-Up
to the Fc receptor interacts with antigen, the
mast cell releases histamine and several of the
mediators of inflammation. This inflammation Selective Ig deficiencies are the most com-
is sometimes protective (perhaps in parasitic mon immune deficiencies of dogs. As men-
infections) and sometimes causes allergic reac- tioned at the outset of this chapter, as many
tions (see chapter 16). as 80 percent of Chinese shar peis have selec-
Because of antibodies and Fc receptors, the tive IgA deficiencies. Their symptoms include
humoral immune response is very effective at recurrent dermatitises (staphylococcal), demo-
dealing with and defending against extracellu- dectic mange, otitis externa, flea allergies, cys-
lar pathogens. Also, investigators have very re- titis, food intolerance, and bronchitis. On the
cently found that some antibodies do get inside basis of this chapter and the preceding one, you
of cells as well. It still appears, however, that should be able to offer an explanation for why
cellular immune responses and Tc cells deal an animal with an IgA deficiency might have
most effectively with intracellular pathogens. the symptoms seen in these dogs. You should
also be able to offer one or more plausible
215

Figure 12.21. Effector mechanisms of antibodies
As shown in the left column, when antibodies bind to toxins (or viruses), the toxins can no longer
bind to specific receptors on host cells and are neutralized. The center column shows how antibody
binding to bacteria (a process called opsonization) allows macrophages to adhere more readily and
facilitate phagocytosis. The right column shows a similar process. Once IgM or IgG antibodies have
bound to a pathogen, the complexes activate complement. Macrophages and other phagocytic cells
also have receptors for complement fragments.

Figure 12.22. Adult German shepherd and puppy (© Lenkadan / Shutterstock)
Selective Ig deficiencies have also been reported in German shepherds.
explanations about how an IgA deficiency pathogens. It may also be that in the absence
might arise and how it could produce the ob- of IgA, responses dominated by IgE might be
served symptoms. more common and lead to flea and food al-
lergies. Clearly, the majority of the symptoms
seen in these dogs result from a deficiency of
Possible Explanations IgA antibodies.
As the result of specific transport mecha- Just how an animal might come to have a se-
nisms that have evolved to deliver IgA anti- lective IgA deficiency is less clear. Several pos-
bodies to epithelial surfaces, as described in sible explanations exist. It could be that these
chapter 11, IgA—especially dimeric IgA—is the animals have an inherited genetic defect in the
predominant antibody at body surfaces, includ- C-gene segment encoding IgA H chains. In this
ing mucosal and other epithelia. No similar case, it would be impossible for B cells to switch
mechanism transports any other isotype across from IgM to IgA under any circumstances. Also,
epithelial surfaces. Furthermore, infections that as shown in Figure 12.14, Th-cell–derived cyto-
occur at epithelial surfaces induce primarily IgA kines (such as TGF-β) direct isotype switches,
antibody responses. including the switch to IgA. A deficiency or de-
In the absence of normal levels of IgA, epi- fect in any one of these cytokines or their re-
thelia are at much greater risk of primary and ceptors on B cells could also lead to a selective
recurrent infections such as dermatitis, de- IgA deficiency. Similarly, defects in the signal
modectic mange, otitis externa, cystitis, and transduction machinery associated with any
bronchitis and are unable to make protective of these cytokine-specific receptors on B cells
IgA antibody responses against the infecting could lead to a selective IgA deficiency.
217

Gerald N. Callahan
Adaptive Immune Responses to

Chapter 13
Infections and Immunological Memory
10.5876_9781607322184.c013
Clinical Correlation: Canine Parvovirus and

the Wolves of Isle Royale 219
Adaptive Immunity: The Big Picture—From
Insult to Recovery 221
Routes of Infection 223
Mechanisms of Pathogenesis 223
Innate Response Cells and Cytokines of Innate
Responses Direct T-Cell Differentiation 225
Effector T Cells Home to Sites of Infection and
Inflammation 226
Antibody Responses Develop in Secondary
Lymphoid Tissues 229
Importance of Different Adaptive Responses
to Clearance of Different Pathogens 233
Immune Responses in the Gut 233
Immunological Memory 237
Primary and Subsequent Immune Reponses 237
Memory B Cells 238
Memory T Cells 238 Figure 13.1. Gray wolf (© Dennis Donohue /
Clinical Correlation Follow-Up 239 Shutterstock)
work and animal populations react to different
stressors.
In about 1900, moose migrated across an ice
bridge onto Isle Royale. Around 1940, a wolf
Canine Parvovirus and the
(Figure 13.1) pack, also via an ice bridge, ar-
Wolves of Isle Royale
rived on Isle Royale. Both wolves and moose
Isle Royale National Park is an island in Lake prospered until the early 1970s (see Figure 13.2).
Superior. The island is roadless, covers about About then, the wolf population began to de-
544 square kilometers (338 square miles), and plete the moose herd.
sits 25 kilometers (15.5 miles) from Ontario. Be- And that was how things continued—increas-
cause of its usual isolation from the mainland, ing wolf population, decreasing moose popula-
ecologists have studied the island for nearly a tion—until 1981. In that year, the wolf popula-
century to learn more about how ecosystems tion fell precipitously, from nearly fifty wolves
219
219

Figure 13.2. Wolf and moose populations on Isle Royale, 1955–2010
to fourteen, and the moose herd began to re- Parvovirus infections occur most frequently
cover. By 1985, the moose population reached when dogs ingest food or other things con-
an all-time high, but the wolf population never taminated by feces from an infected animal.
fully recovered. Many people offered theories Figure 13.3 shows the pathogenesis of canine
about the cause of the wolf population’s crash, parvovirus.
but nothing seemed consistent with all the Approximately 70 percent of dogs infected
facts. Then veterinarians completed a series of with canine parvovirus will die from their in-
wolf necropsies in the early 1980s. Essentially, fections. At necropsy, these dogs exhibit leuko-
every necropsied wolf had died as a result of penia, extensive necrosis of the intestinal epi-
a parvovirus infection. Canine parvovirus was thelium and secondary lymphoid tissues, and
unknown until 1978. By 1979, the virus had severe dehydration.
spread around the globe and ravaged dog and
coyote populations, especially pups. The virus
Learning Objectives
is very similar to feline panleukopenia virus and
some viruses of raccoons and foxes and may After reading this chapter, you should be able to
have evolved from one of them.
Isle Royale National Park was established
• describe mammalian adaptive immune
responses from beginning to end;
in 1940, and among its regulations was the ex-
clusion of domestic dogs (the most common
• understand how the effectiveness of these
responses may vary depending on the nature
reservoir for parvovirus). That means the only
of the pathogen;
plausible explanations for the appearance of
parvovirus on Isle Royale all involve humans.
• understand how and why adaptive immune
responses may fail to provide protection;
Among the possibilities, it seems most likely
that this parvovirus outbreak originated when • describe the unique nature of adaptive
immune responses that arise in the gut;
humans carried the virus onto Isle Royale,
probably stuck to the soles of their boots. Be- • understand the nature of immunological
cause parvovirus is a nonenveloped virus that memory and describe the cells involved;
persists for long periods outside of host cells, • describe how immunological memory arises
this explanation seems entirely plausible. and is maintained.
220 Ad a p t i v e Imm u n e R e s p o n s e s t o In f e c t i o n s a nd Imm u n o l o g i c a l M e m o ry

Figure 13.3. Pathogenesis of canine parvovirus
Canine parvovirus infections occur when canids (especially puppies) ingest food or other material
contaminated with feces from another parvovirus-infected canid. The virus then spreads from the
mouth to the lymph nodes and the gastrointestinal tract. Often, but not always, the virus then causes
a massive bloody diarrhea, leucopenia, gastrointestinal necrosis, dehydration, and death.
Adaptive Immunity: The Big nisms. Until the past decade, most immunolo-
Picture—From Insult to Recovery gists ignored the potential of these innate
defenses and focused on adaptive immunity;
After most encounters with pathogens, a race however, outside of chordates, adaptive im-
ensues—a race between the host animal’s de- mune responses are nonexistent. Because most
fenses and the pathogen. The progress and the animals are not chordates (insects being the
outcome of that competition determine which single largest population of animals), most ani-
survives and which dies. mals, including humans, rely solely on innate
The race begins when the pathogen enters defenses to survive the millions of daily patho-
the host and triggers innate defense mecha- genic assaults they encounter.
Ad a p t i v e Imm u n e R e s p o n s e s t o In f e c t i o n s a nd Imm u n o l o g i c a l M e m o ry 221

221

Figure 13.4. Acute infection
Infections always result in a race between the host defenses and the replicating pathogen. After initial
contact with the pathogen, two things begin to happen: the host’s innate responses engage and the
pathogen begins to replicate. When numbers of pathogens exceed a certain threshold and move
beyond the innate defenses, adaptive responses follow. Over a period of days to weeks, these adaptive
responses usually reach protective levels, and the pathogen disappears from the infected animal.
Clearly, innate immunity can provide a pow- alone. Plus, adaptive immune responses make
erful weapon against infectious agents, and possible one of the greatest achievements in the
even among animals that possess adaptive im- battle against infectious diseases—vaccination.
mune capabilities, innate defenses are terri- It is likely that innate defensive responses to
bly important. First, innate immune responses some pathogens fully control infections. Just
occur much more rapidly than adaptive re- how many is not known. First, innate responses
sponses. Because this is a race between patho- occur so rapidly that clinical signs of infection
gen and host defense, speed is of the essence. may not appear before the pathogen is gone.
Second, few—if any—adaptive responses can Deficiencies in innate defense mechanisms are
develop without some assistance and direction rarer (perhaps an indication of the animals’ ab-
from early innate responses. solute dependence on them) than deficiencies in
Beginning with sharks, however, adaptive adaptive responses. So, fewer opportunities have
immune responses often dominate immune arisen to directly assess the importance of innate
responses. An adaptive immune response pro- defense to overall protection. Regardless, many
vides some things innate immunity cannot, pathogens multiply so rapidly that they outstrip
namely, specificity, memory, and affinity matu- innate defenses. When this happens, adaptive
ration. In a world of evolving pathogens, these immune responses engage (see Figure 13.4).
aspects of adaptive immunity add a level of pro- Most immune responses appear to involve
tection unattainable through innate immunity both innate and adaptive aspects, but all begin

Figure 13.5. Replication of pathogens in various compartments within animals
Different pathogens thrive in different compartments inside of infected animals.
with innate responses. As shown in Figure 13.4, disease. The possible routes of transmission
it appears that once a pathogen replicates be- include inhalation, ingestion, and injection (by
yond the defensive power of innate mecha- mosquitoes or other blood-sucking arthropods);
nisms, adaptive immunity becomes involved. during mating; and through wounds.
Time frames may vary between species and
among individuals, but innate mechanisms gen-
erally engage within seconds to hours, and the Mechanisms of Pathogenesis
development of a primary, full-blown adaptive Once pathogens gain access beyond the bar-
immune response takes a week or longer. riers of skin and mucosal epithelia, they can
thrive and replicate in different compartments
inside of animals (see Figure 13.5). All patho-
Routes of Infection gens exist as either intracellular or extracellular
Infectious agents (i.e., prions, viruses, bac- infections. Extracellular infections may occur in
teria, fungi, and parasites) are also known as any of the body fluids—blood, lymph, intersti-
transmissible agents of disease because, when tial fluid, and urine. Bacteria, protozoa, fungi,
transmitted from an infected to an uninfected and worms are the most common extracellu-
animal, these agents will again induce the same lar pathogens. Pathogenic microorganisms can

223

Figure 13.6. Mechanisms of pathogenesis
Various microorganisms cause diseases in various ways. Bacteria produce exotoxins and endotoxins as
well as directly infect cells and alter normal cell function. Viruses infect cells and kill or interfere with
normal host-cell activities. Fungi grow on tissue surfaces and can enzymatically degrade underlying
cells. Protozoan parasites also often infect host cells and alter these cells’ abilities to perform their
normal functions. Worms can cause diseases in a variety of ways.
also grow intracellularly. Bacteria and viruses LPS is present systemically (as in Gram-neg-
are the most common intracellular pathogens. ative sepsis), however, it can cause endotoxic
Each of these different locations presents par- shock and death (described in chapter 3).
ticular problems for immune systems. Trans- Both Gram-negative and Gram-positive bac-
missible agents also cause diseases in different teria can also produce other types of toxins
ways (see Figure 13.6). called exotoxins. Living bacteria produce exo-
On the basis of their abilities to retain toxins, and unlike endotoxins, there are many
Gram stain, bacteria can be split into two large types of exotoxins with very different sorts of
groups—Gram-negative and Gram-positive. effects on host animals. To exert their effects,
Recall from chapter 3 that the defining feature exotoxins can bind directly to specific mole-
of Gram-negative bacteria is that they have a cules on host cells.
thick outer coat of LPS—a powerful PAMP— For example, as they grow, Clostridium botuli-
on their cell walls. Gram-negative bacteria re- num bacteria release botulinum toxin, possibly
lease LPS into their surroundings, particularly the most potent toxin known (a median lethal
when they die. LPS is endotoxin, and endotoxin dose is about 1 ng/kg). When an animal ingests
causes disease by activation of macrophages botulinum toxin, the toxin moves quickly into
and inducing inflammation. When this same and out of the blood where it binds receptors in

Figure 13.7. Initiation of an adaptive immune response
Antigen gains access to host cells after a break in the skin. DCs ingest this antigen, process it, trans-
port it to the nearest lymph node, and present the antigen to T cells. Adaptive immune responses
begin here and eventually culminate at the site of infection.
neuromuscular junctions. There, it inhibits the Protozoan parasites commonly cause disease
release of acetylcholine from presynaptic nerve by infecting host cells and interfering with nor-
terminals. The neurotransmitter acetylcholine mal host-cell function. Multicellular parasites
is essential for normal muscle activation. In the can cause disease in many different ways—from
presence of botulinum toxin, muscle contrac- simple mechanical obstruction of fluid flow (as
tion becomes impossible, and respiratory and in canine heartworm) to inflammation (as in
cardiovascular systems fail rapidly. sarcoptic mange).
All viruses and some bacteria such as Chla-
mydia grow inside of host cells. The pathogenic
effects of these infections result from the tissue Innate Response Cells and
tropism of the infectious agent and the damage Cytokines of Innate Responses
these pathogens do to those host cells as well Direct T-Cell Differentiation
as from host inflammatory responses. For ex- Adaptive immune responses begin when pro-
ample, in infectious canine hepatitis, the virus fessional (dedicated) APCs ingest antigens and
enters the body through the mouth or nose, transport them to secondary lymphoid tissues
or both, and infects the tonsils and the cervical (see Figure 13.7). Along the way, these APCs
lymph nodes. From there, the virus moves into process the acquired pathogen into pieces, in-
the blood and spreads to (among other places) sert those pieces into MHC class II and MHC
the liver. Here, the virus infects hepatocytes. class I molecules, and relocate these MHC–an-
Some of these hepatocytes die, which stimu- tigen complexes to the surface of the APCs,
lates a host inflammatory response that results where they become available for interaction
in hepatitis. with T cells (see Figure 13.8A–B).
Fungi cause diseases by attachment to host Circulating T cells arrive in lymph nodes via
tissue surfaces and enzymatic degradation of specific interactions with receptors on HEVs.
the underlying host cells. Most often, this at- Initially, in the medullary regions of the nodes,
tachment and degradation occur on the skin. these T cells encounter APCs displaying anti-

225

Figure 13.8A–B. APCprocessing and presentation of antigen in MHC Class I molecules (panel A) and
APC processing and presentation of antigen in MHC class II molecules (panel B)
Panel A: A represents an infectious agent reproducing in the cytosol of the cell. B is antigen being
synthesized in the cytosol of the cell. C is the proteosome where the protein products of A are
digested into small peptides. D shows the peptides being transported into the lumen of the ER. E
shows the peptides being bound by newly formed MHC class I molecules. F shows the movement of
the MHC–Ag complexes inside an endosome. G shows the MHC–Ag complexes on the cell surface for
presentation to T cells. Panel B: A represents exogenous antigen that is ingested by an APC via phago-
cytosis and confined inside of a phagosome (B and C). That phagosome then fuses with a lysosome
to form a phagolysosome. D and E show how, inside of the phagolysosome, enzymes cleave antigenic
proteins into small peptides that bind to MHC II molecules and return to the cell surface as Ag–MHC
complexes (F) for presentation to T cells.
gens they have acquired locally or from remote Effector T Cells Home to Sites
sites. For a few days, antigen-specific T cells of Infection and Inflammation
remain in the lymph node, where at least two Tc effector cells express several different cell-
types of interactions occur. Naive Tc cells in- surface markers. Among these are molecules
teract with MHC class I–antigen complexes on that interact specifically with receptors on vas-
APCs and, with or without Th-cell help, these cular endothelial cells at sites of infection and
naive Tc cells become antigen-activated effector inflammation. Because of this, activated Tc
Tc cells (see Figure 13.9A–B). cells home to sites where these cells are most

Figure 13.10. Effector
Tc-mediated destruction of
pathogen-infected cells
Figure 13.9A–B. Direct activation of Tc cells by After specific recognition of MHC–antigen
highly effective APCs complexes, Tc cells release lytic granules that
After exposure to PAMPs, DAMPs, or both, induce apoptosis in infected host cells.
APCs (like DCs) will express high levels of
MHC classes I and II as well as B7. This, along
with processed antigen, can provide both the
first (MHC–antigen) and the second (B7) signals Also, in secondary lymphoid tissues APCs
to Tc cells (panel A). In response, the T cells
present MHC class II–antigen complexes to Th
begin to express the high-affinity receptor IL
cells. A combination of (1) the nature of the
and produce IL-2 (panel B), which leads to
activation.
APC, (2) the nature of the innate responses, (3)
the cytokines the APC produces, and (4) the
affinity and frequency of TCR binding to the
likely to find pathogen-infected cells, especially MHC–antigen complex determines what hap-
virus-infected cells, and destroy them (see Fig- pens to the Th cell beyond this point (see Figure
ure 13.10). 13.11).

227

Figure 13.11. Alternative pathways of Th-cell differentiation
After interaction with an APC, Th cells may differentiate into several different cell types, including
Th1, Th2, Tf h, Th17, Treg, Th3, and Tr1 cells. The pathway of Th-cell differentiation depends on
what the cytokines produce during antigen presentation (arrows). Each Th cell type secretes a charac-
teristic set of cytokines (far right) and participates in a unique way in the immune response.
Some of these Th effector cells, particularly protective adaptive immune responses (as de-
Th1 and Th17 cells (because of cell-surface scribed in chapter 10), often enhancing inflam-
changes), also home to areas of infection and mation and macrophage-mediated destruction
inflammation. They also bind to receptors of extracellular and intracellular pathogens.
on the vascular endothelium and engage in Other Th cells remain in the lymph nodes and

Figure 13.12. B cells as APCs
B cells acquire antigen through the immunoglobulin portion of their BCRs. After binding antigen
and BCR cross-linking, B cells internalize the antigen–antibody (Ag–Ab) complexes and process both
antigen and antibody into peptides. MHC class II molecules bind these peptides and present them on
B-cell surfaces.
interact with B cells or aid in the activation of germinal center. During these divisions, two
Tc cells. things happen: isotype switching and somatic
hypermutation.
Inside of lymphoid follicles, B cells undergo
Antibody Responses Develop in further genetic rearrangements that couple
Secondary Lymphoid Tissues new H-chain constant-region genes with the
B cells, usually after acquiring antigen, also variable regions generated in the bone marrow,
arrive in the lymph nodes via the HEVs. B-cell which results in a switch from IgM to some
interaction with antigen through the BCR and other isotype, usually IgG or IgA but some-
the B-cell coreceptor activates B cells. After ac- times IgE (see Figure 13.13).
tivation, B cells express more MHC class II and Also during B-cell division inside of lym-
B7 molecules. At the same time, BCR–antigen phoid follicles, mutations rapidly accumulate
complexes enter B cells via receptor-mediated in genes encoding L- and H-chain variable re-
endocytosis, are processed, and are presented gions, a process called somatic hypermutation.
on the surfaces of the B cells in MHC class II With antibody H- and L-chain variable regions,
molecules. All of this makes B cells very effec- most of the variability appears in three CDRs—
tive APCs (see Figure 13.12) CDR1, CDR2, and CDR3. Somatic hypermuta-
After interaction with Th cells, B cells fol- tion generates much of that hypervariability in
low one of three differentiation pathways. The these CDRs (see Figure 13.14).
first wave of B cells (all expressing IgM BCRs) When the B cell exits the cell cycle after so-
migrates to the medullary cords, differenti- matic hypermutation, that B cell must again
ates into plasma cells, and secretes pentameric acquire antigen for presentation to another Th
IgM. A second set of activated B cells migrates cell. If a B cell fails to once again bind antigen,
to the lymphoid follicles, once again interacts that B cell dies. A ready source of antigen in-
with Th cells, and begins to divide, creating a side of lymph nodes is in the form of antigen–

229

Figure 13.13. Cytokine control of isotype switching in germinal centers
In the upper left corner, a B cell first encounters antigen. Inside of a germinal center in secondary
lymphoid tissue, this B cell interacts with a Th cell (upper right). Depending on the cytokines pro-
duced by the Th cell, the B cell switches from expressing IgM to expressing IgG, IgA, or IgE.
antibody complexes on the surface of FDCs. as the immune response progresses, only B cells
For the newly mutant B cell to acquire this an- with increasingly higher-affinity BCRs survive,
tigen, its new BCR must have a higher affinity resulting in affinity maturation: a continuing
for antigen than the antibody on the FDC. So increase in antibody affinity for antigen during

Figure 13.14. Somatic hypermutation during B-cell division inside of lymphoid germinal centers
Late in primary immune responses and throughout secondary and subsequent immune responses
against a given antigen, mutations rapidly arise in B-cell genes encoding Ig molecules. These muta-
tions fall mostly within the CDR1, CDR2, and CDR3 regions of the L and H chains, shown here as
colored vertical bars.
secondary and subsequent immune responses opsonize bacteria and enhance macrophage-
(see Figure 13.15). mediated destruction of these bacteria. By
These newly formed isotype-switched, high- blocking specific molecules, antibodies can also
affinity B cells follow one of two paths. Some neutralize bacterial toxins as well as viruses.
of them migrate out of the lymph nodes and Antibodies also facilitate NK cell–mediated
eventually arrive in the bone marrow as long- destruction of infected host cells via antibody-
lived plasma cells, where they secrete IgG, IgA, dependent cellular cytotoxicity. Finally, it ap-
or IgE. Another group of isotype-switched, pears that some antibodies can have effects in-
high-affinity B cells remain in the secondary side of host cells.
lymphoid tissues as memory B cells (see Mem- By these means, adaptive immune responses
ory B Cells section). have evolved to deal with each of the compart-
Antibodies secreted by B cell–derived plasma ments mentioned at the outset of this chapter
cells enter the blood, the lymph, and interstitial and provide protection from a variety of patho-
tissues and arrive on epithelial surfaces to par- gens. The final results are primary, secondary,
ticipate in several ways in adaptive immune re- etc., adaptive immune responses. The unique
sponses against extracellular pathogens. In par- features of those responses are their specificity,
ticular, IgM and IgG antibodies, after antigen memory, and affinity maturation (see Figure
binding, can activate complement. Comple- 13.16).
ment components aid in protection in several Low-affinity IgM antibodies dominate the pri-
ways, including the direct lysis of microbes, mary immune response, whereas high-affinity
opsonization of pathogens—which enhances IgG antibodies derived from memory B cells
ingestion and destruction by macrophages and dominate most secondary immune responses.
neutrophils—and enhancement of protective After repeated exposures to antigen, the affinity
inflammatory responses. Also, IgM and IgG can of the antibody for its specific antigen continues

231

Figure 13.15. Affinity maturation through somatic hypermutation and selection
Inside of lymph nodes, as they undergo the process of somatic hypermutation, B cells must repeat-
edly bind antigen through their BCRs to survive. The major source of antigen in germinal centers is
in the form of antigen–antibody (Ag–Ab) complexes bound directly or indirectly (through C3b and
C3d receptors) to FDCs. To bind that antigen, hypermutated BCRs must have greater affinity for anti-
gen than the antibody on the FDCs. When this happens, B cells survive as high-affinity B cells.

Figure 13.16. Primary and secondary adaptive immune responses
to increase. All of these arise because of the tective. Generally, antibody responses are most
unique characteristics of B and T cells and their effective against extracellular infections, and
antigen-specific receptors. Th1 and Tc cells are most effective against intra-
cellular infections (see Figure 13.17).
Different antibody isotypes may provide
Importance of Different Adaptive greater or lesser protection against a given
Responses to Clearance of pathogen. Also, antibodies can provide effec-
Different Pathogens tive protection against intracellular pathogens
As mentioned earlier, pathogens arrive inside by neutralizing the pathogens before they can
animals via different routes, multiply in vari- infect their target cells.
ous spaces, and cause disease in various ways.
Because of this, different classes of pathogens
present different challenges to animals’ immune Immune Responses in the Gut
systems, which is important for several reasons. All of the material presented so far in this
First, understanding the process and outcome chapter is most directly relevant to immune re-
of infections depends on understanding how sponses occurring in the lymph nodes and the
particular types of responses protect against or spleen and, therefore, most relevant to antigens
exacerbate diseases caused by different patho- and pathogens in lymph, interstitial fluids, and
gens. Second, effective vaccination must elicit blood. As mentioned at the outset, though,
protective immune responses. With different there are even larger spaces where animals reg-
pathogens, vaccines may have to induce very ularly encounter pathogens—the epithelia and,
different types of immune responses to be pro- specifically, the gut mucosa.

233

Figure 13.17. Different types of immune effector mechanisms are effective against various pathogens
Plus signs indicate the probable importance of different effector mechanisms to protect immunity
against viruses, bacteria, fungi, and parasites. In general, cellular mechanisms are most effective
against intracellular pathogens, and antibodies are most effective against extracellular pathogens. FIV
= feline immunodeficiency virus; SIV = simian immunodeficiency virus.
Scientists have estimated that there are ap- how to completely avoid infection. Instead,
proximately 1029 bacteria on this planet. The each species exists because life requires a com-
mass of that number of bacteria is equivalent promise with bacteria in the form of commen-
to enough aircraft carriers to cover the United sal and mutualistic relationships. Bacteria, espe-
States three deep—that’s seventy stories high. cially the gut bacteria, are essential to animal
No living species exists because it has learned health. Interestingly, in some species (if not all)

Figure 13.18A–B. Mammalian Peyer’s patches
and lymphoid tissues in the small intestine
Panel A shows a diagram of a thin section of
small intestine. Within this section, the dark
purple–rimmed ovoid areas represent special-
ized lymphoid tissue called Peyer’s patches—
sites of immune responses in the intestine.
Panel B shows a diagrammatic representation
of the interaction between the cells of a Peyer’s
patch and an enteric pathogen. Multifenestrated
or microfold (M) cells carry antigens from the
gut lumen into the Peyer’s patch, where DCs
process and present these antigens to the im-
mune system. As with splenic lymphoid tissues,
the patches have discrete T-cell–rich and B-cell–
rich areas.
the composition of the normal flora is as dis-

tinctive as the animal itself. No two are alike.
The other consequence of the enormous
biological success of bacteria is that all natural
foods are teeming with bacteria and fungi as
well as viruses and perhaps a parasite or two.
One of the most important transitions a newly
born animal makes is moving from a sterile gut
to one carrying much of the normal flora. If an
animal fails to acquire these bacteria, that ani-
mal fails to develop normal gastrointestinal and
immune systems. For all animals, bacterial col-
onization is an essential part of early life. Some-
how, the gut immune system, meaning GALT, of the follicle-associated epithelium of Peyer’s
must deal with all the potential threats and, at patches. Unlike enterocytes, these cells have
the same time, maintain healthy normal flora. no glycocalyx and do not secrete mucus. In-
Because of this, gut immune mechanisms stead, M cells have evolved to interact directly
differ some from the immune mechanisms op- with the antigens of the gut. Using phagocy-
erative in lymph and spleen. Perhaps for differ- tosis and endocytosis, M cells take up antigens
ent reasons, the same appears to be true of im- from the gut lumen and transfer these antigens
mune responses in or on respiratory epithelia across the gut epithelium and deliver them into
and the epithelia lining the major body cavities. the extracellular space on the opposite side of
The gut has several specialized lymphoid the epithelium. Here, APCs acquire these an-
tissues, including tonsils and adenoids. At the tigens, process them, and present them to T
moment, we focus on Peyer’s patches, found cells (see Figure 13.19). Beyond M cells, gut-
mostly in animals’ small intestines (see Figure associated DCs have evolved the ability to in-
13.18A–B). sert pseudopodia (or dendrites) between gut
Specialized cells called multifenestrated or epithelial cells and sample the contents of the
microfold (M) cells make up a significant part gut lumen.

235

Figure 13.19. Antigen transfer across the gut epithelium by M cells
M cells acquire antigens by phagocytosis and endocytosis. The basal surfaces of M cells wrap around
lymphocytes and DCs. As antigens cross the gut epithelia, DCs acquire them, process them, and pre
sent them as MHC–antigen complexes. DCs also extend long dendrites into the gut lumen to sample
and present gut contents.
In addition to Peyer’s patches, foci of plasma them to individual epithelia. In addition to

cells and lymphocytes appear scattered along the conventional α/β T cells expressing CD4+
the length of the small intestine. These are the or CD8+, MALTs contain type b lymphocytes,
effector cells of gut immunity. As with all T and which include CD4−, CD8− γ/δ T cells, T cells
B cells, these cells originate in the bone marrow with γ/δ TCRs, T cells with α/β TCRs, and
and the thymus. Because of specific receptors CD8+ T cells with α:α TCRs. These unique
on their surfaces, however, these B and T cells T-cell subsets do not recognize conventional
home to the gut mucosal epithelia and move MHC–antigen complexes on APCs. Instead,
into the Peyer’s patches. Once there, if these these cells react with several different sorts of
cells find their specific antigens, the B and T molecules and with MHC class IB molecules.
cells become active effector cells and migrate— MHC class IB molecules structurally resemble
via the lymphatics—to the mesenteric lymph classical MHC class I molecules; however, MHC
nodes, and from there to the thoracic duct and class IB molecules do not come from genes in
the blood. Using specialized blood vessels that the MHC.
line the MALT and specific surface receptors, Because several classes of type b lympho-
these effector cells return from the blood to the cytes are present in athymic mice, it is clear
lamina propria and the epithelium of the gut that these lymphocytes do not develop in the
and other mucosa. As a result, an immune re- thymus. Also, type b lymphocytes exhibit little,
sponse that began in an isolated area of the in- if any, gene rearrangement. These cells may
testine spreads to the mucosa, throughout the represent an aspect of innate immunity and not
body, and back to the gut. adaptive immunity, or they may function at the
Similar processes occur within many epithe- boundary of both.
lia, and specialized groups of lymphocytes have A variety of unique cells and immune mech-
evolved specific cell-surface markers to target anisms have evolved to deal with the intimate

and intricate relationship between the gut mu- Intestinal epithelial cells have surface recep-
cosa and the gut flora. As you will see, this rela- tors for pathogens (e.g., TLRs), but only on
tionship somehow makes possible normal gut their basal surfaces. For recognition to occur,
activity and normal lives for most animals. the pathogen must somehow first breech the
As we have explained, the predominant anti- defense of the epithelium. In response to a
body found at mucosal surfaces in most mam- detected infection, gut epithelial cells secrete
mals is IgA and in particular dimeric IgA. The several chemokines that attract a variety of
variety of IgA isotypes can vary a lot, with immune cells (in particular, macrophages and
rabbits expressing thirteen different subclasses neutrophils). These recruited cells, after activa-
of IgA. In amphibians, IgX appears to perform tion by pathogen and cytokines, can stimulate
functions similar to IgA in mammals. Reptiles inflammation, antigen presentation, and innate
use a modified form of IgG at mucosal surfaces, and adaptive immune responses.
and chicken IgA looks more like mammalian Beyond that, it is also clear that the gut flora
IgM than IgA. In spite of these variations, it is themselves provide protection from infection.
clear that all species have evolved special types For example, after treatment with wide-spec-
of antibodies to deal with immunity at mucosal trum antibiotics, animals may succumb to se-
surfaces. This specialization is essential because rious infections with Clostridium difficile. C. dif-
each species faces the same dilemma—an abso- ficile produces two exotoxins that interfere with
lute requirement for gut colonization and con- normal water resorption in the gut, resulting in
stant contact with gut microbes, some of which life-threatening diarrhea and sometimes death
could be potential pathogens. In general, muco- from hypovolemic shock. Usually, the normal
sal immunity is a complex mixture of innate and flora occupy most of the available space in the
innate-like mechanisms along with adaptive im- gut and use most of the available nutrients in
mune responses and a touch of benign neglect. the gut, and some normal flora even produce
The gut immune system, interestingly, not antimicrobial compounds. So, even though C.
only fails to react to most ingested antigens, it difficile is a common inhabitant of animal gas-
often becomes permanently tolerant of those trointestinal tracts, it usually causes no prob-
antigens—a thing called oral tolerance. For ex- lem. When antibiotics strip the protective flora,
ample, if one injects a rabbit with ovalbumin however, the gut epithelia become very suscep-
(the major protein in hen-egg whites), the rab- tible to colonization by the normally innocuous
bit will usually make a powerful adaptive im- C. difficile.
mune response to the foreign antigen. How-
ever, if one first feeds a rabbit hen-egg whites
for a period of time and then injects the animal Immunological Memory
with ovalbumin, the rabbit makes no response
Primary and Subsequent
to the foreign protein. It appears that all ani-
Immune Responses
mals have evolved means to suppress immune
reactions to the food they eat. Biologically, this One of the most remarkable features of
makes sense, because it allows for tolerance to adaptive immune responses is memory (see
most food. Figure 13.16). A second encounter with a
Clearly, though, animals can make immune pathogen is rarely as dangerous as the first. In
responses against gut pathogens, including in- secondary and subsequent infections or vacci-
gested gut pathogens. How is this possible in nations, the immune system responds faster;
the face of the overwhelming tendency toward produces higher-affinity, isotype-switched anti-
tolerance? Infection of gut epithelial cells and body; and maintains higher levels of antibod-
inflammation appear to be key. ies and effector cells much longer. All of this

237

is possible because of memory T and B cells.
In essence, immune systems remember things
they have seen before, and when those same
things appear again, immune systems react
very differently—memory.
Up to now, we have focused most closely on
the nature and events of primary adaptive im-
mune responses, and although memory cells
do begin to appear during primary responses,
their effects are most apparent in secondary and
subsequent immune responses.
Memory cells arise as T and B cells undergo Figure 13.20. Comparison of B cells and antibody
multiple divisions (clonal expansion) after anti- from naive and immunized animals (adapted
gen- and cytokine-driven activation. Just what from C. Janeway, P. Travers, M. Walport, and M.
makes a B or T cell a memory cell is unclear, but Shlomchik, 2001, Immunobiology, fig. 10.24)
several changes occur as cells become memory
cells. These changes are mostly very different be-
tween B and T cells. But one thing seems com- frequency of antigen-specific B cells in immu-
mon to all memory lymphocytes: they have very nized animals increases between 10- and 100-
long lives. Trying to explain this, scientists have fold (see Figure 13.20).
proposed very long-lived memory cells, long- In addition, most B cells from immunized
term persistence of antigen, regular reinfection mice express isotypes other than IgM, and the
with the same pathogen, regular reactivation by antibodies they produce show evidence of hy-
cross-reactive pathogens, and cross-activation by permutation and selection, resulting in much
cytokines produced during unrelated primary higher affinities for antigen (see chapter 12).
and secondary immune responses. Generally, these cells seem to follow the same
Presently, the most likely explanations in- circulation patterns as naive B cells. That is,
volve some aspects of the first and last of these memory B cells travel throughout the circula-
hypotheses. Memory T and B cells do appear tion, the spleen, mucosal epithelia, and the lym-
to have unusually long lives, but not as long as phatics. When their antigens reappear, these
immunological memory may last—sometimes B cells settle into lymph nodes and other sec-
decades or more. Recent studies have shown ondary lymphoid tissues, where they undergo
that memory cells, though mostly found in rest- multiplication and differentiation inside of lym-
ing states, do occasionally divide. Scientists do phoid follicles (see chapter 12).
not understand the stimulus for these divisions,
but certain cytokines, such as IL-15 and IL-7,
seem to prolong memory. So it appears that, Memory T Cells
in addition to their very long lives, occasionally During primary immune responses, after
as cells respond to unrelated antigens, cytokine activation T cells follow two distinct pathways
production may drive infrequent memory cell of differentiation. One group becomes effector
division and expansion. T cells, and the other group becomes memory
T cells (see Figure 13.21).
Several cell-surface changes accompany the
Memory B Cells transition from naive to memory T cell. It may
In vitro, memory B cells differ both quantita- be that among memory T-cell populations are
tively and qualitatively from naive B cells. The several subpopulations. Because of that, al-

immune responses. Memory B and T cells exist
in much higher numbers in previously immu-
nized or infected animals, memory B cells have
higher affinities for antigen than naive B cells,
and memory B and T cells respond faster than
naive cells. For all of these (and probably a few
other) reasons, naive T and B cells do not par-
ticipate significantly after a primary response to
their specific antigen.

In 1981 and 1982, the wolf population on Isle
Royale in Lake Superior fell from nearly fifty
animals to fourteen. Parvovirus, carried in by
humans, was the apparent cause. Canine par-
vovirus is highly contagious and often leads to
severe diarrhea and fatal hypovolemic shock.
What aspects of these wolves’ adaptive im-
mune responses could have been relevant to sur-
vival? Why did fourteen animals survive? What
factors may have contributed to the large die-off
Figure 13.21. Encounterwith specific MHC– of wolves after exposure to canine parvovirus?
antigen generates both effector T cells and
memory T cells
Because all of the wolves on Isle Royale were
though the changes described here seem to be and are descendants of one wolf pack with few
characteristic of many memory T cells, they breeding pairs, it is likely that these wolves are
may not be characteristics of all of them. highly inbred. As we discussed in chapter 8 re-
The observed cell-surface changes include garding African cheetahs and Florida panthers,
the loss of receptors for molecules present on inbreeding may have dramatically reduced not
HEVs and the acquisition of receptors for vas- only the MHC variety among these wolves
cular endothelia in inflamed and infected areas (markedly limiting their antigen-presenting di-
of an animal’s body. In addition, effector and versity) but also the amount of genetic varia-
memory T cells have reduced requirements for tion available for the diversification of BCRs
the second signal normally delivered through and TCRs. Any one or all of these might explain
B7 to CD28. As a result, many memory T cells the large number of wolf deaths after exposure
can respond more rapidly to a secondary in- to parvovirus. However, death rates among do-
fection, generate higher numbers of cells in mestic dogs are in the range of 70 percent, so
shorter periods of time, and more quickly elim- it is difficult to evaluate the contribution of in-
inate these secondary challenges. breeding to these wolf deaths.
Because of their characteristics, memory cells As with most, if not all, viruses, parvoviruses
mediate almost all secondary and subsequent are obligate intracellular parasites. To reproduce

239

themselves, parvoviruses must enter the host BCRs, isotype switch signals, or abnormalities
cell. As discussed earlier, Tc cells deal with most in somatic hypermutation and selection might
intracellular parasites, especially viruses. Acti- have prevented the development of specific an-
vation of T cells requires presentation of spe- tibodies to control the infection.
cific antigenic fragments inside of MHC class I With viruses especially, all antiviral antibod-
molecules. To respond appropriately, a Tc cell ies are not equal. Often, a single critical anti-
must have a TCR reactive with that specific genic epitope is involved in virus attachment to
MHC class I–antigen combination. host cells. When host animals produce antibod-
For these reasons, MHC class I homogene- ies to that critical epitope, they survive because
ity could be a problem, as could relative TCR their antibodies neutralize the virus’s ability to
homogeneity. Because death rates in dogs are infect host cells. Because of that, these antibod-
similar to those in the Isle Royale wolf popula- ies are called “neutralizing” antibodies and are
tion, though, it seems more probable that the the most important target of vaccination.
difference between surviving and dead wolves The generation of a protective adaptive im-
was simple genetic variation. Survivors may mune response is a complex process that de-
have expressed MHC class I molecules that pends, at many stages, on the genetic back-
presented the critical parvovirus epitope, and ground of the infected animal. For this reason,
the MHC molecules of the wolves that died the efficiency of these responses varies among
could not. Alternatively, because of genetic dif- individuals. Identifying the critical gene or
ferences, critical TCRs may have been present genes is rarely possible, but understanding how
only among the survivors. Also, because anti- adaptive immune responses develop and pro-
bodies likely play important roles in controlling tect is a critical step in the process of under-
the spread of viruses from cell to cell, critical standing diseases and their therapies.

Gerald N. Callahan
Fetal and Neonatal Immunity Chapter 14

10.5876_9781607322184.c014
involved bacteria most commonly include Esch-

Clinical Correlation: Failure of Passive
erichia coli (most common), Salmonella spp., Lis-
Transfer 241
teria monocytogenes, Pasturella spp., Streptococcus
spp., Leptospira spp., and Actinobacillus spp.
Fetal Development of the Immune System 241
Untreated, many of these calves, foals, lambs,
Maternal Transfer of Immunity 243
and piglets will die. In comparison, other ani-
Prenatal Transfer of Immunity 243
mals from the same sire and dam will flourish
Postnatal Transfer of Immunity 245
and be infection free. What is the difference be-
Neonatal Acquisition of Memory and
Immune Capacity 248
tween the affected and unaffected animals?
Vaccination of Neonates 249
Clinical Correlation Follow-Up 251 Learning Objectives
• describe, in general terms, the fetal develop-
Clinical Correlation: Failure ment of immune competence;
of Passive Transfer • describe species differences in maternal trans-
fer of immunity and the underlying causes of
Currently, veterinarians estimate that as many
these differences;
as 35 percent of dairy calves suffer from at least
one serious and sometimes life-threatening • describe the routes of maternal transfer of
immunity;
bacterial infection during the neonatal period.
Similar infections occur regularly, although less • explain the rationale for neonatal vaccine
frequently, in beef calves, lambs, piglets, foals, protocols;
and likely manatees (Figure 14.1), making these • understand how neonatal immunity is
infections a major biological and economic measured.
issue for the livestock industry. In foals, the
symptoms of these bacterial infections include Fetal Development of
infections of the navel, the joints, and the lungs the Immune System
and septicemia. Animals will also often have
high fevers and be “off suck,” and when the For many animals, immune system develop-
joints are involved, the animals suddenly be- ment is complete, or very nearly complete, at
come lame. Diarrhea is also common. birth, but the prenatal times at which various
For all neonates, the routes of infection for aspects of immune systems mature vary consid-
bacterial invasion include ingestion, inhalation, erably among species. However, some animals
umbilical infection, and in utero infection. The develop immune competence only after days or
241
241

Figure 14.1. Nursing manatee calf (© Liquid Productions, LLC / Shutterstock)
Placentation in manatees, as with that in other sirenians, is endotheliochorial. As a result, transfer of
antibodies across the placenta is limited, and the timely transfer of colostrum is essential for protec-
tion of the calf.
weeks of life. For example, opossums give birth agents such as bovine viral diarrhea virus do in-
to young after only fifteen days of gestation, duce immune responses. Shortly before birth,
and the newborn joeys have no functional im- as cortisol production rises, the cells of the
mune tissues or organs. Only after seven days adaptive immune system (including lympho-
of life do these animals begin to make antibod- cytes, macrophages, and neutrophils) decrease
ies. The opossum is an example of surprisingly considerably.
rapid immune development, but all of it occurs Although immunocompetent, at birth calves
postpartum. As examples of fetal immune de- are mostly protected by maternally transferred
velopment, we focus on the calf and the chick. immunity and innate immune responses—
Other animals are generally similar, but time of rapid and effective reactions to environmental
appearances and completion dates can all vary pathogens. Innate immunity in calves develops
considerably. late during gestation, but just as with adults, it
The gestation period in cattle is approxi- provides first-line, rapid responses to a variety
mately 285 days, or about nine months. Figure of infectious agents, especially bacteria. Figure
14.2 shows a general outline of immune devel- 14.3 shows a more detailed picture of immune
opment in calves. Sometime before birth, fetal development in cattle.
calves acquire the ability to generate adaptive The thymus and bone marrow appear early,
immune responses to antigens. This ability is and lymphocytes and B cells are present after
apparent because some in utero infections by about sixty days of gestation. Granulocytes and
242 F e t a l a nd N e o n a t a l Imm u n i t y

Figure 14.2. The developing immune system of calves
Passive immunity refers to immunity acquired from the mother. Active immunity refers to the calves’
ability to develop adaptive humoral and cellular immune responses as well as memory cells. (Adapted
from C. L. Christopher et al., 2008, Veterinary Clinics of Northern America: Food Animal Practice, 24: 87)
other cells of innate immunity do not appear cause of this, animals have evolved means for
until later. The mucosal lymphoid tissues are transferring immune capacity from mother to
among the very last to appear and are not fully offspring in utero, immediately after birth, or
active until sometime after birth, which is also both.
true of some of the other elements of innate
immunity.
Maternal Transfer of Immunity
The process in chicks is quite different. Incu-
bation time for domestic chicks is about twenty- The process of maternal transfer of immunity
one days. By five to seven days, stem cells have involves physical translocation of elements and
migrated to the thymus and the bursa. Lym- products of the mother’s immune system into
phoid follicles appear in the bursa by about the developing animal. This process varies con-
twelve days. IgM-positive B cells appear by siderably among species.
about fourteen to sixteen days, and inoculated
chick embryos can produce specific antibodies.
In ovo vaccination for diseases such as Marek’s Prenatal Transfer of Immunity
disease is very effective by seventeen days. IgY- In precocial chickens, maternal antibodies
positive B cells appear at twenty-one days, but are transferred across the follicular epithelium
IgA-positive B cells appear in the gut only about into the yolk during oogenesis. As described
a week after hatching. As with cattle, the innate earlier, chickens produce three classes of anti-
immune functions of chicks are not fully active bodies: IgY, IgM, and IgA. IgY (the equivalent
at birth. It is not until sometime after birth that of mammalian IgG) antibodies cross into the
calves and chickens attain immune competence egg yolk at the highest concentrations. The egg
levels approaching those of adult animals. white contains the greatest concentrations of
So in cattle and chickens, as in most other IgA and IgM. Shortly before hatching, all ma-
animals, neonates’ immune systems are active ternal IgY and some IgM move into the embry-
but not yet fully protective. That leaves newly onic circulation. There, they provide protection
born animals at very high risk for infection be- to the newborn (and immunologically imma-
tween birth and full immune development. Be- ture) chick.
F e t a l a nd N e o n a t a l Imm u n i t y 243
243

Figure 14.3. Timeline for development of cells and organs of the immune system in neonatal calves
Maternal antibodies persist in these chicks for nineteen days. Studies have shown that immu-
fourteen to twenty-one days. During that time nization before day seventeen reduces viable
(by three to seven days), the newborns begin to hatching about 1 percent to 2 percent com-
produce their own antibodies. However, they pared with vaccination on day eighteen. The
do not achieve full immunological maturity site of vaccination is also important (see Figure
until about the time maternal antibodies finally 14.4). For embryonic turkey chicks, it appears
disappear from the chicks’ circulation. that vaccine injection into the amniotic fluid is
The timing varies considerably among bird the most efficient route, but injection into the
species. For example, the half-life of maternal embryo itself is also very effective. Targeted in
antibodies in precocial chickens is somewhere ovo vaccination might seem like a difficult and
from three to ten days, whereas in altricial pas- time-consuming approach to chicken protec-
serine birds, such as house sparrows, the re- tion, but in reality, machines routinely perform
ported half-life of maternal antibodies is closer the task at rates exceeding 50,000 eggs per hour.
to two days. Placental mammals exhibit a range of ana-
The optimum time for vaccinating embry- tomical relationships among the placenta, the
onic chicks is when the stalk of the yolk sac be- mother, and the fetus. There are three types
gins its ascent into the abdomen and the head of mammalian placentas: endotheliochorial,
is nestled under the wing until external pipping epitheliochorial, and hemochorial (see Fig-
(near hatching), which occurs at seventeen to ure 14.5A–B). The character of the placenta

Figure 14.4. Effectiveness of vaccination of chick embryos at various sites
Marek’s disease vaccine (a live, attenuated, viral vaccine) was injected into turkey embryos at the
indicated sites on day seventeen or eighteen. At five days posthatch, the chicks were challenged with
Marek’s disease virus (based on data from P. Wakenell et al., 2002, Avian Diseases, 46: 274).
ultimately determines the routes of maternal Here, there is one less cell layer between the
transfer of immunity. mother’s circulation and the fetus’s circulation
In primates and rodents—because the cho- than in animals with endotheliochorial placen-
rionic villi themselves have eroded through tas and one more cell layer than in animals with
the maternal vascular endothelium—the fetal hemochorial placentas. In dogs and cats, some
chorionic epithelium is in direct contact with placental transfer of immunity occurs, but not
maternal blood. As a result, IgG antibodies as much as in primates. As with other species, in
move freely from mother to fetus in these spe- dogs and cats maternal transfer continues after
cies. Therefore, in primates and rodents, much birth through milk.
maternal transfer of immunity occurs before
birth, but transfer of IgG and IgA continues
after birth through the mother’s milk. Postnatal Transfer of Immunity
Three layers of cells separate the maternal Postnatal transfer of immunity occurs dur-
circulation in the fetuses of horses, cattle, pigs, ing nursing, first with colostrum and later with
and others. In these species, no cross-placental milk. Colostrum is the first milk that accumu-
transfer of maternal immunity occurs. For this lates in the udder, and it is rich in proteins, fats,
reason, all maternal transfer of immunity must and carbohydrates. A few weeks before birth—
occur during nursing. as colostrum forms—antibodies begin moving
In between these species are animals (such as from the blood into the cow’s udder. At the same
cats and dogs) with epitheliochorial placentas. time, mammary-associated lymphoid tissues
245

Figure 14.5A–B. Mammalian placentation
Scientists classify mammalian placentas on the basis of the
number of maternal layers retained in the placenta. The
more layers there are, the more restricted the movement of
blood elements between mother and fetus. Panel A shows
the general relationship between maternal and fetal circula-
tion in the chorionic villi. Panel B shows cross-sections of
the chorionic villi and the different layers between maternal
and fetal blood.
also begin to add antibodies to the colostrum. monomeric IgM, IgE, and dimeric IgA, al-
Beyond Igs, bovine colostrum also contains though the amounts of these antibodies vary
neutrophils and macrophages that secrete sev- between species. The relative abundance of Igs
eral defensive proteins and cytokines, including drops dramatically in the milk of both rumi-
antimicrobial proteins and peptides such as lac- nants and nonruminants, but IgG still predomi-
toferrin (a major antibacterial agent), comple- nates in ruminant milk, and IgA is the major Ig
ment, and lactoperoxidase (also an antibacterial in nonruminant milk.
agent), as well as defensins and cathelicidins, The antibodies that accumulate in colostrum
both of which are bactericidal. Mammary epi- reflect the cow’s past and recent exposures to
thelial cells also secrete various immune-related specific environmental agents (local bacteria, vi-
effector compounds. ruses, etc.), which are the same agents the cow’s
The agents of adaptive immunity include calf will face immediately after birth. These
both antibodies and T cells. In ruminants, the maternal antibodies offer the calf the same pro-
primary antibody in colostrum is IgG1. In non- tection they offered the mother—opsonization,
ruminant animals, IgA often predominates in activation of complement, toxin neutralization,
both colostrum and milk (see Figure 14.7). In virus neutralization, and so forth. Furthermore,
addition to IgG, ruminant colostrum contains by vaccinating cows, veterinarians can manipu-

Figure 14.6. Scottish Highland cow and calf (photograph by Jonathan Sutcliffe)
In cattle, maternal immunity transfers solely via colostrum and milk.
late colostral antibodies. One example of this Another mechanism ensures rapid transport
sort of manipulation involves vaccination of of these Igs into the calf ’s blood and other
pregnant cows against rotoviruses and colostral body fluids. On the epithelial cells lining the
transfer to calves of antirotovirus antibodies. surface of the gut are specialized Fc receptors
Newborn calves may ingest from two to that bind the ingested Igs. Intestinal epithelial
more than six liters of colostrum. Two things cells endocytose the bound antibodies and
help to prevent digestion of the ingested an- move them from the gut and eventually to the
tibodies and other proteins. First, in the first blood and lymph. In cattle, essentially all co-
hours after birth, the gastrointestinal tract of lostral antibodies move quickly from the gut
newborn calves has very low protease levels. to the blood, but in other animals (horses and
Second, colostrum contains high levels of tryp- pigs, for example), most colostral IgA remains
sin inhibitors. Both of these factors prevent im- in the gut, providing protection against enteric
mediate destruction of the transferred proteins. pathogens.
247

After ingestion of colostrum, serum anti-
body levels peak between twelve and twenty-
four hours. Because of normal metabolic
breakdown, absorbed maternal antibodies have
a fixed half-life. Different isotypes disappear at
different rates, and the time at which any of
the antibodies may fall below protective levels
depends on the initial concentrations in the
mother’s colostrum and in the calf ’s serum (see
Figure 14.8).
Once gut permeability reaches zero, fur-
ther maternal transfer must occur through the
mother’s milk, which has proportionally more
IgA than does colostrum. During this period,
most of the antibodies in milk remain in the
nursing animal’s gut, providing longer protec-
tion against enteric pathogens.
In addition to antibodies, bovine colostrum
contains about 1 million lymphocytes per mil-
liliter, and nearly half of these are T cells.
These cells may live as long as thirty-six hours
Figure 14.7. Antibodies in serum, colostrum, and in the calf ’s gut. Furthermore, in as little as two
milk hours, some of these T cells cross the intestinal
The predominant antibody in the serum and epithelium and reach the circulation. A variety
colostrum of most livestock is IgG (ranging of studies have shown that these T cells posi-
from 60 to 90 percent). However, the transfer tively affect the immune status of the calf. Milk,
of IgA from serum to milk differs dramatically however, contains very few or no lymphocytes.
between ruminant and nonruminant animals.
Thus, the transfer of cellular immunity contin-
(Adapted from I. R. Tizard, 2009, Veterinary Im-
ues only as long as the calf ingests colostrum.
munology, 229)
Passive transfer failures may occur because
the mother produced too little or antibody-
depleted colostrum, because the newborn failed
Because in all placental animals the special- to ingest sufficient amounts of colostrum be-
ized Fc receptors are present for only a short pe- fore gut closure, or because gut permeability
riod, successful maternal transfer of immunity was never high enough to allow for absorption
must occur inside of a fairly narrow window. of adequate amounts of antibody (which oc-
During the time the Fc receptors appear on the curs in about 25 percent of newborn foals). Re-
gut epithelium, the gut is permeable, and the gardless of the cause, passive transfer failure is
duration of permeability varies some between a life-threatening event and places the newborn
species. In general, gut permeability is greatest animal at great risk.
immediately after birth, begins to decline by six
hours, and ends by twenty-four hours. Colos-
Neonatal Acquisition of
trum itself appears to accelerate gut closure,
Memory and Immune Capacity
because animals that fail to nurse immediately
retain gut permeability for thirty-three hours or The earliest evidence of neonatal immunity is
longer (as much as ninety-six hours in pigs). the appearance of pentameric IgM. Because of

Figure 14.8. Levelsof maternal antibodies in the sera of cow and calf (Adapted from I. R. Tizard,
2009, Veterinary Immunology, 231)
its considerable size, pentameric IgM can nei- group of protozoan parasites—Eimeria—causes
ther pass through the placenta nor find its way coccidiosis in chickens. Coccidiosis is a disease
into colostrum or milk. So the appearance of of considerable economic importance, especially
this antibody in the neonate is definitive evi- in broiler chickens. In one study, chicks were able
dence of active calf immune responses. to mount fully protective immune responses
As mentioned earlier, the production of IgM against three strains of Eimeria (E. tenella, E. max-
often begins before birth, declines briefly be- ima, and E. acervulina) by twenty-five, twenty-
cause of cortisol production, then rises as the four, and twenty-six days, respectively. Parasite
newborn animal generates its first postnatal im- inoculations (in this case, E. tenella) began on
mune responses. Figure 14.9 shows an approxi- the seventh day after hatching (postmaternal
mate time course for neonatal production of immunity). By day seven, dramatically fewer
antibodies. Cattle usually achieve adult serum oocysts were detectable, and by day twenty-
Ig levels of all isotypes before age six months. five, oocyst production ceased. As a result,
Production of the full range of isotypes re- functional immunity is present early, probably
quires fully functional B cells and Th cells and even before hatching, and chicks achieve a fully
indicates the production of memory cells. Tc- protective immune response within sixteen to
cell activity follows a similar time course. GALT twenty-five days.
immunity and strong gastrointestinal immune
responses are often demonstrable within a few
Vaccination of Neonates
days of birth and probably reach adult levels a
little sooner than systemic immunity. Even though some neonates may be capable
Again, things follow a similar order in chick- of many immune responses, the same mater-
ens, but the timetables are very different. A nal antibodies that protect neonates from lethal
249

Figure 14.9. Maternal and calf antibody levels in newborn calves
For the first twelve weeks or so of calves’ lives, they are protected by maternal antibodies transferred
through colostrum. During that time, the calves’ immune systems are beginning to produce their
own antibodies. By week fifteen, calves’ antibodies are all calf-derived and reach adult levels by about
twenty weeks.
infections pose a formidable obstacle for vacci- that, maternal antibodies begin to decline. The
nation. In at least two ways, maternal antibod- rate at which individual isotypes decline var-
ies can interfere with vaccination (see Figure ies within and between species, and the rate of
14.10). Maternal antibodies may bind to injected catabolism of antibodies against different anti-
pathogens or antigens and clear the inoculum gens also varies. Therefore, determining an ap-
before activation of enough cells to stimulate propriate vaccination protocol for a given anti-
a new immune response. Because erythrocytes gen must be an empirical process. For example,
have Fc receptors, these cells bind small anti- the half-life of maternally transferred antidis-
gen–antibody complexes and very quickly clear temper antibodies in dogs is about eight days
such complexes from the blood, preventing and reaches negligible levels after about ten to
effective antigen processing and presentation. twelve weeks. The half-life of maternally trans-
Also, maternal antibodies may block essential ferred rinderpest antibodies in cattle is about
BCR epitopes and prevent the formation of thirty-six days, and the antibody reaches negli-
neutralizing antibodies. gible levels only after about eleven months.
Because of this, timing of vaccination and As a result, neonatal vaccination is a trade-off
boosters in neonates has to be different from between the ideal levels of maternal antibod-
protocols for adult animals. As mentioned ear- ies and the earliest possible protection for the
lier, maternal antibodies absorbed from the gut young animal. For example, in puppies the ideal
reach peak levels within the first 24 hours. After level of maternal antibodies (near zero) against

canine distemper virus occurs between ten and
twelve weeks. Considering only that data, it
might make the most sense to wait until some-
time after twelve weeks to vaccinate for canine
distemper. However, the rate of maternal an-
tibody decline varies a lot between puppies,
and the consequence of distemper infection in
unvaccinated animals can be catastrophic. In
response (for mostly practical reasons, such as
economics and convenience), veterinarians do
not simply vaccinate puppies every week or so
until immune protection is certain. As a com-
promise, canine distemper vaccination begins
at six weeks and is repeated at nine, twelve, and
fifteen weeks, usually as part of a five-way vac-
cine that also includes vaccines for adenovirus
cough and hepatitis, canine distemper, para-
influenza, and parvovirus. Some combination
vaccines may also include leptospirosis (seven-
way vaccines), coronavirus, or both.
In calves, Clostridium infections cause a va-
riety of very serious diseases, including black-
leg, malignant edema, sord, black disease,
red water, and three kinds of enterotoxemia.
Early protection against Clostridium is essen-
tial. As in dogs, using a seven- or eight-way
vaccine, calves may be vaccinated as early as
one month, again four weeks later, and once
more before weaning. The intent is the same
as the vaccination protocol described earlier—
immunize early and often to protect the maxi-
mum number of animals at the earliest pos-
sible date.
Figure 14.10. Maternal antibodies inhibit

vaccination Clinical Correlation Follow-Up
Maternal antibodies may bind to injected Student Considerations
antigens or pathogens and accelerate their
clearance, or maternal antibodies may bind to As mentioned at the outset of this chapter, a
critical antigen epitopes and prevent activation significant percentage of dairy calves are unusu-
of B cells and antibody production. Also, RBCs ally susceptible to early bacterial infections, es-
bind antigen–antibody complexes and quickly pecially E. coli. Grossly, these animals appear no
remove those complexes from the blood. Ab = different from healthy calves; however, blood
antibody; Ag = antigen. work reveals that in comparison with healthy
calves, the affected calves have near-normal lev-
els of IgM but much lower levels of IgG. Similar
251

Figure 14.11 Nursing bison calf (© Leena Robinson / Shutterstock)
Bison, like other bovids, basically have an epitheliochorial placenta, but cells from the trophoblast in-
vade the uterine epithelium, where they form binucleate giant cells. Thus, the ruminant placentation
is most commonly called synepitheliochorial.
lesions likely occur in other ruminants (Figure mother. Therefore, the finding of normal lev-
14.11) els of IgM but low to no IgG suggests passive
transfer failure.
As mentioned earlier in this chapter, passive-
Possible Explanations transfer failures may occur because the mother
The absence of IgG in the calves’ blood could failed to produce sufficient colostrum, failed to
be due to several things, including immuno incorporate sufficient Igs into her colostrum, or
deficiency diseases, but the near-normal lev- lost colostrum before the calf could nurse. The
els of IgM suggest something else—failure of calf, in turn, may not have nursed long enough
passive transfer. Because the newborn calves or failed to adequately absorb the Igs from the
have no memory cells and all initial immune re- ingested colostrum.
sponses will be primary immune responses, the Regardless of the reason, failure of passive
presence of IgM is consistent with a normally transfer of immunity has placed these calves
functioning immune system. Calf-derived IgG at great risk for life-threatening infections. Be-
will only reach normal levels weeks to months cause of this risk, early identification of passive-
later as the animals’ immune systems fully transfer failure and immediate supplementa-
mature. Early calf IgG is all derived from the tion with stored colostrum are essential.

Gerald N. Callahan
Vaccination Chapter 15
10.5876_9781607322184.c015
Remember, neonatal animals’ immune systems

Clinical Correlation: Animal Vaccines 253
contain mostly naive T and B cells. Because of
that, in young animals all immune responses
Vaccines 253
are primary responses, and those take time—
Vaccine Characteristics 253
sometimes too much time. Vaccination helps to
Live-Attenuated Vaccines 255
shorten the time it normally takes to develop
Killed Vaccines 259
rapidly responding memory cells.
Component, Toxoid, Conjugate, Recombinant,
and Naked DNA Vaccines 259
Adjuvants 263 Learning Objectives
Administration 266
Frequency 266
Passive Immunotherapy 267 • describe the properties of effective vaccines
Clinical Correlation Follow-Up 268 and explain the reasons for each of them;
Student Considerations 268 • describe the characteristics of the various
Possible Explanations 268 forms of vaccines and explain the advantages
and disadvantages of each;
Clinical Correlation: • explain the need for as well as the chemistry

Animal Vaccines and immunology of the most commonly
used adjuvants;
Because of their lack of memory cells, puppies • discuss the possible routes of vaccine admin-
(Figure 15.1)—as with the young of most spe- istration and the advantages and disadvan-
cies—are especially susceptible to infectious dis- tages of each;
eases. Even in the developed world, infectious
diseases are among the leading causes of death
• discuss the reasoning behind vaccination
schedules;
among puppies and kittens as well as livestock
and among the leading causes of morbidity and • explain the immunology behind specific
issues for vaccines against specific pathogens.
mortality in feral animal populations. For this
reason, people and their domestic animals re-
ceive a raft of immunizations to protect them Vaccines
from infectious diseases. Figure 15.2 shows the
recommended schedule for dogs. Vaccine Characteristics
As we discussed in chapter 14, as soon as ma- In 1796, Edward Jenner inoculated James
ternally transferred immunity begins to wane, Phipps with cowpox, and the modern era of
young animals become susceptible to infection. vaccination was begun. Sarah Nelmes, a milk-
253
253

Figure 15.1. Puppies (© AnetaPics / Shutterstock)
maid, had contracted cowpox (variola virus) antibodies and T cells that react with similar
from Blossom, a dairy cow. Jenner had noticed antigens of smallpox virus. These anticowpox
that smallpox—then ravaging much of Eu- antibodies provide protection against the more
rope—seemed rare among milkmaids. Jenner virulent pathogen. However, an attempted im-
reasoned that infection with cowpox somehow munization with even a few of the intact small-
prevented later infection with smallpox. To test pox viruses would often be fatal.
his theory, he collected pus from the cowpox This is true of many pathogens. As with
blisters on Nelmes. He then injected this pus re- smallpox virus in humans or parvovirus in dogs,
peatedly into James Phipps (the son of Jenner’s inoculation of even a few virus particles is often
gardener). Finally, Jenner injected young James enough to induce disease. The first require-
with virulent smallpox from an infected person. ment for a vaccine is safety in the majority of
The boy survived, and vaccination (from vaccus, the target animals, which requires identification
Latin for cow) became widespread. or creation of a less pathogenic or nonpatho-
Although it would not be understood for genic form of the disease agent. Underlying
more than a century, Jenner had taken advan- this process is an attempt to copy what Jenner
tage of the antigenic similarity between two found fortuitously—a less dangerous microbe
viruses, cowpox or variola virus, only mildly that shares antigenic epitopes with the virulent
pathogenic in humans, and smallpox virus, pathogen.
which causes a disfiguring and often fatal dis- The other obvious issue for vaccine design is
ease in humans. Because the two viruses have identifying the type of immunity most likely to
some antigenic epitopes in common, an im- be protective. In general, humoral immune re-
mune response against cowpox virus generates sponses are most effective against extracellular
254 V a cc i n a t i o n

pathogens, and humoral plus cellular immune Table 15.1. Essentials for useful vaccines
responses are most effective against intracel- Safe Does not cause illness or death
lular pathogens. For example, many antiviral in even a small percentage of
recipients; minimal side effects
vaccines, such as canine parvovirus vaccines, by
design induce both cellular immunity and pow- Protective Prevents or reduces illness
caused by pathogen
erful antibody responses.
Provides long-lasting Confers sustained resistance on
The cellular immune response that parvovi-
effects vaccinated animals
rus vaccines induce is essential to eliminate in-
Induces formation of Stimulates production of anti-
fected host cells. But cellular immunity alone protective antibodies bodies reactive with appropri-
is ineffective against free viruses (viruses as ate antigenic epitopes
they enter the host are extracellular, as they are Induces formation of Stimulates production of cel-
when they move between host cells). For this protective T cells lular immunity; particularly
reason, the antibodies induced in response to important for intracellular
parvovirus vaccines—because they help limit pathogens
the number of cells infected after virus expo- Other issues Low cost, stable, easily
sure and limit the spread of virus from cell to administered
cell—are also essential for protection. Finally,

because large numbers of animals will receive
the vaccine, it must be immunogenic and non- to produce antiviral vaccines, but live-atten-
toxic for most, if not all, recipients (see Table uated vaccines are also used against bacterial
15.1). agents, such as canine bordetella.
The route and frequency of administration Live-virus vaccines are generally more effec-
are also important. Initially, we consider the com- tive and longer lasting than vaccines contain-
position of the vaccine. Generally, successful ing killed viruses, probably as a result of sev-
vaccines include eral factors. Live viruses continue to replicate
• live-attenuated pathogens; inside of their hosts, which accomplishes two
things: first, viral replication increases the time
• heat-killed or otherwise inactivated forms the immune system is exposed to antigen and
of the agent;
the total antigen load. Also, because live viruses
• subunit or component vaccines containing will replicate in the cytoplasm of infected cells,
only pieces of the pathogen;
peptides from viruses are more efficiently pre-
• toxoid (inactivated bacterial toxins) sented inside of MHC class I molecules. Both
vaccines;
of these factors enhance immunity, including
• conjugate vaccines using an immunostimu- production of CD8+ T cells that are most effec-
lant to induce a response to a harmless tive in eliminating virus-infected cells and virus-
piece of a pathogen; neutralizing antibodies.
• recombinant vaccines using a harmless Again, the initial problem is the virulence of
virus expressing a gene product from the the unattenuated virus—the amount of virus
pathogen; necessary to induce an immune response is also
• naked DNA vaccines. often enough to induce disease and sometimes
death. An attenuated virus retains many of the
physical characteristics (especially antigenic
Live-Attenuated Vaccines
epitopes) of the original virus but is less viru-
One approach to vaccine development is to lent. To achieve attenuation, vaccine makers
produce a less virulent strain of a pathogenic usually grow viruses in cultured cells from an-
agent. This technique has been used most often other species. For example, canine parvoviruses
V a cc i n a t i o n 255
255


Figure 15.2. Canine vaccination schedule
Core vaccines for dogs are those that should be given to every dog. Noncore vaccines are recom-
mended only for certain dogs. Whether to vaccinate with noncore vaccines depends on a number of
things, including the dog’s age, breed, and health status; potential exposure to an animal with the dis-
ease; the type of vaccine; and how common the disease is in the area in which the dog lives or visits.
Source: American Veterinary Medical Association.
have been attenuated by passage in insect cells. host, and that evolution reduces the virus’s abil-
By selecting for the viruses that grow best in ity to effect disease in its natural host.
these insect cells, the investigators were able to When we vaccinate dogs with these live-
isolate a strain of parvovirus that had a lower attenuated virus vaccines, the attenuated infec-
capacity for infection of canine cells (see Figure tion induces sufficient levels of danger signals
15.3). When the virus was repeatedly cultured (particularly DAMPs) to activate APCs. This
on insect cells, mutations began to accumulate, combination induces a protective immune re-
and these mutations allowed the virus to more sponse, but the attenuated viruses infect far
efficiently infect insect cells. These same mu- fewer dog cells and—because of that—induce
tations generally reduced the virus’s ability to milder or no symptoms. Because the attenuated
infect canine cells. In essence, these virions ex- virus retains essential antigenic epitopes, the
perience accelerated evolution in an unnatural immune response to the attenuated parvovirus
257

Figure 15.3. Production of a live-attenuated virus
vaccine
To produce a live-attenuated virus, vaccine
manufacturers grow cultured dog cells along
with virus from an infected dog to produce
large numbers of the virus. They then transfer
this virus to insect cells. Over time, mutations
accumulate in the viral genome. These muta-
tions make the virus more efficient at infect-
ing insect cells and less efficient at infecting
dog cells. Vaccinating dogs with these viruses
may induce immunity without widespread
infection and disease. Because the attenuated
virus retains essential antigenic epitopes, the
immune response to the attenuated parvovirus
induces cross-protection against live, virulent
parvovirus.
induces cross-protection against live, virulent

parvovirus.
Advances in molecular genetics now allow
for precisely directed gene modification—an
ideal approach to the generation of specifically
engineered, attenuated viruses. Viruses pro-
duce disease by affecting essential functions of
specific host cells. Scientists sometimes call the
genes that allow viruses to do this “virulence”
genes. Equipped with molecular techniques
and some knowledge of the pathogenicity
factors of a given virus or bacterium, site-di-
rected mutagenesis within the virulence genes
or complete removal of the virulence genes
from a viral genome can generate attenuated
strains of these agents. These viruses may re-
main highly infective for host cells and induce
very effective host immunity but have little po-
tential for pathogenicity. Because attenuated
virus retains the majority of the components
present in the native virus, the vaccine-induced
immunity is also protective against the native
virus. Because of rapid progress in the realm of
genetic manipulation of transmissible agents, live-attenuated vaccines for dogs include the tri-
vaccine manufacturers either now use or will valent vaccine for herpes virus, calcivirus, and
soon use these techniques to generate the ma- parvovirus; distemper vaccines; and bordetella
jority of live-attenuated vaccines. Examples of vaccines.

Live-attenuated vaccines, in spite of their any consequences. After all, T cells can only
huge successes, have risks. The catch is that “see” protein antigens after these molecules
completely avirulent strains do not make good have been chopped into pieces and presented
vaccines, so the level of attenuation has to inside of MHC molecules. Not so for B cells. B
be titrated to achieve enough disease to elicit cells’ BCRs bind intact antigens, and this bind-
a strong immune response, but not enough ing often depends absolutely on the structure
for it to be clinically apparent. However, if an of the antigenic proteins. When an animal re-
animal’s immune system is somehow compro- ceives a killed vaccine, most of the activated B
mised (e.g., an immunodeficient animal), the cells bind the denatured form of the protein,
virus, even in its attenuated form, can cause se- and ultimately these B cells will produce anti-
vere, sometimes fatal disease. Also, the low po- bodies that react only with denatured proteins.
tential for virulence of a live-attenuated virus Such antibodies are likely to be of little use
depends on the mutations accumulated during when the animal later encounters the native
the attenuation process. As these viruses divide pathogen.
inside of the vaccinated animal, in rare cases Furthermore, as mentioned earlier in this
additional mutations can alter the infectivity chapter, killed pathogens do not replicate inside
of the virus in host cells. Because of this, live- of host cells. Although some crossover between
attenuated vaccines may still cause severe and antigen presentation pathways occurs, MHC
sometimes fatal disease in immunized animals. class I presentation of the antigens from these
However, because they are among the best at killed pathogens is not as efficient as with live
meeting all of the criteria listed in Table 15.1, pathogens. Last, killed microorganisms do not
live-attenuated virus vaccines are among the persist for as long nor do they multiply inside
most common and most effective vaccines in the vaccinated animal.
all species. For these reasons, killed vaccines are gener-
ally not as effective as live-attenuated vaccines.
Regardless, many effective killed vaccines exist,
Killed Vaccines including some canine, feline, and equine ra-
Obviously, inactivated viruses, dead bacteria, bies vaccines.
or other killed pathogens cannot cause disease One major plus for killed vaccines is that
or death (except through allergic responses). the immune status of the animal is less critical.
Because of this, one of the earliest methods Even among immune-compromised animals,
for vaccine preparation involved using chemi- there is essentially no possibility of causing mi-
cal or physical (particularly heat) means to kill croorganism-induced disease. But, as with live
bacteria or inactivate viruses. Pasteur’s rabies vaccines, because killed vaccines contain many
vaccine is one example. potential allergens, there is the danger of aller-
In addition to heat, killing or inactivating gic responses and anaphylaxis.
microorganisms for vaccine preparation has
involved radiation, chemicals, and antibiotics.
The target is, of course, dead but immunogenic Component, Toxoid,
microorganisms. That’s the key. Heating, radia- Conjugate, Recombinant,
tion, and these other procedures are most effec- and Naked DNA Vaccines
tive at killing microorganisms, but not without Obviously, immune responses against all
immunogenic consequences. All of these pro- components of a microorganism are not nec-
cedures result in dramatic alterations in the mi- essary for protection. For example, swine and
croorganisms’ proteins through denaturation. avian influenza A viruses use only two mol-
For T cells, denaturation generally has few if ecules to infect host cells—hemagglutinin and
259

neuraminidase. Vaccination with influenza bacteria involves opsonizing antibodies that aid
A virus will produce antibodies against many macrophages in phagocytosis and destruction
components of the virus, but only when an of the encapsulated bacteria. However, under
animal’s immune system produces antibodies normal circumstances in adult mammals, the
against hemagglutinin and neuraminidase will polysaccharides of bacterial capsules, even
the animal be protected against subsequent in- though they bind to some B-2 or regular B cells,
fection. Vaccination with intact live virus is al- directly activate only B-1 B cells. Th cells do not
ways potentially dangerous. So why not simply recognize and respond to polysaccharide anti-
vaccinate with hemagglutinin and neuramini- gens; thus, there are no antigen-specific T cells
dase molecules, that is, create a vaccine that to help polysaccharide-specific B-2 B cells.
contains only components of the virus—a com- Sometimes, IgM antibodies derived from
ponent vaccine? Then there would be no risk of B-1 B cells are protective, but activation of B-1
inducing influenza with the vaccine. B cells does not result in memory. Moreover,
It turns out that immune systems do not young animals have relatively fewer B-1 B cells
handle isolated proteins very well. Immune re- and are at much greater risk for disease. The
sponses that occur after vaccination with individ- major aim of vaccination is immunological
ual proteins are weak, include only poor cellular memory—a faster and more specific secondary
immunity, and are often ineffective against intact response. So, under normal circumstances, vac-
microorganisms. So until recently, very few ef- cination for protective immunity is impossible,
fective component vaccines existed. However, but conjugate vaccines circumvent this major
one very effective approach uses components block (see Figure 15.4).
of pathogens: toxoid vaccines. Many Gram- As mentioned earlier, even though B-2 B cells
negative and Gram-positive bacteria (such as bind some polysaccharide antigens, because
Clostridium tetani) produce exotoxins that dam- there are no specific Th cells, B-2 B cells do not
age host animals. Toxoid vaccines contain inacti- respond further. APCs effectively process and
vated bacterial toxins. For example, tetanus tox- present only proteins, which means that al-
oid vaccine contains formaldehyde-denatured though many polysaccharide antigens possess
tetanus toxin. In this case, formaldehyde de- epitopes reactive with BCRs (B-cell epitopes),
naturation inactivates the toxin but leaves be- these antigens lack epitopes reactive with
hind enough antigenic epitopes to induce an T cells (T-cell epitopes). To change the immu-
immune response, especially antibodies reac- nogenicity of bacterial-capsular polysaccha-
tive with the native toxin. Vaccinated animals rides, vaccinologists attached an immunogenic
respond very quickly to subsequent exposure protein, tetanus toxoid, to provide a T-cell epi
to tetanus and rapidly neutralize the otherwise tope—a protein portion that APCs can process
fatal neurotoxin. and present to Th cells. When vaccinologists
Because of new understanding about how injected the conjugate into animals, it bound to
immune systems work, it is now also possible BCRs through its polysaccharide portion, was
to make effective conjugate vaccines using mi- internalized, and was processed. Then the B
croorganism components linked (conjugated) cell presented tetanus toxoid-derived peptides
to other T-cell–activating molecules. Vaccines to Th cells. The Th cells produced the requisite
against encapsulated bacterial pathogens are cytokines, and the B cells divided and eventu-
examples of this technology. ally differentiated into both B memory cells and
Several pathogenic bacteria contain polysac- plasma cells expressing antibodies specific for
charide capsules surrounding their cell walls. the bacterial polysaccharide. The resultant an-
These capsules interfere with phagocytosis. The tibodies effectively opsonized the encapsulated
most effective immunity against encapsulated bacteria, preventing disease. At the same time,

Figure 15.4. A conjugate vaccine against capsular polysaccharide
Under normal circumstances, bacterial polysaccharides fail to activate B-2 B cells, primarily because
these polysaccharides do not contain T-cell–activating epitopes. Conjugation of an immunogenic
protein (tetanus toxoid) to the polysaccharide provides the needed T-cell epitope. This conjugate
activates toxoid-specific Th cells and polysaccharide-specific B cells. The end results are production of
protective antipolysaccharide antibodies and memory T and B cells.
memory cells offered vaccinated animals pro- Another approach to vaccine preparation
tection in future encounters with the bacteria. has been to insert genes from pathogens into
Similarly, scientists have identified many relatively innocuous viruses and use this re-
other T-cell epitopes—portions of proteins that combinant vector for vaccination. Called re-
bind to MHC molecules and are recognized by combinant vaccines, these constructs have had
TCRs. As described earlier, when coupled with great success in treating important veterinary
these molecules, antigens or pieces of antigens diseases. Most notable among these is rabies
that bind to BCRs but normally fail to stimulate in wild animals. In 1984, workers at the Wistar
antibody production can effectively immunize Institute in Philadelphia isolated the gene that
animals and elicit memory cells. encoded the essential coat glycoprotein of the
261

proved very effective in stimulating immunity
against rabies virus.
Traditionally, because of the large wild ani-
mal reservoir, control of rabies in domestic
animals had never been very effective. The
availability of a potent oral vaccine offered a
new avenue to rabies control (see Figure 15.5).
Dropped from aircraft, these vaccines dra-
matically lowered the incidence of fulminant
rabies in wild foxes in Europe and raccoons,
coyotes, and foxes in the United States, and re-
sulted in a much lower incidence of rabies in
general.
Another vaccine approach uses structures
called immune-stimulatory complexes (or IS-
COMs). By themselves, many peptides are poor
immunogens. At least one reason for this is that
these peptides do not often enough get inside
Figure 15.5. Oral rabies vaccine
of APCs and into both MHC class I and class II
molecules. One approach to solving this prob-
Packets such as this one contain live recombi-
nant rabies vaccines. Dropped from aircraft, the lem is to incorporate peptide antigens into lipid
packets were very effective in controlling rabies micelles (see Figure 15.6).
in wild animal populations. The lipids serve multiple purposes. They
provide a carrier for the antigenic peptides and
stimulate innate immune responses (see Ad-
juvants section). Finally, lipids easily fuse with
rabies virus. They then inserted this gene into APC cell membranes, delivering the peptides
the genome of vaccinia virus. Vaccinia virus into MHC class I pathways of presentation. Be-
is closely related to cowpox virus (remember cause some of the micelles are also acquired by
Sarah Nelmes) and was first widely used as a endocytosis, the peptides also arrive in MHC
vaccine for smallpox. By itself, vaccinia has very class II antigen-presentation pathways.
low potential for virulence. One of the most unusual approaches to vac-
Vaccinia virus does, however, infect animals cine development involves the use of naked
and induce a strong immune response. By in- protein-encoding DNA. Today, it is often easy
serting the rabies glycoprotein gene into vac- to identify the portions of a microorganism’s
cinia, the scientists created a relatively innocu- DNA that encode essential antigenic proteins
ous virus that expressed an essential rabies or epitopes. In some cases, after injection of this
glycoprotein. Once the vaccinia virus infected DNA into muscle, injected animals make anti-
host cells, it produced not only vaccinia compo- bodies against peptides encoded by the DNA.
nents, but the rabies glycoprotein as well. The underlying mechanisms are not entirely
When APCs phagocytosed these infected clear but, within host cells, the injected DNA
cells, the rabies glycoprotein entered the MHC generates RNA and proteins that APCs eventu-
class II pathway and, through cross-presenta- ally engulf, process, and present to T cells. Pres-
tion, the MHC class I pathway, inducing strong ently, naked DNA vaccines are few, but in the
antibody and T-cell responses. Even when ad- near future these vaccines may become more
ministered orally, that recombinant vaccine common.

Figure 15.6. The use of ISCOMs as vaccines
Combining poorly immunogenic peptides with lipid micelles generates more immunogenic com-
plexes. When injected, these micelles fuse with membranes of APCs, effectively delivering the antigen
in both MHC class I and class II pathways.
In the end, regardless of the approach, the less pathogenic, the need for adjuvants and un-
purpose of vaccination is to place the immuno- derstanding about how they work has become
gens where they will interact with components essential.
of both innate and adaptive immunity, espe- It is often true that the more refined an anti-
cially aspects of both sorts of immunity most gen is, the less immunogenic it is. For example,
critical to protective immunity. effective live-attenuated vaccines may require no
adjuvant at all, but killed and component vac-
cines usually do. As you learned in chapter 10,
Adjuvants APCs present normal host antigens all the time
Immunologists have known for decades that but do so in an unactivated state (low expression
antigens alone were often not enough to induce of costimulatory molecules and proinflamma-
protective immune responses. Good vaccines tory cytokines). Administration of large doses
usually required the addition of an adjuvant— of antigen without proinflammatory stimuli
a substance that enhanced the immunogenicity (e.g., PAMPs, DAMPs, and activating cytokines)
of an antigen without altering the specificity may actually have the opposite effect—T-cell an-
of the immune response. The most common ergy and tolerance to the antigen. As a result,
early adjuvants included oil, emulsifiers, and just delivering the antigen is not enough; the
bacterial cell-wall components, with or without APCs must also receive signals that the adminis-
aluminum salts. At first, no one knew much tered antigen is potentially hazardous.
about what adjuvants did, but vaccine makers The primary goals of vaccine design are to
understood their necessity. This consensual stimulate a strong immune response of the
ignorance once led Charles Janeway, a famous appropriate type (cellular, humoral, or both)
immunologist, to refer to adjuvants as “our inexpensively and with minimum toxicity. Ad-
dirty little secret.” In the past few years, atti- juvants can have major effects on all of these as-
tudes about adjuvants have changed. Particu- pects of vaccination. And because of their im-
larly as vaccines have become more refined and portance to vaccine efficacy, the components of
263

adjuvants are among the most closely guarded juvants, the adsorbed antigens remain mostly
secrets of the vaccine industry. at the site of injections and are phagocytosed
Most adjuvants help to stimulate more ag- more efficiently, considerably enhancing im-
gressive innate immune responses. As men- mune responses.
tioned earlier, innate immune responses play Immunostimulatory and carrier effects re-
major roles in initiating and directing the adap- sult when adjuvants more specifically affect
tive immune response. In general, adjuvants cells of the innate immune system. Most cell
affect the immune response in one of three membranes, including those of APCs, have a
ways. First, some adjuvants—such as aluminum net-negative charge. The net-positive charge of
salts—act by creating a depot effect. That is, cationic liposomes attracts them to APCs and
some adjuvant–antigen combinations dissipate other cells. When cationic liposomes fuse with
very slowly from the site of injection. Without APCs, incorporated antigens readily move into
such an adjuvant, injected antigens spread very antigen-presentation pathways, enhancing im-
quickly in lymph or blood. Because many immune responses. Also, adjuvants containing an-
portant APCs are almost exclusively outside of tigens in oil-in-water or water-in-oil emulsions
lymph and blood, the immunogenic effects of enhance interactions between immune cells
the vaccine are thus dramatically reduced. Sec- and stimulate either Th1- or Th2-directed im-
ond, many adjuvants (particularly liposomes) mune responses. Again, macrophages more ef-
act as carriers, effectively delivering antigen fectively ingest and process emulsified antigens,
into APCs. Last, some adjuvants have immuno- and the emulsions persist longer than simple
stimulating properties (see Figure 15.7). aqueous solutions.
The most commonly used adjuvants include Some of the most potent adjuvants con-
liposomes, microspheres, ISCOMs, minerals tain immunostimulating compounds derived
(particularly aluminum salts such as alum), from pathogens. These adjuvants include ago-
water-in-oil emulsions and oil-in-water emul- nist to PRR, such as the TLR agonists. These
sions, and agonists for PRRs (such as TLRs). All molecules mimic PAMPs or DAMPs that bind
of these act by one (or more) of three means: to PRRs and activate APCs. These synthetic
antigen-depot formation, antigen-carrier effects, copies of pathogen-derived molecules activate
or immune stimulation. mast cells and APCs, enhance antigen presenta-
Antigen-depot effects result from introduction, stimulate inflammation, and initiate spe-
ing antigens in a form that does not quickly cific sorts of Th cells and immune responses.
dissipate into intravascular fluids. Neutral lipo- Because of improved understanding of the in-
somes do this because they are large, remain nate response, many of these sorts of immune
at the injection site, and can fuse with and in- stimulants already exist, and many more are
troduce antigens into APCs. Similarly, attach- likely to be available soon.
ing antigens to relatively inert microspheres or Other sorts of immune stimulants include
other particles slows dissipation and enhances cytokines, such as IL-2 and IL-12, that stimu-
phagoctyosis. Antigens adsorbed onto mineral late Th1 cells. These sorts of stimulants favor
salts, especially alum, not only dissipate slowly very specific responses in immunized animals,
from the injection site but also stimulate the responses that help to push immunity toward
eventual development of Th2 cells and anti- antibody and T-cell responses. For this reason,
body responses. Aluminum salts (especially these immune stimulants may one day allow
alum), perhaps the most commonly used ad- veterinarians to direct immune response along
juvant, are among the best at creating an anti- the most effective pathways.
gen-depot effect (but the effects of aluminum In general, adjuvants include a variety of
salts are likely more complex). With these ad- antigen-unrelated compounds that act to non-

Figure 15.7. Adjuvants and their interactions with antigens
specifically enhance the immune response, adaptive immune response grows, it seems
often through enhancement of aspects of in- clear that immunological adjuvants will come
nate immunity. As understanding of the innate to play an even greater role in vaccination and
immune response and its interactions with the the direction of the immune response.
265

Administration vaccinated animal is most likely to again en-
In some cases, the route of vaccine adminis- counter Bordetella bronchiseptica, one bacterium
tration may make little difference. For example, responsible for pertussis.
Vibrio anguillarum is a Gram-negative bacterium Oral vaccines offer especially attractive ad-
that infects many fish, but especially intensively vantages—they involve a painless procedure for
farmed fish, such as salmon. In one study in- the patient and are easy to administer. As with
volving trout and immunity to V. anguillarum, IN vaccines, oral vaccines directly engage the
investigators found that whether they adminis- mucosal immune system and are effective at in-
tered the vaccine orally, by immersion, or via ducing protective mucosal immunity. The Sabin
intramuscular injection, the vaccinated fish pro- polio vaccine is a good example. One major
duced similar amounts of antibody that reacted, polio transmission route is through feces left
for the most part, with the same antigenic epit- in places such as public swimming pools (fecal–
opes on the bacterium. oral transmission). The virus then enters the
This is not always the case. Often, the route host via the gastrointestinal system. For this rea-
of immunization is very important to the in- son, mucosal immunity is key in defense against
duction of protection. Routes of vaccination polio. Again, because of the predominance
include some surprising options, such as intra- of the mucosal response, the oral vaccine has
ocular, intraperitoneal, intrathecal, intracranial, proven very effective. Similarly, the oral rabies
intravenous, and immersion (as in the preced- vaccine is especially effective because it induces
ing example). In practice, however, the most a mucosal immunity that helps to neutralize the
commonly used routes are intramuscular (IM), virus at epithelial surfaces. It is likely that many
subcutaneous (SC, although sometimes Sub Q), more ways exist in which vaccines might take
oral, and intranasal (IN). advantage of mucosal immune systems, but a
Among these, IM and SC are the most com- necessary first step will be greater understand-
mon. These routes offer several immunologi- ing of how mucosal immunity works.
cal advantages. First, after IM or SC injection,
the inocula tend to form small depots of an-
Frequency
tigen that slowly leak into the surroundings.
Second, muscles and, especially, subcutaneous Frequency of administration is another major
tissues contain large numbers of mast cells and issue with any vaccine. In general, immunity
APCs, including DCs. When properly stimu- induced with live vaccines lasts longer than im-
lated, mast cells help to induce innate immune munity induced by killed vaccines. Two factors
responses that are essential to the development play major roles in veterinary vaccination: the
of adaptive immune responses. The inflamma- status of the animal’s immune system and the
tion that ensues draws macrophages and neu- longevity of the immune response.
trophils and sends DCs to the secondary lym- As discussed in chapter 14, the blood and
phoid tissues. These all help to stimulate robust other fluids of neonatal animals often contain
immune responses. large amounts of maternal antibodies, perhaps
Some vaccines, such as the IN canine pertus- even some maternal T cells. The presence of
sis vaccine, do not necessarily induce as much these antibodies makes effective immunization
inflammation, but they do immediately engage impossible. That is why, under normal circum-
the mucosal immune system, including muco- stances, veterinarians give no vaccines before
sal B cells. As a result of activation, some B cells four to five weeks of age. But even then—be-
ultimately isotype switch and begin producing cause the exact point at which maternal anti-
pathogen-specific IgA, and IgA, in its dimeric bodies become few enough to allow for vacci-
form, returns to mucosal surfaces, where the nation varies from animal to animal and species

Figure 15.8. Cow–calf pair (© Christian Musat / Shutterstock)
to species—the results of early vaccination are Passive Immunotherapy

difficult to predict. For this reason, veterinar- The intent of passive immunotherapy is to in-
ians give most early vaccines repeatedly and at ject enough preformed antibodies to bind and
relatively short intervals. The aim is to provide neutralize antigen (commonly toxins) and pro-
protection as soon as possible after the mater- tect the host animal. Immunologists call it pas-
nal antibodies disappear. sive immunotherapy or passive transfer (some-
Beyond this, efficacy studies should deter- times even passive immunization) because the
mine the frequency of administration of any host animal plays no active role in the develop-
vaccine. Efficacy studies, over long periods of ment of immunity against the toxins. Passive
time, measure the levels of antibodies (anti- transfer induces no host immunity against the
body titers) in vaccinated animals and estab- toxins, produces no memory cells, and lasts
lish when antibody levels fall so low they are only as long as the antibodies persist in the in-
no longer protective. Regardless of the nature jected animal (usually a matter of weeks), just
of the vaccine, there is simply no other way as with the passive transfer of maternal immu-
to establish the necessary frequency of revac- nity discussed in chapter 14.
cination. Unfortunately, vaccination regimens Regardless of its shortcomings, passive im-
are not always determined this way, and rec- munotherapy can be a powerful aid in the
ommended frequencies may be unnecessarily treatment of several diseases, including those
high. For this reason, with animals at risk for induced by toxins (such as tetanus toxin), in-
problems because of excessive vaccinations, halation of anthrax, and even animal cancers.
practitioners often measure antibody titers be- As we have described, antibodies have a wide
fore revaccinating. range of effects, and their sudden introduction
267

Table 15.2. Recommended vaccination schedule for cattle
Timing and goal Vaccination
Prebreeding: The goal is to provide protection against IBR, BVDV, PI-3, BRSV (MLV preferred)
pathogens of general health concern and those that Leptospirosis (five strains; two doses); may also consider
may increase pregnancy wastage. These vaccines should L. hardjo vaccine
be completed 30 days before breeding. Campylobacter fetus (vibrio) if using bulls
Clostridium (seven-way)
Precalving: The goal with precalving vaccinations is to Rotavirus, Coronavirus, E. coli (for calf scours)
enhance colostral antibodies and protect against early
lactation pathogens.
Four to six months—weaning: The goal is to provide IBR, BVDV, PI-3, BRSV (MLV preferred)
protection against common pathogens that may cause Clostridium (seven-way)
problems when colostral antibodies begin to fall off. Mannheimia/Pasteurella (vaccine needs to contain
toxoid component)
Note: IBR = infectious bovine rhinotracheitis; BVDV = bovine viral diarrhea virus; PI-3 = parainfluenza virus; BRSV =
bovine respiratory syncytial virus; MLV = modified live vaccine.
into an animal can dramatically change the contain adjuvants. The recommended vaccine
course of some diseases. Possible risks always for pasteurella contains a toxoid.
include type III hypersensitivities and anaphy- On the basis of the material in this chapter,
lactic shock, but prophylactic treatments such you should be able to offer plausible explana-
as anti-inflammatory and immunosuppressive tions for the differences in the composition and
drugs can greatly reduce these risks. routes of administration of these vaccines.
Clinical Correlation Follow-Up Possible Explanations

Student Considerations Each potential cattle pathogen presents a
Obviously, calves and adult cattle (Figure unique set of problems to the animals’ im-
15.8) face many infectious challenges. To help mune system and to vaccine designers. Cattle
protect cattle, clinical researchers recommend will most likely encounter parainfluenza virus
a series of vaccinations throughout the lives at the respiratory mucosa. Protective immune
of these animals. (Table 15.2 shows the rec- responses against parainfluenza virus will likely
ommended vaccination schedule for cattle.) require both cellular and humoral immune re-
Each infectious agent presents a particular set sponses—antibodies to slow or prevent virus
of challenges, and the vaccines for each agent from infecting epithelial cells and Tc cells to
must address those immunological issues. For destroy virus-infected cells. Furthermore, the
this reason, each vaccine is different in essential best antibodies for the job are IgA antibodies
ways. because of their presence and actions at muco-
At least one parainfluenza virus (PI-3) vac- sal surfaces. For these reasons, the vaccine con-
cine contains live-attenuated virus and is admin- tains live virus to stimulate both T-cell and hu-
istered IN. The virus in this vaccine is a temper- moral responses. Because the viruses are live,
ature-sensitive mutant of the pathogenic para no adjuvant is needed, and by administering the
influenza virus. This mutant will grow only in vaccine IN, the potential for production of IgA
the relatively cooler respiratory tissues of cattle. antibodies is greater.
Most leptospirosis vaccines contain inactivated Leptospirosis, however, is caused by a spi-
virus and are administered IM or SC, and most rochete, Leptospira interrogans. The bacterium

grows extracellularly and moves from animal Finally, the pasteurella vaccine contains a
to animal through urine, milk, aborted fetuses, toxoid. Pasteurella multocida, the Gram-nega-
and contaminated water. The most effective tive organism responsible for pasteurellosis,
immune responses against extracellular bacte- produces an exotoxin that interferes with as-
ria include antibodies, primarily IgG and IgM, pects of signal transduction and causes a se-
because both can opsonize bacteria and both vere respiratory syndrome in cattle. Effective
can activate complement—essential elements vaccination for pasteurellosis requires the in-
of antibacterial immune responses. The vac- clusion of the toxoid (inactivated Pasteurella
cine includes an adjuvant as well, most likely multocida toxin) to provide toxin-neutralizing
one that activates macrophages and helps to in- antibodies.
duce both Th1 and Th2 responses—important Because each organism challenges animals’
for antibody production, activation of macro- immune systems in particular ways, so must
phages, and inflammation, and all essential in each vaccine engage animals’ immune systems
antibacterial immune responses. in correspondingly specific ways.
269

Gerald N. Callahan
Immune Deficiencies and

Chapter 16
Immune-Mediated Diseases
10.5876_9781607322184.c016
ply from prolonged contact. Instead, cats shed

Clinical Correlation: Feline Immuno-
FIV in saliva and transmit the virus through bite
deficiency Virus 271
wounds. Vertical transmission appears rare.
FIV infects, among others, CD4+ T cells—Th
Immune Deficiencies 272
cells. As with HIV, FIV-induced disease pro-
Congenital Immune Deficiencies 272
gresses through five stages:
Acquired Immune Deficiencies 273
Immune-Mediated Diseases 274 1. acute infection (four to sixteen weeks);
Hypersensitivities 274 2. asymptomatic carrier (months to years);
Type I hypersensitivities 274 3. persistent generalized lymphadenopathy
Type III hypersensitivities 279 (usually very short and sometimes missed
Type IV hypersensitivities 282 by cat owners);
Type II Hypersensitivities and Autoimmune 4. AIDS-related complex (including chronic
Diseases 283 oral fungal infections and gastrointestinal
Clinical Correlation Follow-Up 284 and respiratory disease);
Student Considerations 284 5. AIDS, including respiratory infections
Possible Explanations 284 (canine herpes and calciviruses); oral infec-
tions (mostly fungal); ocular disease, includ-
ing glaucoma; chronic diarrhea (bacterial,
Clinical Correlation: Feline parasitic, malignant); and skin and ear
Immunodeficiency Virus infections (parasites, yeasts, and bacteria).
After the development of AIDS-related
Feline immunodeficiency virus (FIV) infects complex or AIDS, infected cats survive less
about 2 percent of domestic cats in the United than one year.
States. Infection rates are higher among free-
roaming and feral cats—around 8 percent (see
Figure 16.1). FIV is also enzootic in some large Learning Objectives
cats, such as snow leopards, tigers, Florida pan- After reading this chapter, you should be able to
thers, bobcats, and African lions. The effects
of FIV infection on these wild populations are • describe the nature and possible causes of
mostly unknown, but the virus appears to have congenital immune deficiencies;
much less impact in the wild. • describe the nature and possible causes of
Like HIV, FIV is a retrovirus—an RNA virus acquired immune deficiencies;
that uses reverse transcriptase to generate a • classify and explain the bases of types I, III,
DNA copy of its RNA and inserts that copy and IV immune-mediated hypersensitivities;
into the host’s genome. Unlike feline leukemia • understand the nature of autoimmune dis-
virus, transmission of FIV does not result sim- eases, including type II hypersensitivities.
271
271

Figure 16.1. Impact of feline immunodeficiency virus in the United States
Infection rates among symptomatic and high-risk cats. (Data from the National FeLV/FIV Awareness
Project, sponsored by the IDEXX Corporation.)
Immune Deficiencies tious agents, as well as other drugs. Exposure

of mother or fetus to any of these agents can
Perhaps the most familiar immune deficien-
lead to suppression of the maternal and fetal
cies are acquired immune deficiencies (AIDS)
immune systems, which can result in severe im-
induced by retroviral infections in some mam-
mune deficiencies in the neonate. In general,
mals. However, immune deficiency disorders
these environmental agents damage immune
come in many forms.
systems through interference with or destruc-
tion of essential elements of the innate or adap-
Congenital Immune Deficiencies tive immune responses. For example, steroids
Congenital immune deficiencies are those (especially glucocorticoids) suppress produc-
present at birth. These deficiencies may result tion of several important cytokines, includ-
from the mother’s exposure to environmental ing IL-1, IL-2, IL-4, IL-6, and gamma IFN. All
agents such as over vaccination, vaccinating of these play critical roles in the development
during the mother’s heat cycle or pregnancy, of innate (especially inflammatory) and adap-
antibiotics, steroids, protein malnutrition, too tive immune responses. Glucocorticoids also
few calories, vitamin or mineral insufficien- induce apoptosis in some T-cell populations
cies, hormones, and viruses and other infec- and directly affect B cells. The result is a pro-
272 Imm u n e D e f i c i e nc i e s a nd Imm u n e - M e d i a t e d D i s e a s e s

nounced immune suppression. Sex hormones Acquired Immune Deficiencies
may also have similar effects on immunity, as Acquired immune deficiencies arise later in
can malnutrition. life, sometime after birth. Some of the best-
The most common causes of congenital known immune deficiencies result from viral
immune deficiencies, though, are genetic mu- infections, such as AIDS, but animals can ac-
tations. We have already described several of quire immune deficiencies in a variety of ways.
these diseases, including SCID in Arabian foals, The most common causes include
XLSCID in basset hounds, and IgA deficiencies
affecting several dog breeds. • nutritional deficiencies;
Each of these diseases results from a muta- • drugs;
tion that affects a gene critical to the develop- • stress;
ment of immune responses. Foals with SCID • aging;
lack a functional DNA-dependent protein ki-
nase; dogs with XLCID lack the γc chain, an
• microbes.
essential component of receptors for IL-2, As we have described, clonal expansions
IL-4, IL-7, IL-9, IL-15, and IL-21; and IgA-de- among both T and B cells are essential parts of
ficient dogs lack the genes necessary for func- immune responses, as are multiple rounds of T-
tional isotype switches to IgA and effective and B-cell division that generate effector popu-
mucosal immunity. Each of these diseases re- lations essential for defense against infection,
sults in reduced or no ability to defend against which takes a lot of ATP and a lot of nutrients.
infections, sometimes resulting in premature As a result, immune systems have high nutri-
death. tional requirements. Because of these require-
These examples are probably just the tip of ments, what might seem like a relatively minor
the iceberg. Effective immune responses rely on nutritional deficiency can result in a significant
complex series of genes, cytokines, receptors, immune deficiency that manifests as repeated
MHC molecules, cells, tissues, and organs. A infections or lack of response to vaccination.
defect in any element of the series can diminish Glucocorticoids such as hydrocortisone,
an animal’s ability to generate effect defensive prednisone, and dexamethasone are strongly
responses. immune suppressive and anti-inflammatory.
Sometimes these defects are subtle, and In addition, both chemotherapeutic and radio-
sometimes not. Recognizing something such therapeutic cancer drugs, such as methotrexate
as SCID in Arabian foals is easy because of and cisplatin, are immunosuppressive. Metho-
the dramatic recurrent infections and deaths trexate prevents the normal synthesis of thy-
among affected animals. Detecting minor midine, a base essential for DNA replication.
changes is much harder. Animals’ abilities to Cisplatin causes cross-linking of DNA, which
respond to infectious threats vary considerably, leads to apoptosis. Both chemicals are effective
even within a single species or within a single against cancers because these drugs have their
litter. Some of this variation reflects subtle dif- greatest impact on the DNA of rapidly divid-
ferences in immune competence, changes that ing cells, as does radiation therapy. Immune re-
can range from undetectable to fatal. The same sponses also depend on rapid cell division, and
can be said about animals’ responses to vaccina- for that reason, these drugs are often powerful
tion; individual animals can show great varia- immune suppressants.
tion in immunity after vaccination. Although In animals as divergent as horses and eels,
this variation is not usually considered to reflect cortisol is a major adrenal glucocorticoid. When
underlying congenital immune deficiencies, to an animal is stressed (ranging from perceived
some extent it clearly does. dangers to prolonged and repeated physical
Imm u n e D e f i c i e nc i e s a nd Imm u n e - M e d i a t e d D i s e a s e s 273

273

stress), the adrenal glands produce higher lev- In spite of all the checks and balances and all
els of cortisol. Through its actions on T and B of the selections that occur, immune systems
cells, cortisol, like other glucocorticoids, sup- sometimes overreact to innocuous agents and
presses the immune system. Under normal cir- sometimes turn on self. The diseases that result
cumstances, this suppression may offer impor- are immune-mediated diseases or hypersensi-
tant protection against autoimmunity, but in tivities (see Figure 16.2).
repetitive stressful situations, cortisol can lead Although several immune-mediated diseases
to pronounced immune suppression. do not fall neatly into any one category, immu-
Also, as all animals age, they experience a nologists have divided hypersensitivity reac-
progressive decrease in immune capacities. The tions into four major categories—types I–IV.
exact rate of decline and character of these de- Types I–III are the immediate-type hypersen-
creases can be hard to predict. In general, older sitivities, all of which involve antibodies. After
animals are less responsive to vaccination and initial exposures, these antibodies reach high
more susceptible to infections. levels in sera and tissues and initiate responses
Finally, infectious agents cause acquired im- very rapidly (immediate type). T cells medi-
mune deficiencies. The most familiar of these ate type IV responses, and even after initial ex-
is virus-induced AIDS, such as the feline AIDS posures, it takes time to generate sufficient T
that arises after infection with FIV (discussed cells to mount an apparent response. For this
at the beginning and end of this chapter). But reason, immunologists also call type IV hy-
with the apparent exception of prions, immu- persensitivities delayed-type hypersensitivities
nosuppressive examples are found among every (DTHs).
class of transmissible agent. Type I hypersensitivities are true allergies
For example, after infection with viruses, and involve IgE antibodies. Type II hypersen-
such as canine parvovirus, animals often be- sitivities involve antibodies produced against
come more susceptible to bacterial infections. self components and are autoimmune diseases.
This increase in susceptibility is the result of Type III hypersensitivities, also known as im-
the immunosuppressive effects of the virus. In mune complex diseases, often arise when large
truth, many infections result in varying levels amounts of preformed antibodies encounter
of immune suppression; the ones bearing the large amounts of antigens and form immune
acronym AIDS simply represent the most se- complexes too large to remain soluble in
vere of these syndromes. plasma.
Immune-Mediated Diseases Hypersensitivities Type I, III, and IV
Immune responses are, of necessity, toxic. Im- Type I hypersensitivities

mune responses have evolved to destroy things: As we have discussed, type I hypersensitivi-
bacteria, viruses, fungi, parasites, infected host ties are the true allergies, marked by the in-
cells, perhaps even tumor cells. Because of that, volvement of IgE, mast cells, and sometimes
all animals have evolved sophisticated means basophils and eosinophils. Signs of allergies can
for controlling immune responses—Treg cells, range from corn-induced gastritis and vomiting
cytokines, receptors and their ligands, apopto- in dogs to toe tapping in parrots, from an an-
sis, the thymus itself, positive selection, nega- noying dermatitis to a fatal anaphylactic shock,
tive selection, central tolerance, and peripheral but the underlying immune mechanisms are
tolerance all help to control immune responses alike, and all involve IgE.
and try to limit the opportunities for animals’ Antigens that cause allergies are called aller-
immune systems to turn against their hosts. gens. When an animal encounters an allergen

Figure 16.2. Immune hypersensitivities
Immune-mediated diseases fall into four major categories, types I–IV. Antibodies mediate the first
three of these categories, and they are immediate-type hypersensitivities. T cells mediate type IV
responses, including delayed-type hypersensitivities (DTHs). Ag = antigen; CTL = Tc cells.
for the first time, no grossly observable re- as an allergen-specific mast-cell receptor. This
sponse occurs. However, inside of an allergy- point is essentially where the primary response
prone animal, much is happening. First, APCs ends, with thousands upon thousands of armed
process and present the allergen to Th cells (in mast cells and probably also basophils and
particular Th2 cells), which in turn interact eosinophils.
with antigen-presenting B cells. Those B cells On secondary and later exposures, the al-
then undergo an isotype switch from IgM to lergen binds directly to the IgE on the mast
IgE (see Figure 16.3), which is key. Clearly, some cells, which causes the cells to degranulate, re-
nonallergic animals also make antibody releasing large amounts of histamine and other
sponses against allergens, but they do not make mediators. After this, the mast cells begin to
IgE responses. synthesize and secrete more proinflammatory
The IgE produced in the primary exposure mediators—such as prostaglandins and leuko
moves from the blood into the perivascular trienes—just as they do during other inflamma-
spaces, where it binds to specific Fc receptors tory responses (see Figure 16.4 and chapter 5).
on mast cells. These specialized Fc receptors The release of these factors leads to vasodi-
(named FcεRs) have a very high affinity for lation, increases in vascular permeability, swell-
IgE and stably maintain IgE on the surface of ing, smooth-muscle contraction plus bron-
mast cells for long periods, essentially acting chospasm, and recruitment and activation of

275

Figure 16.3. Type I hypersensitivity
On first encounter (upper left), APCs acquire the allergen, process it, and present it to CD4+ T cells
that eventually differentiate into Th2 cells. In response, the Th2 cells interact with and activate B cells.
In the presence of IL-4, these B cells switch from IgM to IgE production. The IgE antibodies migrate
from the blood into surrounding tissues, where they bind to specific Fc receptors on mast cells. On
the second encounter, the allergen binds to the IgE on mast cells and induces mast-cell degranulation,
which leads to inflammation, swelling, smooth-muscle contraction, and recruitment and activation of
leukocytes.
leukocytes. These cells further enhance the on- tered this current vaccine, the cat’s mast cells
going inflammation (see Figure 16.5). were armed with IgE. The antigens in the vac-
The results of this process can be annoying cines bound to the IgE on the cat’s mast cells
things such as flea allergies or the following and caused the release of large amounts of
scenario: histamine, prostaglandins, and leukotrienes
and other proinflammatory cytokines, which
On a routine well visit for a six-year-old
caused systemic vasodilation and increased
indoor–outdoor cat, the veterinarian adminis-
vascular permeability. Because the volume of
tered a vaccination for rabies and decided, as
the circulatory system had suddenly increased
long as the cat was in, to give it a feline leu-
dramatically, the cat’s blood pressure crashed,
kemia virus vaccination a couple of months
leading to anaphylactic shock. Fortunately, the
early. Ten minutes later, the cat was lying flat,
veterinarian recognized this as an allergic re-
purple, struggling to breathe, with no detect-
sponse, implemented critical care measures,
able blood pressure.
and saved the cat’s life.
It was not the first time the cat had received Why the cat responded this way is unclear. Al-
either vaccine, but it was the first time the though clear genetic predispositions to allergies
animal had ever reacted this way. After earlier exist, allergies may arise suddenly at any time,
vaccinations, the cat had formed IgE antibod- and many animals acquire them later in life. At
ies with one or more of the components of some point, this cat switched from making IgG
the vaccines. When the veterinarian adminis- to making IgE in response to the vaccines, and

Figure 16.4. Products of activated mast cells
After antigen binds to and cross-links IgE molecules on the surface of mast cells, the cells release
their stored granules. The first two rows show the contents of these granules. The bottom three rows
show synthesized mast-cell products. The net effect is to initiate and maintain inflammation. MIP =
macrophage inflammatory protein.
at this same point, the animal became at risk for on the immune status of the animal, the dose
vaccination-induced anaphylactic shock. of the allergen, and the route of exposure (see
Clearly, the manifestations of allergic re- Figure 16.6A–B).
sponses can vary dramatically. The eventual Intravenous exposure to large amounts of
outcome of an exposure to an allergen depends antigen holds the greatest risk for sudden,

277

Figure 16.5. Mast cells and type I
hypersensitivities
When allergens bind to IgE attached to
mast cells, the mast cells release granules
and begin to synthesize several other
factors. Factors in the granules as well
as those synthesized later have effects on
the gastrointestinal tract, the airways,
and the blood vessels.
systemic anaphylaxis. Generally, subcutaneous Th2-cell development, and Th2 cells are essen-
exposure poses less of a threat. Inhaled and tial for the isotype switching of B cells to IgE.
ingested allergens can induce widely varying Also important are the cytokines produced dur-
responses—from rhinitis and diarrhea to ana- ing interactions between Th2 cells and antigen-
phylactic shock—depending on the amount presenting B cells. Again, no way currently ex-
of allergen and the immune status of the host ists to predict the antibody isotype an animal
animal. will produce after exposure to an antigen.
Why some animals produce IgE in response Beyond allergies, it seems that IgE antibodies
to certain antigens and other animals do not is must have a more productive role in defense.
not clear. Two things favor IgE production: dif- Evidence suggests that IgE antibodies are im-
ferentiation of Th0 cells into Th2 cells and the portant elements for defense against infections
actions and products of those Th2 cells. by multicellular parasites (such as insects and
Factors important to the production of Th2 helminths). As a result of their interactions
cells include the cytokines produced during an- with Fc receptors on mast cells, IgE antibodies
tigen presentation and during the subsequent end up distributed at the sites where animals are
development of those Th2 cells, the intrinsic most likely to encounter such parasites—skin,
properties of the antigen, the dose of antigen, lung, and gut epithelia. Another important fac-
and the route of exposure to antigen. The pres- tor seems to be inflammation. In the presence
ence of IL-4, IL-5, IL-9, IL-10, and IL-13 favors of inflammation, Th1-cell development pre-

Figure 16.6A–B. The route of allergen exposure affects the clinical manifestation of the allergy
Mast cells appear in large numbers in two places in an animal’s body: in the connective tissues sur-
rounding blood vessels (panel A) and in the mucosa of the gut and airways (panel B). The severity of
the response to an allergen depends on which and how many mast cells are activated.
dominates; without inflammation, Th2-cell de- Currently, among several animal popula-
velopment is most common. The fact that most tions the incidence of allergies is increasing sig-
allergens appear at epithelial surfaces and are nificantly. In the developed world, the most af-
unlikely to initiate inflammation may help to fected populations are house pets and humans.
explain allergic responses. However, by them- This observation prompted several studies, and
selves, these observations are not enough to together they suggest that early exposure to
explain allergic reactions because these factors some infectious agents, particularly bacteria, is
often do not differ between allergic and nonal- essential for normal immune development. An-
lergic animals. Mast cells and basophil products imals intentionally shielded from infections do
also enhance IgE production, but again there not develop normal immune or gastrointestinal
are no observable differences between afflicted systems. People’s inclination toward sterilizing
and unaffected animals. their and their pets’ environments may be hav-
Some animals have clear genetic predisposi- ing pathological consequences.
tions. Offspring of allergic animals are more
likely to develop allergies themselves. But be-
cause so many factors are involved in immune Type III hypersensitivities
responses, just what those genetic variations Type III hypersensitivities are also known as
might be remains largely unknown. Environ- immune complex diseases because the underly-
mental factors also contribute to the risk of ing cause of type III hypersensitivities is antigen–
allergies. antibody complexes. Here is a hypothetical

279

Figure 16.7. Type III hypersensitivities and immune complexes
When an excess of antibodies exists, each antigen is completely coated with antibodies and not acces-
sible for binding into large complexes (left panel). When an excess of antigens exists, one or no anti-
bodies bind most antigens and no large complexes form (right panel). When antibodies and antigens
are equal or nearly so, these molecules can link together to form large complexes (center panel).
scenario (based on an actual scenario) that in- and rush to the emergency veterinary clinic.
volves a type III hypersensitivity and a veteri- Again the veterinarian begins therapy and
narian’s major oversight: supportive care for the snakebite and initiates
antivenin therapy. The next morning the dog
You regularly take your dog into the foothills
seems fine, and the veterinarian discontinues
for long, uneventful (except for the occasional
antivenin treatment. Several hours later, the
rabbit) walks, but today is different. Today,
dog’s temperature rises, her blood pressure
your dog spooks up a rattlesnake and gets
crashes, she goes into kidney failure, and her
bitten. You pick up the dog and carry her to
breathing becomes very labored.
your car, cover her with blankets, and head
to the nearest emergency veterinary clinic. This scenario describes a type III hypersen-
There, the veterinarian begins therapy for sitivity called serum sickness. Type III hyper-
the snakebite and administers antivenin, sensitivities occur when large and roughly
a concoction containing antibodies to the equivalent amounts of antibody and antigen
snake venom antigen. After a few days in the abruptly come together. Because antibodies are
hospital, your dog seems to have shaken off multivalent, each antibody molecule can bind
the snakebite. multiple antigens, and because most natural
You move to another town and several antigens are also multivalent, each antigen can
months later you are out walking with your bind to antibody at multiple positions, mean-
dog again. Again, she rousts an angry rattler ing that, under the right conditions, antibodies
and gets bitten. Again, you gather up the dog can link multiple antigens together into massive

chains of antigen–antibody complexes (see Fig-
ure 16.7).
These complexes quickly become insoluble
because of their size. They then fall out of solu-
tion in the blood and deposit onto the vascular
endothelium, where they activate complement.
As a result, C3b fragments appear and attach to
the endothelium. Neutrophils, via their Fc and
C3b receptors, bind to the IgG and C3b of the
complexes, which, in turn, stimulates release of
enzymes that damage vessel walls (see Figure
16.8).
In addition to C3b, complement activation
results in the formation of C5a, a powerful ana-
phylatoxin (inducer of anaphylaxis; see Figure
16.9). C5a acts in several ways to enhance in-
flammation, up to and including anaphylactic
shock. C5a induces blood vessel dilation and in-
creased vascular permeability, activates macro-
phages and epithelial cells to produce TNF (an-
other major proinflammatory cytokine), and
activates neutrophils, all of which combine to
produce the outcome in the hypothetical case
described earlier.
Most antivenins are not simply antivenoms.
Most are horse sera containing antivenom an-
tibodies. Administration of antivenin after the
first snakebite not only blocked the effects of
the snake venom, but also induced immune
responses to all of the protein components of
the horse serum. At the time of the second an-
tivenin treatment, the possibility of a type III
hypersensitivity reaction was high—lots of pre-
formed antibodies and lots of antigens. As the
antibody levels rise and the serum protein an-
tigens wane (see Figure 16.10), however, serum
sickness can occur even on a dog’s first expo-
sure to horse serum.
Once the veterinarian discontinued the anti- Figure 16.8. Immune complexes and blood vessel
venin in this dog, antigen levels began to fall. damage
When the serum antigen and antibody levels Large immune complexes are insoluble and,
reached the point of equivalence, large im- as they form, deposit onto blood vessel walls.
mune complexes formed and precipitated There, these complexes activate complement,
which results in the appearance of C3b. Neutro-
onto blood vessel endothelia, which triggered
phils bind to IgG and C3b and release destruc-
complement activation, neutrophil-induced tive enzymes.
blood vessel damage (leading to glomerulo

281

Figure 16.9. Actions of C5a
The C5a fragment of C5 stimulates a variety of cells, including epithelial cells, endothelial cells, mac-
rophages, and neutrophils. The net result is a dramatic increase in inflammation. Both antigen–anti-
body complexes and Gram-negative sepsis activate complement and release C5a.
nephritis), and systemic anaphylaxis, including ceptible to poison ivy dermatitis. APCs ingest,
bronchospasms. process, and present the catechol-modified pro-
Because of this possibility, veterinarians rou- teins to Th1 and Tc cells. Once they emigrate
tinely scratch-test animals to assess sensitivity from the secondary lymphoid tissues, effector
before initiating antivenin therapy. Even after T cells migrate to the affected skin. On second-
negative scratch testing, therapy with immuno- ary exposure, local Th1 cells induce inflam-
suppressive drugs often accompanies antivenin mation, and newly created Tc cells migrate to
therapy. the inflamed areas to induce apoptosis in cells
presenting catechol-modified peptides in MHC
class I molecules.
Type IV hypersensitivities In this case, the results are the classic rash,
Type IV hypersensitivities are unique among itching, and pain of poison ivy, but a number of
the hypersensitivities, because T cells, not anti- other agents can—using the same means—also
bodies, are the underlying causes of the clini- induce contact hypersensitivities. These agents
cal manifestations. Because of this, type IV hy- include
persensitivities take longer to develop. Type IV
hypersensitivities have two major forms: DTHs
• insecticides (flea collars, sprays, and dips);
and contact hypersensitivities. Clinically, con- • wood preservatives;
tact hypersensitivities are the most relevant. • carpet dyes;
Th1 cells mediate DTH responses. A classic • dermatology creams and ointments;
example is a positive response to tuberculin • leather;
skin testing. CD8+ Tc cells cause contact hyper- • paints;
sensitivities, and the classic example is poison
ivy dermatitis. In this case, oils (catechols) on
• some house plants.
the poison ivy leaf surface absorb into animal Essentially, anything that contains chemi-
skin and attach to host proteins. Dogs’ fur cals capable of binding to and modifying
helps to protect them from this phase, but hair- skin proteins is a potential cause of a contact
less areas, such as the abdomen, are very sus- hypersensitivity.

Figure 16.10. Serum sickness
After a first or subsequent injection of foreign serum, the host animal begins to make antibody to
serum components. When, during the course of treatment, the antigens and antibodies reach the
point of equivalence, large immune complexes form and precipitate onto blood vessel walls.
Type II Hypersensitivities and macrophages. Under these conditions, erythro-

Autoimmune Diseases poiesis cannot keep pace, and the red cell count
Antibodies also mediate type II hypersensi- drops dramatically.
tivities, but these antibodies are different from As is the case with all autoimmune diseases,
those described earlier. The antibodies of type scientists do not know why animals with IMHA
II hypersensitivities are anti-self antibodies, produce self-reactive antibodies. A complete
which makes most type II hypersensitivities au- list of animal autoimmune diseases would fill
toimmune diseases. For example, immune-me- a page or two, and although some of these dis-
diated hemolytic anemia (IMHA) is a relatively eases are not antibody mediated (type II hyper-
common disorder of dogs, especially in cocker sensitivities), many are. Some examples include
spaniels, old English sheepdogs, poodles, and diabetes in dogs, cats, gorillas, horses, and oth-
Irish setters. Its most common symptom is se- ers; systemic lupus erythematosus (SLE) and
vere anemia. During IMHA, these animals’ im- thyroiditis in dogs; and immune-mediated ar-
mune systems are making antibodies against thritis in many species.
their own RBCs. When those antibodies bind to Obviously, antibody-mediated autoimmune
their antigens, complement activates, enhanc- diseases come in many forms and are far from
ing both inflammation and phagocytosis by rare in animals. It is clear that both environmen-

283

tal and genetic factors (as demonstrated by breed
predispositions in dogs) contribute to these dis-
eases. In most cases, the essential genetic factors
remain unknown. Also, with one exception (ce-
liac disease), the environmental factors for auto-
immune diseases of animals are unknown.
Other autoimmune diseases result from the
actions of self-reactive T cells and still others
from the combination of self-reactive antibod-
ies and T cells. At the root of them all, though,
are self-reactive Th or Tc cells. Self-reactive B
cells by themselves do not pose an equivalent
risk, because most B cells cannot proliferate
or differentiate into antibody-secreting plasma
Figure 16.11. Affected cat
cells without the aid of Th cells.
In chapter 9, we discussed central and pe-
ripheral means for maintaining tolerance. Ob- uses reverse transcriptase to make a DNA copy
viously, these processes are inadequate to elimi- of the viral RNA. That DNA inserts itself into
nate all autoimmune responses in animals. Re- the nuclear genome inside of the infected cell,
cent evidence has suggested that certain types where it resides until an APC or some other fac-
of self-recognition may even be essential for tor activates that Th cell. After activation, using
the maintenance of normal populations of T host ribosomes, bases, amino acids, and en-
and other cells. Regardless of the mechanisms, zymes, viral DNA transcribes and new viruses
some animals clearly retain the ability to re- assemble.
spond to their own tissues. FIV is an enveloped virus, so it exits the cell
along with a bleb of host-cell membrane and
Clinical Correlation Follow-Up seeks other cells to infect. Cats’ immune sys-
tems do respond to FIV infections, but during
Student Considerations those responses, many Th cells die because
FIV is a retrovirus that infects CD4+ T cells infecting viruses have depleted essential fac-
(Th cells). Months to years after exposure, most tors. In addition, reverse transcriptase (unlike
cats infected with FIV will develop chronic in- other polymerases) has no editing function,
fections—fungal, bacterial, or viral infections. so mutations accumulate rapidly. As a result,
Eventually, these cats succumb to their infec- an animal’s immune system is aiming at a con-
tions (Figure 16.11). In effect, after FIV infection, stantly moving target. Over time, FIV outstrips
the cats disappear and are replaced by a host of its host’s immune system and destroys most of
other living organisms. After reading this and the CD4+ Th cells, and the cat becomes severely
preceding chapters, you should be able to ex- immune deficient. That acquired immunodefi-
plain the cause and results of this immunodeficiency leaves the cat at considerable risk. Op-
ciency disease. portunistic infections usually follow, and even-
tually the cat dies. Immune deficiencies, both
congenital and acquired, put animals at great
Possible Explanations risk for debilitating and often fatal infections.
FIV resides, among other places, inside the Moreover, although FIV causes a pronounced
host cat’s Th cells. After infection, the virus immunodeficiency that results in major clinical

symptoms, all immunodeficiencies are not so animals. Some of that variance is likely due to
obvious. Because of the complexity of immune subclinical immunodeficiencies. The veterinar-
systems, even under the best of circumstances, ian’s challenge is to be aware of that as part of
immune competence varies among individual diagnosis and therapy.

285

Gerald N. Callahan
The Immune System and Cancer Chapter 17

10.5876_9781607322184.c017
lower counts of lymphocytes, CD3+ T cells

Clinical Correlation: Canine Cancer 287 and CD4+ T cells, than normal dogs, and that
Learning Objectives 287 these counts became much lower as the tumor
Background 287 stage progressed.
Evidence That Mammalian Immune Systems
(Itoh et al., 2009, Journal of Veterinary
Affect Tumors 289
Immunology and Immunopathology, 132: 85)
Tumor–Host Interactions 290
Outcomes 292 Clearly, tumor progression correlated with sub-
Clinical Correlation: Follow-Up 293 stantial changes in the immune status of these
Student Considerations 293 dogs. Why?
Learning Objectives
Clinical Correlation: After reading this chapter, you should be able to
Canine Cancer
• understand the concept of immune
In 2009, Hiroshi Ito and colleagues carried out surveillance;
a study of 65 dogs of various breeds, includ-
• explain why tumor cells might trigger host
ing Irish wolf hounds (Figure 17.1). Nineteen of
immune responses;
the dogs were healthy, and the rest had tumors
of various cell origins. During the course of
• describe several ways in which host animals’
immune systems may limit tumor-cell
the study, Ito collected blood from both nor-
growth;
mal and tumor-bearing dogs and analyzed his
samples for several markers of immune status. • describe several ways in which tumor cells
Here is part of his description of his findings: may interfere with the development of host
immune responses;
We found that tumor-bearing dogs had higher
• explain the relationships between host
leukocyte counts than normal dogs, and that
animals and their tumors;
the counts increased as the tumors became
more advanced. In addition, tumor-bearing
• describe the possible outcomes of host–
tumor interactions.
dogs had higher differential counts of leuko-
cytes and ratios of inflammatory cells such as
neutrophils, acidophils and monocytes. This
Background
finding was suggestive of an inflammatory
reaction at sites of tumor development and In the early 1900s, Paul Ehrlich first proposed
infection resulting from decreased immunity. the idea that immune systems help to eliminate
We also found that tumor-bearing dogs had cancer cells. In fact, Ehrlich even went so far as
287
287

Figure 17.1. Irish wolfhound (© foaloce / Shutterstock)
Irish wolf hounds have an increased risk of developing osteosarcoma, the most common bone tumor
in dogs.
to propose that cancer might have provided a genes from cancer cells to normal cells and
part of the selective force that led to the evolu- cause the normal cells to behave more like
tion of modern mammalian immune systems. cancer cells. Because of their apparent role in
About sixty years later, Lewis Thomas and Sir the malignant phenotype, these genes came
MacFarlane Burnett formalized these concepts to be called oncogenes, after the process of
under the title “immune surveillance”—the oncogenesis (tumorigenesis). Other research-
idea that many tumors never fully mature into ers found that some genes from normal cells
life-threatening cancers because of immune- could cause cancer cells to behave a little more
mediated destruction of nascent malignancies. like their normal-cell counterparts. These
The basic idea behind the theory of immune genes were named tumor-suppressor genes be-
surveillance was the recognition that the driv- cause of their ability to suppress the malignant
ing force behind malignant transformation— phenotype.
the conversion of a normal host cell into one It is now clear that all oncogenes have their
with the potential to invade and metastasize— counterparts in normal cells, and all tumor-
is genetic mutation. At its root, cancer results suppressor genes have their counterparts in
from imbalances in the rates of cell division and cancer cells. The normal-cell versions of onco-
cell death. When cells divide more rapidly than genes are called proto-oncogenes. The differ-
they die, tumors arise. ences between proto-oncogenes and oncogenes
Decades ago, research on cancer cells dem- are mutations that alter the amino acid se-
onstrated that it was possible to transfer some quences and functions of the protein products
288 T h e Imm u n e Sy s t e m a nd C a nc e r

of these genes. Similarly, the tumor-suppressor suppressor genes as well as changes arising
genes of tumor cells no longer suppress ma- from any other sorts of mutations in cancer
lignancy because of mutations that produce cells, with the hope that these proteins might
proteins with altered amino acid sequences and provide new avenues for cancer diagnosis and
altered function. therapy. Among all the research ignited by the
This is important for two reasons. In general, National Cancer Act, cancer immunology be-
proto-oncogenes are genes that regulate cell di- came one of the fastest-growing fields.
vision—genes that produce cell growth factors, By the early 1990s, though, little had come
receptors for growth factors, second messen- from this massive effort. In addition, the avail-
ger system proteins, and so forth—and tumor- able evidence indicated that humans and other
suppressor genes, in general, are genes whose mammals with severe immune deficiencies,
products are involved in regulating cell death such as AIDS, developed only a few tumors
and DNA repair. So, first of all, mutations in during the course of their diseases, and those
proto-oncogenes and tumor-suppressor genes tumors were virus-induced tumors, such as Ka-
provide the basis for the observed imbalance in posi’s sarcoma. These data did not fit very well
the rates of cell division and cell death among with the idea of immune surveillance.
tumor cells. Second, these genetic changes
should produce altered self proteins—proteins
Evidence that Mammalian
with distinct amino acid sequences recogniz-
Immune Systems Affect Tumors
ably different from the corresponding normal
cellular proteins. Also, these proteins arise in As a result, some tumor immunologists began
ways that potentially circumvent all of the nor- to believe that, under normal circumstances,
mal means for generating self tolerance. There mammalian immune systems had little or no
is, then, every reason to imagine that these al- effect on developing tumors. However, by the
tered proteins could potentially trigger protec- mid-1990s, some investigators found that trans-
tive immune responses, responses that could planted tumors grew more aggressively in mice
slow the division of or eliminate the newly treated with antibodies against IFN-γ. Similarly,
transformed malignant cells. mice that lacked parts of the IFN-γ receptor de-
These ideas suggested a most attractive new veloped more tumors after exposure to chemi-
approach to cancer therapy. Most cancer thera- cal carcinogens. However, because these stud-
pies are relatively indiscriminate, killing many ies focused on chemical carcinogenesis, the rel-
host cells as well as tumor cells. Immune at- evance of immunity to spontaneously arising
tacks, however, are specifically focused on for- tumors remained open to question. To address
eign elements of invading pathogens and do this issue, other investigators showed that mice
relatively minor damage to host cells. The com- lacking either IFN-γ responsiveness or RAG2
bination of foreign proteins on and in tumor genes (which results in the absence of T, B, and
cells, coupled with the specificity and efficiency NK T cells) developed more spontaneous tu-
of immune responses, made cancer immunol- mors and more aggressive chemically induced
ogy a particularly attractive approach to tumor tumors.
therapy. But what about therapy? Could the power
This new approach, along with President and specificity of mammalian immune systems
Richard Nixon’s declaration of the war on can- be exploited to change the course of developed
cer in 1971 and the passage of the National Can- cancers? In support of this idea, humans treated
cer Act, launched nearly innumerable research with immune stimulants, such as antibodies
projects based on identifying and exploiting that block CTLA-4 (a receptor on T cells that,
mutations in proto-oncogenes and tumor- when coupled to its ligand, slows T-cell division),
T h e Imm u n e Sy s t e m a nd C a nc e r 289
289

have been shown to have improved prognoses Figure 17.2. Effects
of innate and adaptive
and fewer tumor recurrences. immunity on tumor cells
The reality of tumor immunity seemed clear. TRAIL—TNF-related apoptosis-inducing
How, then, were many tumors regularly avoid- ligand—is produced by neutrophils and
ing host immune responses? It is now clear that stimulates apoptosis in tumor cells; NKG2D is
some tumors actively subvert a host immune an activating receptor on NK cells, powerful ele-
response. For example, at least some tumor ments of innate and adaptive immunity; during
tumor progression; TGF-β induces prolifera-
cells are very poor presenters of endogenous
tion, angiogenesis, invasion, and metastasis and
antigens. The evidence for this came from
suppresses the antitumor immune response;
experiments in which tumor cells transfected indoleamine 2,3-dioxygenase (IDO) is produced
with a gene encoding a TAP were used to ef- by DCs and inhibits T-cell proliferation; galec-
fectively immunize host animals against their tin-1, produced by a variety of cells, inhibits T-
own tumors, suggesting that recognizable cell activation; vascular endothelial cell growth
tumor antigens (i.e., modified self proteins) factor (VEGF) is produced by some tumor cells
were present all along but were not effectively and stimulates angiogenesis; programmed cell
presented in MHC class I molecules on the death ligand 1 (PD-L1), a B7 analogue produced
tumor cells. by some macrophages, binds to a death receptor
Also, certain groups of γ/δ T cells have re- on T cells; myeloid-derived suppressor cells
cently been shown to recognize and respond to (MDSCs) are induced by tumor-secreted and
host-secreted factors, many of which are proin-
proteins expressed by some tumor cells. These
flammatory molecules and inhibit both adaptive
T cells not only bind to tumor-specific proteins
and innate immunity. (Adapted from Vesley et
and peptides but also can express MHC class II al., 2011, Annual Review of Immunology, 29: 235)
molecules and act as professional APCs in anti-
tumor immune responses.
For decades, it had been apparent that many
intrinsic factors acted to limit the develop-
ment and spread of cancer cells. These fac- Tumor–Host Interactions
tors included tumor-suppressor genes, proto-
oncogenes, and the general processes of cellu- A general model of how host immunity may
lar senescence and apoptosis. But these studies limit or even promote the development, prolif-
of mice and humans with modified immune eration, and spread of malignant cells is shown
systems were among the first to demonstrate in Figure 17.2.
that host immune systems might produce As shown in Figure 17.2, after malignant
tumor cell–extrinsic factors that limited the transformation many tumors may be elimi-
formation, proliferation, and spread of malig- nated by intrinsic mechanisms, but some are
nant cells. not. One of the roles of the immune system is
Currently, most of the evidence in support to deal with these transformed and potentially
of immune surveillance comes from studies deadly cells. These host responses involve a va-
on mice and humans. Therefore, the immedi- riety of both innate and adaptive mechanisms.
ate relevance of these findings to veterinary Neutrophils, major elements of the innate im-
medicine remains unclear. However, the gen- mune response, produce TNF-related apoptosis-
eral significance of these findings to cancer inducing ligand, which stimulates apoptosis in
biology and therapy seems apparent, as does tumor cells; NK cells, important factors in both
the possibility that, in the near future, these innate and adaptive immunity, have a cell-sur-
findings will affect both human and veterinary face receptor called NKG2D. When bound to
practices. its ligand, this receptor stimulates strong NK-

cell activation. Two NKG2D ligands, MICA and in MHC class I molecules on tumor cells) can,
MICB, appear on many tumor cells. using perforins and granzymes, directly lyse
Also as shown in Figure 17.2, CD8+ T cells tumor cells. Also, CD4+ T cells may be activated
(activated by tumor-specific peptides presented by APCs presenting tumor-specific antigens,
291

and these T cells may give rise to any or all of press aspects of both innate and adaptive host
the types of T cells and responses described in immune responses.
chapter 10. The activation of these cells may,
then, lead directly or indirectly to destruction of
Outcomes
the tumor. Particularly important among these
T-cell effector functions is production of IFN-γ In one sense, then, the relationship between
(also produced by NK cells), which is a potent host and tumor may be thought of as a com-
stimulator of macrophages and immune func- petition in which the power and specificity of
tions in general. NK T cells are a heterogeneous host immune mechanisms are pitted against
group of T cells that recognize (among other the evasive tactics of the tumor cells. In some
things) modified self antigens presented by cases, host immunity may triumph and elimi-
nonclassical MHC class I molecules on tumor nate the tumor. In other instances, all the im-
and other cells. This aspect of innate immunity mune system can throw at tumors is only
appears to be particularly important during enough to maintain a sort of equilibrium, or
tumor progression. CD8+ DCs cross-prime for steady state, in which tumor cells remain but
presentations of endogenously derived peptides do not progress. In these cases, tumor cells are
in MHC class II molecules, ensuring activation not completely eliminated, but neither do they
of a full range of adaptive immune responses proliferate—unless something happens to alter
against tumor cells. the immune competence of the host animal or
Although an impressive arsenal of weapons the nature of the tumor cells. Evidence of this
may be activated by tumor cells, it is now clear steady-state situation comes from newer stud-
that some tumor cells actively suppress host ies of longer-living AIDS patients in whom the
immune responses. TGF-β, produced during frequency of spontaneous tumors is higher.
tumor progression, induces tumor cell prolif- In still other instances, all the potential innate
eration and angiogenesis (providing essential and adaptive mechanisms the immune system
blood vessels for tumor growth) and acceler- can muster are not sufficient to prevent tumor
ates tumor invasion and metastasis. TGF-β cell proliferation. In one sense, developing
also suppresses elements of the host antitumor tumor cells are evolving. As immune mecha-
immune response. Tumor-activated DCs, in nisms eliminate some tumor cells, others un-
nearby lymph nodes, secrete indoleamine dergo mutations that allow them to escape both
2,3-dioxygenase, which suppresses T-cell pro- adaptive and innate responses either directly
liferation. Tumor cells also often secrete ga- (e.g., by preventing tumor antigen presentation)
lectin-1, which stimulates angiogenesis, again or indirectly through production of factors that
causing nourishing blood vessels to grow into suppress immunity. These tumor cells, even in
the developing tumor. In addition, some tumor the face of a host immune response, may prog-
cells secrete vascular endothelial cell growth ress to local invasion and metastasis.
factor, another stimulator of angiogenesis. In- Under still other conditions, it appears that
hibitory factors, such as programmed cell death immune mechanisms, especially inflammation,
ligand 1, secreted by tumor cells may dramati- may not only fail to prevent tumor cell growth
cally slow immune responses. Programmed cell and proliferation but actually, through the ef-
death ligand 1 is an analogue of B7 and binds fects of proinflammatory factors (as described
to CD28, but instead of stimulating prolifera- earlier), promote both. Here again, the out-
tion as B7 sometimes does, PD-L1 induces T- come is progressively growing, potentially fatal
cell apoptosis. Last, myeloid-derived suppressor tumors.
cells, induced by some tumor cells as well as At this point, immunotherapy—with exog-
some host-derived inflammatory factors, sup- enous agents such as cells, antibodies, and im-

munoactivating cytokines—is the only option.
Immunotherapy is the area in which cancer
immunology is undergoing some of its most
promising developments. Also, although most
laboratory and clinical trials of immunothera-
pies have been limited to mice and humans,
immunotherapy is the area of cancer biology
most likely to affect veterinary practitioners
soonest.
Currently, immunotherapy appears to be
most effective in combination with chemothera-
pies and radiation therapies. Positive effects have
been found using adoptively transferred tumor-
specific T cells as well as passively transferred
antibodies. Antibodies against immunosuppres
sive agents, such as CTLA-4 (mentioned earlier)
are also improving prognoses, and tumor-spe-
cific vaccines, prepared from host tumor cells
themselves, have slowed development of and
in some cases eliminated established tumors.
Thus, as of now, immunotherapy, especially in
combination with other antitumor therapies,
offers considerable promise. The potential out-
comes of the interactions between developing
tumors and host immune systems are shown in
Figure 17.3.
That developing tumors face a variety of
immune attacks now seems evident. Just how
those immune mechanisms may be manipu- Figure 17.3. Immunity and cancer
lated to change cancer prognoses for veterinary Interactions between tumor cells and mamma-
patients remains to be clarified, but the poten- lian immune systems can have several out-
tial seems enormous. comes. Immune attack may result in complete
elimination of tumor cells (elimination) or an
equilibrium state in which tumor cells remain
Clinical Correlation: Follow-Up but cannot proliferate (equilibrium), or in some
cases, immune mechanisms, such as inflamma-
tion, may promote changes that favor tumor
We found that tumor-bearing dogs had higher cell proliferation. Either because of such an
leukocyte counts than normal dogs, and that immune assist or through mutation and evolu-
the counts increased as the tumors became tion, the tumor cells may escape innate and
more advanced. In addition, tumor-bearing adaptive immune responses and proliferate and
dogs had higher differential counts of leuko- metastasize (tumor escape and growth). Even
cytes and ratios of inflammatory cells such as after escape from immune surveillance, im-
munotherapeutic interventions may stimulate
neutrophils, acidophils and monocytes. This
a new set of effective immune responses that
finding was suggestive of an inflammatory
eliminates the tumor.
reaction at sites of tumor development and
293

infection resulting from decreased immunity. intrinsic factors may have played a role in gen-
We also found that tumor-bearing dogs had erating the observed changes.
lower counts of lymphocytes, CD3+ T cells, The elevated white cell counts and the in-
and CD4+ T cells than normal dogs and that crease in those elevations over the course of
these counts became much lower as the tumor the study suggest inflammation. As the authors
stage progressed. suggested, some of the elevation might be due
to infections as a result of tumor-induced im-
(Itoh et al., 2009, Journal of Veterinary
Immunology and Immunopathology, 132: 85) mune suppression. Tumors themselves, how-
ever, are also often inflammatory, and that in-
The important elements in the tumor-bear- flammation may serve to promote tumor cell
ing dogs here are the elevated white cell counts proliferation. Elevated neutrophil and mono-
and the increase in these counts as the tumors cyte counts are also compatible with increased
progressed. Also, the increases in neutrophils inflammation. CD3 is present on all T cells, and
and acidophils over the course of the study are CD4 is characteristic of Th cells. Decreases in
noteworthy. Finally, the decreases in CD3- and T-cell numbers during tumor progression fit
CD4-bearing lymphocytes are important to with tumor cell production of T-cell toxins,
consider. Also, not included in this excerpt, Itoh such as PD-L1 (mentioned earlier). Last, eleva-
et al. described elevations in the levels of TGF-β tions in TGF-β could be both immunosuppres-
in the tumor-bearing dogs. sive (because of the known roles of TGF-β in
establishing immune tolerance) and supportive
of angiogenesis (providing additional blood
Possible Explanations vessels to support continued tumor growth and
The results of the study described at the be- accelerating tumor invasion and metastasis).
ginning of this chapter are clouded by the fact The host–tumor relationship is complex, and
that the tumor-bearing dogs were receiving the role of host immunity in tumor progres-
antitumor therapy throughout the course of sion is still unfolding. It is clear, though, that a
the study. Those therapies might have been re- full understanding of veterinary oncology must
sponsible for some of the observed differences include an appreciation of the role and nature
in immune status between the experimental of host antitumor immune responses as well as
and control groups. Regardless, on the basis of the means by which tumor cells may manipu-
the material presented in this chapter, several late those antitumor responses.

Amy L. Warren
Veterinary Clinical
Chapter 18
Laboratory Immunology
10.5876_9781607322184.c018
Clinical Correlation: Transfusion Reactions 295
Transfusion Reactions
Principles of Antibody-Based Techniques 298 In several clinical situations, the transfusion of
Generating Antibodies: Polyclonal and blood or blood products is critical to saving the
Monoclonal Antibodies 298 life of an animal (Figure 18.1). Most of these
Production of polyclonal antibodies 298 situations involve the loss of one of the major
Production of monoclonal antibodies 299 blood components (RBCs, platelets, or clotting
Detecting Antibody–Antigen Reactions 300 factors), either directly by hemorrhage or indi-
Specific Techniques 300 rectly in an immune-mediated process (such as
Enzyme-linked immunosorbent assays IMHA or immune-mediated thrombocytope-
(ELISAs) 300 nia). In the case of anemia, the animal presents
Immunoblotting 302 with hypoxia. Animals with thrombocytopenia
Immunofluorescent microscopy 303 and coagulation factor deficiencies typically
Precipitation assays 304 present with bleeding, either as microhemor-
Flow cytometry 305 rhages (petechiae) or overt hemorrhages.
Immunohistochemistry 306 One of the most direct ways to restore nor-
Diagnosis of Blood Type Incompatibilities 307 mal blood oxygen levels and coagulation factors
Blood Typing 307 is to transfer blood from another animal. How-
Blood typing cards 308 ever, as we have discussed, several factors, in-
Typing gel 308 cluding MHC molecules, present immunologi-
Membrane dipstick 309 cal barriers to tissue (including blood) transfer
Crossmatching 309 between individuals.
Diagnosis of Immunodeficiencies 312 Even though RBCs do not express either
Serum Immunoglobulin Quantification 312 MHC I or MHC II molecules, they do express
Phenotypic Analysis of Lymphocytes 312 a less diverse group of potentially antigenic
Other Immunodeficiency Tests 313 molecules called erythrocyte or red blood cell
Diagnosis of Autoimmunity 313 (RBC) antigens, which are genetically deter-
Antinuclear Antibody Test 313 mined immunogenic markers on the surface
Coombs Test 314 of erythrocytes. If an animal with a particular
Antiplatelet Antibody Test 314 (RBC antigen) blood type is transfused with
Antithyroglobulin Autoantibody Test 315 blood of a different type, a severe immune
Clinical Correlation: Follow-Up 316 transfusion reaction can develop.
Student Considerations 316 Each species has several defined groups of
Possible Explanations 316 erythrocyte antigens, and these groups differ in
295
295

species. During transfusions, these alloantibod-
ies attack the donor’s RBCs and also initiate in-
flammatory and other reactions.
In dogs, the most important antigens in-
volved in blood group incompatibility are the
dog erythrocyte antigen 1 (DEA-1) group. Un-
transfused dogs do not have immunologically
significant amounts of naturally occurring al-
loantibodies to DEA-1, but when exposed to
DEA 1.1–positive blood cells, dogs lacking DEA
1.1 will produce large amounts of anti–DEA 1.1
antibodies, which on secondary challenge will
agglutinate and lyse RBCs. Other blood groups
exist in dogs but are less commonly implicated
in clinical disease (Table 18.1).
In cats, there are three main blood groups,
types A, B, and AB, and a newly recognized
group, Mik (Table 18.1). Unlike dogs, some un-
transfused cats have naturally occurring anti–
blood group antibodies. Specifically, even be-
fore a primary transfusion, cats that have type B
Figure 18.1. Domestic short hair cat receiving a
blood produce high titers of anti-A antibodies.
blood transfusion (photograph courtesy of Dr.
For this reason, cats with type B blood will have
Josiane Houle)
a strong transfusion reaction to type A blood on
the first transfusion, reportedly with as little as
1 ml of type A blood. Why type B cats naturally
their antigenicity. In light of all we have covered produce anti-A antibodies is unknown.
in this book, one would anticipate that, once Horses have a number of different erythro-
an animal is transfused with foreign RBCs, the cyte antigens, with types Aa, Qa, and to a lesser
transfused animal would produce antibodies degree Ca being the most clinically important
against the foreign RBC antigens. (Table 18.1). Unlike dogs and cats, blood trans-
Because of this, one might also expect that fusion of horses is less common. The most im-
animals receiving their second or third transfu- portant disease process that arises from blood
sion would be at greater risk of a transfusion group–antigen incompatibilities in horses is
reaction—antibodies binding to RBC antigens, neonatal isoerythrolysis. During delivery of
destruction of RBCs, and inflammation (much a first blood group–incompatible foal, some
as in hypersensitivity reactions). That certainly of the foal’s blood enters the maternal circu-
does happen, but surprisingly many animals lation and induces an immune response that
are at considerable risk of transfusion reac- produces anti–blood group antibodies. After
tions even when they receive blood for the first a second pregnancy with an additional blood
time. These adverse reactions happen because group–incompatible foal, the mare’s colostrum
many animals have naturally occurring anti- will contain large amounts of anti–blood group
bodies to RBC antigens called alloantibodies, antibodies. After nursing, those antibodies can
which are literally antibodies of one individual enter the foals’ circulation and induce what is
that react against the alloantigens (homologous essentially a transfusion reaction that is poten-
molecules) of another individual of the same tially life threatening.
296 V e t e r i n a ry C l i n i c a l L a b o r a t o ry Imm u n o l o gy

Table 18.1. Major known blood groups in dogs, cats, and horses
Blood groups Genetics Naturally occurring alloantibodies Special considerations
Dog
DEA 1.1, Codominant None Most important group in transfusions as
DEA 1.2 RBC positive or nega- causes acute hemolytic reaction
tive for each 1.1 and 1.2
DEA 3 DEA 3 (dominant) and In some DEA-3 negative, but Rare
null results in delayed RBC survival 23% of greyhounds are
DEA 3–positive
DEA 4 DEA 4 (dominant) and None ~98% of dogs in the United States are
null DEA 4–positive
DEA 5 DEA 5 (dominant) and In some DEA-5 negative, but 10% of dogs; as many as 30% of grey-
null results in delayed RBC survival hounds are DEA 5–positive
DEA 7 DEA 7 (dominant) and None 99% of dogs in the United States are DEA
null 6–positive
Dal None 100% of the general dog population;
absent from Dalmatians
Cat
A AA, Aaab, or Ab* Low anti-B titer ~90% of cats are A positive
B Bb High anti-A titer Agglutinins and hemolysins resulting in
severe transfusion reaction
AB aabb or aabaab Most have no alloantibodies Rare
Mik Mik-negative cats might have Most type A cats are Mik-positive
alloantibodies to Mik
Horse
EAA group Minute amounts Aa important in neonatal isoerythrolysis
Aa most important
EAC group Anti-Ca in horses that are
C-negative
EAQ group Minute amounts Qa important in neonatal isoerythrolysis
Qa most important
*The A allele in cats is dominant to b and aab (Bighignoli et al., 2007, BMC Genetics 8: 27).
Cattle, sheep, and pigs have a wide variety of • describe the immunological principles
erythrocyte antigens, so many that blood typing underlying antibody-based tests;
and transfusions are rarely practical. Because of • list and describe different examples of
the nature of veterinary care in these species, antibody-based tests used in veterinary
transfusions are rare anyway, so transfusion diagnostics;
reactions are generally not an issue. If a trans-
fusion is warranted, cross-matching is a more • describe and explain blood typing and
practical way to avoid transfusion reactions. cross-matching;
• describe the diagnostic tests available for
Learning Objectives diagnosing immunodeficiencies;
After reading this and the preceding chapters,

• describe the diagnostic tests available for
diagnosing autoimmune diseases.
you should be able to
V e t e r i n a ry C l i n i c a l L a b o r a t o ry Imm u n o l o gy 297
297

Principles of Antibody- Both begin with inoculation of animals with
Based Techniques the antigen of interest. The simplest method
produces a mixture of Igs that detect multiple
As discussed in previous chapters, the humoral
epitopes of an antigen. This mixture is often
immune response generates antibodies (Igs)
referred to as polyclonal antibodies (usually an-
that bind to antigens with great specificity.
tisera; anti for antibodies in sera). The term poly-
Aside from protecting animals from invading
clonal means that these preparations contain an-
microbes, the humoral immune systems of do-
tibodies derived from many different clones of
mestic species can be exploited to produce an-
B cells. The alternative method uses cell-culture
tibodies for laboratory research and diagnostic
techniques to propagate a single B-cell clone
tests. Indeed, many of the immunologic tech-
from an immunized animal. These monoclonal
niques used in diagnostic veterinary medicine
antibodies are homogeneous and react strongly
use antibodies purposefully raised against par-
with only a single antigenic epitope.
ticular antigens.
Because it is easy to collect and often con-
tains disease-defining antibodies and antigens, Production of polyclonal antibodies
serum is the most common diagnostic sample Polyclonal antibodies are sera from animals
used. Serum is the portion of the blood left immunized with a relevant antigen (e.g., a mol-
after clotting (which removes platelets, white ecule from distemper virus) plus an adjuvant
blood cells, RBCs, and clotting factors). For (Figure 18.2). Beyond their diagnostic potential,
the most part, antibody-based diagnostic tests antisera are also important in a process known
either directly detect antigens of infectious as passive transfer. In certain situations (e.g.,
agents (bacteria, virus, or parasite) or detect after a snakebite), an animal may have an im-
the presence of antibodies that recognize a par- mediate need for antibodies (in this case, an-
ticular antigen (such antibodies are evidence of tivenom antibodies). Infusion of antisera can
a previous exposure to the relevant antigen or provide a source for these antibodies. In some
pathogen). cases, antisera can be used directly as diagnostic
Many different types of antibody-based di- tools.
agnostic tests are available, but they all require Because raw serum is full of antibodies di-
two components: (1) antibodies that are specifi- rected against hundreds of different antigens
cally generated to detect a particular antigen (not just the immunizing antigen), an addi-
(e.g., antibodies may be generated that recog- tional purification step is often used to purify
nize the Fc portion of a dog Ig [antiglobulins], only antigen-specific antibodies from antisera.
which are useful when one is trying to detect This purification step involves passing the
dog Ig in serum) and (2) a method of “visual- serum through a solid-matrix column with
izing” the antibody–antigen reactions. For visu- linked specific antigen molecules. Under these
alization, radioisotopes, fluorescent dyes, or en- conditions, the unwanted serum products, in-
zymes are commonly coupled to the diagnostic cluding the nonspecific antibodies, pass freely
antibodies, or the precipitation of antibody–an- through the column. However, the antibod-
tigen complexes is observed. ies that bind the specific antigen remain in the
column. Using a variety of techniques, the an-
tigen-specific antibodies can then be recovered
Generating Antibodies: Polyclonal as purified polyclonal antibodies. Such antigen-
and Monoclonal Antibodies specific, polyclonal antibodies usually still con-
Two common methods are used to generate tain a mixture of different Ig isotypes that bind
antibodies for research and diagnostic purposes. multiple antigenic epitopes.

Figure 18.3. Production of monoclonal antibodies
A mouse is inoculated with an antigen and
mounts an acquired humoral immune response.
B cells from the mouse’s spleen and lymph node
are harvested and fused with immortal neoplas-
tic plasma cells (myeloma cells), which produces
an immortal B cell that produces an antibody
directed against a single antigen epitope.
strongly reactive with a single antigenic epi

tope. Monoclonal antibodies provide a consis-
tent and predictable source of antibodies for
Figure 18.2. Production and purification of antibody–antigen assays.
polyclonal antibodies As with the production of antisera, the gener-
After injection of canine distemper virus ation of monoclonal antibodies starts with the
antigen, a goat mounts an adaptive humoral im- inoculation of an animal (typically a mouse, rat,
mune response. The polyclonal antibodies pro- or rabbit) with a specific antigen along with an
duced are then harvested from the goat’s serum adjuvant (Figure 18.3). After the animal mounts
as an antiserum and purified using antigen– a humoral immune response to the antigen, B
matrix columns. These antibodies can then be cells are isolated from the lymph nodes, spleen,
used in diagnostic tests for canine distemper or both. In tissue culture, these B cells are fused
virus infections. with myeloma cells (an immortal neoplastic
plasma cell line) to produce antibody-secreting,
Although the production of polyclonal anti- hybrid, immortalized cells called hybridomas.
bodies is relatively simple and cheap, it requires Hundreds, sometimes thousands, of these
the continued use of animals and, because of hybridomas must be screened to identify the
animal-to-animal and batch-to-batch variations, ones that secrete antibodies reactive with the
the products are not always predictable. How- antigen of interest. Once one such hybridoma
ever, many antigen-specific reagents (especially is identified, this immortalized plasma cell can
anti-Igs) are still produced this way. be propagated and grown in cell-culture media
indefinitely, producing copious quantities of a
monoclonal antibody. This process produces
Production of monoclonal antibodies antibodies of a particular isotype directed to-
Monoclonal antibodies are products of a ward a single epitope of one antigen, and the
single B-cell clone (hence the term monoclonal) process does not require the continuing use of
and thus contain only a single type of antibody animals.
299

Detecting Antibody–
Antigen Reactions
Generating polyclonal or monoclonal an-
tibodies reactive with specific antigens is only
half the battle when designing antibody-based
diagnostic tests. The other half involves design-
ing a means to detect these antibodies when
bound to their specific antigens. Tests that ex-
ploit antigen–antibody reactions can either di-
rectly detect the bound antibody or indirectly
detect it through the use of secondary antibod-
ies. In both direct and indirect reactions, labels
such as enzymes (including horseradish peroxi-
dase or alkaline phosphatase) or fluorescent or
radioactive chemicals allow for detection of the
specific antigen–antibody interaction. Figure 18.4. A multiwell plate for ELISA
Antibody–enzyme complexes typically react The wells that are yellow have detectable an-
with a substrate to produce a detectable color tibody; wells that are clear do not. The yellow
change or chemiluminescence detectable using color is produced by an enzymatic reaction
either photometric methods or photographic mediated by the enzyme attached to the binding
film. When illuminated, fluorescent chemicals antibody.
attached to antibodies produce a specific wave-
length of light observable with a fluorescent tions of ELISA are also available, including a
microscope or fluorometer. Because of the sandwich ELISA (so named because it features
dangers of working with radioactive chemicals, an antigen sandwiched between two different
radioactive labeling has become less common. antibodies).
Indirect ELISAs detect antibodies in the
serum of an animal. For example, if a veterinar-
Specific Techniques
ian wanted to know whether a dog has Lyme
Enzyme-linked immunosorbent assays disease, he or she could use an ELISA to detect
Enzyme-linked immunosorbent assays (ELI- serum antibodies against the Lyme disease bac-
SAs) use antibodies or antigens labeled with an terium Borrelia burgdorferi. In this example, an-
enzyme that, when combined with a substrate, tigens of B. burgdorferi are bound to the base of
causes a detectable color change. The color the microwells. The dog’s serum is then added
change is typically measured using a spectro- to the wells, and if the dog has antibodies
photometer. In recent years, fluorescent labels against B. burgdorferi, those antibodies will bind
have also been used in ELISAs. ELISAs are to the bacterial antigens. Unbound antibodies
typically performed in multiwell plates (Figure are washed away, and the bound antibodies are
18.4), allowing for multiple simultaneous assays. detected using a second antibody labeled with
In veterinary diagnostics, ELISAs are most a marker, such as the fluorescent streptavidin
commonly used to detect antibodies in the phycoerythrin. The amount of fluorescence
serum of an animal to quantitate that ani- emitted from the well reflects the amount of
mal’s immune response to a given pathogen. specific antibody in the dog’s serum (Figure
Usually, a variant of the ELISA known as the 18.5). ELISA tests are available for a multitude
indirect ELISA is involved. Several other varia- of different infectious agents important in vet-

Figure 18.5. IndirectELISA to detect serum Figure 18.6. Sandwich ELISA to detect antigens
antibodies against B. burgdorferi (Lyme in the sera
disease) A monoclonal antibody to the antigen of
B. burgdorferi antigen is coated on the bottom of interest (the capture antibody) adheres to the
a well. The dog’s serum is added, and if the dog bottom of the well. The serum (containing
has antibodies against B. burgdorferi, they will the antigen) is added, and any specific antigen
bind. The antibodies are then detected using present binds to the capture antibody. A second
fluorescently labeled antidog antibodies. antibody to the antigen (the detecting antibody)
is added and reacts with bound antigen. A
third, labeled antibody specific for the second
antibody, allows for visualization of the bound
erinary practice, including many for use in the complex.
clinic or field.
Indirect ELISAs are useful for detecting
pathogen-specific antibodies in patients’ sera,
but there are times when one may wish, in- body fluid samples). A second antibody (the
stead, to know the amount of antigen in patient detecting antibody) that recognizes a different
sera. This amount can be determined using a epitope on the same antigen is then added to
variant of the ELISA technique called the sand- detect bound antigen. Finally, a third antibody,
wich ELISA. In a sandwich ELISA, an antibody labeled with an enzyme or fluorescent probe,
against the test antigen (the capture antibody) is added to detect the second antibody (Figure
is attached to the bottom of microwells in a mi- 18.6). If all three antibodies used in this test are
crotiter plate. If the specific antigen is present, from different species, then no possibility exists
it will bind to the capture antibodies attached that the tagged third antibody will give a false-
to the well after addition of serum (or other positive result.
301

Figure 18.7. Immunofiltration technique
This technique is a variant of a sandwich ELISA using antibodies attached to a membrane to which
the sample containing the antigen is added. A second labeled antibody forms the sandwich, which is
detected by an enzymatic change in color of an added substrate. The right side of the figure depicts a
well where the serum is added and two dots in the detection panel. One dot represents the test’s posi-
tive control; the second dot is the positive reaction to the antigen (in this case, canine parvovirus).
Using the sandwich ELISA technique, sev- tor antibody (i.e., typically labeled with colloi-
eral companies have also developed “pet-side” dal gold to produce a pink color or colloidal se-
in-clinic assays, including the immunofiltra- lenium to produce a blue color). The antibodies
tion technique (used in SNAP ELISAs) and and antigens form complexes that continue to
lateral flow tests (also called immunochroma flow up the porous strip until the complexes
tog r aphy). come to a line of bound capture antibodies that
The immunofiltration technique is similar “capture” the antigen–antibody complex, form-
to a standard sandwich ELISA. An immobi- ing a sandwich among the detector antibody,
lized capture antibody attached to a membrane antigen, and capture antibody. This reaction is
binds to the antigen in the sample (blood, urine, visible as a line (pink or blue) on the strip. Nega-
or serum), and a labeled detector antibody is tive samples will not bind the detector antibody,
added to measure the bound first antibody (Fig- so no line will be produced (Figure 18.8).
ure 18.7).
The lateral flow test uses the capillary ac-
tion of a porous strip dipped into a sample. The Immunoblotting
sample (containing the antigen) flows along the Immunoblotting (also called western blot-
strip until it comes to a line of unbound detecting) is a common method used in research, but

Figure 18.9. Image of an immunoblot designed to
detect IgG
The line corresponding to the 150-kDa marker
Figure 18.8. Lateral flow test is IgG.
A porous strip is dipped into the antigen-
containing sample and flows up the strip by
capillary action. If antigen is present, it will
bind to an unbound detector antigen, forming brane is washed and then placed into a solution
an antigen–antibody complex that will bind containing a secondary antibody. The second-
farther up the strip to a bound capture antigen ary antibody is an Ig able to bind the primary
to produce a colored line or dot. antibody (antiglobulin) and is coupled to a de-
tection enzyme (usually horseradish peroxidase
or alkaline phosphatase). These enzymes can
because of the length of time required to pro- convert a substrate into a colored product that
cess immunoblots, it has limited use in routine stains the membrane or creates light (chemilu-
diagnostics. Before immunodetection, protein minesence) that can be detected on film or by a
mixtures (from serum or a tissue homogenate) camera (Figure 18.9).
are first separated on the basis of size using
polyacrylamide gel electrophoresis, which
stretches the sample over the length of the gel Immunofluorescent microscopy
and in the process separates the proteins, with Another antibody-based technique used in
small proteins migrating farther than larger both diagnostics and research is immunofluo-
ones. It essentially produces a bar code–like pat- rescent microscopy. This method of immuno-
tern, with every band or stripe being composed detection uses fluorescently labeled antibodies
of a different-sized protein. The separated pro- to highlight protein antigens in cells or within
teins are then transferred from the gel onto a ni- histological sections (in situ). Immunofluores-
trocellulose membrane (which tightly binds to cence involves a primary antibody raised against
protein bands). Areas of the membrane that do the protein of interest, in combination with a
not bind a protein from the sample are blocked secondary antibody (anti-Ig or anti–primary
with a generic irrelevant protein (such as albu- antibody) carrying a fluorescent tag. The fluo-
min or casein) so they cannot further bind pro- rescent tag, when illuminated by a particular
tein nonspecifically. To detect which band con- wavelength of light, emits light at a longer
tains the protein of interest, the membrane is wavelength. Through the use of specific filters
incubated in a solution containing an antibody within the microscope, only the wavelength
(either monoclonal or polyclonal) raised against that is emitted by the given fluorescent tag on
the particular protein of interest. The mem- the secondary antibody passes through to the
303

Figure 18.11. Precipitation of antigen and
antibody complexes in a precipitation assay
exploits the concentrations of antibody and
antigen (zone of equivalence) to measure their
relative quantities (titers)
Figure 18.10. A merged image generated by

immunofluorescent microscopy showing the To form a precipitate, the antigen of interest
locations of two different cytoskeletal proteins must be multivalent (i.e., contain multiple epi-
(actin and tubulin) within two macrophages. topes to which multiple antibodies can bind),
Actin is shown in green, tubulin is shown in red and the relative proportions of antibodies and
and nuclei are stained blue. antigen must be optimal (the zone of equiva-
lence). With too much of either antigen or
antibody, the precipitate will not form (Figure
eyepiece, giving an image corresponding to the 18.11).
location of the protein of interest. A type of precipitation test used in diagnos-
One advantage of this technique is that, tics follows the diffusion of antigen and anti-
using multiple antibodies tagged with different body within agar and is aptly called an immu-
fluorochromes, multiple proteins can be iden- nodiffusion assay. Usually, the antigen and anti-
tified in a single tissue sample. By overlaying bodies (typically serum samples or antisera) are
these images, it is possible to locate the various deposited in separate wells in an agar gel. As the
proteins relative to one another (Figure 18.10). two proteins diffuse toward each other, their
interactions form a visible area of antigen–
antibody precipitate—the zone of equivalence.
Precipitation assays Where the precipitate forms relative to the an-
As you have learned in previous chapters, tigen and antibody wells gives an indication of
under the right conditions antibodies and an- the concentration (or titer) of antibody, relative
tigens can form immune complexes consisting to the antigen (Figure 18.12). A specific example
of aggregates of antigen cross-linked with anti- of an immunodiffusion assay is the Coggin’s
bodies. Usually, these complexes are small and test. The Coggin’s test is specifically used to
quickly disposed of via Fc receptors on RBCs, detect antibodies against equine infectious ane-
but, as with type III hypersensitivities, under mia virus (a negative Coggin’s test is required
the right conditions these antigen–antibody for horses to travel into or between most states
complexes can form large insoluble aggregates. in the United States).
In vivo, such aggregates can be life threatening, Radial diffusion is a variant of the multi-
but in vitro, the complexes can form visible pre- well immunodiffusion assay, where either the
cipitates useful for diagnostic analyses. antigen or the antiserum is incorporated into

fluorescently labeled antibodies, which are in-
creasingly being used in veterinary diagnostics
to diagnose hematopoietic cancers (leukemia
and lymphoma).
Flow cytometry analyzes a stream of cells
suspended in fluid by passing them through a
laser beam one by one. As the cells pass, they
scatter the laser light in characteristic ways.
There are two detectors. The first detector is
positioned in line with the laser beam and de-
tects light (forward scatter) blocked by a cell as
it passes by. A second detector perpendicular to
the laser detects light (side scatter) deflected by
the cell.
In basic terms, forward scatter measurement
Figure 18.12. Example of an agar gel
gives information about the cell’s volume (size),
immunodiffusion assay (also known as
Ouchterlony double immunodiffusion assay)
and side scatter measurement gives information
about the cell’s internal complexity (granularity
Here, an unknown serum sample is being tested
and nuclear conformation). This information is
to see whether it contains antibodies that rec-
ognize four different antigens (A, B, C, and D). then used to characterize immune cells in the
In this example, a precipitate forms in the zone blood. For instance, lymphocytes are small (low
of equivalence between the serum well and the forward scatter) and have simple round nuclei
well that contains antigen D, indicating that the with no cytoplasmic granularity (low side scat-
serum contains antibodies that can recognize ter). Neutrophils, however, are large (high for-
and precipitate antigen D, but not antigens A, ward scatter) and have complex nuclei and a
B, and C. high degree of cytoplasmic granularity (high
side scatter; Figure 18.13).
Using forward and side scatter, flow cytom-
the agar itself and forms a precipitate with the eters characterize and count the various types
test serum or antigen deposited in a well. The of cells in a blood sample, providing very accu-
precipitate forms in a circle around the well as rate information on the circulating population.
the sample diffuses out. The area of the circle Both in research and in the diagnosis of he-
is proportional to the concentration of the rel- matopoietic neoplasms, more detailed infor-
evant antigen or antibody within the sample. mation is needed on the immune cells than
the cytometry of unstained cells provides. For
example, one might want to know not only
Flow cytometry whether lymphocytes are present but also
Flow cytometry is a powerful technique whether they are CD4+ or CD8+ lymphocytes.
used to characterize cells in fluid. In veterinary For these sorts of analyses, forward and side
diagnostics and research, it is most frequently scatter data are combined with analyses of
used to quantitate different immune cells in fluorescent antibodies attached to white blood
blood samples. Most basic complete blood cell cells. For example, CD4 is unique to Th lym-
counts in veterinary laboratories are now ana- phocytes, so fluorescently tagged anti-CD4
lyzed using flow cytometers and are typically antibodies can be used to label those cells. As
done on unstained blood. Further information the cells pass by the laser beam, the fluores-
can be gathered by labeling immune cells with cent tags on the antibodies are excited by light,
305

Figure 18.13. Flow cytometry of a blood sample
Large cells produce high forward-light scatter (size). Cells with a high degree of internal nuclear com-
plexity and cytoplasmic granularity produce high side-light scatter.
causing the antibodies to fluoresce and emit a paraffin-embedded tissues; thin sections of fro-
certain wavelength of light. This emitted light zen tissues; or cells attached to slides.
is concurrently detected along with forward When antigen-specific antibodies labeled with
and side scatter data (Figure 18.14). an enzyme (typically a peroxidase) are added to
the section or slide, they bind to any antigens
present. The peroxidase is then visualized by a
Immunohistochemistry chemical reaction that produces a brown or red
Immunohistochemistry is used principally color change. The advantage of immunohisto-
on formalin-fixed tissues collected at necropsy chemistry is that it does not disrupt the tissue
or biopsy and submitted for histopathologic architecture, so the location of the antibody re-
evaluation. It has become one of the most pow- flects the in situ location of the antigen.
erful tools pathologists have for identifying and In veterinary diagnostics, immunohistochem-
characterizing tissues, structures, and infec- istry is most often used to categorize tumors
tious agents in tissue samples. Immunohisto- and to identify or detect the presence of infec-
chemistry uses thin sections of formalin-fixed, tious agents. Immunohistochemical analyses of

incompatibilities is to know the recipient’s and
donor’s respective blood types and their com-
patibility. This can be achieved through blood
typing or by cross-matching the patient’s and
donor’s blood.
Blood Typing
Blood typing uses monoclonal or poly-
clonal antibodies that recognize the common
erythrocyte antigens of a species. When these
antibodies bind to the blood-type antigens on
the surface of erythrocytes, they can start to
cross-link these cells and cause them to visibly
agglutinate. If the erythrocytes do not express
that particular antigen, the antibody will have
no effect on them. Thus, by simply observing
Figure 18.14. Flow cytometry using fluorescently
labeled antibodies to detect subsets of cells
whether erythrocytes agglutinate on a slide
or in a test tube in the presence of a particular
Anti-CD8 fluorescent antibodies bind to CD8+
lymphocytes and not to CD4+ lymphocytes.
anti–erythrocyte antigen antibody, one can de-
Anti-CD4 fluorescent antibodies bind to CD4+ termine an animal’s blood type. For example,
lymphocytes and not CD8+ lymphocytes. FL2 to type feline blood, a drop of blood is added
detects the green fluorescence, and FL1 detects to a card or gel containing anti-A antibodies.
red fluorescence. When the erythrocytes are added, the anti-A
antibodies bind to any erythrocyte A antigens
present and cause the blood to clump or ag-
tumors use antigens unique to certain cell types. glutinate, confirming the blood to be type A.
For instance, CD3 is unique to T cells and char- If type B blood (that does not possess an A an-
acteristic of T-cell lymphomas. Similarly, von tigen) is added, the anti-A antibodies will not
Willebrand factor is characteristic of endothelial bind and thus no agglutination will take place.
cells in hemangiosarcomas (Figure 18.15A). Similarly, blood added to a card with anti-B anti-
Immunohistochemistry is especially useful bodies will cause agglutination of type B blood
for detecting infectious agents when no suitable but not of type A (Figure 18.16).
sample is available for more traditional bacte- With the increased use of blood products and
riological, virological, or parasitological analy- blood banks in veterinary practice, blood typing
ses. Immunohistochemical analysis uses perox- for dogs and cats has become more available—
idase-labeled antibodies specific for an antigen offered in specialized diagnostic laboratories
unique to a particular organism. These types of as well as in-clinic tests. Commercial, in-clinic
analyses also pinpoint the tissue and cellular lo- blood group tests are available for canine DEA
calization of pathogens (Figure 18.15B). 1.1 and feline groups A, B, and AB. For horses,
cattle, and other production species, blood typ-
ing is specialized and needs to be referred to a
Diagnosis of Blood Type
diagnostic laboratory. Currently, three methods
Incompatibilities
are available for the in-clinic typing of blood:
One of the best ways of preventing or antici- blood typing cards, typing gel, and membrane
pating transfusion reactions due to blood type dipstick.
307

Figure 18.15A–B. Immunohistochemical staining of a hemangiosarcoma using anti–von Willebrand
factor antibodies (panel A) and transitional epithelium containing canine distemper virus (panel B)
(courtesy of Dr. E. G. Clark)
Antigen-specific antibodies labeled with horseradish peroxidase allow for visualization of antigens in
situ as brown-colored areas on the cells.
Panel A shows anti–von Willebrand factor antibodies adhered to the cells of a hemangiosarcoma.
Panel B shows anti–canine distemper virus antibodies attached to ureteral transitional epithelium.
Inside the cytoplasm of many of these cells (and in some of the nuclei) are dark brown–staining ag-
gregates of the canine distemper virus.
Blood typing cards placed at the top of the tube is allowed to filter
Blood typing cards are available for dog DEA through the gel. If the erythrocytes possess the
1.1 and cat AB blood group typing. These cards antigen that the monoclonal antibody is directed
have monoclonal antibodies to DEA 1.1 or to fe- against, the erythrocytes will agglutinate (simi-
line A, B, and AB lyophilized in the card wells. lar to that on the cards). Agglutinated erythro-
Blood positive for the antigen being tested will cytes move less easily through the gel than in-
bind to the lyophilized antibodies and visually dividual erythrocytes and become suspended.
agglutinate (Figure 18.17). Thus, negative samples pass through the entire
tube unimpeded and end up forming a button
on the bottom, whereas positive blood remains
Typing gel as a suspended line halfway up the tube (Figure
Typing gels use monoclonal antibodies sus- 18.18). Typing gels are available for DEA 1.1 typ-
pended in gel matrices in test tubes. Blood ing in dogs and A, B, and AB typing in cats.

Figure 18.16. Blood typing in a cat
In a cat with type A blood, type A antibodies will bind to the A antigen on the surface of the erythro-
cytes and cause visible agglutination. Cats with type B blood will have no agglutination with a type
A antibody. Similarly, type B cats will have agglutination using type B antibodies, and type A cats will
have no agglutination. The images in the lower panels are of blood smears with no agglutination (left)
and with agglutination (right).
Membrane dipstick negative blood will move through, leaving no

The membrane dipstick method uses mono- line (Figure 18.19).
clonal antibodies bound to lines on a dipstick.
The dipstick is dipped into diluted blood, and
erythrocytes move up the dipstick by capillary Crossmatching
action. As the blood moves up the dipstick, anti- A more thorough and versatile method for
gen-positive RBCs will bind to the monoclonal testing for blood compatibility is a cross-match.
antibodies at the corresponding line. Antigen- Even when the blood types of two animals are
309

Figure 18.18. Typing gel for feline blood typing
The suspension of erythrocytes halfway down
the tube indicates the presence of that antigen
Figure 18.17. Typing card for feline blood on the surface of the erythrocyte. The blood
Macroscropic agglutination can be observed in from the cat in this image has both A and B anti-
the upper box labeled Type A, which is absent gens and thus has an AB blood type.
from the box labeled Type B, indicating that the
A antigen is present on the erythrocytes and B
antigen is absent. This cat has type A blood.
known to be the same, a transfusion reaction

(mediated by other types of antibodies) may
still occur. For example, a DEA 1.1–positive dog
may be transfused with DEA 1.1–positive blood
but still have an immune reaction because of
DEA 1.2 dissimilarity. A cross-match allows the
veterinarian to prescreen for transfusion reac-
tions before a transfusion and is a much more
accurate determinant of compatibility. The
disadvantage of cross-matching is that a com-
patible cross-match does not mean that both
the donor and the recipient animals necessarily
have the same blood type, just that no antibod- Figure 18.19. Membrane dipstick blood typing for
ies to that blood type are currently circulating. a cat
Thus, the recipient animal may still be suscep- The red lines on the stick correspond to spots
tible to a transfusion reaction the next time that where erythrocytes have bound to monoclonal
blood is used. Some dogs are also still suscep- antibodies. The first line, “A,” contains anti-A
tible to delayed and nonhemolytic transfusion antibodies; the second line, “B,” contains anti-
reactions. Thus, a cross-match is necessary be- B antibodies; and the third line is the control.
fore any transfusion to prevent a transfusion Thus, this cat has an AB blood type.

Figure 18.20. Cross-matching of feline A, B, and AB blood types
The areas shaded in pink indicate cross-reactions evidenced by agglutination. In the white boxes, no
cross-reaction occurs, indicating that the blood would be safe to transfuse.
reaction, and even then the cross-match is a rocytes are then mixed in a major and minor
relatively insensitive test and may fail to detect cross-match. In a major cross-match, the recipi-
circulating antibodies in certain circumstances. ent’s serum is mixed with the donor erythro-
In ruminants, in which a wide variety of eryth- cytes. This process will determine whether the
rocyte antigens exist, a cross-match is generally recipient has any significant alloantibodies to
more practical because it does not require spe- the blood about to be transfused. In a minor
cific antibodies raised against each of the many cross-match, the donor’s serum is mixed with
erythrocyte antigens. the recipient’s erythrocytes, which will assess
A saline-agglutination cross-match is the most for alloantibodies in the donor’s serum that may
common type of cross-match performed in lyse the recipient’s erythrocytes. Both the major
veterinary laboratories. Both major and minor and the minor cross-matches are examined for
cross-matches are performed. Briefly, EDTA- hemolysis and agglutination, both macroscopi-
anticoagulated blood from both the donor and cally and microscopically (Figure 18.20). Some
the recipient are individually centrifuged, and laboratories further evaluate the cross-match
the erythrocytes removed. The centrifuged by adding in the Coombs reagent (see Diagno-
erythrocytes from both the donor and the re- sis of Autoimmunity section), which allows a
cipient are then washed and resuspended in more sensitive assessment of bound antibodies
saline. Serum samples (which contain possible than the cross-matches alone.
alloantibodies) from both the donor and the In the circumstance of equine neonatal iso-
recipient are also obtained. The sera and eryth- erythrolysis (a type of blood incompatibility
311

between a mare and a newborn foal, in which
antibodies against the foal’s erythrocytes are
in the colostrum; see the Clinical Correlation
section at the beginning of this chapter), the
cross-match method has been modified into
the “jaundiced foal agglutination test.” Here,
instead of mixing the foal’s erythrocytes with
the mare’s serum, the foal’s erythrocytes and
the mare’s colostrum are mixed.
Diagnosis of Immunodeficiencies
In general, immunodeficiencies are diagnostic
possibilities when animals present with chronic
or recurrent infections or infections with un-
usual agents that do not typically cause disease
in an immunocompetent animal. Getting a pre-
cise diagnosis of immunodeficiency and where
the defect lies can be difficult, but several tests
Figure 18.21. Radial immunodiffusion to measure
can aid in the diagnostic process.
serum levels of Igs
The greater the antibody concentration in the
Serum Immunoglobulin serum, the larger the ring formed around the
Quantification well.
Several Ig deficiencies are relevant to veteri-

nary medicine, including congenital hypo- or
agammaglobulinemia (reduced or no Igs of all with a standard curve provides a measure of
classes), selective IgG deficiencies in cattle and the serum concentration of a particular Ig (IgG,
cavalier King Charles spaniels, selective IgA de- IgM, or IgA; Figure 18.21).
ficiencies in German shepherds, and acquired
failure of passive transfer in foals. To diagnose
these diseases, veterinarians need to be able to Phenotypic Analysis of Lymphocytes
quantitate the total and relative quantities of Immunodeficiencies related to leukocyte
Igs in the serum. abnormalities usually manifest as either a de-
The radial immunodiffusion assay is the most ficiency of a cell type or a deficiency of cell-
common assay used to quantitate IgG, IgM, and surface receptors. In general, if a deficiency of a
IgA in sera. This assay is a precipitation test (see specific leukocyte type is present, it is apparent
Principles of Antibody-Based Techniques sec- in a routine complete blood cell count and bone
tion) in which anti-Igs against species-specific marrow examination. Morphologic abnormali-
IgG, IgM, and IgA antigens are incorporated ties in some immunodeficiency diseases, such
into an agar plate. The patient’s serum is added as Chediak-Higashi syndrome, are apparent on
into wells in the plate, where it then diffuses blood smears. Lymphocyte subset deficiencies
into the antiglobulin-impregnated gel. At the are detected in flow cytometric analysis using
zones of antigen–antibody equivalence, im- antibodies reactive to specific lymphocyte-sub-
mune complexes precipitate and form a visible set markers (CD4+ T cells, CD8+ T cells, and B
ring. The width of the precipitation compared cells).

Other Immunodeficiency Tests
BLAD and canine leukocyte adhesion defi-
ciency (CLAD) result from inadequate levels of
cell-surface proteins CD11b and CD18. With-
out these cell-surface proteins, neutrophils can-
not adhere to endothelial cells and exit blood
vessels. In diagnostic tests, this deficiency is
apparent as the inability of neutrophils to ad-
here to plastic surfaces or to ingest particles
opsonized with the complement component
C3b. These deficiencies can also be diagnosed
by flow cytometry using anti-CD11b and anti-
CD18 markers.
Diagnosis of Autoimmunity
Figure 18.22. ANA positive serum sample
Antinuclear Antibody Test
The nuclei of the test cells fluoresce green
The antinuclear antibody test (ANA) mea- because of bound serum ANA antibodies. This
sures circulating antibodies against nuclear fluorescence is a diffusely strong reaction and
antigens. Although a number of different auto- would be supportive of a diagnosis of SLE if
immune diseases can produce antinuclear anti- other clinical features were present.
bodies, the ANA test is most commonly used
to establish the diagnosis of SLE—in veterinary cent marker. The antidog IgG antibodies will
medicine, a disease seen almost exclusively in bind to any antibodies bound to the cell nuclei
dogs. SLE is one of the more enigmatic im- in the plates and will fluoresce green when the
mune-mediated diseases in veterinary species, appropriate wavelength of light is shone on
principally because its clinical presentation is so them (Figure 18.22). Different patterns of stain-
variable. Animals with SLE produce antibodies ing may be seen, including diffuse, speckled,
reactive with a number of self-antigens, includ- rim, or nucleolar staining. It appears that dif-
ing components of the animals’ nuclei. These fuse (homogeneous) staining may be linked to
components include antibodies that bind to the diagnosis of SLE, whereas speckled staining
DNA, ribosomes, RNA, ribonuclear proteins, is more often seen with SLE-related diseases.
and components of the nuclear membrane. A Regardless, the diagnosis of SLE on the basis
strongly positive ANA reaction is useful in diag- of a positive ANA should be made with cau-
nosing this disease. tion. ANA positivity is associated with a wide
The ANA test is an indirect immunofluores- range of other autoimmune diseases and can be
cence assay (see Immunofluorescent Micros- weakly positive in normal animals or animals
copy section). Serial dilutions of patients’ sera with chronic disease.
are added to specialized plates with attached A variety of secondary assays are available to
nucleated cells. If the dog has circulating anti- detect the specific autoantibodies present in the
bodies reactive with nuclear antigens, the antiserum of ANA-positive dogs (remember, the
bodies will bind to the nuclei of the cells on the ANA test measures a variety of different auto-
bottom of the plates. The serum samples are antibodies that may target DNA, histones, RNA,
then removed from each plate and replaced by ribonuclear proteins, or nuclear membranes).
an antidog IgG antibody tagged with a fluores- These tests include ELISAs, immunodiffusion
313

assays, radioimmunoassays, and immunoblots, antiglobulin reagent is added to the washed
which use specific lyophilized nuclear antigens erythrocytes and then incubated. If the eryth-
embedded in a well, gel, or film. rocytes are coated with bound IgG, IgM, or C3,
the added antibodies will bind to the erythro-
cytes and cause agglutination. The aggluti-
Coombs Test nation can be evaluated macroscopically and
The Coombs test is one of the oldest tests for microscopically as a positive reaction (Figure
immune-mediated diseases in existence. It was 18.23).
developed in 1945 by Cambridge immunologist The indirect Coombs test (indirect anti-
Robin Coombs and his colleagues. The Coombs globulin test) differs from the direct Coombs
test, used in the diagnosis of IMHA, detects an- test because it measures serum levels of anti-
tibodies directed against erythrocytes. Depend- erythrocyte antibodies. In this test, erythro-
ing on the reagents used, the Coombs test can cytes are washed and incubated with the test
also measure cell-surface–bound complement serum (in the case of neonatal isoerythrolysis,
proteins. There are two Coombs tests that dif- washed erythrocytes would be from the foal
fer in their mechanism of action: the direct and the test serum would be from the mare).
Coombs test (also known as a direct antiglobu- If anti-erythrocyte antibodies are present in
lin test, or DAT) and the indirect Coombs test the serum, they will bind to the RBCs. The
(also known as an indirect antiglobulin test). second step of the indirect antiglobulin test is
The DAT detects antibodies and complement much like the direct antiglobulin test. After in-
directly bound to erythrocytes, and the indirect cubation, the erythrocytes are washed again (to
antiglobulin test detects antibodies that are un- remove any unbound antibody) and are incu-
bound and is more useful in antibody screen- bated with an antiglobulin antibody (e.g., in a
ing for blood transfusions and cross-matching. horse, an antihorse IgG). If antibody binds dur-
Both tests work on the principle that antibodies ing the first step, the anti-IgG antibody will bind
bound to erythrocytes can result in their agglu- in the second step and cause agglutination of
tination. This agglutination can be visualized, the erythrocytes (Figure 18.23).
which is the end result of this test. (One of the
interesting effects of IMHA is that erythrocytes
can also autoagglutinate. When autoagglu- Antiplatelet Antibody Test
tination occurs, the Coombs test is no longer Immune-mediated thrombocytopenia is a
useful.) common disease in dogs. Although a number
In IMHA, most of the Igs that bind to the of tests for this disease have been developed,
erythrocyte surface are IgG and, less commonly, the most sensitive (but least specific) are the
IgM. Many of these are incomplete antibodies. direct immunofluorescent assays. These as-
They sensitize and coat the erythrocyte and tar- says use platelet-rich plasma (prepared from an
get it for premature destruction (by antibody- EDTA anticoagulated sample) to obtain puri-
dependent complement activation, particularly fied platelets. The platelets are then washed and
C4 and C3) but do not cause agglutination. incubated with a fluorescein-labeled monoclo-
Polyreactive Coombs reagents in veterinary nal antibody against canine or feline IgG. If au-
medicine commonly contain anti-IgG, anti- toantibodies are bound to the platelets, the anti-
IgM, and anti-C3 antibodies. IgG fluorescently labeled antibody will bind to
In the direct Coombs test (direct antiglobulin them and can be detected by flow cytometry.
test), erythrocytes are first washed in saline (to This test detects only Igs bound to platelets. It
remove unbound antibodies and complement cannot differentiate which antigen the anti-
that may produce false-positive reactions). The body is binding to and cannot differentiate auto

Figure 18.23. Direct and indirect Coombs tests
In the direct Coombs test, erythrocytes from an animal that has bound antibodies are washed. Anti
dog antibodies are added that will bind to the antibodies already bound to the erythrocytes, which
will cross-link the erythrocytes and cause visible agglutination. In the indirect Coombs test, the
antibodies to erythrocytes are not bound to the erythrocytes but are free in the serum (usually in the
serum of a blood donor). The washed erythrocytes are added to the serum and antibodies are allowed
to bind to the erythrocyte. Antidog antibodies are then added that will cross-link any bound antibod-
ies and cause visible agglutination.
immune thrombocytopenia from other causes ing with thyroglobulin are observed. Antithyro-
of immune-mediated thrombocytopenia. globulin antibody assays are available in some
laboratories to aid in the diagnosis of hypo
thyroidism. This test is based on an indirect
Antithyroglobulin Autoantibody Test ELISA (see Principles of Antibody-Based Tech-
Hypothyroidism is a common endocrine niques section), in which the patient’s diluted
disorder of dogs. Approximately 50 percent of serum is added to purified canine thyroglobulin
canine hypothyroidism cases result from lym- bound to the bottom of plastic wells in a mi-
phocytic thyroiditis, the pathogenesis of which crotiter plate. If autoantibodies to thyroglobu-
is generally thought to be the autoimmune de- lin are present in the serum, they will bind to
struction of the thyroid gland. In some dogs the thyroglobulin. A second antidog IgG with
with hypothyroidism, serum antibodies react- an alkaline phosphatase tag is added to allow
315

visualization of any bound antithyroglobulin
IgG. Although a positive result here is indica-
tive of an autoimmune disease, because of the
potential importance of autoimmune T cells, a
negative result does not rule out autoimmunity.

After reading this chapter and knowing that
an animal can develop an acquired immune
reaction against a blood type different from its
own, you should be able to predict what will
happen when incompatible blood is transfused. Figure 18.24. MAC formed by complement
You should also be able to select two pretrans- proteins lysing a red blood cell (RBC)
fer diagnostic techniques you could use to avoid
this reaction.
the production of a MAC. The MAC literally

Possible Explanations punches holes in the membrane of the eryth-
Transfusion reactions are similar to any im- rocyte, resulting in lysis, usually intravascularly
mune response to a foreign antigen, in that (Figure 18.24).
antibodies bind to foreign antigens, in this case Antibodies, either alone or in combination
on the surfaces of erythrocytes. As mentioned with complement proteins, can also opsonize
earlier, these antibodies can be naturally occur- erythrocytes. Bound antibodies or complement
ring alloantibodies (i.e., present before the ani- fragments (C3b) then enhance the phagocyto-
mal has any exposure to the foreign blood type, sis of RBCs by leukocytes (principally macro-
e.g., in cats) or created during a previous expo- phages). Either intravascular or extravascular
sure to incompatible blood type (in an acquired hemolysis can result.
immune reaction, e.g., in dogs and horses). Hemolytic transfusion reactions can be acute
Regardless of whether the antibodies are natu- or delayed. Acute hemolytic transfusion reac-
rally occurring or generated, they are generally tions typically occur within twenty-four hours
either IgG or IgM. Clinically, transfusion reac- of transfusion and result from preformed serum
tions can be either hemolytic transfusion reac- antibodies in the recipient animal, against the
tions (in which transfused erythrocytes are de- donor blood type (either naturally occurring,
stroyed) or nonhemolytic transfusion reactions. as in type B cats, or as a result of prior trans-
Hemolysis of erythrocytes occurs as the re- fusion). Delayed transfusion reactions occur
sult of antibodies binding to the surfaces of more than twenty-four hours after the transfu-
erythrocytes and can be either intravascular sion and typically result from an acquired im-
(occurring within the lumen of blood vessels) mune response to a previously exposed blood
or extravascular (mostly in the spleen and liver). antigen in which the reactive alloantibody was
The severity of the hemolysis depends on the weak or in very low concentrations. Alter-
type of antibody and its ability to fix comple- natively, delayed reactions can occur several
ment. For example, the binding of the hemo- weeks after transfusion—sufficient time for the
lysin antibody to an erythrocyte surface acti- body to mount a new humoral response against
vates the complement system, which results in the transfused erythrocytes. Typically, delayed

transfusion reactions are clinically milder than associated with an immune reaction resulting
acute reactions. from preformed recipient antibodies reactive
Less common are the nonhemolytic transfu- with donor plasma proteins. As with most aller-
sion reactions. The two basic types of nonhe- gic (type I hypersensitivity) reactions, the onset
molytic transfusion reactions are fever and al- of an allergic reaction is acute (within fifteen
lergic (hypersensitivity type I) reactions. Fever minutes of transfusion) and can include urti-
reactions are typically thought to be due to caria, pruritis, and erythema as well as vomit-
the recipient’s alloantibodies reacting to leuko- ing, nausea, diarrhea, abdominal pain, and ana-
cyte antigens (rather than erythrocyte antigens) phylactic shock.
on the donor’s lymphocytes, granulocytes, or Although not all reactions can be predicted,
platelets. Cytokines in the plasma of the do- particularly those that are nonhemolytic, most
nor’s blood, believed to be released from plate- transfusion reactions can be anticipated and
lets and leukocytes during storage, are also often avoided by performing blood typing or
blamed. Allergic responses are thought to be cross-matching.
317

Glossary
acute-phase protein (APP). A serum protein antigen-presenting cells (APCs). Cells capable
whose abundance is altered in response to an of presenting antigenic peptides to T-helper cells.
acute inflammatory process.
antigens. Molecules that can stimulate an
adaptive immune system. A collection of organs immune-specific adaptive response.
and cells involved in generating adaptive immune
antiglobulin. Antibodies that bind to another
responses, that is, those that involve T-helper and
immunoglobulin (antibody), for example, an
cytotoxic T cells.
antidog IgG.
adjuvant. Any of a number of chemical
antiserum. Blood serum that contains polyclonal
compounds or components of microorganisms
antibodies.
that nonspecifically enhance innate and adaptive
immune responses. Adjuvants are common basophils. Granulocytic white blood cells of low
components of vaccine formulations. abundance thought to assist in the coordination
of inflammation in a manner similar to that of
affinity maturation. The increase in antibody
mast cells.
affinity to antigen observed during secondary
and subsequent adaptive immune responses. B-cell receptor (BCR). Antigen-specific receptor
molecule found on B cells. It is composed of
alternative pathway of complement
two heavy chains and two light chains (an
activation. A pathway that leads to the
antibody molecule plus a membrane anchor) and
activation of the complement cascade in
Igα and Igβ, two molecules involved in signal
response to the spontaneous deposition of
transduction.
complement (C3) on non host surfaces.
B cells. Bone marrow–derived lymphocytes that
anaphylatoxin. Products of complement
express antigen-specific receptors (BCRs) and,
activation (mostly C3a and especially C5a)
under appropriate circumstances, differentiate
that cause local or systemic anaphylaxis—
into antibody-secreting plasma cells.
inflammation locally and anaphylactic shock
systemically. blood typing. A group of techniques that
determine the antigenic blood type of an
antibody-dependent cell-mediated
animal before transfusion, reducing the risk of a
cytotoxicity. NK cell–mediated killing of target
transfusion reaction.
cells decorated with antibodies.
bovine leukocyte adhesion deficiency (BLAD).
antibody titer. The dilution at which a solution
An autosomal recessive disease that affects
of monoclonal or polyclonal antibody generates
Holstein cattle as a result of a mutation in the
one-half its maximum activity.
gene that encodes β2-integrin (CD18), which
319
319

results in an inability to effectively recruit clonal expansion. The antigen-induced expansion
leukocytes to areas of inflammation. of a single T or B cell into a large clone of
identical cells.
bronchus-associated lymphoid tissues.
Secondary lymphoid organs found in the colony-stimulating factors (CSFs). Produced
mucosal epithelia of the bronchi and the site of by various cells, CSFs are cytokines that induce
adaptive immune responses in the lungs. proliferation and differentiation of certain
hematopoietic cells.
B7. A molecule found on APCs. B7 comes in two
forms, B7.1 and B7.2, also known as CD80 and colostrum. Milk produced by mammals early
CD86. When B7 on the APC binds to CD28 on T in lactation that has high concentrations of
cells, it provides the second signal necessary for maternal antibodies.
the activation of T cells.
complement. A group of about sixteen plasma
β2-microglobulin. A small protein that associates proteins arranged in a biochemical cascade
with MHC class I α chains to form complete that can be triggered in response to microbial
MHC class I molecules. surfaces or immune complexes. Activation
of the complement cascade can result in the
cathelicidins. Cationic antimicrobial peptides with
release of inflammatory mediators, opsonization
an α-helical structure. Cathelicidins are secreted
of activating surfaces, and the direct lysis of
by certain immune and epithelial cells and
microbes.
contribute to the killing of engulfed microbes
within the phagolysosomes of phagocytes. complementarity-determining regions
(CDRs). Portions of antibody molecules that
CD28. Found on T cells; interacts with B7 to create
bind directly to antigens. The character of the
the second signal necessary for activation of T
CDRs determines the complementarity of the fit
cells.
between antigen and antibody.
central tolerance. Elimination of self-reactive
complement receptors. A group of proteins
lymphocytes in the thymus.
expressed on the surface of a variety of cells
chemokines. Cytokines that induce chemotaxis in that can bind complement bound to surfaces or
nearby cells. antigen complexes.
chemotaxis. The directed movement of a cell in conjugate vaccines. Vaccines composed of

response to a chemical gradient (typically toward two or more molecules conjugated together to
a chemoattractant such as a chemokine). enhance immunogenicity.
classical pathway of complement activation. C-reactive protein. An APP that can bind to the
A pathway that leads to the activation of the surface of a pathogen or a damaged host cell,
complement cascade in response to antigen– where it can activate complement or directly act
antibody complexes. as an opsonin.
class II–associated invariant chain peptide cross-match. A technique that mixes the
(CLIP). Class II–associated invariant chain binds blood and serum of donor and recipient
to nascent MHC class II molecules to prevent animals before a blood transfusion to predict
peptide binding in the endoplasmic reticulum. a transfusion reaction. A major cross-match
CLIP is the last portion of the invariant chain tests for alloantibodies in the recipient’s
that remains bound until MHC class II molecules serum by mixing the recipient’s serum with
are in the endosomal compartment. the donor erythrocytes. A minor cross-match
tests for alloantibodies in the donor’s serum by
320 G l o s s a ry

mixing the donor’s serum with the recipient’s Variants include the indirect ELISA and sandwich
erythrocytes. ELISA.
cross-presentation. The process whereby eosinophils. Granulocytic white blood cells

extracellular antigens taken up by dendritic cells important principally for defense against
(which would normally be presented on MHC II parasites such as helminths.
molecules) are processed and presented in MHC
fever (pyrexia). The elevation of the core body
I molecules.
temperature as a result of a higher-than-normal
cytokines. Cell-derived soluble molecules that thermoregulatory set point within the anterior
act on the cells that produce them and on other hypothalamus. Fever is commonly induced
receptive nearby cells. through the systemic release of proinflammatory
cytokines (e.g., tumor necrosis factor–α and
damage-associated molecular patterns
interleukin-1).
(DAMPs). Molecular structures indicating tissue
injury that are released from damaged and flow cytometry. A technique that characterizes cell
necrotic host cells, which can be detected by the types in a fluid by passing a thin stream of cells
innate immune system. by a laser.
defensins. Cationic antimicrobial peptides with a γ-interferon. A major proinflammatory cytokine.

β-pleated sheet structure. Defensins are secreted
granzymes. Serine proteases found in the
by certain immune and epithelial cells and
cytoplasmic granules in cytotoxic T and NK cells
contribute to killing of engulfed microbes within
that induce apoptosis once delivered to target
phagolysosomes of phagocytes.
cells.
delayed-type hypersensitivities.
gut-associated lymphoid tissues (GALT).
Hyperreactivities that involve T-helper or
Secondary lymphoid structures found in the
cytotoxic T cells but not antibody molecules and
mucosal epithelia of the gastrointestinal tract
take days to develop.
and the sites of gut-associated, secondary
dendritic cell. Any one of several cell types with a immune responses.
dendritic morphology that play important roles
haptens. Small molecules that by themselves
in antigen handling and antigen presentation
are not immunogenic but may, when coupled
during immune responses.
with other molecules, induce specific immune
D-gene segments. DNA segments present in the responses.
TCR β gene and Ig H-chain gene families that
hematopoiesis. The production in the bone
add to the variety of TCRs and BCRs of T and
marrow of red blood cells, platelets, and white
B cells.
blood cells from hematopoietic stem cells.
eicosanoids. A group of signaling molecules
high endothelial venules (HEVs). Specialized
derived from the oxidation of fatty acids. Both
postcapillary venous swellings found in
leukotrienes and prostaglandins are eicosanoids
secondary lymphoid tissues. These structures are
that play important roles in mediating
the sites where lymphocytes exit the blood and
inflammation.
move into secondary lymphoid tissues and the
enzyme-linked immunosorbent assay (ELISA). lymphatic system.
A technique that uses antibodies labeled with
humoral immune response. An adaptive
enzymes, radioisotopes, or fluorescent markers
response characterized by the production of
to detect an antigen or, indirectly, an antibody.
antibodies.
G l o s s a ry 321
321

hypersensitivities. Adaptive immune response IL-17. A cytokine that is a powerful stimulator of
directed toward innocuous substances such as inflammation.
self or pollen that induce damage to the host.
immediate-type hypersensitivities.
IgA. An isotype of antibodies characterized by α Hypersensitivities mediated by antibodies,
heavy chains. IgA appears in both monomeric including type I, type II, and type III
(in the blood) and dimeric (mucosal secretion) hypersensitivities.
forms.
immune complex diseases. Type III
IgE. An isotype of antibodies characterized by ε hypersensitivities involving large antigen–
heavy chains and of particular importance in antibody complexes that precipitate onto vessel
allergies and immunity to parasites. walls and activate complement.
IgG. An isotype of antibodies characterized by γ immune mediated. Any of a series of effects that
heavy chains. It is the most common antibody in result from the actions of innate or adaptive
many mammals. immune responses.
IgM. An isotype of antibodies characterized by µ immune-stimulatory complexes. Combinations

heavy chains. It is the predominant antibody in of antigens and other molecules (such as
most primary adaptive immune responses. lipid micelles) that enhance the host immune
response.
IL-1. Any one of a family of cytokines that are
strongly proinflammatory and some of which immunoblotting (western blotting). A technique
induce fever. that detects specific proteins in a fluid. Proteins
are first separated using gel electrophoresis and
IL-2. A cytokine that stimulates T-cell division
are then transferred onto membranes, where
and is necessary for the development of most
they are incubated with labeled antibodies that
adaptive immune responses.
can bind and identify specific proteins.
IL-3. A cytokine that stimulates the proliferation of
immunodiffusion assay. A technique that
bone marrow progenitor cells.
determines antibody or antigen concentrations
IL-4. A cytokine that stimulates B-cell division and in a fluid by diffusing them through an agar gel
induces production of certain immunoglobulin and detecting their zones of precipitation.
isotypes.
immunogens. Molecules or molecular complexes
IL-6. A major proinflammatory cytokine. that induce immune responses.
IL-10. A cytokine with diverse effects in immunoglobulin (Ig). Antibody molecules.

immunoregulation and inflammation. It
immunohistochemistry. A technique used to
suppresses the expression of T-helper 1 cytokines,
detect antigens of cells or infectious agents in a
MHC class II antigens, and costimulatory
histologic section using labeled antibodies.
molecules on macrophages. It also extends B-cell
survival, proliferation, and antibody production. inflammasome. A multiprotein complex that can
form in immune cells, such as macrophages,
IL-12. A cytokine that stimulates the differentiation
in response to inflammatory stimuli (typically
of naive T cells into T-helper 1 cells. It can also
PAMPs and DAMPs). The inflammasome
stimulate inflammation by enhancing production
catalyzes the maturation of proinflammatory
of IFN-γ and tumor necrosis factor–α from T
cytokines IL-1β and IL-18.
and NK cells.
322 G l o s s a ry

inflammation. A term used to describe the process neutrophils, eosinophils, basophils, NK cells, and
whereby immune cells, fluid, and protein lymphocytes), it is commonly used to describe all
accumulate in a tissue in response to infection or immune cells, including those not typically found
injury. in the blood (e.g., macrophages, mast cells, and
dendritic cells).
innate immune system. Complex collections of
cells and molecules that preexist contact with leukotrienes. Lipid-based signaling molecules
pathogens and interact to produce protective derived from arachidonic acid synthesized
immune responses such as inflammation. and released by sentinel cells and other cells.
Leukotrienes play important roles in mediating
integrins. Cell-surface proteins that mediate
inflammation.
cellular adhesion to extracellular matrix or to
other cells. lipopolysaccharide (LPS). A significant
component of the outer membranes of Gram-
interferons (IFNs). A collection of cytokines that
negative bacteria, consisting of a complex of
interfere with viral infection (IFN-α and IFN-β)
lipid and sugar residues. LPS is a major PAMP of
and modulate immune responses (most notably
the innate immune system and is often referred
IFN-γ).
to as endotoxin.
Interleukins (ILs). Cytokines that are produced
lymph. The fluid that flows through the
by one white blood cell (leukocyte) and act in
lymphatics.
autocrine and paracrine modes to alter leukocyte
actions. lymph nodes. Small, oval, filtering secondary
lymphoid organs that appear throughout the
invariant chain (Ii). A polypeptide that occupies
lymphatics.
MHC class II molecules’ binding sites to prevent
peptide binding while the nascent MHC class lymphoid. Used to describe cells that are derived
II molecules are resident in the endoplasmic from the common lymphoid progenitor cells
reticulum. during hematopoiesis; includes B cells, T cells,
and NK cells.
J gene segments. DNA segments present in TCR
and BCR gene families that add to the variety of lymphoid follicles. Regions found in secondary
possible TCRs and BCRs with individual animals. lymphoid tissues where T and B cells interact.
junctional diversity. Addition and subtraction of macrophages. Large phagocytic immune

DNA bases that occur during the formation of cells found throughout tissues and at sites of
junctions between variable-joining and variable- inflammation; can function as sentinel cells,
diversity–joining portions of TCRs and BCRs. APCs, and antimicrobial effector cells.
It is responsible for dramatically increasing the
major histocompatibility complex (MHC). The
variety of TCRs and BCRs found in individual
segment of animals’ genomes that contains
animals.
genes encoding MHC class I and MHC class II
lectin pathway of complement activation. molecules.
A pathway that leads to the activation of the
mast cells. Granular immune cells found
complement cascade in response to simple sugar
predominantly in connective tissues surrounding
residues commonly found on microbial surfaces.
vasculature and nerves that act to initiate
leukocytes. White blood cells. Although this and amplify inflammation through the
term technically encompasses only white cells release of vasoactive compounds and other
that can be found in the blood (e.g., monocytes, proinflammatory lipids and cytokines. Mast cells
G l o s s a ry 323
323

are also the principal cells associated with type I monocytes, neutrophils, eosinophils, basophils,
hypersensitivity reactions (allergy). mast cells, macrophages, and myeloid dendritic
cells.
membrane attack complex (MAC). A structure
that is assembled in membranes (typically of naive T cells. A mature T cell expressing a TCR,
microbes) after the activation of the complement CD3, and CD4 or CD8 that has not encountered
cascade. The MAC, which is composed an antigen reactive with the cell’s TCR.
predominantly of ten to sixteen molecules of C9,
natural killer (NK) cells. Large granular
forms a barrel-shaped pore that compromises the
lymphocytes that function as a part of the
integrity of the target membrane.
innate immune response against intracellular
MHC class I molecules. Molecules composed pathogens. These cells can recognize and kill
of complex MHC class I α chain (derived virally infected host cells or tumor cells before an
from the MHC genes) and a smaller molecule, adaptive immune response is mounted.
β2-microglobulin. MHC class I molecules bind
negative selection. The process by which
cytosol-derived peptides and present them to
self-reactive lymphocytes are eliminated in the
CD8+ T cells.
thymus.
MHC class II molecules. Molecules composed
neutralizing antibodies. Antibodies that bind to
of a complex of MHC class II α and β chains
critical sites on toxins, viruses, and sometimes
(both derived from MHC genes). MHC class
bacteria and prevent the toxin, virus, or
II molecules bind peptide antigens present in
bacterium from binding to its receptor on host
cellular endosomes (generally derived from the
cells.
extracellular spaces) and present these peptides
to CD4+ T cells. neutrophil extracellular traps. Structures
secreted from activated neutrophils at the
MHC class III genes. A classification sometimes
site of inflammation that consist of DNA and
used to describe MHC class I– and II–unrelated
antimicrobial proteins that can trap and kill
genes located among or between the MHC class
extracellular microbes.
I and II genes. Some MHC class III gene products
are involved in immune responses but are in no neutrophilia. The increased abundance of
way similar to the antigen-presenting activities of neutrophils in the peripheral blood. Neutrophilia
MHC class I and II molecules. is said to have a left shift when increased
numbers of less mature neutrophils (band
monoclonal antibodies. Antibodies derived from
neutrophils) are released into circulation.
a single clone of hybridoma cells producing a
single type of antibody with a single (or nearly neutrophils. Granulocytic white blood cells
so) antigenic specificity. important for innate defense against extracellular
microbes such as bacteria. Neutrophils are
monocytes. Large circulating, mononuclear white
rapidly recruited in acute inflammation when
blood cells that give rise to a variety of other
they can phagocytose and kill microbes and
cells, including tissue macrophages.
release antimicrobial products.
mucosal-associated lymphoid tissue (MALT).
nonsteroidal anti-inflammatory drugs
Secondary lymphoid tissues found in lung,
(NSAIDs). A class of pharmaceutical agents
gastrointestinal, and other mucosal epithelia.
that aim to reduce inflammation, fever, and
myeloid. Used to describe cells that are derived pain. Many NSAIDs are inhibitors of the
from the common myeloid progenitor cells cycloxygenase enzymes that are responsible for
during hematopoiesis. These cells include the synthesis of prostaglandins.
324 G l o s s a ry

nuclear factor kappa-light-chain-enhancer perforins. Cytolytic proteins found in the granules
of activated B cells (NFκB). A transcription of cytotoxic T lymphocytes and NK cells.
factor that controls the expression of genes
periarteriolar lymphoid sheath. Secondary
associated with proinflammatory responses and
lymphoid tissue of the spleen.
cell survival.
peripheral tolerance. Maintenance of tolerance
nucleotide-binding oligomerization domain
to self antigens beyond the thymic mechanism
(NOD)–like receptors. A family of intracellular
of negative selection, including Treg cells.
PRRs that can detect pathogen-associated
molecular patterns, such as peptidoglycan, phagocytes. Cells (generally white blood cells)
within the cytosol of a cell. These receptors capable of phagocytosis.
are particularly important for the detection of
phagocytosis. Engulfment of pathogens or other
intracellular bacteria.
particles into intracellular phagosomes.
opsonization. Coating (generally of bacteria) with
phagolysosome. A hybrid organelle resulting
antibodies, C3b, or both to enhance phagocytosis
from the fusion of a phagosome with lysosomes.
and destruction of pathogens.
The lumen of a phagolysosome is antimicrobial
passive immunotherapy. Neutralization of and degradative, which acts to kill and digest
toxins, viruses, or bacteria by injection of phagocytosed microbes and debris.
preformed antibodies, for example after a
plasma. The liquid component of blood that
snakebite.
has the red blood cells, white blood cells, and
passive transfer. Movement of preformed platelets removed. Clotting factors are still
antibodies from mother to fetus (transplacental present.
or yolk sac passive transfer) or offspring (via
polyclonal antibodies. Usually sera-containing
colostrum and milk).
antibodies of multiple Ig isotypes with multiple
pathogen-associated molecular patterns antigenic reactivities, produced by multiple
(PAMPs). Evolutionarily conserved molecular clones of plasma cells in immunized animals,
structures that are common to groups of also known as antisera.
microbes and pathogens but are absent from the
positive selection. The thymic process that
host species. The innate immune system can
results in elimination of T cells with unreactive
detect the PAMPs through PRRs.
TCRs and selection for T cells with functional
pattern-recognition receptors (PRRs). TCRs.
Germline-encoded receptors expressed on or in
primary lymphoid organs. Lymphoid organs
many immune cells, particularly sentinel cells,
in which lymphocytes arise or differentiate; in
that detect PAMPs, DAMPs, or both. These
mammals, they include the thymus and the bone
receptors include TLRs, NOD-like receptors, and
marrow.
RLRs.
proinflammatory cytokines. Cytokines that
peptidoglycan. A meshlike component of
promote inflammation.
bacterial cell walls consisting of chains of
alternating sugar residues cross-linked with prostaglandins. Lipid-based signaling molecules
short chains of amino acids. Peptidoglycan is derived from arachidonic acid synthesized
recognized by the innate immune system as a and released by sentinel cells and other cells.
PAMP. Prostaglandins play important roles in mediating
G l o s s a ry 325
325

inflammation as well as in homeostatic genes that encode CDR1, CDR2, and CDR3
processes. portions of the BCRs of B cells in lymphoid
follicles.
proteasomes. Large molecular complexes that
degrade proteins into their component peptides T-cell receptors (TCRs). T-cell surface molecular
and amino acids. complexes that bind antigens often in association
with MHC class I or MHC class II molecules on
pyrexia. See fever.
APCs.
pyrogen. Any substance that induces fever.
Th1 cells. CD4+ T cells involved in inflammatory
Pyrogens can be derived exogenously (e.g., LPS)
and antibody responses.
or endogenously (e.g., tumor necrosis factor–α).
Th2 cells. CD4+ T cells involved primarily in
RAG1. A protein that acts in concert with RAG2
antibody responses.
to induce and direct recombination of TCR and
BCR gene segments during T-cell and B-cell Th17 cells. CD4+ T cells involved primarily in
differentiation. inflammatory responses.
RAG2. A protein that acts in concert with RAG1 to Thymocytes. T cells in the thymus.
induce and direct recombination of TCR and
thymus-independent antigens. Antigens capable
BCR gene segments during T-cell and B-cell
of inducing T-helper cell–independent antibody
differentiation.
production.
recombinant vaccines. Immunogens composed
toll-like receptors (TLRs). A major family of
of antigen-encoding DNA incorporated into the
intracellular PRRs that can detect a variety of
genome of a viral vector. The purpose of the
PAMPs as well as DAMPs, which leads to the
vector is to introduce the antigen-encoding DNA
activation of proinflammatory responses.
into the cytosol of host APCs.
transfusion reaction. A potentially life-
RIG-like receptors (RLRs). A family of
threatening reaction after a blood transfusion
intracellular pattern-recognition receptors that
in which the animal’s immune system destroys
can detect the presence of viral RNA in the
transfused red blood cells from another (donor)
cytosol of host cells.
animal.
secondary lymphoid organs. Sites of immune
transporters associated with antigen
responses, including lymph nodes, spleen, and
presentation. Molecular complexes that
MALTs.
deliver cytosolic peptides into the endoplasmic
selectins. Cell-surface proteins that can bind reticulum for binding by MHC class I molecules.
specific glycoproteins displayed on other
T-regulatory cells (Tregs). Extrathymic CD4+ T
cells. Selectins can be expressed on vascular
cells that help to regulate immune reactions to
endothelial cells as well as several immune
self.
cells and are important in the recruitment of
leukocytes to areas of inflammation. V gene segments. DNA segments found in BCR
and TCR gene families. These segments join
serum. Plasma (from the blood) with the clotting
with joining or diversity-joining gene segments
factors removed.
to form the N-terminal, variable regions of the
somatic hypermutation. Rapid and random BCR heavy and light chains, and the TCR α/β or
mutation of DNA bases in the regions of Ig γ/δ chains.
326 G l o s s a ry

Index
Page numbers in italics indicate illustrations.
acidity, and microbial growth, 20, 21 alternative pathway of complement antibody titer, 319
acquired immune deficiencies, 2, 272, activation, 58–60, 319 antigen-antibody complexes, 279–81
273–74 aluminum salts, as adjuvants, 264 antigen-depot effects, in vaccines, 264
acquired immunodeficiency syn- amino acids, 124; and MHC mol- antigen-presenting cells (APCs), 10,
drome, 16 ecules, 131–32 14, 29, 132, 137, 138, 157, 170,
Actinobacillus spp., 241 amphibians, 237; B-cell development, 235, 263, 319; allergens and, 275,
activating PRRs. See signaling 182; BCR gene arrangement, 276; B cells as, 203–4, 229; CD4/8
pattern-recognition receptors 183–84 interactions, 164–66; CD8 activa-
active immunity, 243 ANA. See antinuclear antibody test tion, 173–74; characteristics of,
acute infection, adaptive responses anaphylactic shock, 276–77, 280, 281 133–35; Tc-cell activation, 168–69;
to, 222 anaphylatoxin, 281, 319 T-cell response, 12, 225–26; TCRs
acute inflammation: initiators and anemia, autoimmune hemolytic, 156 and, 153, 154; tumor apoptosis
mediators in, 70–72; leukocyte ankylosing spondylitis, 121 and, 291–92
recruitment in, 75–80; mastitis, 67, anorexia, 82 antigens, 9, 64, 99, 111, 117, 120,
85–88; purposes of 68–69; signs of, anthrax, passive immunotherapy 122, 125, 319; adaptive immune
69–70; systemic effects of, 80–85; for, 267 responses to, 10–12, 225–26; aller-
vascular changes in, 72–75 anthrax bacteria, phagocytosis of, 94 gic responses to, 274–75, 277–78;
acute phase proteins (APPs), 82, antibiotics, and gut flora, 237 and antibodies, 199–201, 202; and
84–85, 86(table), 319 antibodies, 3, 8, 11, 13, 64, 143, 250, B-1 B cells, 193, 194; erythrocite,
adaptive immune responses 316; and antigens, 122, 199–201, 295–97; in gut immune system,
(acquired immune responses), 202; B cells and, 182, 193, 231; 236–37; and MHC, 130–39, 140,
9, 14–15, 28, 91, 110, 222–23; an- complement system, 54, 55; 239; recognition of, 39–40; T-cell
tigens and, 225–26; in secondary diversity in, 183, 186, 212–13; gen- recognition of, 162–64; T-cell
lymphoid tissues, 229–33; T cells erating, 298–99; immunoglobulins response to, 164–66; TCR recogni-
and, 226–29 and, 198, 217; maternal transfer tion of, 144–45
adaptive immune system, 5, 18, 31, of, 243–48; opsonization, 213–14; antigen-specific receptors, 10–11, 114
122, 319; features of, 6(table), 7, 8, in passive immunotherapy, antiglobulin, 319
9–14; memory in, 237–39 267–68; primary and secondary anti-inflammatory drugs, 268
adenovirus, 143; vaccination against, responses, 9–10, 194–196, 206, 256; antimicrobial effector cells, 29, 50
251 self-reactive, 283 antimicrobial enzymes, 95
adipocytes, 85 antibody-antigen reactions, detection antimicrobial mechanisms, 6, 14
adjuvants, for vaccines, 263–65, 269, of, 300–307 antimicrobial peptides, 99, 246
319 antibody-based diagnostic tests: anti- antimicrobial products, 90
affinity maturation, 198–99, 211, 232, body generation, 298–99; ELISAs, antimicrobial proteins, 100, 246; in
319; of B cells, 230–31 300–302; flow cytometry, 305–6; phagosomes, 94–95; in secretions
agammaglobulinemia, 312 immunoblotting, 302–3; immu- 18–20
aging, and immune deficiencies, 274 nofluorescent microscopy, 303–4; antimicrobial toxins, 25
AIDS, 1, 272, 273, 274 immunohistochemistry, 306–7, antinuclear antibody (ANA) test,
AIRE. See autoimmune regulator 308; precipitation assays, 304–5 313–14
allergens, responses to, 274–79 antibody-dependent cell-mediated antiparasitic mechanisms, 14
allergic responses, 27, 71, 317 cytotoxicity, 32, 105, 319 antiparasitic products, 6
allergies, 24, 28, 100; mast cells and, antibody effector functions, 213–15 antiparasitic proteins, 27
31, 71; types of, 274–79 antibody-mediated autoimmune antiplatelet antibody test, 314–15
alloantibodies, 316 diseases, 283–84 antipyretics, 82
allografts, 125 antibody responses, and Th2 cells, 170 antiserum, 319
327
327

antithyroglobulin autoantibody test, hypermutated, 207, 209–10; im- development, 182, 186, 188, 189,
315–16 munoglobulin isotypes in, 189–91; 214; hematopoiesis, 21–25, 110–11,
antivenins, 280, 281, 283 MZ B cells, 194, 203 113; immune cell production,
antivenin therapy, type III hypersensi- B cells (B lymphocytes), 3, 9, 114, 111–12, 152
tivities, 280, 281–82, 283 117, 118, 120, 135, 236, 243, 259, bordetella, vaccines against, 258, 266
antiviral capabilities, 27 275, 319; affinity maturation of, Bordetella bronchiseptica, 266
antiviral vaccines, 255 207–10, 230–31; antigen interac- Borrelia burgdorferi, ELISA for, 300,
APCs. See antigen-presenting cells tions, 199–203; as APCs, 133, 301
apoptosis: NK cells n, 7, 32; of tu- 203–4, 229; and BCRs, 182–83; in botulinum toxin, 224–25
mors, 290–92 calves, 242, 249; clonal expan- bovine leukocyte adhesion deficiency
APPs. See acute phase proteins sion of, 10–11; and complement (BLAD), 77, 313, 319–20
acquired immune responses. See system, 63–64; development of, bovine respiratory syncytial virus
adaptive immune responses 188–89, 214; genetics of, 183–86; infections, 84
arthritis: in goats, 139, 140; suppura- Ig isotype switching in, 206–7; and bovines, 44, 84, 252; acute mastitis,
tive, 70 immunoglobulin isotypes, 189–91; 67, 68, 85–88; basophils, 27; BLAD
Aspergillus sp., 42 marginal-zone, 194–95; matura- in, 77. See also cattle
aspiration, bone marrow, 24–25 tion of, 33, 112–13; memory, 12, bovine viral diarrhea, 242
atopy, in dogs, 197 212, 231, 238; production of, 23, Brittany dog (Brittany spaniel), 54; C3
autocatalysis, 58 111; somatic hypermutation, 231, deficiency in, 53, 64–65
autoimmune diseases, autoimmunity, 232; Th-activated, 204–7, 210‒13; bronchitis, in dogs, 197, 215
121, 132, 156; antibody-mediated, and Th cells, 170, 171; variety bronchopneumonia, 84
283–84; diagnostic tests for, 313–16 of, 191–95; in XLSCID dogs, bronchus-associated lymphoid tis-
autoimmune hemolytic anemia, 156 162(table), 178–79. See also by sues, 119, 320
autoimmune regulator (AIRE), 156, number designation Bruton’s tyrosine kinase (BTK), 181,
157 BCRs. See B-cell receptors 195–96
autosomal recessive diseases, 77 bile, 20 BTK. See Bruton’s tyrosine kinase
birds, 43, 156; B cell development in, Burnett, MacFarlane, 124, 288
B-1 B cells, actions of, 191–94 182, 212; BCR gene diversity in, bursa of Fabricius, 113, 114, 243; B-
β2-microglobulin, 320 184, 186, 187; in ovo vaccination, cell development, 182, 189
B-2 B cells, 182, 260; BCR diversity 244, 245; maternal transfer of im-
in, 194–95 munity in, 243–44 C3b, 53–63, 90–91, 198, 207, 210, 228,
B7 molecules, 168–69, 320 black disease, 251 277, 281, 311, 315
bacteremia, in neonatal foals, 37–38 BLAD. See bovine leukocyte adhesion C3 convertase, 55
bacteremia-induced systemic inflam- deficiency C3 deficiency, 53, 64–65
matory response syndrome blackleg, 251 C5a, 54–62, 69, 71, 76, 277, 281, 282
(SIRS), 37 bladders, urinary, 20 C5 convertase, 55
bacteria, 14, 19, 21, 40, 44, 119; blood, 114, 182: crossmatching, cachexia, 82
antibodies and, 13, 194; exotoxins 309–12; erythrocyte antigens in, CAEV. See caprine arthritis encepha-
of, 224–25, 260; Gram-negative, 295–97; innate immune system litis virus
37, 38, 224; Gram-positive, 224; cells in, 25–28; typing of, 307–9 calcivirus, live-attenuated vaccines
gut, 234–35, 237; phagocytosis of, blood cells, 3, 34; production of, and, 258
93, 94, 95 21–23, 111 California Mastitis Test (CMT), 67, 69
bacterial infections, 77, 274; in dairy blood counts, 24 calves. See cattle
cattle, 67, 68, 86–87, 241, 251–52; blood flow, and acute inflammation, camelids, BCR genetics, 183, 186
in dogs, 17, 53, 161; exotoxins and, 73 cancer, 84; cell development, 288–89;
224–25; in foals, 37–38, 89–90, 181, blood groups, 296–97 and immune system, 287–88;
195–96 blood smears, monitoring hemato- immunotherapy for, 267, 292–93;
barriers, anatomical and physiologi- poiesis, 24 research on, 124–25; tumor
cal, 18–21 blood typing, 319; crossmatching, growth, 290–92
basophils, 3, 4, 27–28, 274, 319; pro- 309–12; methods of, 307–9, 310 cancer drugs, and acquired immune
duction of, 22, 23, 112 blood typing cards, 308, 310 deficiencies, 273
basset hounds, 163; XLSCID in, blood vessels, 236; immune com- cancer therapy, 289, 292–93; immune
161–62, 177–79 plexes and, 281–82; and inflamma- stimulants as, 289–90
B-cell coreceptors, 202–203 tory mediators, 72–73 Candida albicans, 16
B-cell receptors (BCRs), 9, 111, B lymphocytes. See B cells candidiasis, oroesophageal, 1, 16
182, 201, 211, 229, 230, 252, 299, bobcats, FIV in, 271 canine leukocyte adhesion deficiency
319; antigen binding, 199–200; bone marrow, 34, 82, 85, 171, 212, (CLAD), 313
diversity of, 183, 187, 194–95, 239; 242; aspiration of, 24–25; B-cell canine pertussis vaccine, 266
328 Ind e x

canines: C3 deficiency, 53, 64–65; can- cellular immune responses, 255 55–57; deficiencies in, 64–65; in
cer, 287; cyclic neutropenia in, 17, central tolerance, 156, 320 lectin pathways, 57–58
18, 33–35; distemper vaccination, Chediak-Higashi syndrome, 312 complement receptors, 320
251; eosinophil degranulation, cheetahs, 128 complement system, 6, 8, 231, 320;
100; hepatitis, 225; lymphatics and chemoattractants, 77–78 activation of, 54–60, 78; outcomes
lymph nodes, 14, 116; mast cells, chemokines, 4, 50, 237, 320; and of activation in, 62–64; regulation
32; myleoid innate immune cells, acute inflammation, 70, 76, 77, 79 of activation in, 60–62
25, 26; NK cells, 33; parvovirus chemotaxis, 320; leukocyte, 25, 63, complex life, evolution of, 1–2
in, 219–20, 239–40; spleen in, 117, 78, 79 component vaccines, 259–60, 263
118; X-linked severe combined im- chemotherapy, for cancer, 293 congenital immune deficiencies,
munodeficiency in, 161–62 chickens, 43; B-cell development, 186, 272–73, 312
capillaries, lymph leakage from, 115, 212; BCR gene diversity, 184, 187; conjugate vaccines, 260–61, 320
117 γ/δ T cells, 156; immune system connective tissues, mast cells and, 31
caprine arthritis encephalitis virus development in, 243–44, 249 contact hypersensitivities, 282
(CAEV), 121, 139–41 Chlamydia, 225 convertases, C3 and C5, 55, 62–63
cardiovascular system, 115 chronic granulomatous disease, 97 Coombs, Robin, 314
carrier effects, 264 chronic infections, from FIV, 284 Coombs tests, 314, 315
cathelicidins, 19, 320 chronic inflammation, 80 coreception, by B cells, 202
cats: allergic responses in, 27, 276; cilia, in respiratory tract, 20–21 cortisol, and acquired immune defi-
autoimmune hemolytic anemia, ciliary dyskinesia, 20–21 ciencies, 273–74
156; blood groups, 296, 297(table); CIO. See hypochlorite ion cowpox, and vaccination, 253–54
blood typing, 307, 308, 309, 310, cisplatin, 273 COX. See cyclooxygenase enzymes
311; bone marrow hematopoiesis, CLAD. See canine leukocyte adhesion coyotes, rabies vaccines for, 262
110–11; feline immunodeficiency deficiency C-reactive proteins (CRPs), 46, 85, 320
virus in, 271, 284–85; feline classical pathway of complement cross-matching, blood, 309–12,
lymphadenopathy, 109, 110(table), activation, 55–57, 60, 320 320–21
119–20; maternal transfer of im- class II-associated invariant chain cross-presentation, 321
munity, 245 peptide (CLIPs), 320 CRPs. See C-reactive proteins
cattle, 27, 44, 77, 123(table), 186, cleavage products, 55 crypt Paneth cells, 20
297, 312; acute mastitis in, 67, 68; CLIPs. See class II-associated invari- CSFs. See colony-stimulating factors
colostrum in, 246–47; immuno- ant chain peptides CXCL8 (IL-8), 70
competency in, 242–43, 249–50; clonal expansion, 320; of lympho- cyclic neutropenia, in collies, 17, 18,
leptospirosis in, 268–69; maternal cytes, 10–11, 14–15 33–35
transfer of immunity, 245, 246, Clostridia, 53, 251; botulinum, 224–25; cyclooxygenase (COX) enzymes, 71
247; neonatal infections in, 241, difficile, 237 cystitis, in dogs, 197, 215
251–52; vaccination of, 268(table) CMT. See California Mastitis Test cytokines, 3–4, 5(table), 6(table), 23,
CCL11. See eotaxin coccidiosis, in chickens, 249 69, 217, 238, 264, 321; in isotype
CD4, 151, 163, 294; and CD8, 164–66; Coggin’s test, 304 switching, 209, 230; proinflamma-
flow cytometry, 305, 307; T cells, collies, cyclic neutropenia in, 17, 18, tory, 30, 50, 51, 70, 81–82, 86; Th
151, 152, 153, 154, 155, 171, 173, 33–34 cells, 171, 174
236, 291–92 colon, epithelial cells in, 18 cytokine storm, 52
CD5+ B cells, 191 colony-stimulating factors (CSFs), 4, cytotoxins, 99
CD8, 163; activation of, 168–69, 23, 82, 320 cytotoxic T (Tc) cells. See Tc cells
173–74; and CD4, 164–66; flow colostrum, 248, 296, 320; immunity cytotoxicity: antibody-dependent
cytometry, 305, 307; T-cell devel- transfer through, 245, 246–47 cell-mediated, 32, 105, 319; NK
opment and selection, 151, 152, commensalism, 21, 234 cell-mediated, 32, 90, 101–5, 231
153, 154, 155, 236; tumor apoptosis communication, among cells, 3–5
and, 291, 292 complementarity-determining dairy cattle, neonatal infections, 67,
CD28, 320 regions (CDRs), 182, 199, 201, 207, 68, 86–87, 241
CD40 molecules, B cells and, 203–4 229, 320; and TCR chains, 163–64 damage-associated molecular pat-
CD55. See decay-accelerating factor complement cascades, 55, 61–62, terns (DAMPs), 52, 70, 169, 321;
CDRs. See complementarity-deter- 214, 320 innate immune system and,
mining regions complement components, 53, 55 46–49; and macrophage activa-
cecal tonsils, GALT in, 119 complement control proteins, 60–62 tion, 50, 51; production of, 47–48
cell-adhesion molecules, 75 complement proteins, 54–55; activa- Daniel, M. D., 1
cells, communication between, 3–5. tion of, 213–14; activation control, DAMPs. See damage-associated mo-
See also by type 60–62; in alternative pathways, lecular patterns
cellular debris, 29 58–60; in classical pathways, DAT. See direct antiglobulin test
Ind e x 329
329

DCs. See dendritic cells; myeloid edema, 69, 251 extracellular microbes, 26, 90
dendritic cells effector cells: antimicrobial, 29, 50; in extravasation, of leukocytes, 25, 75–79
DEA-1. See dog erythrocyte antigen gut immune system, 236 extravascular fluids, 182
1 group effector cytotoxic T cells, 174–76 exudate, 73–74
decay-accelerating factor (CD55), 61 effector mechanisms, 12, 13, 26; of
defensins, 19, 20, 321 antibodies, 213–15, 216 fatty acids, 71
degranulation, 71; by neutrophils and effector T-helper cells, 169–71 Fc receptors, 213, 247, 248, 250, 275;
eosinophils, 99–100; by NK cells, efferocytosis, 91 distribution and function, 214–15
102, 104, 105 Ehrlich, Paul, on cancer and immune FDCs. See follicular dendritic cells
delayed-type hypersensitivities system, 287–88 feline immunodeficiency virus (FIV),
(DTHs), 274, 282, 321 eicosanoids, 71, 321 271, 272, 274, 284–85
demodetic mange (demodicosis; red Eimeria, and chicken coccidiosis, 249 feline leukemia virus, allergic re-
mange), 197, 199, 215 ELISAs. See enzyme-linked immuno- sponse to vaccine, 276
denaturation, 259, 260 sorbent assays feline lymphadenopathy, 109,
dendritic cells (DCs), 3, 6, 28, 30, encephalitis, in goats, 121, 139 110(table), 119–20
33, 111, 117, 235, 321; as APCs, endocytosis, 30; antigen acquisition feline panleukopenia virus, 220
133–34; myeloid, 23, 31; in tumor and, 134–35 felines, 271; lymphadenopathy, 109,
growth, 292 endoplasmic reticulum (ER), 136, 146 119–20; MHC polymorphism in,
dermatitises: in dogs, 197–98, 215; endosomes, 94, 137 128–29; monocytes, 28. See also
poison ivy, 282 endothelial cells, 71; in inflammation, cats
detergents, in bile, 20 73–74 fetuses, immune system develop-
D-gene segments, 321 endothelia: leukocyte recruitment ment, 241–48
diacylglycerols, 71 and, 75–76; vascular, 228, 239; vas- fever, 83, 321; functions of, 82, 84
diapedesis, 77 cular permeability and, 73–74, 75 ficolins, 58
diarrhea: antibiotic-induced, 21; bo- endotoxic shock, from LPS, 38 fish, 182, 183‒84, 186, 203, 213, 266
vine viral, 242; from Clostridium enterotoxemia, 251 FIV. See feline immunodeficiency
difficile, 237; in neonatal livestock, enzyme-linked immunosorbent virus
241 assays (ELISAs), 300–302, 321; for flea allergies, 197, 215, 276
dilation, vascular, 62–63 hypothyrodism, 315–16 flies, biting, 128
direct antiglobulin test (DAT), 314 enzymes, 20, 71, 95, 97–98, 148 flies, fruit. See Drosophila melanogaster
dismutation, 97 eosinophils, 3, 6, 14, 26–27, 63, 100, flow cytometry, 305–6, 307, 321
distemper, canine, 161–62, 251, 258 274, 321; in acute inflammation, flushing, as barrier, 20
DNA, 7; in B-cell receptor chains, 68, 79; production of, 22, 23, 112 foals. See horses
183–86; hairpins, 158; naked eotaxin (CCL11), 79 follicular dendritic cells (FDCs), 33,
protein-encoding, 262; in T-cell epidermal cells, 18 230, 232; and affinity matura-
receptor chains, 145–50 epithelial cells, epithelia, 54, 220, tion of B cells, 207–10, 211; and
dog erythrocyte antigen 1 (DEA-1) 233; ciliated, 20, 21; gut, 236, 237; complement system, 63, 64; Fc
group, 296, 308 as microbial barriers, 18, 19; and receptors and, 214–15
dogs, 46, 54, 85, 116; allergies in, 274; serum sickness, 281–82 food intolerance, in dogs, 197, 215
autoimmune diseases in, 156, 313; epitopes, 122, 260; antigenic, 122, formyl peptide receptors (FPR), 46
blood groups in, 296, 297(table); 125; T-cell, 260–61 fowl, bursa of Fabricius, 113, 114. See
blood typing, 307, 308; C3 defi- equine X-linked agammaglobulin- also chickens; turkeys
ciency, 53, 64–65; cyclic neutro- emia (EXLA), 181, 195–96 foxes, rabies vaccines for, 262
penia in, 17, 18, 33–35; genetic equines, 143, 181; neonatal sepsis, FPR. See formyl peptide receptors
disorders in, 17, 33–35; hypothy- 37–38 Francisella tularensis, 119–20
roidism, 315–16; IMHA in, 283; ER. See endoplasmic reticulum free radicals, 97, 98, 99
immune deficiencies in, 197–99, erythrocyte antigens (RBC antigens), function, loss of (functio laesa),
215, 217, 312; and live-attenuated types of, 295–97 69–70
vaccines, 257, 258; maternal erythrocytes (red blood cells), 3, 22; fungal infections, in Drosophila, 42
transfer of immunity, 245; tumors transfusion reactions, 316–17 fungi, 14, 225
in, 287, 293–94; type III hypersen- Escherichia coli, 39, 53; neonatal infec-
sivitiy in, 280, 281–82; vaccination tions, 37, 241 GALT. See gut-associated lymphoid
of, 250–51, 253, 256–57; XLSCID evolution, of complex life, 1–2 tissues
in, 161–62, 177–79, 273 EXLA. See equine X-linked γ/δ T cells, 33, 156–58, 290; T-cell
Drosophila melanogaster, fungal infec- agammaglobulinemia selection in, 150–53
tions in, 42 exotoxins, bacterial, 224–25, 237, 260 γ-interferon, 321
DTHs. See delayed-type extracellular innate effector mecha- gastrointestinal tract, 20, 21, 85. See
hypersensitivities nisms, 99–101 also gut; intestine
330 Ind e x

G-CSF. See granulocyte CSF hematopoietic growth factors, 23, 35 ICAM-1. See intercellular adhesion
genetic disorders: in horses, 143, hepatitis, canine, 161, 162, 225, 251 molecule 1
158–59, 181, 195–96; in dogs, 17, hepatocytes, 85, 225 IFN-α, 33
18, 33–35, 161–62, 177–79 herpes virus, 101, 258 IFN-β, 4
genetic bottlenecking, 128 HEVs. See high endothelial venules IFN-γ, 4, 171, 206, 289
genetic mutations, and congenital high-affinity B cells, 212 IgA, 193, 229, 231, 237, 243, 312, 322;
immune deficiencies, 273 high endothelial venules (HEVs), 204, development of, 198, 248; isotypes,
genetics: B cell, 183–86; of cancer 229, 239, 321 189–91; deficiencies of, 197–99,
cells, 288–89; goat MHC alleles, histamine, in acute inflammation, 215, 217, 273; maternal transfer
140–41; of MHC, 125–29; recom- 71, 73–74 of, 245, 246
binant vaccines, 261–62; of T-cell histocompatability, TCR and MHC Igα, 182–83
receptors, 145–50, 158–59 complex interactions, 164–66 Igβ, 182–83
germinal centers, in lymph nodes, HLAs. See human leukocyte antigens Ig (immunoglobulin) chains, 182,
207–10 Holsteins: acute mastitis in, 87; 183; quantification of, 312. See
germline chromosomal DNA, 7 BLAD in, 77 also antibodies; by various Ig letter
glomeruli, damage to, 53, 64, 65 horses, 144, 182; allergic responses in, designations
glomerulonephritis, 53, 64–65 27; blood groups, 296, 297(table); IgE, 31, 193, 198, 231, 246, 322; al-
glucocorticoids: and acquired im- eosinophilic inflammation in, 79; lergic reactions and, 274, 276; Fc
mune deficiencies, 273–74; and equine agammaglobulinemia, receptors and, 215, 275; isotypes,
congenital immune deficiencies, 181, 195–96; maternal transfer of 189–91; production of, 278–79
272–73 immunity in, 245; MHC, 126; neo- IgG, 56, 92, 193, 214, 231, 237, 243,
glycoproteins, 54 natal infections in, 241; neonatal 312, 322; antibody development,
GM-CSF. See granulocyte-monocyte isoerythrolysis, 296, 311–12, 314; 198, 248; in calves, 251, 252; iso-
CSF neonatal sepsis in, 37–38, 50–52; types, 189–91; maternal transfer
goats, 122; CAEV in, 121, 139–41 rattles in, 89–90, 107; Rhodococ- of, 245, 246
grafts, skin, 110, 112 cus equi, 106–7; SCID in, 143, Ig isotypes, 189–91; switching in B
Gram-negative bacteria, 224; LPS in, 158–59, 273; suppurative arthritis, cells, 206–7, 209
37–38, 39 70 IgM, 56, 193, 198, 206, 214, 229, 243,
Gram-positive bacteria, 224 host cells, 101, 169, 213 246, 252, 275, 312, 322; in calves,
granulocyte CSF (G-CSF), 23, 35, human leukocyte antigen-Bw27, 121 251, 252; isotypes, 189–91; penta-
111–12 human leukocyte antigens (HLAs), meric, 248–49
granulocyte-monocyte CSF (GM- 125, 137 IgX: in gut mucosa, 237; IgNAR and
CSF), 23 humans, 1, 146, 181, 184; cancer IgW, 186
granulocytes: in calves, 242–43; therapy in, 289–90; leukocyte IgY, in chicks, 243
production of, 22, 171 antigens, 101, 125, 137; MHC, 126, ILs. See interleukins; see also by various
granzymes, 176, 321 127–28; vaccination of, 253–54 IL number designations
Grist, Roy, 109 human T-lymphotropic virus III, 1 IL-1, 322
gut: immune responses in, 233–37; humoral immune responses, 182, IL-2 receptor, 166, 167, 171, 173, 322;
impacts of colostrum on, 247, 248 200, 298, 321; and vaccines, 254–55 lack of, 177–78
gut-associated lymphoid tissues hybridomas, 299 IL-3, 322
(GALT), 119, 235, 321; B-cell hydrochloric acid, 20 IL-4, 206, 322
development in, 186, 212, 214; in hydrocortisone, 273 IL-6, 322
cattle, 249 hydrogen peroxide, 99, 100; phago- IL-10, 322
gut mucosa, 233; and gut flora, some generation of, 97, 98 IL-12, 322
236–37 hypermutation, 215; somatic, 207, IL-17, 322
211, 231, 232 IM. See intramuscular vaccination
H chains (BCR), 182, 217; and hypersensitivities, 322; type 1, 31, 71, IMHA. See immune-mediated hemo-
antigens, 199–200; B-cell, 188, 207, 274–79; type II, 283–84; type III, lytic anemia
214; genetics of, 183–86; immuno- 64, 279–82, 283; type IV, 282 immediate-type hypersensitivities, 322
globulin, 189–91, 206 hypochlorite ion (CIO), 97, 98 immune competence, development
heart, APP synthesis, 85 hypogammaglobulinemia, 312 of, 241–43
heartworms, canine, 225 hypotension, severe, 37. See also immune complex diseases, 322;
helminths, eosinophil attacks on, 26, shock causes of, 279–82
27, 79 hypothalamus, and fever, 82, 83 immune deficiencies (immunodefi-
hematopoiesis, 34, 321; bone marrow hypothyroidism, in dogs, 315–16 ciencies), 2, 284–85, 289; acquired,
and, 110–12, 113; cell types, 21–23; 273–74; in Chinese shar peis,
coordinating and monitoring, I-CAMs. See intracellular adhesion 197–98, 215, 217; congenital,
23–25 molecules 272–73; diagnosis of, 312–13
Ind e x 331
331

immune effector mechanisms, 234, infectious disease, lymphadenopathy, Isle Royal National Park, wolves of,
236 120 219–20, 239–40
immune mechanisms, gut, 235–37 inflammasome, 48–49, 71, 322 isotypes: Ig, 189–91
immune-mediated diseases, 322; inflammation, 24, 28, 31, 323; com- isotype switching, 206–7, 209, 215,
autoimmune diseases and, 283–84; plement activation and, 62–63; 230, 231, 273
hypersensitivities, 274–83 purposes of, 68–69; at tumor sites, Ito, Hiroshi, 287
immune-mediated hemolytic anemia 293–94. See also acute inflamma-
(IMHA), 283, 314 tion; chronic inflammation Jenner, Edward, vaccination, 253–54
immune memory, 3, 12, 212, 231, inflammatory mediators, 51, 69; and
237–38, 260 vascular system, 72, 73–74 keratin, as barrier, 18
immune responses, 124, 213, 274; de- inflammatory processes, mediat- kidneys, 85, 182
termining, 300–301; gut, 233–37; ing, 23 killed vaccines, 259, 263, 266
primary, 206, 207; to tumors, inflammatory responses, 9, 171 killer cell immunoglobulin-like recep-
290–92; typical, 14–15. See also influenza A virus, vaccination tors (KIRs), 101
adaptive immune response; innate against, 259–60 killer lectin-like receptors (KLRs), 101
immune response innate cellular effector mechanisms, kinase: DNA-dependent, 158; tyro-
immune stimulants, as cancer classification of, 90–91. See also sine, 181, 191
therapy, 289–90 phagocytosis KIRs. See killer cell immunoglobulin-
immune-stimulatory complexes innate defense mechanisms, 221—22 like receptors
(ISCOMs), 262, 263, 264, 322 innate immune responses, 12, 14–15, Klebsiella, 53; pneumoniae, 101
immune surveillance, 288 222–23, 264 KLRs. See killer lectin-like receptors
immune system: activation of, 38–40; innate immune system, 18, 323; and
components of, 2–5; fetal develop- damage-associated molecular pat- lactobacilli, commensal, 21
ment of, 241–43; gut, 233–37; terns, 46–49; features of, 5–9; he- lactoferrin, 20, 246
neonatal development of, 248–49. matopoiesis, 21–25; lymphoid cells lamina propria, mast cells and, 31
See also adaptive immune system; in, 31–33; myeloid cells in, 25–31; L-amino acids, 124
innate immune system pattern recognition receptors and, large granular lymphocytes, 32
immunization, 266; B cells and, 41–44; SIRS and, 37–38, 50–52 lateral flow test, 302, 303
238; in ovo, 243, 244. See also in ovo vaccination, 243, 244, 245 L chains (B-cell receptor), 182, 199;
vaccination insects, live-attenuated vaccine devel- B-cell, 186, 188, 207; genetics of,
immunoblotting (western blotting), opment and, 257 183–86; and immunoglobulin
302–3, 322 integrins, 323 isotypes, 189–91
immunodeficiencies. See immune intercellular adhesion molecule 1 lectin pathway of complement acti-
deficiencies (ICAM-1), 77 vation, 57–58, 60, 323
immunodiffusion assay, 304–5, 312, interferons (IFNs), 4, 323. See also by lectins, 55. See also selectins
322 IFN letter designation leopards, snow, 271
immunofiltration technique, 302 interleukins (ILs), 4, 23, 323; in acute Leptospira, 241; interrogans, 268–69
immunofluorescent microscopy, inflammation, 73, 75, 81–82; in ad- leptospirosis, 268–69
303–4 juvants, 264; and B cells, 206; and lethargy, 82
immunogens, 322 immune memory, 238; and NETs, leukocyte-dependent injury, vascular
immunoglobulin (Ig), 206, 322; 100; receptors, 166, 167; and Th2 permeability and, 74
isotypes, 189–91, 193; isotype cells, 171, 174, 278; and Th17 leukocyte function-associated anti-
switching, 215; maternal transfer cells, 175; in XLSCID-affected gen-1 (LFA-1), 175
of, 247; quantification of, 312. See dogs, 177–79. See also by IL number leukocytes (white blood cells), 3,
also by Ig letter designation designation 4, 20, 85, 100, 287, 323; in acute
immunohistochemistry, 306–7, 308, interstitial fluid, 114 inflammation, 67, 68; in allergic
322 intestine, 20, 21; epithelial cells in, reactions, 275–76; and comple-
immunostimulating compounds, 264 18, 19, 220; GALT in, 119; immune ment system, 62–63; extravasation
immunostimulatory effects, 264 responses in, 233–37 of, 75–79; increased production of,
immunosuppressive agents, 293 intracellular adhesion molecules (I- 23–24; neutrophils, 25–26; recruit-
immunosuppressive drugs, 268, 282 CAMs), 175, 176 ment of, 79–80
immunotherapy, 201; cancer, 292–93; intramuscular (IM) vaccination, 266 leukocytosis, 82
passive, 267–68 intranasal (IN) vaccination, 266 leukopenia, in parvovirus infections,
IN. See intranasal vaccination in utero infections, in calves, 242 220
infections, 4, 17, 114, 143, 223; invariant chain (II), 323 leukotrienes, 4, 71, 323
and antibody diversity, 212–13; iron homeostasis, 29 LFA-1. See leukocyte function-
chronic, 284; opportunistic, 1, 16, ISCOMs. See immune-stimulatory associated antigen-1; lymphocyte
143, 284; responses to, 14–15, 222 complexes function-associated antigen-1
332 Ind e x

licensing, of NK cells, 31 lysozyme, 19 mastitis: bovine acute, 67, 68, 84,
lions, 128, 271 lytic granules, 102 85–88; in goats, 139
lipid mediators, 50 maternal transfer of immunity, 242,
lipid micelles, in immune-stimulatory MAC. See membrane attack complex 252; postnatal, 245–48; prenatal,
complexes, 262 macaques, 1, 2 243–45; and vaccination, 250–51
lipopolysaccharide (LPS), 39, 100, macrophage CSF (M-CSF), 23 MBL. See mannose-binding lectin
323; and immune system activa- macrophages, 3, 6, 23, 28, 29–30, 54, MBL-associated serine proteases
tion, 37–38; and SIRS, 50–52 63, 85, 111, 133, 134, 171, 210, (MASPS), 57–58, 61
liposomes, as adjuvants, 264 237, 246, 323; activation of, 50, 51; M cells. See multifenestrated cells,
lipoxygenase, 71 and acute inflammation, 70, 71, 119
Listeria monocytogenes, 44, 241 79–80; opsonization, 213, 214, 231; M-CSF. See macrophage CSF
live-attenuated vaccines, 266, 268; phagocytosis by, 13, 91–99, 210; MDSCs. See myeloid-derived suppres-
development of, 255, 257–59 and Rhodococcus equi, 89, 106–7 sor cells
liver, 6, 54, 82, 85; B-cell develop- major histocompatability complex mediators: in acute inflamation, 71–
ment, 186, 192, 214 (MHC), 121, 122, 123(table), 72; inflammatory, 51, 69, 72–75;
LPS. See lipopolysaccharide 239, 259, 295, 323; antigens and, proinflammatory, 50, 51, 77
lungs, acute injury to, 99 131–39, 163–64, 176, 239; CD4/8 melanocytes, in canine cyclic neutro-
Lyme disease, ELISA for, 300, 301 interactions, 164–66; genetics of, penia, 34
lymph, 115, 182, 323 125–29; history of, 124–25; reac- membrane attack complex (MAC),
lymphadenitis, 120 tion with TCRs, 153–54, 163–64. 60, 61, 64, 316, 324
lymphadenopathy, feline, 109, See also MHC class I molecules; membrane dipsticks, 309, 310
110(table), 119–20 MHC class II molecules; MHC membrano-proliferative glomerulo-
lymphatics (lymphatic system), 115, class III molecules nephritis, 64, 65
117; canine, 14, 116 malnutrition, and congenital im- memory, immunological, 9, 237–39,
lymphatic system. See lymphatics mune deficiencies, 273 260
lymph nodes, 31, 114, 115, 117, 195, MALT. See mucosa-associated lym- memory B cells, 210, 212
206, 236, 323; and adaptive im- phoid tissue memory T cells, 210
mune responses, 12, 110; antigen- mammals: B-1 B cells in, 192–93; mesangio-proliferative glomerulone-
antibody complexes in, 229–30; B BCR genetics, 183–86, 201; Ig iso- phritis, in Brittany dogs, 53, 64
and Th cells in, 204–5, 232; canine, types, 189–91; maternal transfer metabolic acidosis, 82
14, 116; FDCs in, 33, 64; germinal of immunity, 244–48; MHC genet- methotrexate, 273
centers in, 207–10; SCID-affected, ics in, 125–29; mucosal antigens, MHC. See major histocompatability
143, 161, 162; T-cell activation, 237; opsonins in, 92; placentas, complex
225–26 244–45, 246; proteasomes, 136; MHC class I molecules, 102, 129–30,
lymphocyte function-associated rabies vaccines in, 261–62; spleen 131, 324; antigen processing and
antigen-1 (LFA-1), 76–77 in, 118; T-cell development, 151; presentation, 135–36, 137, 138,
lymphocytes, 3, 23, 31, 32, 68, 236, TCR genes in, 146; thymus in, 157; APCs and, 173–74, 226, 236;
238, 242, 248, 294, 312; flow 113–14, 115 in immune-stimulatory complexes
cytometry tests and, 305–6, 307; mammary glands: inflammation of, (IS-COMs), 262; in parvoviruses,
in immune response, 14–15; T-cell 67, 68; lymphoid tissues in, 245–46 240; in tumors, 292
development, 151–53; in XLSCID, MAMPs. See microbe-associated MHC class II molecules, 130–31, 132,
177–78. See also B cells; T cells molecular patterns 133, 227, 324; antigen processing
lymphoid follicles, 229, 323 manatees, 241, 242 and presentation, 136–37, 138, 139,
lymphoid germinal centers, B-cell mange: demodetic, 197, 199, 215; 140, 157; and B cells, 203, 229
division in, 231 sarcoptic, 225 MHC class III molecules, 324
lymphoid lineage, 111–12; of im- mannose-binding lectin (MBL), 46, mice, 97, 101, 124–25, 126, 184, 238,
mune cells, 22, 23 57–58 289; nude, 109–10, 111, 112
lymphoid neoplasia, 84 mannose receptors (MRs), phagocy- microbe-associated molecular
lymphoid organs: bone marrow, tosis by, 46, 47, 91 patterns (MAMPs), 40. See also
110–12; secondary, 114–19; thy- Marek’s disease, 243, 245 pathogen-associated molecular
mus, 112–14 marginal-zone B (MZ B) cells, 194, patterns
lymphoid tissues, 31, 177, 189, 210, 203 microbes, innate immune response
220, 235; antibody responses in, MASPS. See MBL-associated serine to, 6, 7, 26
229–31; in mammary glands, proteases microbial invasion, anatomical and
245–46; in spleen, 117–18. See also mast-cell precursor cells, 31 physiological barriers to, 18–21
gut-associated lymphoid tissues mast cells, 4, 22, 23, 31, 62, 71, 100, microfold cells. See multifenestrated
lysosomes, 137; in phagocytosis, 277, 323–24; and allergic reactions, cells
94, 97 274, 275, 278 microspheres, as adjuvants, 264
Ind e x 333
333

milk: antimicrobial proteins in, 19, neonatal isoerythrolysis, 296, 311–12, nursing, maternal transfer of immu-
20; detecting mastitis in, 67, 69, 314 nity through, 245–48
87; transfer of immunity through, neonates: immune memory and ca- nutritional deficiencies, 273
245, 248, 249 pacity in, 248–49; immune system
milkmaids, smallpox and, 254 development, 241–43; maternal oil-in-water emulsions, as adjuvants,
minerals, as adjuvants, 264 transfer of immunity, 243–48; vac- 264
molecular genetics, 258 cination of, 149–51, 266 oncogenes, 288–89
monkeys, 1, 2, 16 neoplasia, bone marrow, 25 oncogenesis (tumorigenesis), 288
monoamines, 4 NETS. See neutrophil extracellular opossums, immune system develop-
monoclonal antibodies, 299, 324 traps ment, 242
monocytes, 3, 14, 28–29, 54, 324; in neuropeptides, 69 opsonic phagocytic receptors,
acute inflammation, 68, 79–80; neutralization, pathogen and toxin, 93(table)
macrophage, 31, 111; production 213 opsonins, 92
of, 22–23 neutralizing antibodies, 324 opsonization, 13, 55, 92, 93, 213–14,
MPO. See myeloperoxidase neutropenia, cyclic, 17, 18, 33–35 231, 325; complement activation
MRs. See mannose receptors neutrophil extracellular traps (NETs), and, 57, 63
mucociliary clearance, 20 26, 100–101, 324 oral tolerance, 237
mucosa, 20, 237 neutrophilia, 24, 82, 85, 324 oral vaccination, 266
mucosa-associated lymphoid tissue neutrophils, 3, 6, 14, 17, 25–26, 34, osteoblasts, hematopoiesis, 23
(MALT), 12, 110, 118–19, 236, 324 237, 246, 324; acute inflammation osteosarcoma, in Irish wolf hounds,
mucosal surfaces, 20–21; as infection and, 68–69, 75–79, 87; chemotaxis, 288
location, 114, 118–19 63, 78; opsonization, 213, 231; otitis externa, in dogs, 197, 215
mucous membranes, 18, 19 phagocytosis by, 91–99; produc- Ouchterlony double immunodiffu-
mucus, 19; as mechanical barrier, tion of, 22, 23, 24, 85, 112 sion assay, 305
20–21 NFAT. See nuclear factor of activated oxidation, in phagocytosis, 97, 98
multifenestrated (microfold; M) cells, T cells oxydative burst, 97
119, 235, 236 Nf-κB. See nuclear factor kappa-B
multiple myeloma, 84 N-formylmethionine, 78 PAMPs. See pathogen-associated
murine, myleoid cells, 29, 30. See also nicotinamide adenine dinucleotide molecular patterns
mice phosphate-oxidase (NADPH pancreas, enzymes from, 20
mutations, 207; and vaccine develop- oxidase), 97, 100; and Rhodococcus panleukopenia virus, feline, 220
ment, 257, 258 equi, 107 parainfluenza virus (PI-3), 251, 268
mutualism, of gut bacteria, 234 nitric oxide (NO), 97 parasites, 2, 6, 14, 90, 225, 249; eo-
Mycobacterium tuberculosis, 89 Nixon, Richard, 289 sinophils and, 26–27, 100
myeloid cells, 324; in innate immune NK cells. See natural killer cells parasitic infections, 2, 18, 28, 79, 128
system, 25–31 NK immunological synapse, 102, 105 parrots, allergies in, 274
myeloid dendritic cells (DCs), 23, 31 NK T lymphocytes, 31 parvoviruses, 9; canine, 219–20,
myeloid-derived suppressor cells NLRs. See nucleotide-binding oligo- 239–40, 274; vaccination against,
(MDSCs), 290, 291 merization domain (NOD)-like 251, 255, 257, 258
myeloid lineage, 111; of immune receptors passive immunity, 243, 252
cells, 22–23 NO. See nitric oxide passive immunotherapy, 267–68, 325
myeloperoxidase (MPO), 97, 99 NOD. See nucleotide-binding oligo- passive transfer, 267–68, 325
MZ B. See marginal-zone B cells merization domain (NOD)-like passive transfer failure, 248, 252, 312
receptors Pasteurella, 241; multocida, 269
NADPH oxidase. See nicotin- nonsteroidal anti-inflammatory pasteurellosis, 268, 269
amide adenine dinucleotide drugs (NSAIDs), 71, 82, 324 pathogen-associated molecular pat-
phosphate-oxidase NSAIDs. See nonsteroidal anti-inflam- terns (PAMPs), 40, 41, 44, 46, 48,
naive T cells, 324 matory drugs 52, 57, 70, 78, 100, 169, 325; and
naked DNA vaccines, 262 nuclear factor kappa-B (Nf-κB), 166; macrophage activation, 50, 51; and
naked protein-encoding DNA, 262 activation of, 43–44, 45 TLRs, 42–43
National Cancer Act, 289 nuclear factor of activated T cells pathogenesis, 213; mechanisms of,
natural killer (NK) cells, 3, 7, 8, 111, (NFAT), 166 223–25
324; cytotoxicity by, 90, 101–5, nuclear factors (Nfs), 166, 325. See pathogen neutralization, 213
231; in innate immune system, also by type pathogens, 13, 14, 16, 122; extracel-
31–33; tumor apoptosis and, nuclear proteins, and DAMPs, 47, 48 lular and intracellular, 8, 223–24;
290–91, 292 nucleotide-binding oligomerization innate immune responses to, 6, 8
negative selection, 324 domain (NOD)-like receptors pattern-recognition receptors (PRRs),
Nelmes, Sarah, cowpox and, 253–54 (NLRs), 42, 44, 325 6, 40, 45–46, 48, 70, 135, 264, 325;
334 Ind e x

in phagocytosis, 91–92; signaling, polio vaccine, Sabin, 266 rabies: oral vaccine for, 261–62, 266;
41–44. See also by type polyarthritis, in goats, 121 Pasteur’s vaccine, 259
PDCs. See plasmacytoid dendritic polyclonal antibodies, 325; produc- raccoons, rabies vaccines for, 262
cells tion of, 298–99 radiation therapy, 273, 293
pentameric IgM, 248–49 polypeptide chains: in B-cell recep- radicals, 97, 98, 99
peptides, 78, 157, 262; antimicrobial, tors, 182; and T-cell receptors, RAG1, 326
99, 246; cationic, 19–21; and MHC 144–45 RAG2, 289, 326
molecules, 131–32; and protea- polysaccharides, capsular, and conju- RAGE. See receptor for advanced
somes, 135, 136 gate vaccines, 260–61 glycation end products
peptidoglycan, 40, 44, 325 positive selection, 325 rapid-acting transcription factors,
perforins, 176, 325 precipitation assays, 304–5 43–44
peripheral tolerance, 156, 325 precursor cells, in hematopoiesis, rattles, in foals, 89–90, 106–7
peritoneal cavity, B-1 B cells in, 23, 24 rattlesnake bite, antivenin treatment,
192–93 primary antibody responses, 9, 10 280
permeability, vascular, 62, 63 primary immune responses, 9, RBC antigens. See erythrocyte
peroxidase, eosinophil, 100 194–196, 206, 233, 237–38, 256 antigens
peroxynitrite, 97, 98 primary lymphoid organs. See also reactive nitrogen intermediates
pertussis, canine, 266 bone marrow; thymus (RNI), 97
Peyer’s patches, 119, 204, 235 primates, 46, 85, 245 receptor clustering, 94
pH, and microbial growth, 20, 21 pro-biotic supplementation, 21 receptor for advanced glycation end
phagocytes, 6, 8, 90, 325; phagocyto- proenzymes, complement, 54–55 products (RAGE), 48
sis and, 91–99 professional phagocytes, phagocyto- receptors, 6, 9, 101; antigen acquisi-
phagocytic pattern-recognition sis by, 91–99 tion and, 134–35; antigen-specific,
receptors (PRRs), 44, 46, 91, 92, proinflammatory chemokines, 76 10–11, 114; pattern-recognition,
93(table) proinflammatory cytokines, 70, 71, 40–46; phagocytic, 93(table), 94
phagocytosis, 6, 26, 30, 63, 90, 210, 171, 325; in acute inflammation, recombinant vaccines, 261–62, 326
325; antigen acquisition, 134–35; 81–82, 86 recombination-activating gene
and MRs, 46, 47; phagosomes proinflammatory mediators, 50, 51, (RAG). See RAG1; RAG2
and phagolysosomes in, 94–99; 77, 275 red blood cells (RBCs; erythoro-
and PRRs, 44, 46; recognition and proinflammatory processes, 30, 31 cytes), 3, 117, 283; production of,
adhesion in, 91–94; and Rhodo- proinflammatory PRRs. See signaling 21, 22
coccus equi, 106–7 pattern-recognition receptors red water, 251
phagolysosome, 6, 91, 96, 99, 325 proinflammatory stimuli, 263 renal disease, in dogs, 53, 64–65
phagosomal maturation, 94–99 prophylactic treatment, 268 reproductive tracts, commensal
phagosome: creation of and matura- prostaglandins, 4, 69, 71, 82, 325–26 organisms, 21
tion of, 94–99; response to Rhodo- prostate cancer, 84 reptiles, mucosal antigens, 237
coccus equi, 106–7 protozoa, 225 respiratory burst, 97
Phipps, James, vaccination of, 253, proteases, in phagocytosis, 97, 99 respiratory tract, cilia in, 20–21
254 proteasomes, 135–36, 326 response speeds, 9–10
phosphokinase, in SCID-affected protein complexes, 56 retroviruses: FIV, 271; simian T-
horses, 143 proteins, 14, 23, 32, 42, 61, 99, 124, lymphotropic virus III, 1, 16
phospholipids, 71 127, 182; altered self, 289; and Rhodococcus equi, 89, 106–7
PI-3. See parainfluenza virus proteasomes, 135–36; and vaccina- RIG-like receptors (RLRs), 42, 44, 326
pigs, 46, 85, 241, 245, 248, 297 tion, 259–60. See also by type RLRs. See RIG-like receptors
pinocytosis, 30 proteolysis, 135 RNA, in retroviruses, 271
placentas, 246, 252; and immunity proto-oncogenes, 288–89 RNA helicases, and RIG-like recep-
transfer, 244–45 provirus, SIV, 16 tors, 44
plasma, 57, 325 PRRs. See pattern-recognition RNI. See reactive nitrogen
plasma cells, 11, 231, 236 receptors intermediates
plasmacytoid dendritic cells (PDCs), Pseudomonas spp., in Brittany dogs, rodents, maternal transfer of im-
33 53 munity, 245
plasma proteins, 6, 54 pumas, panthers: FIV in, 271; Florida, rotoviruses, vaccination against, 247
platelets, production of, 21, 22 128–29 ruminants, 156, 246
pleural cavities, B-1 B cells in, 192–93 pus, 25, 70
Pneumocystis carinii, 143 pyrexia. See fever Sabin polio vaccine, 266
pneumonia: in dogs, 161, 162; in pyrogens, 82, 326 saline-agglutination cross-match, 311
horses, 89–90 saliva, 19, 20
poison ivy dermatitis, 282 rabbits, 46, 85, 119, 186, 212, 237 SC. See subcutaneous vaccination
Ind e x 335
335

SCID. See severe combined immuno- sleep patterns, altered, 82 11; response to tumors, 291–92,
deficiency disease smallpox, vaccination against, 254 294; self-reactive, 155–56, 284;
sebaceous glands, 18 somatic hypermutation, 207, 232, 326 signal transduction and activation,
secondary antibody responses, 10 sord, 251 166–68; and Type IV hypersensi-
secondary immune responses, spleen, 31, 110, 114–15, 182; B-cell tivities, 274, 282
194–196, 206, 233, 237–39, 256 development, 186, 194; lymphoid TCR α chains, 166; antigen recogni-
secondary lymphoid organs, 326. See system in, 117–18 tion, 162–64; assembly of, 145,
also lymph nodes; spleen; mucosa- SQ. See subcutaneous vaccination 146–48, 149
associated lymphoid tissue starvation, APPs diagnosis of, 84 TCR β chains, 166; antigent recogni-
secondary lymphoid tissues, antibody stem cells, 34, 21, 111 tion, 162–64; assembly of, 145–48
responses in, 229–31 steroids, and congenital immune TCRs. See T-cell receptors
secretions, as barriers, 18–20 deficiencies, 272–73 tears, 19, 20
selectins (lectins), 75, 326 Streptococcus, spp., 241; suis, 84 terminal membrane attack pathway,
self: immunological sense of, 110, stress, and acquired immune deficien- 60
114, 169; proteasomes and, 136; cies, 273–74 tetanus, passive immunotherapy for,
expression in thymus, 157 stromal reticular cells, hematopoi- 267
self-presentation, 132 esis, 23, 24 tetanus toxoid vaccines, 260
sentinel cells, 29, 30, 31, 42, 70, 77; strongyle infection, 79 Tf h cells. See T-follicular helper cells
danger signal recognition, 50–52 subcutaneous (SC; SQ) vaccination, T-follicular helper (Tf h) cells, 169–71,
sepsis, septicemia, in neonatal foals, 266 173
37–38, 50–52 superoxide, 100; in phagocytosis, Th cells. See T-helper cells
septic shock, 99 97, 98 Th effector cells, 227–29
serine protease proenzymes, 54–55 systemic inflammatory response T-helper (Th) cells, 3, 8, 16, 138,
serotonin, 4 syndrome (SIRS), in foals, 37–38, 163, 173, 174, 228, 249, 260, 284;
serum, 326; antibody-based diagnos- 50–52 in adaptive immune response,
tic tests of, 298–307 166, 233; and B cells, 203, 204–13;
serum immunoglobulin quantifica- Tc (cytotoxic T) cells, 3, 8, 174, 176, effector, 169–71, 172, 227–29; and
tion, 312 177, 210, 226, 282, 284; activation Tc-cell activation, 168–69. See also
serum proteins, 54; in immune of, 168–69, 233; and CD8 mol- by number designation
response, 14–15 ecules, 163, 164–65 Th0 cells, 169–71
serum sickness, 280, 281–82, 283 Tc effector cells, homing of, 226–27 Th1 cells, 169–71, 173, 279, 326
severe combined immunodeficiency T-cell gamma and delta chains, Th2 cells, 28, 169–71, 278, 326
disease (SCID), 143, 273; causes of, 152–154 Th3 cells, 171, 173
158–59; X-linked, 161–62, 177–79 T-cell receptors (TCRs), 9, 111, 122, Th17 cells, 169–71, 326; and IL-17,
sex hormones, and congenital im- 227, 239, 240, 326; α and β chain 175
mune deficiencies, 273 assembly, 145–46, 147, 149; antigen theory of immune surveillance,
shar peis, Chinese, immune deficien- recognition, 162–64; and B cells, 124–25
cies in, 197, 198, 215, 203, 204; and CD4/8 interactions, Thomas, Lewis, 124, 288
sheep: B-cell development, 186, 212; 164–66; genetics of, 145–53; and thymocytes, 114, 159, 162, 326;
erythrocyte antigens, 297; neona- MHC-antigen complexes, 176, positive and negative selection of,
tal infections, 241 227; and SCID, 158–59; structure 153–56
shock: anaphylactic, 276–77, 280, 281; of, 144–45; and T-cell selection, thymus(es), 109–10, 115, 178, 193,
hypovolemic, 237; septic, 37, 99 153–56 242, 243; SCID-affected, 143, 159,
signaling pathways, and TLRs, 43–44 T cells (T lymphocytes), 3, 4, 5, 8, 161, 162; T-cell development in,
signaling pattern-recognition recep- 16, 30, 33, 99, 118, 120, 124, 125, 151–53; T-cell selection in 153–54;
tors, 41–44 236, 240, 248, 259, 324; adaptive T lymphocytes in, 113–14
signal transduction, 195, 199; and immune responses, 9, 46; antigen thymus-dependent antigens, 193
T-cell activation, 166–68 recognition by, 162–64; and thymus-independent antigens, 326
simian immunodeficiency virus APCs, 10, 31, 138, 225–26; CD4/8 thyroid disease, in dogs, 197
(SIV), 1, 2(table), 16 interactions, 164–66; develop- tigers, feline immunodeficiency virus
simian T-lymphotropic virus III, 1 ment of, 151–53; effector, 12, 178; (FIV) in, 271
SIRS. See systemic inflammatory effector cytotoxic, 174–76; effector tissue damage, 71; acute inflamma-
response syndrome regulatory, 171, 173; epitopes, tion and, 68–69; DAMPs and,
SIV. See simian immunodeficiency 260–61; functional, 153–54; in 46–49
virus lymph nodes, 117, 204; matura- tissue repair and remodeling, 4, 29
skin, 18, 19, 21; fungal infections, 225; tion of, 113–14; as memory cells, TLRs. See toll-like receptors
grafting, 110, 112 12, 238, 238–39; production of, T lymphocytes. See T cells
SLE, diagnosis of, 313 23, 111; response to antigens, 10, TNF-α. See tumor necrosis factors
336 Ind e x

TNF-β. See tumor necrosis factors istry categorization of, 306–7; and vasodilation: during acute inflam-
tolerance, 156 theory of immune surveillance, mation, 69, 72–73, 75; in allergic
toll-like receptors (TLRs), 42–44, 326 124–25; therapies against, 289–90 reactions, 275–76
toxic metabolites, 27 tumor-suppressor genes, 289 VEGF. See vascular endothelial cell
toxin neutralization, 213 turkeys, in ovo vaccination of, 244, growth factor
toxins, 13; anaphylactic, 281, 319; 245 V gene segments, 326
antimicrobial, 25; botulinum, typing gel, 308, 310 Vibrio anguillarum, 266
224–25; tumor-produced, 292, 294 viral infections, 33, 84; NK cells, 32,
toxoid vaccines, 260, 269 urinary tract flushing, 20 101–2; in wolves, 219–20
transcytosis, 74–75 virulence genes, 258
transforming growth factor-β vaccination(s), 222, 253; administra- viruses, 4, 7, 14, 121, 138, 171, 225,
(TGF-β), 4, 171; and tumor cell tion of, 266–67; and colostral 274; in live-attenuated vaccines,
proliferation, 290–91, 292 antibodies, 246–47; and congenital 255, 257–58; reproduction of,
transfusion reactions, 295–97, 326; immune deficiencies, 272; history 239–40
hemolytic, 316–17 of, 253–54; immune responses
transmigration. See diapedesis to, 233; in ovo, 243, 244, 245; war on cancer, 289
transplants, response to, 124–25 of neonates, 249–51; recom- water-in-oil emulsions, as adjuvants,
transporters associated with antigen mended for dogs, 256–57. See also 264
presentation, 326 immunization western blotting. See
Treg cells. See T-regulatory cells vaccines, 268–69, 276; adjuvants, immunoblotting
T-regulatory (Treg) cells, 170, 171, 263–65; administration of, 266–67; white blood cells. See leukocytes
173, 326 design of, 253–55; live-attenuated, Wistar Institute, recombinant vac-
trout, vaccination in, 266 255, 257–59; other types of, cines, 261–62
trypsin inhibitors, 247 259–63 wolf hounds, Irish, and osteosar-
tuberculin skin testing, DTH re- vaccinia virus, 262 coma, 288
sponses to, 282 vacuoles, phagosome, 94 wolves, parvovirus in, 219–20, 239–40
tularemia, 119 vagina, commensal lactobacilli, 21
tumorigenesis (oncogenesis), 288 vascular dilation, 62–63 X-linked agammaglobulinemia, 181,
tumor immunity, eosinophils and, 27 vascular endothelia, 228, 239 195–96
tumor necrosis factors (TNF-α; vascular endothelial cell growth fac- X-linked severe combined immu-
TNF-β), 4, 75, 171, 173, 206, 281; tor (VEGF), 290, 291 nodeficiency (XLSCID), in dogs,
in acute inflammation, 81–82; vascular permeability, in inflamma- 161–62, 177–79, 273
apoptosis of tumors, 290–91 tion, 73–75 XLSCID. See X-linked severe com-
tumors, 32, 287; host interactions vascular system, during acute inflam- bined immunodeficiency
with, 290–92; immunohistochem- mation, 69, 72–75
Ind e x 337
337


Basic Veterinary Immunology - Callahan

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Basic Veterinary Immunology - Callahan

Uploaded by

Copyright:

Available Formats

Basic

callahan text.indd 1 3/11/14 1:40 PM

University Press of Colorado

callahan text.indd 3 3/11/14 1:41 PM

Published by University Press of Colorado

All rights reserved

The University Press of Colorado is a proud member of

Library of Congress Cataloging-in-Publication Data

callahan text.indd 4 3/11/14 1:41 PM

Chapter 1: Overview of Chapter 2: Overview of the

callahan text.indd 5 3/11/14 1:41 PM

callahan text.indd 6 3/11/14 1:41 PM

callahan text.indd 7 3/11/14 1:41 PM

callahan text.indd 8 3/11/14 1:41 PM

callahan text.indd 9 3/11/14 1:41 PM

callahan text.indd 10 3/11/14 1:41 PM

callahan text.indd 11 3/11/14 1:41 PM

Overview of Mechanisms of Defense Chapter 1

was a retrovirus—simian T-lymphotropic virus

callahan text.indd 1 3/11/14 1:41 PM

callahan text.indd 2 3/11/14 1:42 PM

Cells of Defense Communication through cytokines

callahan text.indd 3 3/11/14 1:42 PM

callahan text.indd 4 3/11/14 1:42 PM

callahan text.indd 5 3/11/14 1:42 PM

callahan text.indd 6 3/11/14 1:42 PM

callahan text.indd 7 3/11/14 1:42 PM

callahan text.indd 8 3/11/14 1:42 PM

callahan text.indd 9 3/11/14 1:42 PM

callahan text.indd 10 3/11/14 1:42 PM

callahan text.indd 11 3/11/14 1:42 PM

callahan text.indd 12 3/11/14 1:42 PM

callahan text.indd 13 3/11/14 1:42 PM

callahan text.indd 14 3/11/14 1:43 PM

callahan text.indd 15 3/11/14 1:43 PM

Clinical Correlation Follow-Up

callahan text.indd 16 3/11/14 1:43 PM

Overview of the Innate Immune System Chapter 2

susceptibility to bacterial infections. Within

callahan text.indd 17 3/11/14 1:43 PM

Only vertebrates have adaptive immunity; in

callahan text.indd 18 3/11/14 1:43 PM

Figure 2.3A–C. Epithelial barriers

callahan text.indd 19 3/11/14 1:43 PM

callahan text.indd 20 3/11/14 1:43 PM

callahan text.indd 21 3/11/14 1:43 PM

callahan text.indd 22 3/11/14 1:43 PM

callahan text.indd 23 3/11/14 1:43 PM

certain leukocytes, including their immature

callahan text.indd 24 3/11/14 1:43 PM

callahan text.indd 25 3/11/14 1:43 PM

callahan text.indd 26 3/11/14 1:43 PM

callahan text.indd 27 3/11/14 1:43 PM

circulating in the blood. Although rare, their Monocytes

callahan text.indd 28 3/11/14 1:43 PM

callahan text.indd 29 3/11/14 1:43 PM

callahan text.indd 30 3/11/14 1:43 PM

callahan text.indd 31 3/11/14 1:43 PM

Name: Mast cells

callahan text.indd 32 3/11/14 1:43 PM

Name: Natural killer cells

callahan text.indd 33 3/11/14 1:43 PM

callahan text.indd 34 3/11/14 1:44 PM

callahan text.indd 35 3/11/14 1:44 PM