Michael T.

Lotze The grateful dead: damage-

Herbert J. Zeh
Anna Rubartelli
associated molecular pattern
Louis J. Sparvero molecules and reduction/oxidation
Andrew A. Amoscato regulate immunity
Newell R. Washburn
Michael E. DeVera
Xiaoyan Liang
Mahmut Tör
Timothy Billiar

Authors’ address Summary: The response to pathogens and damage in plants and animals
Michael T. Lotze1, Herbert J. Zeh1, Anna Rubartelli2, Louis J. Sparvero1, involves a series of carefully orchestrated, highly evolved, molecular
Andrew A. Amoscato1, Newell R. Washburn3, Michael E. DeVera1, mechanisms resulting in pathogen resistance and wound healing. In
Xiaoyan Liang1, Mahmut Tör4, Timothy Billiar1, metazoans, damage- or pathogen-associated molecular pattern molecules
1 (DAMPs, PAMPs) execute precise intracellular tasks and are also able to
Departments of Surgery and Bioengineering, G.27A
Hillman Cancer Center, University of Pittsburgh Cancer exert disparate functions when released into the extracellular space. The
Institute, Pittsburgh, PA, USA. emergent consequence for both inflammation and wound healing of the
2 abnormal extracellular persistence of these factors may underlie many
Cell Biology Unit, National Cancer Research Institute,
Genova, Italy. clinical disorders. DAMPs/PAMPs are recognized by hereditable receptors
3
Department of Biomedical Engineering, Department of including the Toll-like receptors, the NOD1-like receptors and retinoic-
Chemistry, Carnegie Mellon University. acid-inducible gene I-like receptors, as well as the receptor for advanced
4
Warwick HRI, University of Warwick, Wellesbourne, UK. glycation end products. These host molecules ‘sense’ not only pathogens
but also misfolded/glycated proteins or exposed hydrophobic portions of
Correspondence to: molecules, activating intracellular cascades that lead to an inflammatory
Michael T. Lotze response. Equally important are means to not only respond to these
Departments of Surgery and Bioengineering molecules but also to eradicate them. We have speculated that their
G.27A Hillman Cancer Center destruction through oxidative mechanisms normally exerted by myeloid
University of Pittsburgh School of Medicine cells, such as neutrophils and eosinophils, or their persistence in the
5117 Centre Avenue setting of pathologic extracellular reducing environments, maintained by
Pittsburgh, PA 15213, USA exuberant necrotic cell death and/or oxidoreductases, represent important
Tel.: 11 412 623 5977 molecular means enabling chronic inflammatory states.
Fax: 11 412 623 1212
e-mail: lotzemt@upmc.edu Keywords: DAMPs, PAMPs, redox, HMGB1, TLR4, RAGE

Acknowledgements Long distance runner what you standing there for?

This project was funded by a grant from the NIH 1 PO1 CA
101944-01 (Michael T Lotze) Integrating NK and DC into
Cancer Therapy National Cancer Institute, and a grant with
the Pennsylvania Department of Health. The Department
specifically disclaims responsibility for any analyses, inter-
pretations, or conclusions.
Get up, get off, get out of the door
You’re playing cold music on the bar room floor,
drowned in your laughter and dead to the core
There’s a dragon with matches loose on the town
Take a whole pail of water just to cool him down.
(Fire on the Mountain, Grateful Dead)

Introduction

Immunological Reviews 2007 Two papers appearing 14 years ago presaged the deeper under-
Vol. 220: 60–81
standing of innate immune reactivity, dictating the subsequent
Printed in Singapore. All rights reserved
nature of the adaptive immune response. The first (1) came
r 2007 The Authors from transplant surgeons who conducted a prospective rando-
Journal compilation r 2007 Blackwell Munksgaard
Immunological Reviews mized double-blind placebo-controlled trial. Administration of
0105-2896 recombinant human superoxide dismutatase (rh-SOD) in

60

recipients of cadaveric renal allografts demonstrated prolonged
patient and graft survival with improvement in both acute and
chronic rejection events. They speculated that the effect was
related to its antioxidant action on the initial ischemia/
reperfusion injury of the renal allograft, thereby reducing the
immunogenicity of the allograft and the ‘grateful dead’ or
stressed cells. Thus, free radical-mediated reperfusion injury
was seen to contribute to the process of innate and subsequent
adaptive immune responses. The second publication (2) sug-
gested the possibility that the immune system detected ‘dan-
ger’ through a series of what we now call damage-associated
molecular pattern molecules (DAMPs), working in concert
with both positive and negative signals derived from other
tissues. Thus, these two papers presaged the modern sense of
the role of DAMPs and reduction/oxidation (redox) reviewed
here, important apparently for both plant and animal resistance
to pathogens and the response to cellular injury or damage.
Although initially suspected in animals as a result of
initiating events preceding the adaptive immune response,
local and systemic responses in plants have identified solely
innate mechanisms including membrane-bound receptors
such as FLS2 (3), PEPR1 (4), and WRK (5), and members of
kinase cascade such as wounding-induced protein kinase
(WIPK) (6). Hypersensitive response, a form of localized
apoptotic cell death in plants, is also associated with disease
resistance (7). The prototypic Gram-negative-derived
exogenous pyrogen, lipopolysaccharide (LPS), or endotoxin
and flagellin represent pathogen-associated molecular pattern
molecules (PAMPs). In addition, lipoteichoic acid (Gram-
positive) and hypomethylated DNA/single-or double-
stranded RNA are also characteristics of pathogens. PAMPs are
recognized in animals by hereditable receptors such as the Toll-
like receptors (TLRs), the NACHT (domain present in NAIP,
CIITA, HET-E, and TP1)-leucine-rich repeat (NLRs)/NOD1
family, retinoic-acid-inducible gene I-like receptors, and the
receptor for advanced glycation end products (RAGE). Similar
to animals, plants recognize PAMPs by cell surface receptors
including receptor-like kinases (RLKs) (8), receptor-like
proteins (9), and polygalacturonase-inhibiting proteins (10),
or within the cytoplasm by NBS-LRR proteins or so-called R-
genes (11).
Response to cell damage and stress in prokaryotes
and plants
In response to environmental cues including nutrient depriva-
tion, changes in electrolytes or temperature, or proximate cell
death, prokaryotes respond with multicellular integrative re-
Lotze et al
DAMPS and redox regulate immunity
sponses with the release of intracellular constituents. These
include migration, increased nutrient uptake, and biofilm
formation (12–14). Likewise, quorum-sensing behavior, a
bacterial cell–cell communication system dispersal, and regu-
lated cell death of microorganisms such as Dictyostelium in
response to intracellular debris enable coordinate responses to
stressful events such as death in local organisms, nutrient
deprivation, or major changes in extracellular temperature.
Dictyostelium discoideum harbors sentinel cells that engulf bacteria,
sequester toxins, and utilize Toll/interleukin-1 receptor (IL-
1R)-like domains to enable phagocytosis (15). Little is known,
however, about how prokaryotic mechanisms relate to com-
plex interactions that occur within multicellular eukaryotes.
Recently, in animals lacking defined adaptive responses such
as the sea urchin, expansion of these innate TLRs 10-fold
over those found in vertebrates with adaptive responses en-
hanced the encoded means to recognize cognate pathogenic
molecules (7).
Plant pathogens are classified as biotrophs, necrotrophs, and
hemibiotrophs according to their mode of nutrition.
Biotrophic pathogens feed on living cells and cause limited
injury mainly at the cell wall during penetration. Necrotrophs
cause the maximum damage by killing the cell and consuming
the released materials, and hemibiotrophs have an initial
biotrophic phase that is followed by a necrotrophic stage (16,
17).
In eukaryotes, disrupted or injured cells communicate their
damage to the tissue or to recruited innate inflammatory cells
by releasing intracellular factors. Interestingly, some of these
factors are redox-sensitive proteins that are central to many of
the earliest endogenous responses to microbial stress as they
are for their hosts. Plants develop a systemic response to
pathogen attacks as well as to other injuries caused by DAMPs
(Fig. 1). These responses are usually mediated by salicylic acid
(SA), jasmonic acid (JA), or ethylene (ET) (18). Damaged
tomato leaves, for example, produce prosystemin, a protein
that is found in parenchyma cells of vascular bundles and is
cleaved to form systemin. This initial cleaved 18 amino acid
peptide, systemin, acts as a primary signal for systemic defense
in Solanaceae species (19–21). Arabidopsis has at least seven
PROPEP proteins that act as precursors for peptides that act as
endogenous elicitors. For example, AtPep1, a 23 amino acid
peptide derived from PROPEP1, is induced in response to
wounding and associates with the innate immune responses
through JA and ET signaling pathways (22, 23).
Plants have also evolved complicated regulatory systems to
control responses to damage and pathogens (24). Both positive
and negative signaling pathways interplay to coordinate
Immunological Reviews 220/2007 61
Lotze et al
DAMPS and redox regulate immunity

Fig. 1. The innate immune response is initiated by pathogen-associated molecular pattern molecules (PAMPs) and damage-associated
molecular pattern molecules (DAMPs). Both in plants and animals, characteristic innate immune responses, regulated by reduction/oxidation,
promote the response to pathogens and damage. Pathogens display their presence with recognized PAMPs such as bacterial flagellin, fungal/oomycete
chitin, or viral coat proteins. Recognition of these PAMPs by cell surface, vesicular, and cytosolic receptors including TLRs/RLRs/NLRs in animals and
RLP/RLKs in plants activates innate immune responses. Although in some animals this process leads to downstream activation of adaptive immune
responses, plants do not have such adaptive systems. Activation of innate immunity by DAMPs in animals promotes recruitment of inflammatory cells,
oxidation of extracellular debris, and internalization and cross-presentation of antigen by specialized antigen-presenting cells. These ultimately drive
the development of B and T-cell adaptive responses. In plants, in addition to pathogens, external stimuli including ozone, ultraviolet light, tissue
damage by insects, herbivores, and wounding, and internally programmed processes such as senescence induce the generation of reactive oxygen
species (ROS). This in turn promotes various functions including secondary signaling, cross-linking of cell walls, DNA laddering, protein modification,
cell death, and the release of endogenous elicitors, here termed DAMPs, such as oligogalacturonides, systemins, and AtPep1. DAMPs are also recognized
by cell surface receptors and contribute to innate immune responses by activating the signaling cascade. Both PAMPs and DAMPs could activate salicylic
acid (SA), jasmonic acid (JA), and ethylene (ET) signaling pathways that express PR-1 and defensins. TLR, Toll-like receptors; RLK, receptor-like kinases;
RLR, retinoic-acid-inducible gene I-like receptors; NLR, NOD1-like receptors; RLP, receptor-like proteins.

development of a resistance response with the appropriate
intensity and duration. Kinase signaling pathways in plants
also play an important role in response to pathogen attack and
tissue damage. For example, in tobacco the mitagen-activated
protein kinase WIPK (6) and salicylic acid-induced protein
kinase (SIPK) (25) are involved in the regulation of JA- and SA-
mediated systemic communication and response to wounding.
NtMEK2, a tobacco mitogen-activated protein kinase (MAPK), is
the upstream kinase of SIPK and WIPK, both stress-responsive
MAPKs (9). Both nitric oxide (NO) and SA activate SIPK but not
WIPK (26). Thus, plants have evolved similarly complicated
regulatory systems to control the response to both infection
and damage. AtNUDT7, a protein recently identified in
Arabidopsis that hydrolyzes nucleotides, is a negative regulator
of the innate immune response, and its loss-of-function
mutation enhances resistance to infection by the bacterial
pathogen Pseudomonas syringae (27). In vitro, both adenosine
diphosphate (ADP)-ribose and nicotinamide adenine

62 Immunological Reviews 220/2007


dinucleotide (NADH) are preferred substrates of NUDT7,
important in cellular redox homeostasis. Thus, alteration in
cellular antioxidant status when NUDT7 is mutated primes the
cells for and amplifies defense responses. The nudt7 mutation
enhances both SA-dependent and -independent defense
response pathways. Thus, the nudt7 mutation perturbs cellular
redox homeostasis and leads to a higher level of NADH in
pathogen-challenged leaves. However, the role of AtNUDT7 in
response to tissue damage caused by other factors including
wounding has not been demonstrated.
Generation and accumulation of reactive oxygen species
(ROS) are central to the plant response to microbial pathogens
and tissue damage. Membrane-bound nicotinamide adenine
dinucleotide phosphate oxidize activity, cytosol-located
oxidases, and apoplastic peroxidases have been suggested to
generate hydrogen peroxide (H2O2) (28). The role of ROS in
secondary signaling, cross-linking of cell walls, DNA
laddering, protein modifications, cell death, and the release of
endogenous elicitors (described here as DAMPs) is well known
(29). To counterattack ROS and limit damage and maintain
homeostasis, the cellular machinery in plants relies on
antioxidants such as carotene, tocopherols, and ascorbate
networks (30), scavenging systems including metallothionein
(31), and enzymatic and non-enzymatic degradation of ROS.
In plants, chloroplasts are among the main sources responsible
for the production of ROS. Therefore, degradation of
chlorophylls is an important step in the reduction of ROS-
mediated damage. In Arabidopsis, chlorophyllase 1, encoded by
AtCLH1, is one of the enzymes that is responsible for the
degradation of chlorophyll, thereby controlling damage (32).
Ascorbate peroxidase 2 (APX2) encodes a key enzyme of the
antioxidant network in Arabidopsis (33). Glutathione (GSH) and
homoglutathione play a significant role in protecting cells
against the toxicity of free radicals and heavy metals (34). The
scavenging enzyme catalase (35) works closely with SOD (36)
to prevent free radical damage to the cell. SOD converts the
dangerous superoxide radical to H2O2, which catalase converts
to harmless water and oxygen. Thioredoxins also play an
important role in the defense and repair of tissue damage in
plants. They regulate the redox status of proteins during
oxidative stress by sustaining the reducing power to
reductases, repairing oxidized proteins involved in scavenging
mechanisms, and as components of signaling pathways in the
plant antioxidant network (37).
During normal conditions, oxidized proteins or protein
aggregates, damaged proteins, and organelles from the
affected cells have to be removed as a normal housekeeping
function. Apoptotic cell death in animals or programmed cell
Lotze et al
DAMPS and redox regulate immunity
death and hypersensitive reactions at the infection sites in
plants generate excessive cellular debris, and the removal of
these becomes more important. Although plants do not have
caspase homologues, caspase-like proteolytic activity during
programmed cell death has been reported (38).
Autophagy has been known for some time to be important
for nutrient remobilization during sugar and nitrogen
starvation and leaf senescence in plants (39). Recent studies
have shown that autophagy, a conserved mechanism of bulk
processing of protein and organelle turnover, also plays a
significant role in innate immunity and stress response (40,
41). Molecular and genetic studies in Arabidopsis have identified
some of the genes that are involved in autophagy and revealed
their role in defense responses (41).
In addition to autophagy, ubiquitin-mediated proteolysis has
also been conserved across the eukaryotic kingdoms including
yeast, insects, plants, and mammals (42–44) and plays a
significant role in the removal of foreign, abnormal, or
misfolded proteins and maintaining the balance between
anabolism and catabolism. The involvement of ubiquitin-
mediated proteolysis in many aspects of plant defense,
development, and physiology has been well documented
(42, 45).
Response to damage and pathogens in metazoans
Much of the initial response to damage and pathogens in
metazoans involves pathways similar to those in prokaryotes
and plants. Interestingly, molecules with defined functions
intracellularly are also able to exert disparate functions when
released into the extracellular space following cell death. The
emergent consequences for both inflammation and wound
healing of the abnormal extracellular persistence of these
factors may occur during pathological states. This could be
quite important for many clinical disorders. The TLRs are a
family of evolutionarily conserved pattern recognition recep-
tors (PRRs) that recognize and respond to a variety of bacterial
products (46, 47). Some members of the TLR family, notably
TLR2, TLR4, and TLR9, recognize multiple endogenous
DAMPs that are released following cellular stress or injury
(48–51). These host molecules ‘sense’ stress including mis-
folded proteins, genomic alterations, or altered membrane
fluidity, and activate intracellular cascades that lead to an
inflammatory response (52, 53). In animals lacking defined
adaptive responses such as the sea urchin, expansion of these
innate TLRs to 10-fold over those vertebrates with adaptive
responses enhanced the encoded means to recognize cognate
pathogenic molecules in such species (54).
Immunological Reviews 220/2007 63
Lotze et al
DAMPS and redox regulate immunity

DAMPs phage inhibitory factor, are oxidoreductases found in several

pathological states including autoimmunity and cancer (57).
In addition to PAMPs, there are cytosolic proteins that, besides
In healthy tissues, the intracellular space is highly reducing, but
having an intracellular localization and function and lacking a
the extracellular space is normally oxidizing. With damage or
secretory leader sequence, can be secreted by specialized
injury, oxidoreductases and non-protein thiols, usually con-
myeloid cells [macrophages, dendritic cells (DCs), neutrophils,
fined within cells, can be released and alter the extracellular
eosinophils], lymphoid cells [B, T, natural killer (NK)], and
redox state. Cells recruited to the site of injury, including
possibly parenchymal cells to act extracellularly to induce
macrophages, myofibroblasts, mast cells, DCs, and eosinophils,
inflammatory responses (47, 48). These proteins are released
promote healing. The patterns of immune reactivity, so-called
at high levels by injured cells and therefore have been termed
polarized states, likely occur in response to environmental cues
DAMPs (Fig. 1). The receptors involved in recognizing DAMPs
including both the DAMPs/PAMPs as well as, we hypothesize,
in plants include extracellular LRR RLK-type cell surface
the redox state of the injured tissue (Table 3).
receptors. For example, the systemin in tomato and AtPep1 in
The cytosol and the extracellular milieu are very different
Arabidopsis are recognized by the RLK-encoding tBRI1/SR160 and
environments. The cytosol is highly reducing due to several
PEPR1 genes, respectively (4, 55, 56). In animals, these
thiol-regulating enzymatic systems, including the thioredoxin-
receptors, in addition to TLR4 and other TLRs, include RAGE,
thioredoxin reductase and glutaredoxin-GSH systems. In
which is present on activated endothelium and inflammatory
both plants and animals, the extracellular space is normally
cells. In most cases, acute inflammation results in healing,
oxidized due to oxidizing agents including oxygen itself
initiated by either DAMPs (primary intention) or PAMPs
(Fig. 1). Non-protein thiols, including GSH and cysteine, are
(secondary intention). Higher numbers of microorganisms
mostly reduced intracellularly, whereas in the disulfide form
are associated with a longer period of healing and greater
outside the cell. Thiol-disulfide oxidoreductases are present in
scarring. Molecules important for healing include high-mobi-
the endoplasmic reticulum (ER) and promote disulfide bond
lity group box 1 (HMGB1) and the degraded matrix compo-
formation. Within the cytosol, the reduced and oxidized GSH
nents hyaluronan and heparin sulfate, which are found within
molecules are found at a ratio of approximately 100:1 (highly
the extracellular matrix. Recruited macrophages, myofibro-
reducing). Within the ER lumen, it is approximately 3:1 (more
blasts, mast cells, and eosinophils, depending on the site and
oxidizing).
the nature of the insult, promote healing. A series of coordinate
The reduced or oxidized form of proteins thus depends on
interactions initiating (Signal 0) and culminating (Signal 5) in
the compartments where they are found. Cytoplasmic protein
the tissue allow development of the adaptive immune response
cysteine residues typically have free sulfhydryl groups, located
(Table 1). Peptide or lipid antigens are presented to immune
in binding pockets of substrates, coenzymes, or metal
cells (Table 2) with resultant generation of immune effectors.
cofactors, and take part directly in catalytic reactions. They are
inactivated by oxidation and remain reduced in the presence of
DAMPs and redox
thiol-regulating systems. The amino-terminal signal leader
Some secreted proteins, such as thioredoxin, interferon-g sequence proteins are targeted to the ER linked in disulfide
(IFN-g)-induced lysosomal thiolreductase (GILT), and macro- bonds that ensure stability outside the cell. GILT, expressed

Table 1. Signals 0–5: Immune and inflammatory response to tissue environmental stress
Signal Mediators Cellular source Outcome
Signal 0 PAMPs and DAMPs TLR, NLR, RLR ligands Stressed, damaged cells Recruitment of inflammatory cells
Signal 1 Class I, Class II, and CD1 loading Peptide and lipid antigens Endogenous, exogenous antigen TCR engagement
Signal 2 Costimulatory and coinhibitory CD80, CD86, PDL1 and 2; DCs, other APCs Intensity of immune response
molecule upregulation B7H1-4
Signal 3 Environmental polarization IL-6, IL-12, IL-23, IL-27 DCs, other APCs Nature of the immune response
Signal 4 Tissue localization Integrins, chemokines DCs, other APCs Site of the immune response
Signal 5 PAMPs and DAMPs; inflamma- IL-10, TGF-b; TLR, NLR, RLR Epithelia, stroma, inflammatory Integrated inflammatory response
tory cell products ligands cells and wound-healing response
The response to environmental stressors is to recruit inflammatory cells (Signal 0), transmit signals to cells of the adaptive immune response (Signals 1–4)
via DCs, and then promote an integrated inflammatory response at the site of tissue stress (Signal 5).
PAMP, pathogen-associated molecular pattern molecule; DAMP, damage-associated molecular pattern molecule; TLR, Toll-like receptor; DC, dendritic
cell; IL, interleukin; NLR, NOD1-like receptor; RLR, retinoic-acid-inducible gene I-like receptors.

64 Immunological Reviews 220/2007

 Lotze et al
DAMPS and redox regulate immunity

Table 2. Classes of molecules that promote innate response – Signal 0 – tissue environmental stress
Signal 0
Sentinel Stress Antigen Target Signal 5
NK Genomic NKp30 ligands; MICA/B ligands MICA, MICB; NKp30 HMGB1, S100s, HSP molecules; ATP, uric acid
Metabolic
NKT Cell membrane Glycolipid CD1d ???
CD4 Cell membrane 13–20 peptide Class II ???
CD8 ER stress 8–10 peptide Class I k? Neut
apoptosis
The earliest events in immunity/inflammation are recruitment and activation of innate and adaptive immune cells; specialized sentinel cells respond to
Signals 0/5, depending on the nature of the stress, redox, and type of antigen.
ER, endoplasmic reticulum; NK, natural killer; ATP, adenosine triphosphate; HMGB1, high-mobility group box 1; redox, reduction/oxidation; ???, unknown.

Table 3. Classes of molecules that promote inflammation and immunity

T-cell response pattern Signal 0 Redox Inducer Transcription factor, miRNAs Products References
Th1 HMGB1, LPS, ?DNA Oxidizing IL-12 T-bet IFN-g (58, 59)
mmiR 146, kmiR 150
Th2 Most PAMPs Reduced IL-4 GATA3 IL-4, IL-5, IL-13 (60)
kmiR 146, kmiR 150
Th3 Most DAMPs Reduced IL-10, TGF-b FOXP3 IL-9, IL-10, TGF-b (61)
?miRNA
Th4 (Th17) PAMPS neutrophil apoptosis Oxidizing IL-6, TGF-b, IL-23 RORg IL-6, IL-7, IL-16, IL-17 (62)
?miRNA
gdT cells DAMPs Oxidizing IL-7, IL-15 Sox13 IL-4, IFN-g (27)
?miRNA
One of the critical aspects for vertebrate immunology is the linkage between innate and adaptive immune responses. Chronic inflammation is associated
with many diseases with substantial unmet clinical need. The role of initiating and perpetuating chronic infection or DAMP release is important to
understand how these might promote and drive inflammation and targeting them for elimination by antibodies or oxidative pathways. Seen this way, in
concert with the redox state, helps to define emergent patterns of immunity dictated by such environmental cues. The role of miRNA (micro RNA) has
only been suggested as important in murine T-helper 1 (Th1)/Th2 cells to date (63).
DAMP, damage-associated molecular pattern molecule; PAMP, pathogen-associated molecular pattern molecule; IL, interleukin; IFN, interferon; LPS,
lipopolysaccharide; redox, reduction/oxidation; HMGB1, high-mobility group box 1; ? miRNA (unknown).

constitutively in DCs, also reduces disulfide bonds to facilitate
antigen presentation and can be secreted as an active enzyme.
Upregulation of GILT levels in the serum of mice injected with
LPS and in patients with sepsis has been demonstrated
(64–71).
Ischemia–reperfusion injury and the host response
Intracellular molecules including HMGB1 are mediators of
inflammation in models of sterile injury, including hemorrha-
gic shock, hepatic ischemia/reperfusion, and femur fracture
(52, 53, 72–74). Although HMGB1 interacts with PRRs
including TLR2 and RAGE, in injury models it appears to
mediate its effects through an interaction with TLR4 (52, 53).
HMGB1 indeed interacts with TLR4 (75–77), although the
precise molecular nature of this interaction remains unclear.
Specifically, whether HMGB1 binds TLR4 directly or with a
costimulatory complex including such molecules as MD2 and
CD14, as appears likely, is unknown. Engagement of TLR4 by
LPS involves the formation of a signaling complex including
the co-receptor molecules MD2 and CD14, as well as multiple
intracellular molecules such as myeloid differentiation factor
88 (MyD88) and IL-1R-associated kinase (78). This interaction
facilitates an intracellular signaling cascade terminating in
translocation of the transcription factor nuclear factor kB to
the nucleus (79). LPS responsiveness is enhanced by dimeriza-
tion of TLR4 molecules and mobilization of the signaling
complex to the lipid raft (80–83).
Although LPS can bind CD14, this interaction alone is not
sufficient to induce signaling (84, 85). CD14 shuttles LPS to
MD2, in turn activating TLR4. Recruitment of the TLR4
signaling apparatus into the lipid raft enables receptor
engagement and activation. Recruitment of signaling
molecules to the raft leads to internalization of both TLR4 and
LPS, a process required for an adequate inflammatory response
to LPS (86). The importance of this process is demonstrated by
the attenuation of LPS-dependent TLR4 activation upon
disruption of the raft complex (80). Although HMGB1 has
been demonstrated to display TLR4-dependent activity, it is
unclear what role the lipid raft plays in this process. We have

Immunological Reviews 220/2007 65

Lotze et al
DAMPS and redox regulate immunity

recently demonstrated that HMGB1-dependent TLR4 activation
in vitro in macrophages requires the co-receptors MD2 and
CD14, interacting closely with the raft ganglioside GM1.
HMGB1-dependent TLR4 activation is attenuated upon
disruption of the lipid raft.
Inflammation and cancer are linked through the innate
immune response to DNA and DAMPs
Cancer is fundamentally a genetic disease (87–90). Develop-
ment of an invasive cancer, however, is not simply a result of
the genetic changes in the tumor cell but also the result of
genetic and epigenetic changes within the host. Host cells,
including inflammatory cells, endothelial cells, and fibroblasts,
are recruited and activated in the microenvironment of trans-
formed cells. The acute inflammatory response might succeed
in eliminating the malignant cells, but if not, a chronic
inflammatory process develops in conjunction with the dying
tumor cells. The subsequent reciprocal interactions between
these responding normal host cells and genetically altered cells
result in the development of an invasive cancer. There is a
significant role for innate immune cells in this process (57,
91–96). Innate immune cells contribute to the development
and progression of invasive cancer by producing a large
number of cytokines, chemokines, growth factors, and protei-
nases that lead to the cardinal features of invasive cancer:
stromagenesis, angiogenesis, and suppression of the adaptive
immune response. While there is a growing appreciation that
innate immune cells may potentiate tumor development and
progression, the precise molecular factors that regulate this
complex response remain largely unexplored. We have hy-
pothesized that chronic unscheduled tumor cell death (necro-
sis) results in the release of DAMPs and DNA into the tumor
microenvironment and systemic circulation (72, 97–100).
These DAMPs, best exemplified by the molecule HMGB1 as
well as release of DNA, lead to subsequent activation of innate
immune effectors and establishment of a chronic inflammatory
state that favors tumor progression, growth, and metastasis.
Unscheduled cell death results in the release of DAMPs
and DNA
Eukaryotic, multicellular organisms evolved rather sophisti-
cated mechanisms of programmed cell death that are necessary
for the orderly elimination of effete cells, which are no longer
required or that have become damaged. Essential to scheduled
cell death is careful husbanding of adenosine triphosphate
(ATP) to drive the apoptotic process, fragmentation of cellular
macromolecules, and the speedy subsequent phagocytosis and
clearance of the apoptotic corpse. Apoptotic cell death indeed
provides cell membrane signals to the host that result in ready
identification and rapid, ordered clearance of the cell. How-
ever, it has only recently become evident that cells dying by
non-apoptotic pathways, principally necrosis, release factors
that elicit a host response. These DAMPS communicate the
nature and magnitude of cellular injury to the host in addition
to mobilizing repair mechanisms (2, 101–110). What DAMPs
are has been a matter of conjecture for a decade, but several
promising candidates have been revealed in the last few years:
(i) the nuclear protein HMGB1; (ii) the S100 family of
molecules; (iii) the purinergic metabolites ATP, adenosine
monophosphate, adenosine, and uric acid; and (iv) heat shock
proteins. These factors are usually found in the cytosol and,
when released into the extracellular space, result in significant
host responses. Acting on specialized receptors including TLR-
2 (111–114), TLR-4 (115–119), and RAGE (120–137), they
recruit inflammatory cells, which promote wound healing and
associated stromagenesis, angiogenesis, epithelial prolifera-
tion, and modulation of the immune response. These repair
processes create an ideal environment for tumor progression.
DAMPs and DNA are released by tumor cells
Many of these newly recognized DAMPs released upon necrotic
cell death are overexpressed in tumor cells. HMGB1 expression
is greater in tumor cells than normal surrounding epithelium
in a large variety of human neoplasms, including breast (138),
colorectal (129, 139, 140), hepatocellular/biliary (140, 141),
lung (128, 142–145), and pancreatic cancer (127, 136), as
well as melanoma (97, 146). Higher HMGB1 levels have also
been found in tumor cells with greater metastatic potential.
Increased expression of the HMGB1 receptor RAGE has been
associated with increased cancer progression and metastatic
potential. Similar overexpression of a variety of the S100
protein family members has been described in numerous
studies of various tumors including breast, lung, pancreatic,
and colorectal cancer (142, 147–182). Elevated serum levels of
S100A4 are associated with a worse prognosis in breast cancer
patients. Recent studies have confirmed the role of extracellular
S100A4 in angiogenesis, stromagenesis, and innate immune
effector infiltration into tumors. Interestingly, loss of S100A4
results in decreased innate immune effector infiltration and
decreased metastatic potential of a highly aggressive murine
adenocarcinoma. These proteins also have an intracellular role
in response to stress, serving as anti-apoptotic factors (de-
scribed for both HMGB1 and S100). Aneuploidy and poly-
ploidy are characteristics of tumor cells, thus generating greater

66 Immunological Reviews 220/2007


amounts of DNA/cell when released. DAMPs and DNA are thus
increased in response to intracellular stress and, when released,
also serve as extracellular signaling molecules. This role is
consistent with the evolving paradigm of these agents in
biology. It is expected that overexpression of these factors in
transformed cells would lead to a relatively greater release of
these factors when tumor cells die by non-apoptotic pathways.
Binding of DNA to DAMPs may very well be a central
component to cancer progression.
HMGB1 is the best-characterized DAMP
A rapidly growing body of literature supports the dual function
of the DNA-binding protein HMGB1 as a regulator of intracel-
lular transcription and as an extracellular cytokine/inflamma-
tory mediator. HMGB1 can be released into the extracellular
space under two conditions: (i) HMGB1 is acetylated or ADP-
ribosylated and translocated into the cytosol and is actively
secreted from macrophages that have been exposed to either
LPS, tumor necrosis factor-a (TNF-a), or IL-1 (183–187), and
(ii) HMGB1 is released by cells undergoing non-apoptotic
(unscheduled) cell death (144, 188–193). HMGB1 is loosely
associated with chromatin in normal cells, but in cells under-
going apoptosis, histone 2B phosphorylation of the DNA
results in tight binding that prevents its release into the
extracellular space (M. Bianchi, personal communication).
Cells undergoing alternative deaths (necrotic) do not modify
their DNA to bind HMGB1, and consequently HMGB1 is
liberated into the extracellular milieu. Once liberated into the
extracellular space, HMGB1 mediates endothelial cell activa-
tion, angiogenesis, stem cell migration, and recruitment and
activation of innate immune effectors. The host response to
unscheduled cell death, with the release of HMGB1, is a
paradigmatic signaling pathway within the immune system.
Factors released upon unscheduled cell death serve as danger
signals, activating the innate immune response.
HMGB1 stimulation of endothelia
As one of the earliest events following tissue damage, the
release of HMGB1 and S100 family molecules leads to a dose-
dependent increase in the expression of intercellular adhesion
molecule-1, vascular cell adhesion molecule, and RAGE and
increased secretion of TNF-a, IL-8, monocyte chemotactic
protein-1, plasminogen activator inhibitor 1, and tissue plas-
minogen activator (123, 135, 137, 194–196). Through poly-
gamous receptors, including RAGE, TLR2, and TLR4, HMGB1
signals upregulation of adhesion molecules in human endothe-
lial cells, resulting in increased neutrophil recruitment. Re-
Lotze et al
DAMPS and redox regulate immunity
combinant, highly purified HMGB1 delivery also leads to
mesangioblast stem cell migration to injured tissues. Similarly,
chronic HMGB1 delivery to normal muscle promotes endothe-
lial cell permeability, proliferation, and angiogenesis. Primitive
mesangioblasts and bone marrow-derived stem cells injected
into mice preferentially migrate to sites of HMGB1 delivery.
HMGB1 from necrotic cells leads to increased angiogenesis
through endothelial cell sprouting.
HMGB1 release results in the activation of multiple
innate immune cells
Macrophages, neutrophils, and myeloid and plasmacytoid DCs
(PDCs) are activated by HMGB1 release. The delivery of
recombinant HMGB1 to human and murine macrophages
results in the release of TNF-a, IL-1b, IL-1a, IL-1RA, IL-6, IL-
8, macrophage inflammatory protein-1a (MIP-1a), and MIP-
1b. The 60 amino acid B box domain of HMGB1 upregulates
CD40, CD54, CD80, CD83, and major histocompatibility com-
plex class II molecules on the surface of immature myeloid DCs
(Fig. 1). HMGB1 also increases the production of the pro-
inflammatory cytokines/dendrikines IL-12, IL-6, IL-1a, and IL-
8. When released from necrotic cells, HMGB1 serves as an
endogenous adjuvant, enhancing cellular and humoral immune
responses. Treatment with recombinant HMGB1 converts poorly
immunogenic apoptotic lymphomas into efficient vaccines. In
our hands, recombinant HMGB1 suppresses IFN-a release from
PDCs. We hypothesize that redox and PAMPs/DAMPs are the
environmental cues that promote the four T-helper phenotypes
(Fig. 2). These roles of HMGB1 are in part related to their ability
to bind DNA and signal through cognate receptors on the cell
membrane (TLR2/4, RAGE) and inside the cell (TLR9). Its
ability to be oxidized (Fig. 3) modifies its intracellular and
extracellular activity. Unlike LPS, recombinant HMGB1 fails to
elicit a remarkable increase in IL-10 or nitric oxide but does
enhance IL-12 production (Fig. 4). Similarly, activated T or NK
cells promote the release of HMGB1 from tumor cells (Fig. 5)
(197) and promote migration of tumor leukocytes (Fig. 6). Most
chemotherapeutics cause HMGB1 release, with the exception of
the platinums (Fig. 7). Similarly, treatment of human neutrophils
with HMGB1 results in the release of inflammatory cytokines IL-
6, IL-8, IL-1b, and TNF-a. HMGB1 matures myeloid DCs alone
and synergistically with TNF-a or ATP (Fig. 8).
Platinums cross-link DNA and enhance DNA binding
to HMGB1
The platinums have been widely applied in chemotherapy
regimens to patients with cancer. The anti-cancer activity of
Immunological Reviews 220/2007 67
Lotze et al
DAMPS and redox regulate immunity

Fig. 2. Classes of molecules that initiate adaptive immune responses: Signal 0. Although Signals 1 and 2 have been recognized for some time as
important for initiation of the adaptive immune response, the so-called Signal 0, hypothesized by the late Charles Janeway, appears to be prompted by
damage to host or microbial cells and release of molecules that promote characteristic initiation of innate and adaptive immune responses.

Fig. 3. Protein structure of high-mobility group box 1 (HMGB1) revealing oxidation-sensitive unpaired cysteines at positions 23, 45, and 106.
Following tissue damage or injury, HMGB1 is released locally, possibly binding to tissue matrix components, recruiting inflammatory cells that
themselves can release HMGB1 into the local microenvironment. Time- and distance-dependent inactivation are mediated in part, we postulate, by
oxidation of damage-associated molecular pattern molecules at unpaired cysteines, such as those found in HMGB1.

the platinums as a class arises from their ability to cross-link HMGB1/2 domain (HMG) proteins (198–203) and other
DNA, with the metal adducts forming largely intrastrand DNA-binding proteins, including poly-ADP ribosyl polymerase
d(GpG) and d(ApG) cross-links. These cross-links bend and (204), histone H1 (205), mitochondrial transcription factor A
unwind the duplex, and the altered structure attracts the (206), p53/p73, upstream binding protein (207), and the

68 Immunological Reviews 220/2007

 Lotze et al
DAMPS and redox regulate immunity

Fig. 4. Recombinant high-mobility group box 1 (HMGB1) and lipopolysaccharide (LPS) differentially stimulate interleukin (IL)-12p40 and
nitric oxide (NO) production. Perhaps the most important means of distinguishing HMGB1 and LPS responses is the differential production of tumor
necrosis factor or other cytokines or factors in response to them. Shown here are the responses illustrative of murine bone marrow-derived
macrophages in response to HMGB1 and LPS (S Dave, MT Lotze, S Plevy, manuscript submitted).

Fig. 6. Migration of human leukocytes to high-mobility group box 1

Fig. 5. Interleukin (IL)-2/LAK-induced high-mobility group box 1
(HMGB1) release from tumor cells. Cytolysis of human melanoma cells
by immune effectors (both natural killer and T cells) is associated with
release of the nuclear chromatin protein HMGB1. Extracellular HMGB1
mediates a number of important functions including endothelial cell
activation, stromagenesis, recruitment and activation of innate immune
cells, and also dendritic cells’ maturation that, in the setting of cancer,
leads to a chronic inflammatory response. This reparative inflammatory
response promotes tumor cell survival, expansion, and metastasis.
TATA-binding protein (208, 209). Binding of HMGB1 could
indeed mediate the anti-tumor properties of the platinums,
although it is not clear that this is due solely to intranuclear
binding (Fig. 7). Many of the known HMG-domain proteins,
including the suppressor of cytokine signaling and sry pro-
teins, recognize altered DNA structures such as four-way
Holliday junctions (210) and cisplatin-modified DNA. The
HMGB1 A box binds to the minor groove of a 16 base-pair
DNA duplex containing a site-specific d(GpG) adduct. Phos-
(HMGB1). HMGB1 serves as a potent chemoattractant and survival factor
for human granulocytes and eosinophils. We demonstrated that HMGB1,
illustrative of other damage-associated molecular pattern molecules,
serves as a chemoattractant as well as a survival factor for both neutrophils
and eosinophils.
phorothioate modifications of DNA are highly selective for
platinum in oligonucleotides (ODNs), something that has been
known for quite some time, and could thus be useful in
creating a new class of DNA therapeutics and associated
diagnostics (211).
The pro-inflammatory effect of HMGB1 is best
demonstrated in concert with other cytokines. We
demonstrated that it promoted immune reactivity with IFN-g
production with IL-1 and IL-2 and most recently with the
application of other IL-1 family members (Fig. 9). The role of
CpG or other motifs in HMGB1 stimulatory activity has been
suggested by the fortuitous finding of DNA remaining in even

Immunological Reviews 220/2007 69

Lotze et al
DAMPS and redox regulate immunity

Fig. 7. Chemotherapy differentially affects high-mobility group box 1 (HMGB1) release. The release of HMGB1 after chemotherapy-induced
cytotoxicity has not been well characterized. We measured the release of HMGB1 after chemotherapy or immune cytolysis and demonstrated
that this did not correlate with conventional markers of apoptosis and necrosis in several human colorectal, pancreatic, and melanoma tumor cell lines.
Rather, we observed that tumor cells incubated with the platinating agent oxaliplatin retained HMGB1 within the nucleus for significantly longer
periods than other agents used at comparable cytotoxic concentrations or even with potent cytolytic cells. Thus, the release of HMGB1 from dying
tumor cells treated with chemotherapy or cells with lymphokine-activated killer cell activity is not dependent solely on the mode of cell death.
Sequestration of the damage-associated molecular pattern molecules HMGB1 may play a role in the clinical efficacy of platinating agents and
suggests this as a superior agent for coupling with immunotherapeutic strategies, possibly enhancing their effectiveness. (A) Melphalan treatment,
(B) Oxaloplatinum treatment.

70 Immunological Reviews 220/2007

 Lotze et al
DAMPS and redox regulate immunity

Fig. 8. Maturation of human immature dendritic cells (DCs) by high-mobility group box 1 (HMGB1). HMGB1 matures myeloid DCs alone and
synergistically with tumor necrosis factor (TNF)-a or adenosine triphosphate (ATP). The 60 amino acid B box domain of HMGB1 upregulates CD40,
CD54, CD80, CD83, and major histocompatibility complex class II molecules on the surface of immature myeloid DCs. HMGB1 also increases the
production of the pro-inflammatory cytokines/dendrikines interleukin (IL)-12, IL-6, IL-1a, and IL-8. When released from necrotic cells, HMGB1
serves as an endogenous adjuvant, enhancing cellular and humoral immune responses. Treatment with recombinant HMGB1 converts poorly
immunogenic apoptotic lymphomas into efficient vaccines.

the most highly purified preparations of HMGB1 (T. Coyle,
personal communication).
Clinical test of ODNs
The major applications of ODNs have been as antisense
constructs of 20–30 mers, CpG ODNs (212–215), and purified
porcine gut-derived ODNs. The latter natural product is
composed of approximately 50–60 mers as defibrotide
(216–221), a treatment for hepatic venocclusive disease
(VOD), approved in Europe with pending approval in the
United States. VOD is a lethal condition associated with
intensive cancer chemotherapy alone (particularly busulfan-
or melphalan-containing regimens) and is observed in both
autologous and allogeneic bone marrow transplantation set-
tings. The mechanisms by which defibrotide mediates its
salutary effects are obscure, but proposed mechanisms have
included a thrombolytic effect or interaction with adenosine
receptors. Antisense ODNs have been approved for the treat-
ment of patients with cytomegalovirus retinitis and are being
explored by Isis, Genta, and a number of biotechnology/
pharmaceutical companies. Several antisense drugs with appli-
cation to cancer patients are in development at Lilly in
conjunction with Isis; these include Phase III studies in non-
small-cell lung cancer for protein kinase Ca and survivin in
Phase I studies. Earlier in development are antisense molecules
targeting signal transducer and activator of transcription 3,
eukaryotic translation initiation factor 4E, and myeloid cell
leukemia sequence 1. Improvements including chemical mod-
ifications of antisense ODNs, which increase resistance to
nuclease digestion, prolong tissue half-lives, and improve
current scheduling strategies, are in development as so-called
second-generation (2-O-methoxyethyl, 2-O-methyl, and
locked nucleic acid) and third-generation (morpholina and
peptide nucleic acid) chemistries. Recent clinical trials confirm
the ability of antisense ODNs to target and suppress gene
expression (222). The immunostimulatory capability of ODNs
with hypomethylated CpG (and a class of ODNs with immu-
noinhibitory CG) within DNA has been known for a decade.
Class A ODNs contain a central phosphoester CpG motif and
phosphorothioate-stimulating IFN-a. Class B phosphor-
othioated backbone ODNs promote TNF-a and IFN-inducible
protein 10 production, and Class C ODNs stimulate antibody
and IL-6 production (215, 223–228). Klinman and colleagues
(229–231) have demonstrated that suppressive ODNs with the
TTAGGG motif, patterned after the mammalian telomeres, and
hypomethylated CG sequences, blocking the TLR9 ligand CpG,
have immunosuppressive properties and protect mice from
lethal endotoxic shock, glomerulonephritis in the setting of
lupus erythematosus, and enhance resistance to Listeria. PDCs
are innate immune cells recruited to sites of chronic inflamma-
tion, where they modify the quality and nature of the adaptive
immune response. PDCs modulate adaptive immunity in
response to signals delivered within the local inflammatory
milieu by PAMPs, DAMPs, and activated immune cells (includ-
ing NK, T, and myeloid DCs). We have investigated the effect of
HMGB1 on the function of PDCs (Fig. 10) and have shown that
it inhibits PDC responses to CpGs, suggesting a critical role in

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DAMPS and redox regulate immunity

Fig. 9. IL-1F1, F2, F4, and F5 but not F6-F10 synergize with IL-2/high-mobility group box 1 (HMGB1). Following genotoxic
or metabolic stress, cells release or induce a series of inflammatory molecules, specifically the IL-1 family. HMGB1 enhances interferon-g release
from macrophage (but not dendritic cell)-stimulated natural killer cells. IL-1F5 appears in other systems to predominantly antagonize IL-1F6,
F8, and F9 when appropriately N-terminally cleaved (J. Sims, personal communication). This is effective only when coupled with other pro-
inflammatory cytokines such as IL-2 in combination with IL-1 or IL-12. Of the IL-1 family members that we have tested, only some of those noted
above can promote this immune reactivity. IL-33, the latest identified member of the IL-1 family, is now being tested. These observations suggest
that HMGB1, which also promotes epithelial migration and proliferation, drives repair in the absence or inhibition of other factors but enhances
inflammation in their presence. The implications for tumorigenesis and tumor progression are quite important, as they may be for other states of
chronic inflammation.

immune reactivity that may be important in the setting of
tumors where it is released (Fig. 11) or other chronic inflam-
matory settings.
Uncertain role of eosinophils
Eosinophilic granulocytes are often found in the setting of
asthma, atopy, and parasite infections. These circumstances
have led to the assumption that these cells played a crucial role
in the pathogenesis of this disease, disregarding the fact that
not only eosinophils but also many other leukocytes (T-helper
2 lymphocytes, type II DCs, neutrophils, NK cells, and macro-
phages) are found in high numbers in these settings. DCs are
required for the development of chronic eosinophilic inflam-
mation in response to provocation in several murine models
(233). Even though eosinophilia disappears following the
corticoid-based treatment of asthma, it does not really demon-
strate a crucial role of eosinophils in the pathogenesis, as
corticosteroids have a broad suppressive effect on practically
all leukocytes without major preference for any specific
subpopulation. The anti-IL-5 therapy, which is much more
eosinophil specific and significantly reduces the airway and
blood eosinophil levels, failed to reduce the symptoms of
asthma (234), an observation that is consistent with our
hypothesis of considering factors other than eosinophils as
being responsible for initiating and perpetuating asthma or
chronic inflammatory states.

72 Immunological Reviews 220/2007


DAMPs including HMGB1 activate granulocytes
and eosinophils
HMGB1 is a highly conserved, ubiquitous protein present in
the nuclei and cytoplasm of nearly all cell types (72, 97). It is
released upon necrotic but not apoptotic death of normal cells
and is secreted by a variety of activated immune and non-
immune cells. Administration of HMGB1 to normal animals
causes inflammatory responses, including fever, weight loss,
Fig. 10. High-mobility group box 1 (HMGB1) inhibits plasmacytoid
dendritic cell (PDC) maturation. HMGB1 suppresses PDC cytokine
secretion and maturation in response to Toll-like receptor (TLR)9 agonists
including the hypomethylated ODN CpG- and DNA-containing viruses,
shown in inhibiting several pro-inflammatory cytokines including inter-
feron (IFN)-a, interleukin (IL)-6, tumor necrosis factor-a, IFN-inducible
protein 10, and IL-12. In addition, HMGB1 prevented the CpG-induced
upregulation of costimulatory molecules on the surface of PDCs and their
ability to induce IFN-g-secreting T cells. Our observations suggest that
HMGB1 may play a critical role in regulating the immune response during
chronic inflammation and tissue damage through modulation of PDC
function.
Fig. 11. Elevated serum levels of high-mobility group box 1 (HMGB1) in spontaneous tumor-bearing male mice. In animals bearing the SV40-
promoted a-amylase promoter gene, tumors develop within one to two months in the liver of only transgenic mice. Mice expressing the SV40 large T
antigen oncoprotein as a transgene develop hepatomas coincident with the appearance of immunoreactive HMGB1 in their serum. HMGB1 was not
detected in the serum of female mice, which do not develop tumors (232).
Lotze et al
DAMPS and redox regulate immunity
and anorexia, acute lung injury, epithelial barrier dysfunction,
arthritis, and even death. Moreover, HMGB1 given intratrache-
ally produces acute inflammatory injury within the lungs, with
neutrophil accumulation, development of lung edema, and
increased pulmonary production of IL-1b, TNF-a, and MIP-2
(235). Significantly, anti-HMGB1 treatments with antibodies
or specific antagonists rescue mice from lethal endotoxemia or
sepsis and ameliorate the severity of collagen-induced arthritis
and endotoxin-induced lung injury (236). Activated macro-
phages, mature DCs, and NK cells are able to secrete HMGB1.
Thus, in addition to its role as a constituent of chromatin
proteins, HMGB1 acts to promote leukocyte chemotaxis and
activation, including the amplification of adaptive immune
responses (72, 237), e.g. DC maturation (187, 238). Specifi-
cally, HMGB1 induces the migration and activation of human
DCs and acts as an alarmin (239). These activities position
HMGB1 as a unique danger signal identifying regions of cell
stress/necrosis and may serve to attract and activate granulo-
cytes (including eosinophils) within damaged lung tissue.
Indeed, in addition to Stenfeldt’s observations (240), our own
findings support the role of DAMPs including HMGB1 as a
chemoattractant for neutrophils and eosinophils (Fig. 6) as well
as a survival factor.
RAGE is a receptor on neutrophils for HMGB1
In addition to TLR2 and TLR4 (76), RAGE serves as a receptor
for HMGB1. We have shown that both neutrophils and
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Lotze et al
DAMPS and redox regulate immunity

eosinophils express RAGE. RAGE is a member of the immuno-
globulin superfamily of cell surface proteins that has been
implicated as a progression factor in a number of pathologic
conditions from chronic inflammation to cancer to Alzheimer’s
disease. In such conditions, RAGE facilitates pathogenic pro-
cesses. Its secreted isoform, soluble RAGE (sRAGE), has the
ability to prevent RAGE signaling by acting as a decoy. sRAGE
has been used successfully in animal models of a range of
diseases to antagonize RAGE-mediated pathologic processes
(241). sRAGE binds HMGB1 and neutralizes its effects in many
models (242). sRAGE is mainly found in lung tissues; the
hyperresponsiveness to DAMPs may be due to a lower sRAGE
concentration in the lung of asthma patients. If so, the
application of sRAGE or other HMGB1-neutralizing factors
may reduce inflammation in asthma.

Eosinophils and neutrophils migrate into necrotic tissue

Fig. 12. Hydrogen peroxide (H2O2) limits damage-associated mole-
cular pattern molecules biologic function and eosinophil peroxidase
(EPO) release. Oxidized forms of high-mobility group box 1 (HMGB1)
can be detected by mass spectrometry. The native, reduced state could be
maintained during storage by the addition of dithiothreitol or other
reducing agents. Here we show that two epithelial cell lysates, CaCO2 and
HCT116 promoted EPO release in their reduced but not in their oxidized
forms when H2O2 is added.
Eosinophils migrate into necrotic tissue. Cormier et al. (243)
demonstrated a massive migration of eosinophils and neutro-
phils into the injured lung tissue of ozone-exposed animals.
Necrotic cells and HMGB1 induce degranulation of
eosinophils with the subsequent release of eosinophil
peroxidase (EPO) and major basic protein (MBP) (Fig. 12).
Recombinant HMGB1 is reduced in the presence of
dithiothreitol (Fig. 13). Incubating eosinophils for 2 h with

Fig. 13. Recombinant high-mobility group box 1 (HMGB1) is only biologically active when reduced. One test of the reduced state of a molecule
is to examine the ability of reducing agents such as dithiothreitol (DTT) to alter the mass spectrum of tryptic peptides. In this instance, oxidized HMGB1
was digested by trypsin and then either reduced with DTT or not. The resultant peptides are consistent with induction of covalent disulfide bonds
between peptide thiols that is reversible. HMGB1 is a substrate of glutaredoxin. It contains three cysteines: Cys23, Cys45, and Cys106 (see Fig. 1). Under
mild oxidative conditions, Cys23 and Cys45 mostly form an intramolecular disulfide bridge, whereas Cys106 mostly remains in the reduced form. The
disulfide bond between Cys23 and Cys45 can be reduced in the presence of DTT. Cys23 and Cys45 induce conformational changes in response to
oxidative stress, whereas Cys106 is critical for nucleocytoplasmic shuttling (244).

74 Immunological Reviews 220/2007

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DAMPS and redox regulate immunity

two individual lysed epithelial cells (colorectal cell lines with Summary
repeated freeze/thaw cycles) and with HMGB1 isolated from
That dead and dying cells could play a major role in promoting
normal human liver results in a decrease of intracellular MBP, as
reactive cellular proliferation, angiogenesis, stromagenesis,
assessed by flow cytometry after intracellular staining with
and wound repair has been hinted at in biology for some time,
monoclonal murine anti-human MBP. We have shown the
but the synthesis of disparate biologic themes from prokar-
release of EPO following similar stimulation. EPO in
yotes, plants, and metazoans allows a new emergent paradigm.
supernatant was measured using a colorometric assay with O-
Within cells, reductive mechanisms maintaining free thiols
phenylenediamine as a peroxide substrate (245). Similarly, in
enable biologic processes disparate from those outside the cell,
the setting of acute injury (246–248), HMGB1 plays a role
where oxidative mechanisms are critical for tissue homeostasis
along with coordinate oxidative mechanisms to upregulate and
and free thiols are rare (249). Both neutrophils and eosinophils
drive TLR signaling. Together, these observations suggest that
provide a missing link for the adaptive immunologist as a
the earliest events in response to necrotic death drive
means for limiting through oxidation, signals derived from
development of pro-oxidant mechanisms designed to clear
within the cell, DAMPs, to promote tissue remodeling and
debris and drive the wound-healing process. In chronic
repair. These mechanisms, presaged 14 years ago, now appear
inflammation, regulatory adaptive cellular elements promote
to be central to the tissue-centric role of inflammation and
either containment and elimination or control of pathogens, or
immunity and open up new areas for investigation and
suppress effector cells to promote immune-mediated tissue
development of novel therapeutics.
restitution.

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