The Resolution of Inflammation

Progress in Inflammation Research
Series Editor
Prof. Michael J. Parnham PhD

Director of Preclinical Discovery
Centre of Excellence in Macrolide Drug Discovery
GlaxoSmithKline Research Centre Zagreb Ltd.
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Advisory Board
G. Z. Feuerstein (Wyeth Research, Collegeville, PA, USA)

M. Pairet (Boehringer Ingelheim Pharma KG, Biberach a. d. Riss, Germany)
W. van Eden (Universiteit Utrecht, Utrecht, The Netherlands)
Forthcoming titles:
New Therapeutic Targets in Rheumatoid Arthritis, P.-P. Tak (Editor), 2008

Inflammatory Cardiomyopathy (DCM) Pathogenesis and Therapy, H.-P. Schulthei,
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Matrix Metalloproteinases in Tissue Remodelling and Inflammation, V. Lagente, E. Boichot
(Editors), 2008
Angiogenesis in Inflammation: Mechanisms and Clinical Correlates, M.P. Seed, D.A. Walsh
(Editors), 2008
Microarrays in Inflammation, A. Bosio, B. Gerstmayer (Editors), 2008
Bone Morphogenetic Proteins: From Local to Systemic Therapeutics, S. Vukicevic,
K.T. Sampath (Editors), 2008
Natural Products for Joint Health, M.J.S. Miller (Editor), 2008
(Already published titles see last page.)

The Resolution of Inflammation
Adriano G. Rossi
Deborah A. Sawatzky
Editors
Birkhuser
Basel Boston Berlin
Editors
Dr Adriano G. Rossi
Dr Deborah A. Sawatzky
MRC Centre for Inflammation Research
Queens Medical Research Institute
University of Edinburgh
47 Little France Crescent
Edinburgh, EH16 4TJ
Scotland
United Kingdom
Library of Congress Control Number: 2007934182
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Contents
List of contributors ................................................................. vii
Preface .............................................................................. xi
Derek Gilroy and Toby Lawrence

The resolution of acute inflammation: A tipping point in the development
of chronic inflammatory diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Moira K.B. Whyte, Christopher Haslett and Edwin R. Chilvers

Granulocyte apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Andrew Devitt and Christopher D. Gregory

Innate immune mechanisms in the resolution of inflammation .................. 39
Ian Dransfield, Sandra Franz, Kim Wilkinson, Aisleen McColl,

Martin Herrmann and Simon P. Hart
Cell surface molecular changes associated with apoptosis . . . . . . . . . . . . . . . . . . . . . . . 57
Geoffrey J. Bellingan and Geoffrey J. Laurent

Fate of macrophages once having ingested apoptotic cells:
Lymphatic clearance or in situ apoptosis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Charles N. Serhan
Novel lipid mediators in resolution and their aspirin triggered epimers:
lipoxins, resolvins, and protectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Paola Maderna and Catherine Godson

Beyond inflammation: Lipoxins; resolution of inflammation
and regulation of fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Dalip J.S. Sirinathsinghji and Ray G. Hill
Contents
Mauro Perretti and Roderick J. Flower

Anti-inflammatory glucocorticoids and annexin 1 ................................ 141
Garry M. Walsh and Catherine M. McDougall

The resolution of airway inflammation in asthma and
chronic obstructive pulmonary disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
David C. Kluth and Jeremy Hughes

Resolution of glomerular inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
John L. Wallace and Philip M. Sherman

Resolution of mucosal inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Index ................................................................................ 235
vi
List of contributors
Geoffrey J. Bellingan, Centre for Respiratory Research, University College London,

Rayne Institute, London, WC1E 6JJ, UK; e-mail: geoff.bellingan@uclh.nhs.uk
Edwin R. Chilvers, Respiratory Medicine Division, Department of Medicine, Uni-

versity of Cambridge, School of Clinical Medicine, Addenbrookes and Papworth
Hospitals, Cambridge, CB2 2QQ, UK, e-mail: erc24@cam.ac.uk
Andrew Devitt, School of Life & Health Sciences, Aston University, Aston Triangle,
Birmingham, B4 7ET, UK; e-mail: a.devitt1@aston.ac.uk
Ian Dransfield, MRC Centre for Inflammation Research, University of Edinburgh

Medical School, Teviot Place, Edinburgh EH8 9AG, UK;
e-mail: i.dransfield@ed.ac.uk
Roderick J. Flower, William Harvey Research Institute, Barts and the London,
Queen Mary School of Medicine and Dentistry, Charterhouse Square, London
EC1M 6BQ, UK
Sandra Franz, Institute for Immunology, FAU Erlangen-Nuremberg, Glckstrasse

4a, 91054 Erlangen, Germany
Derek Gilroy, Rayne Institute, Centre for Clinical Pharmacology, University College
London, 5 University Street, London WC1 6JJ, UK; e-mail: d.gilroy@ucl.ac.uk
Catherine Godson, UCD School of Medicine and Medical Science, UCD Conway
Institute, University College Dublin, Belfield, Dublin 4, Ireland;
e-mail: catherine.godson@ucd.ie
Christopher D. Gregory, MRC Centre for Inflammation Research, The Queens

Medical Research Institute, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK
vii
Simon P. Hart, MRC Centre for Inflammation Research,University of Edinburgh

Medical School, Teviot Place, Edinburgh EH8 9AG, UK
Christopher Haslett, MRC Centre for Inflammation Research, Queens Medical

Research Institute, University of Edinburgh Medical School, Edinburgh, EH16 4TJ,
UK; e-mail: c.haslett@ed.ac.uk
Martin Herrmann, Institute for Immunology, FAU Erlangen-Nuremberg, Glck-

strasse 4a, 91054 Erlangen, Germany
Jeremy Hughes, MRC Centre for Inflammation Research, University of Edinburgh,

Queens Medical Research Centre, 47 Little France Crescent, Edinburgh EH16 4TJ,
UK
David C. Kluth, MRC Centre for Inflammation Research, University of Edinburgh,

Queens Medical Research Centre, 47 Little France Crescent, Edinburgh EH16 4TJ,
UK; e-mail: david.kluth@ed.ac.uk
Geoffrey J. Laurent, Centre for Respiratory Research, University College London,

Rayne Institute, London WC1E 6JJ, UK
Toby Lawrence, Institute of Cancer, Centre for Translational Oncology, Barts and
The London School of Medicine and Dentistry, Charterhouse Square, London
EC1M 6BQ, UK; e-mail: t.lawrence@qmul.ac.uk
Paola Maderna, UCD School of Medicine and Medical Science, UCD Conway Insti-
tute, University College Dublin, Belfield, Dublin 4, Ireland;
e-mail: paola.maderna@ucd.ie
Aisleen McColl, MRC Centre for Inflammation Research,University of Edinburgh

Catherine M. McDougall, School of Medicine, Institute of Medical Sciences, Uni-

versity of Aberdeen, Foresterhill, Aberdeen, UK
Mauro Perretti, William Harvey Research Institute, Barts and the London, Queen
Mary School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ,
UK; email: m.perretti@qmul.ac.uk
Charles N. Serhan, Center for Experimental Therapeutics and Reperfusion Injury,

Brigham and Womens Hospital and Harvard University, 75 Francis St., Thorn
Building for Medical Research, Boston, MA 02115, USA
viii
Philip M. Sherman, Hospital for Sick Children and University of Toronto, 555 Uni-
versity Avenue, Toronto, Ontario, M5G 1X8, Canada
John L. Wallace, Department of Pharmacology and Therapeutics, University of Cal-

gary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, Canada;
e-mail: wallacej@ucalgary.ca
Garry M. Walsh, School of Medicine, IMS Building, University of Aberdeen, For-

esterhill, Aberdeen AB25 2ZD, UK; e-mail: g.m.walsh@abdn.ac.uk
Moira K. B. Whyte, Academic Unit of Respiratory Medicine, School of Medicine

and Biomedical Sciences, University of Sheffield, Royal Hallamshire Hospital, Shef-
field S10 2JF, UK; e-mail: m.k.whyte@sheffield.ac.uk
Kim Wilkinson, MRC Centre for Inflammation Research,University of Edinburgh

ix
Preface
It was with tremendous enthusiasm that we endeavoured to compile and edit this
volume for Progress in Inflammation Research describing novel findings and devel-
opments pertaining to the processes governing the resolution of inflammation. It
is perhaps surprising that this topic had, to our knowledge, not previously been
covered as a separate subject area in a dedicated monograph given what now seems
such an obvious thing to do. Historically, researchers have focussed and have made
great advances on the initiation and propagation of inflammation. Little atten-
tion had been specifically devoted to elucidating the mechanisms orchestrating the
resolution of inflammation, although a variety of mechanisms that limit the inflam-
matory response had been described (e.g., mediator dissipation and deactivation;
exogenous mediator removal or reduction; receptor, cell and tissue desensitisation to
mediators; identification of agents with anti-inflammatory potential such as IL-10,
IL-1 receptor antagonists, TGF-`, etc).
It is now believed that manipulation of more recently described processes, recog-
nised as being actively involved in resolution, are therapeutically manipulatable for
the treatment of inflammatory diseases. Indeed, patients with chronic inflamma-
tory diseases are by necessity treated in order to reduce established and persistent
inflammation with the added hope of preventing further progression of the inflam-
matory response. It has recently become evident that many of the anti-inflammatory
agents currently used in the clinical setting influence inflammatory resolution. For
example, glucocorticoids have been shown to influence processes now recognised as
being important mechanisms allowing resolution to occur; namely glucocorticoids
trigger apoptosis (programmed cell death) in most leukocytes (the neutrophil how-
ever is a notable exception) and augment apoptotic cell clearance by phagocytes.
Similarly, aspirin, the most widely used NSAID, is involved in an unorthodox bio-
synthetic pathway yielding important lipid mediators (e.g., 15-epi-lipoxin A4 and
15-epi-lipoxin B4) actively involved in the resolution process.
This volume contains major contributions from an international panel of experts
who describe the basic processes regulating the resolution of inflammation including
apoptosis, macrophage clearance of apoptotic cells and novel pro-resolution lipid
xi
Preface
mediators. In addition, there are sections that describe how existing anti-inflam-
matory drugs such as aspirin and glucocorticoids may influence these resolution
processes. There are three chapters devoted to describing fine examples of clinically
relevant inflammatory disease areas where much progress has been made in under-
standing resolution. We feel that we are at the beginning of a rapidly burgeoning
and exciting area of inflammation research where new advances are being made in
understanding the resolution of inflammation. It is without doubt that continued
research will fully elucidate the mechanisms whereby existing anti-inflammatory
drugs influence resolution. Furthermore, there is now emerging experimental in vivo
evidence indicating that by pharmacologically and selectively inducing apoptosis of
inflammatory cells, specifically enhancing non-phlogistic clearance of apoptotic cells
by phagocytes, and administration of pro-resolution lipids (e.g., lipoxins, resolvins
and protectins), inflammatory resolution is achievable. Consequently, we believe
that better designed and novel classes of drugs that specifically target resolution
processes will be forthcoming in the not too distant future.
October 2007 Adriano G. Rossi

Deborah A. Sawatzky
xii
The resolution of acute inflammation: A tipping point in the
development of chronic inflammatory diseases
Derek Gilroy1 and Toby Lawrence2
1
Rayne Institute, Centre for Clinical Pharmacology, University College London, 5 University
Street, London WC1 6JJ, UK; 2Institute of Cancer, Centre for Translational Oncology, Barts
and The London School of Medicine and Dentistry, Charterhouse Square, London EC1M
6BQ, UK
The scope of this chapter
Evolution has given us inflammation, a formidable ally in the constant battle against
infection, cancer and tissue injury. It is a primordial response that protects against
injury and restores damaged tissue to its normal physiological function. In fact, our
well-being and survival depends upon its efficiency and carefully balanced control.
In general, the innate inflammatory response initiates within minutes and, if all is
well, resolves within hours. In contrast, chronic inflammation persists for weeks,
months or even years. Here, we are going to discuss the key endogenous checkpoints
necessary for mounting an effective, yet limited, inflammatory response and the
crucial biochemical pathways necessary to prevent its persistence. Figure 1 depicts
what we understand today about the endogenous soluble mediators that control
the severity of inflammatory onset as well as its longevity. In doing so, we wish to
underline the consequence to the host of failing to adequately control inflammatory
resolution.
Acute inflammation is characterised by leukocyte recruitment from the circula-
tion, classically defined by the initial trafficking of polymorphonuclear granulo-
cytes, followed by monocytes, which differentiate locally into macrophages [1].
Invariably, this response is triggered by tissue mast cells and resident macrophages,
whose degranulation and activation sequentially release a battery of inflammatory
mediators, including bioactive amines (histamine and 5-HT), cytokines, chemokines
as well as lipid mediators that collectively recruit and activate inflammatory cells,
which also results in oedema formation. While this system has an enormous capac-
ity for synergy and redundancy, over the years it has served as the stable basis for the
development of anti-inflammatory drug discovery, typified by the development of
nonsteroidal anti-inflammatory inhibitors of eicosanoid synthesis beginning in the
1960s to more recent times with the inhibition of the actions of TNF-_. These early
days of inflammation research that focused on elucidating the nature of soluble pro-
inflammatory mediators have now given way to the view that inflammation is far
more complex and sophisticated than originally appreciated, not least muddied by
The Resolution of Inflammation, edited by Adriano G. Rossi and Deborah A. Sawatzky

2008 Birkhuser Verlag Basel/Switzerland 1
Figure 1
Schematic depicting the cellular and molecular components of resolving inflammation.
Acute inflammation is characterised by the accumulation of neutrophils and oedema
early in the response. Later, mononuclear cells and macrophages accumulate and help
prepare the tissue for resolution. In both (A) and (B) we depict the role that specific
molecular mediators play in these events. In (A), sequentially released pro-inflammatory
mediators are released very early in response to injury/infection, which initiate and aug-
ment the acute-phase of the response (green lights). However, this is counterbalanced
by endogenous anti- inflammatory signals such as corticosterone, which serve to temper
the severity and limit the duration of this early onset phase. As inflammation progresses,
certain stop signals prevent further leukocyte traffic into tissue. These stop signals
include the lipoxins, resolvins and prostaglandins (PGs) of the D series, and pave the way
for monocyte migration and their differentiation to phagocytosing macrophages. These
remove dead cells and then exit the site of inflammation. Stromal cells such as fibroblasts
also contribute to the resolution of inflammation by the withdrawal of survival signals
and the normalisation of chemokine gradients, thereby allowing infiltrating leukocytes to
undergo apoptosis or leave the tissue through the draining lymphatics. This sequential set
of responses leads to complete resolution and, importantly, the restoration of the inflamed
tissue to its prior physiological functioning. This is the ideal sequence of events in physi-
ological inflammation, which contrast to the situation in pathological inflammation (B),
where some of the factors that initiate the resolution program lead to the inappropriate
accumulation of leukocytes in the wrong place at the wrong time (from [69]).
2
The resolution of acute inflammation
the multiple protective and destructive roles the eicosanoids, for instance, that are
now known to play in orchestrating the inflammatory response. Such clear diversity
is not the preserve of lipid mediators but it extends to cytokines, chemokines and
the expression of both activating and inhibitory receptors by inflammatory cells. On
this theme of biological diversity, recent evidence suggests that alternative pattern
recognition receptors of the scavenger receptor and C-type lectin families may play
equally important roles in the recognition of microbes and the regulation of the host
inflammatory response. Thus, the C-type lectin, Dectin-1 [2], was recently shown to
act in concert with the Toll-like receptor (TLR)-2 to activate macrophages exposed
to `-glucans from the yeast Candida albicans [3]. A number of these receptors also
recognise endogenous inflammatory ligands including the scavenger receptors SR-
A and CD36, both of which have been described to mediate the phagocytosis of
apoptotic cells, leading to a down-regulation of macrophage activation [46] (see
the chapter by Dransfield et al.). Thus, many of the factors that drive inflammation
also double-up in bringing about its resolution and it is this theme of inflammatory
resolution that is going to be the focus of this chapter. In current day inflammation
research, one of our objectives must be to understand whether known inflammatory
mediators that ignite inflammation also trigger its resolution as well as highlight-
ing resolution. In addition, resolution must be highlighted as a critical facet of the
inflammatory response and, at the very least, to underline the importance of not
altering its normal course of action when developing novel anti-inflammatory drugs.
Ultimately, it is proposed here that resolution is controlled by endogenous pro-reso-
lution factors, which may represent new possibilities for drug discovery in terms of
designing modalities that mimic their mode of action or enhance their synthesis. In
the course of doing so, we hope to argue that resolution is as active process, whose
failure may predispose the host to chronic inflammatory diseases and autoimmunity,
such as that typified by rheumatoid arthritis, inflammatory bowel disease, systemic
lupus erythematosus and asthma.
Resolution of acute inflammation
The receptors and signalling pathways that initiate and promote the inflammatory
response have become increasingly well characterised; however, relatively little is
known about how acute inflammation resolves to prevent chronic inflammatory
diseases. We have discussed above the intracellular checkpoints that limit the activa-
tion of inflammatory cells either directly in response to infection or tissue injury or
through paracrine activation by proinflammatory cytokines. If we were to define the
fundamental requirements for the successful resolution of inflammation it is becom-
ing increasingly clear that the most simple but absolutely critical determinant for
the inflammatory response to switch off is the neutralisation and elimination of the
injurious agents that initiated it. Failure to achieve this first step will invariably lead
3
to chronic inflammation, with the nature of the agent in question almost certainly
dictating the aetiology of the developing chronic immune response. For example,
chronic granulomatous disease is characterised by severe, protracted and often fatal
infection, which results from a failure of the phagocytic NADPH oxidase enzyme
system to produce superoxide and kill invading infections, leading to a predisposi-
tion to recurrent bacterial and fungal infections and the development of inflamma-
tory granulomas [7]. Successfully dispensing with the inciting stimulus will signal
a cessation to pro-inflammatory mediator synthesis (eicosanoids, chemokines,
cytokines, cell adhesion molecules, etc.) and lead to their catabolism. This would
halt further leukocyte recruitment and oedema formation. These are probably the
very earliest determinants for the resolution of acute inflammation, the outcome of
which signals the next stage of cell clearance. The clearance phase of resolution,
be it innate immunity [polymorphonuclear leukocyte (PMN) or eosinophil driven]
or adaptive immunity (lymphocyte mediated), also has a number of mutually
dependent steps. The clearance routes available to inflammatory leukocytes include
systemic recirculation [8] or local death of influxed PMNs, eosinophils or lympho-
cytes followed by their phagocytosis by recruited monocyte-derived macrophages
[9]. Once phagocytosis is complete, macrophages can leave the inflamed site by
lymphatic drainage [10] with evidence that a small population may die locally by
apoptosis [11]. If all of these pathways are strictly followed then acute inflammation
will resolve without causing excessive tissue damage and give little opportunity for
the development of chronic, non-resolving inflammation. As with the onset phase
of the acute inflammatory response, which is driven by a cohort of well-described
endogenous factors, the resolution phase of the response is also highly coordinated
and under the tight control of what may be called pro-resolution factors. In con-
trast to onset, however, these resolution phase factors are less well described.
Controlling the early phase of acute inflammation
Inflammation is a reaction of the microcirculation that is characterised by the move-

ment of serum proteins and leukocytes from the blood to the extravascular tissue
with PMNs or eosinophils predominating at the early onset phase, giving way to
phagocytosing macrophages leading to resolution. One well-described event in the
transition towards resolution is the replacement of PMNs or eosinophils by mono-
cytes and phagocytosing macrophages. However, until recently our understanding
of the signals that control this cell profile switch was unclear. Studies addressing
this issue of leukocyte infiltration in peritoneal inflammation have suggested that
the interaction between interleukin (IL)-6 and its soluble receptor, sIL-6R, forms one
of the major determinants of this switch from PMNs to monocytes [12, 13]. It was
shown that sIL-6R, produced by the infiltrating PMNs, forms a complex with IL-6,
which in turn, directly modulates CC and CXC chemokine expression. Thus, CXC
4
chemokine synthesis, induced by IL-1 and TNF-_, was suppressed, whereas the CC
chemokine CCL2 (MCP-1) was promoted. This chemokine shift suppresses further
neutrophil recruitment in favour of sustained mononuclear cell influx. In addition
to chemokines, the eicosanoids also orchestrate the early transition to resolution
in acute inflammation. Transcellular metabolism of arachidonic acid by lipoxy-
genase/lipoxygenase interaction pathways gives rise to the lipoxin (LX) family of
eicosanoid metabolites [14]. LXs display selective actions on leukocytes that include
inhibition of PMN chemotaxis [15], PMN adhesion to and transmigration through
endothelial cells [16], as well as PMN-mediated increases in vascular leakage [17]. It
is unclear at this point whether there is any cross-talk between the LXs and IL-6/sIL-
6R complex signalling in the control of leukocyte profile switching. Nonetheless, it
seems that when acute inflammation needs to resolve the IL-6/sIL-6R, chemokines
and LXs represent some of the earliest signals that control the switch from very
early PMNs to monocyte/macrophage.
The transition to resolution
Once PMNs and eosinophils have done their job and their help is no longer needed,
what happens next? At this juncture it must be borne in mind that these are a for-
midable cell lineage and if left unchecked could do untold damage to an already
inflamed site. After all, these cells are designed to combat infection by releasing
hydrolytic and proteolytic enzymes as well as generating reactive oxygen species.
Therefore, PMNs and eosinophils must be disposed of in a controlled and effective
manner. To oversee this, nature has come up with an ingenious way of defusing such
potentially explosive cells called programmed cell death or apoptosis. Apoptosis of
inflammatory cells is a physiological process for the non-phlogistic removal of cells.
During apoptosis, cells maintain an intact membrane and, therefore, do not release
their potentially histotoxic agents. Necrosis of inflammatory leukocytes, on the
other hand, involves a loss of membrane integrity, leading to the release of poten-
tially toxic intracellular contents [9]. Moreover, apoptotic cells express a repertoire
of surface molecules that allow their recognition and phagocytosis by macrophages
[18]. Despite stating above that once the injurious agent has been neutralised PMNs
and eosinophils are redundant, in fact, the way in which these cells die helps the
resolution process enormously. Recognition of these apoptotic cells by macrophages
does not liberate pro-inflammatory agents from the macrophages themselves but
can release anti-inflammatory signals such as IL-10 and transforming growth factor-
` (TGF-`) [19]. Thus, not only is apoptosis a non-inflammatory way of disposing of
cells, but this method has the added advantage of conferring upon macrophages an
anti-inflammatory phenotype conducive to resolution. It is important to note that
if not recognised and disposed of, apoptotic cells will eventually undergo secondary
necrosis releasing damaging intracellular contents and amplifying the inflammatory
5
response. Therefore, increasing the rate of apoptosis, as a potentially anti-inflam-

matory strategy, must be matched by a mechanism that up-regulates macrophage
phagocytic clearance capacity [20]. Thus, the removal process might also be suscep-
tible to selective modulation by pharmacological agents for therapeutic gain.
As mentioned above, the strategy of enhancing leukocyte apoptosis must also be
paralleled with enhancing their phagocytosis by macrophages and other non-profes-
sional phagocytes. On this theme, there are an increasing number of factors that aid
the phagocytic clearance of apoptotic granulocytes. Ligation of the matrix receptor
CD44, for instance, results in the rapid and specific internalisation of apoptotic
PMNs [21]. Besides controlling PMN trafficking, LXs also stimulate monocyte
chemotaxis and adherence. Certainly, this may seem dangerous for inflammation
as too many monocyte-derived macrophages can be a bad thing, but these LX-che-
moattracted macrophages accelerate resolution by enhancing phagocytosis of apop-
totic PMNs in a non-phlogistic manner [22]. In addition to a role in granulocyte
apoptosis, glucocorticoids facilitate the phagocytic response. It was recently found
that exposure of peripheral blood monocytes to glucocorticoids during the first 24
h of the 5-day culture period induced a highly phagocytic monocyte-derived mac-
rophage phenotype [23]. Functional and morphological homogeneity was matched
by cell surface phenotype, including specific induction of expression of the haemo-
globin scavenger receptor, CD163 following glucocorticoid treatment. A potentially
pro-resolution role for CD163 was demonstrated recently in both in vitro and in
vivo models of resolving inflammation [24]. Here, the authors showed that human
peripheral blood monocyte-derived macrophages either in culture medium or in
resolving phase cantharidin-induced skin blisters express CD163. These authors
also found elevated levels of CD163 on circulating monocytes in cardiac surgical
patients during the resolution phase of the systemic inflammatory response to car-
diopulmonary bypass surgery. In each case, binding of the haemoglobinhaptoglo-
bin complex to CD163-bearing cells elicited potent IL-10 secretion, which in turn
enhanced hemeoxygenase 1, widely shown to have anti-inflammatory and tissue
protective properties. Such induction of hemeoxygenase 1 was observed in vivo
2448 h after the onset of cardiopulmonary bypass surgery. This is coincident with
the observations of Willis and colleagues [25], who showed that hemeoxygenase 1
was expressed during and essential for the resolution (2448-h phase) of a rat car-
rageenin-induced pleurisy. Thus, apoptosis and the phagocytosis of apoptotic cells
are crucial to the resolution process, failure of which may predispose, as mentioned
earlier, to chronic inflammation and possibly autoimmunity. This has been proposed
in the case of systemic lupus erythematosus (SLE); an autoimmune syndrome that is
associated with the presence of autoantibodies to endogenous antigens exposed on
dead or dying cells, which failed to be cleared and disposed of. Studies in mice, for
instance, have established a role for the complement receptor C1q on macrophages
in the development of this disease. C1q was discovered to be important for the
phagocytic clearance of apoptotic cells; in the absence of this receptor mice devel-
6
oped a lupus-like syndrome [26]. The persistence of apoptotic cells and necrotic
bodies led to the development of an inappropriate immune response to endogenous
antigens. Evidence has also been established in human SLE patients for an associa-
tion between C1q deficiency and disease [27]. The phagocytosis of apoptotic cells
has been suggested to play an important role in the negative regulation of macro-
phage activation, apoptotic leukocytes may well fit into the category of endogenous
anti-inflammatory mediators, therefore the mechanisms of apoptosis and the clear-
ance of apoptotic cells may be critical in the development of chronic inflammation
(as discussed further by Dransfield et al. in this book).
Soluble mediators of resolution: Opportunities for drug discovery
Returning to eicosanoids, prostaglandin (PG) D2, a metabolite of the action of

haematopoietic PGD2 synthase on COX-derived PGH2, has emerged recently as
an eicosanoid with both pro- and anti-inflammatory properties. PGD2 undergoes
dehydration in vivo and in vitro to yield biologically active PGs of the J2 series,
including PGJ2, 612,14-PGJ2 and 15-deoxy-612,14-PGJ2 (15d-PGJ2). In addition to
being a high-affinity natural ligand for anti-inflammatory peroxisome prolifera-
tors-activated receptor gamma (PPARa), 15d-PGJ2 also exerts its effects through
PPARa-dependent as well as -independent mechanisms to suppress pro-inflamma-
tory signalling pathways and the expression of genes that drive the inflammatory
response. 15d-PGJ2 also preferentially inhibits monocyte rather than PMN traffick-
ing through the differential regulation of cell-adhesion molecule and chemokine
expression [28]. We have shown that COX 2-derived PGD2 metabolites contribute
to the resolution of acute inflammation (pleuritis) through the preferential synthe-
sis of PGD2 and 15d-PGJ2 [29], which, along with the alternative DNA-binding
p50p50 homodimers complexes of NF-gB [30] bring about resolution by induc-
ing leukocyte apoptosis [11]. Recently, we extended these studies to examining the
role of PGD2 metabolites in the resolution of adaptive immunity and lymphocyte
function [31]. Indeed, there is an increasing body of evidence detailing the differ-
ential effects of PGD2 metabolites on leukocyte apoptosis as well as the signalling
pathways involved [32, 33].
In addition to the well-known eicosanoids, there is a new generation of lipid
mediators showing real promise as endogenous anti-inflammatories. Resolvins and
docosatrienes are fatty acid metabolites of the COX/lipoxygenase pathways, where
the omega-3 fatty-acid constituents of fish oils docosahexaenoic acid and eicosa-
pentaneoic acid are the substrates and not arachidonic acid. These resolvins and
docosatrienes were identified in inflammatory exudates during the resolving phase
of acute inflammation and shown to be potent inhibitors of PMN transendothelial
migration and microglial-cell cytokine expression, and to ameliorate experimental
models of dermal inflammation and leukocyte accumulation in peritonitis at nano-
7
gram doses [34]. These studies on resolvin metabolism are uncovering surprising
new avenues in anti-inflammation research, putting fatty acid metabolites right at
the forefront of potential drug therapy. These studies are also challenging existing
dogma that not all eicosanoids are detrimental to inflammation and are putting a
balanced view of their role in pathophysiology. To add fuel this notion, a recent
and very surprising paper has shown that eicosanoids of the LXs family, described
above, are orally active in models of acute inflammation [35].
Signalling pathways that regulate inflammation
The inflammatory response is characterised by coordinated activation of various

signalling pathways that regulate expression of both pro- and anti-inflammatory
mediators in resident tissue cells and recruited leukocytes (see Fig. 1). Currently
most of our knowledge of signalling in inflammation is gained from studying mem-
bers of IL-1 and TNF receptor families and the Toll-like microbial pattern recogni-
tion receptors (TLRs), which in fact belong to the IL-1R family. IL-1 and TNF-_
represent the archetypal pro-inflammatory cytokines that are rapidly released upon
tissue injury or infection. TLRs recognise microbial molecular patterns, hence the
term pattern-recognition receptor (PRR), and therefore TLRs represent a germline-
encoded non-self recognition system that is hard-wired to trigger inflammation.
However, there is some suggestion that endogenous ligands may trigger TLRs dur-
ing tissue injury and certain disease states, which may act to promote inflammation
in the absence of infection [36]. Although structurally different, these receptors use
similar signal transduction mechanisms. Receptor engagement results in recruitment
of adaptor proteins that possess either TollIL-1 receptor (TIR) domains in the case
of TLRs and IL-1R or death domains (DD) in the case of the TNFR family, linked
to the regulation of cell survival [37]. Once recruited these adaptors recruit further
signalling proteins that belong to the TRAF family [38, 39] and various protein
kinases, including IRAK1 and 4 in the case of TIR signalling [40] and RIP kinases
in the case of TNFR signalling [41, 42]. These molecules activate several effector
pathways, the most important of which lead to activation of mitogen-activated
protein kinases (MAPK) [43, 44], including JNK [45] and p38 MAPK [46], as well
as IgB kinases (IKK) [47]. The MAPKs lead to direct and indirect phosphorylation
and activation of various transcription factors, especially those that belong to the
bZIP family: AP-1 [48] and CREB [49], which bind to the promoters of pro-inflam-
matory genes. MAPKs also regulate pro-inflammatory gene expression through
post-transcriptional mechanisms such as mRNA turnover, mRNA transport and
translation [5052]. The IKKs, which form a complex composed of two catalytic
subunits-IKK_ and IKK` and a regulatory subunit IKKa/NEMO, are responsible for
activation of the NF-gB transcription factor [47], which has emerged as a central
regulator of inflammatory and immune responses [53, 54]. Target genes for the IKK
8
and MAPK pathways include IL-1 and TNF-_, generating a feed-forward mecha-
nism to amplify the inflammatory response. The pro-inflammatory cytokines IL-6,
IL-12 and type I interferons (IFNs), which are also target genes for IKK and MAPK
regulation, signal via receptor-associated tyrosine kinases (RTKs) that belong to the
JAK group, whose activation results in phosphorylation and nuclear translocation
of STAT transcription factors [55]. Engagement of cytokine receptors, as well as
TLRs, can also lead to activation of phosphoinositide-3-kinases (PI3K), which in
turn activate other proteins kinases such as AKT [56]. Collectively, these proteins
kinases coordinate the expression of a large number of pro-inflammatory mediators
to initiate and maintain the inflammatory response.
Negative regulation of pro-inflammatory signalling
All of the intracellular signalling pathways described above, which contribute to the
onset of innate immunity and inflammation are also subject to negative regulation.
PI3K signalling is inhibited by the PTEN phosphatase that belongs to the protein
tyrosine phosphatase (PTP) family; some of its other members, for instance SHIP,
SHP1/2 and CD45, are responsible for negative regulation of TK signalling [57].
MAPK kinase phosphatases (MKPs), which also belong to the PTP family, control
the duration of MAPK activation as recently shown for TNF-_-mediated JNK acti-
vation [58]. Inducible suppressors of cytokine signalling (SOCS), which function
as ubiquitin ligases, are responsible for the negative feedback control of JAK-STAT
signalling [59]. A20 is another inducible ubiquitin ligase, which functions as a nega-
tive feedback regulator of TLR and TNFR signalling to IKK and NF-gB. A20 is also
a direct target gene for the NF-gB pathway constituting a negative feedback loop for
NF-gB activation [60]. Recently, a new pathway for negative regulation of IKK/NF-
gB was described from observations made in mice that harbour a variant of IKK_,
IKK_AA, that can not be activated by upstream regulators. Although IKK_AA mice
do not develop spontaneous inflammation, they develop an exaggerated inflam-
matory response when challenged with bacteria, fungal cell wall particles, or even
immune complexes [61]. These studies established that, while IKK` catalytic activ-
ity is important for the activation of NF-gB through phosphorylation of endogenous
inhibitory (IgB) proteins [62], IKK_ is required for termination of NF-gB activation
through phosphorylation of the transcription factors RelA (p65) and c-Rel [61].
IKK-mediated phosphorylation results in polyubiquitination of the target protein,
leading to its accelerated degradation via the 26S proteasome. However, while IgB
degradation is essential for NF-gB activation and nuclear translocation, the accel-
erated degradation of nuclear Rel proteins via IKK_-mediated phosphorylation is
important for controlling the duration of NF-gB activation. The evolution of two
catalytic subunits in the IKK complex with opposing, yet complimentary, activity
therefore ensures rapid and transient activation of NF-gB.
9
As discussed below, pro-inflammatory signalling pathways have the capacity

of inducing the parallel expression of anti-inflammatory mediators, such as IL-10.
Recent studies reveal that the signalling pathway used by TLRs to activate expres-
sion of pro- and anti-inflammatory cytokines diverges at the level of the adaptor
proteins TRAF3 and TRAF6, such that TRAF3 is critical for induction of IL-10
expression and in its absence, expression of the TRAF6-dependent pro-inflamma-
tory cytokines IL-6 and IL-12 is dramatically increased [63]. The balance between
the TRAF3- and TRAF6- generated signals may therefore play an important role in
controlling the inflammatory response and its perturbation may interfere with the
proper resolution of inflammation.
Some of these signalling pathways and their respective negative regulators are
illustrated in Figure 2; a deficiency in any one of these negative regulators may result
in either spontaneous chronic inflammation, perhaps reflecting host cell activa-
tion by PAMPs present in endogenous microflora, or an exaggerated inflammatory
response to insult or injury that culminates in severe inflammation and damage to
the host. Although all of these negative regulatory mechanisms affect different sig-
nalling pathways, genetic studies in mice have shown that even the absence of one
negative regulator is sufficient to result in serious inflammatory disorders. Undoubt-
edly, aberrations in such negative regulatory pathways will be found to contribute
to the development of chronic inflammatory diseases.
Resolution of T cell-driven inflammation
Inflammation has an important role in instructing the adaptive immune response,

in particular the maturation and migration of dendritic cells (DCs) from the site of
inflammation, where they pick up antigens and traffic to the secondary lymphoid
organs where they can prime antigen-specific immune responses. There have been
major advances in the study of DC biology in the past few decades that clearly show
that both pro-inflammatory cytokines, including TNF-_, and microbial products
drive DC maturation and migration to draining lymphoid tissue. The immature
DC at the site of inflammation has the capacity to efficiently take up and process
antigens. Specific signals in the inflammatory environment trigger the expression of
chemokines and receptors that promote the migration of antigen-loaded DCs to the
local lymphoid organs where they can present their antigens to cells of the adap-
tive immune system. Although cross-talk between the innate and adaptive immune
system is extensively reviewed elsewhere [64], it suffices to underline that the switch
from an inflammatory response driven by short-lived granulocytes to a lymphocyte-
or macrophage-dominated event is classically associated with chronic inflammation.
Various investigators have examined the resolution of lymphocyte-driven adaptive
immune responses including Type III hypersensitivity (Arthus reaction) or Type IV
delayed-type hypersensitivity (DTH) reactions, which are clinically relevant models
10
Figure 2
Schematic illustration of the co-ordinated activation of pro-inflammatory signalling path-
ways by TLR ligands and the pro-inflammatory cytokines TNF-_, IL-1 and IFN.
Adaptor molecules (MyD88, TRADD, TRAF) and receptor associated kinases (RIP, IRAK, JAK)
couple to downstream kinase cascades (MAPK; JNK, p38; TAB/TAK, IKK), which regulate
the activation of transcription factors (AP-1, NF-gB, CREB, STAT) and the expression of pro-
inflammatory genes. A number of negative regulatory mechanisms (broken lines) limit the
activation of specific signalling pathways; SOCS targets JAK/STAT and TLR signalling; PTEN
and SHIP phosphatases block PI3K; A20 and IKK_ negatively regulate the NF-gB pathway;
the MAPK phosphatase MKP limits activation of JNK and p38.
of adaptive immune diseases. For instance, in a purified protein derivative-induced

DTH response, it was shown that the induction and resolution of this response
may depend on the expression of cytokines, such as IL-2 and IL-15, that regulate
both proliferation and apoptosis in T cells [65]. Failure to control either of these
phases of the reaction may contribute to the chronicity of T lymphocyte-mediated
inflammatory reactions. In another important series of studies the endogenous fac-
tors that control the longevity of granulomatous autoimmune thyroiditis revealed
that the ratio of CD4+/CD8+ T cells are critical determinants of its resolution. In
this disease process, CD4+ T cells outnumber CD8+ T cells when lesions progress to
11
fibrosis, while CD8+ T cells outnumbered CD4+ T cells in thyroids that resolve [66].
Recently, we found that haematopoietic PGD2 synthase (hPGD2S) transgenic mice,
bearing a DTH reaction, display an exaggerated inflammatory response that fails
to resolve [67]. While hPGD2S-derived PGD2 and the cyclooxygenase-derived PGs
possess potent but diverse biological roles in host defence, the suppressive effects of
hPGD2S on T lymphocyte functioning appears to be mediated by 15d-PGJ2 and its
inhibition of NF-gB DNA binding, with no contribution from PGD2 and its actions
on either of its receptors namely DP1 or DP2/CRTH2. These findings suggest an
important role for hPGD2S as a checkpoint controller in the progression from acute
to resolving inflammation. Whether the absence of hPGD2S predisposes to chronic
inflammation or autoimmunity has yet to be determined. Nonetheless, the clear lack
of inflammation in animals that over-expressed hPGD2S further reinforces the criti-
cal role that this down-stream PGH2 metabolising enzyme plays in the aetiology of
T lymphocyte-driven immune responses.
Unblocking the drains!
The role of the lymphatic system in the context of the resolution of acute innate
inflammation is enormously understudied given its essential function in draining
inflammatory mediators and effete leukocytes away from the inflamed site [68]. We
have already discussed the importance of PMN clearance to the resolution of acute
inflammation, but it is equally important that phagocytosing inflammatory macro-
phages are cleared away from the inflamed site to prevent local macrophage-induced
tissue damage, potential granuloma tissue damage and the development of chronic
inflammation. However, despite the need to understand the endogenous control of
macrophage clearance during acute inflammatory resolution, little is known about this
field. There is increasing evidence that macrophage clearance from an inflamed site is
a highly regulated event. Using an experimental model of acute resolving peritonitis,
it was shown that macrophages adhere specifically to mesothelium overlying drain-
ing lymphatics and that their emigration rate is regulated by the state of macrophage
activation [10, 68] providing the first evidence that macrophage emigration from the
inflamed site is controlled by adhesion molecule regulation of macrophagemesothe-
lial interactions. This report highlights the importance of adhesion molecules control-
ling clearance of inflammatory macrophages into the draining lymphatic circulation,
thus highlighting new pathways in the resolution of acute inflammation.
Conclusions
In conclusion, it is clear the inflammatory response has a number of built-in check-

point controls that limit the duration and magnitude of acute inflammation. Defects
12
in these endogenous anti-inflammatory pathways will undoubtedly predispose to

the development of chronic inflammatory diseases. A further understanding and
analysis of these pathways in the pathogenesis of chronic inflammation is required,
this will allow the pursuit of therapeutic strategies to correct possible defects in these
feedback control systems or manipulate these pathways to suppress inflammation.
However, it is equally clear that the resolution of inflammation is driven by a com-
plex set of pro-resolution mediators that regulate specific cellular events required
to clear inflammatory cells from the site of injury or infection and restore tissue
homeostasis. These endogenous anti-inflammatory and pro-resolution mechanisms
are clearly intimately linked; however, the true goal in the treatment of chronic
inflammatory diseases must be to inhibit persistent inflammation and restore tissue
function. To achieve this goal we must improve our understanding of the resolu-
tion of inflammation and identify possible approaches to promote this process in
combination with anti-inflammatory therapy. Perhaps the, as-yet-unidentified, anti-
resolution factors that may prevent the proper resolution of inflammation would
represent appropriate targets to achieve this goal.
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18
Granulocyte apoptosis
Moira K. B. Whyte1, Christopher Haslett2 and Edwin R. Chilvers3
1
Academic Unit of Respiratory Medicine, School of Medicine and Biomedical Sciences, Uni-
versity of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2JF, UK; 2MRC Centre for
Inflammation Research, Queens Medical Research Institute, University of Edinburgh Medical
School, Edinburgh, EH16 4TJ, UK; 3Respiratory Medicine Division, Department of Medicine,
University of Cambridge, School of Clinical Medicine, Addenbrookes and Papworth Hospi-
tals, Cambridge, CB2 2QQ, UK
Introduction
Neutrophil apoptosis (programmed cell death) is now recognised to play a funda-

mental role in the physiological resolution of innate immune responses. Early work
by Metchnikoff correlated the ingestion of microphages (neutrophils) by macro-
phages with the resolution of acute inflammation, but nearly 100 years elapsed
before the key role of apoptosis in determining the lifespan of granulocytes and
their clearance from sites of inflammation was described [1]. Further work showed
that apoptosis leads to down-regulation of neutrophil pro-inflammatory functions
[2] and this, together with evidence that macrophage clearance of apoptotic granu-
locytes was anti-inflammatory [3], suggested that apoptosis induction could be a
powerful therapeutic strategy to turn off neutrophilic inflammation [2].
By contrast, eosinophil clearance mechanisms appear to be more diverse and
include primary cytolysis, migration to regional lymph nodes and transepithelial
migration as well as apoptosis. Hence, allergen challenge in animals induces a rapid
and highly co-ordinated exit of eosinophils into the airway lumen [4] and, once
present in the lumen, these cells are expectorated and/or undergo apoptosis trigger-
ing; this latter event triggers phagocytic removal by alveolar or inflammatory mac-
rophages, or bronchial epithelial cells [5]. While eosinophils are clearly capable of
undergoing constitutive apoptosis both in vitro and in vivo [6, 7], studies conducted
in a variety of models of allergic airways inflammation have shown little evidence of
eosinophil apoptosis in the airway wall itself. It is possible that the highly efficient
coupling of granulocyte apoptosis to macrophage recognition and removal may
result in an underestimation of the true extent of eosinophil apoptosis in these cir-
cumstances; these data, however, support the concept that for the eosinophil, which
serves little, if any, physiological function in the host, apoptosis may represent a
relatively minor clearance mechanism from tissues [8].
The balance between neutrophil survival and apoptotic cell death is exqui-
sitely regulated and involves multiple input signals, including both host-derived and

Moira K. B. Whyte et al.
pathogen-derived molecules [9, 10]. Our current understanding of these events pre-
dicts that, in the setting of acute infection or inflammation, granulocyte apoptosis
is delayed until essential host functions such as pathogen clearance are successfully
performed, but thereafter proceeds promptly to avoid the tissue damage that could
arise from a perpetuated response. Given the extent of co-ordinated granulocyte
clearance that is occasionally required (e.g. in the context of lobar pneumonia or
eosinophilic pneumonia) and the delicate balance between such pro-survival and
pro-apoptotic signals, it is not surprising that there exists considerable potential for
pathological perturbation of these physiological events. The cellular processes of
apoptosis can also be dysregulated by certain pathogens as an immune evasion strat-
egy, whereas delayed apoptosis and prolonged granulocyte survival is important in
persistence of tissue inflammation.
External factors regulating granulocyte apoptosis
It is now well established that the rate at which granulocytes undergo apoptosis, at
least in vitro, can be modified by several extra-cellular stimuli. For the neutrophil
these include certain cytokines and growth factors (e.g. GM-CSF, IL-1, IFN-a, C5a,
IGF-1) [913], cell adhesion or interaction with particulate material, including
bacteria and bacterial products (e.g. VCAM-1, LPS) [10, 14, 15] and various physi-
cochemical perturbations (e.g. UV irradiation, hypoxia, acidosis, hyper-osmolarity)
[16, 17]. A similar spectrum of apoptosis regulators has also been described in the
eosinophil (see Tab. 1).
In addition to such physiological and pathological stimuli, a wide array of
pharmacological agents have been reported to modulate granulocyte apoptosis, the
most classical being the pro-apoptotic effect of glucocorticosteroids in eosinophils
[6] and protein synthesis inhibitors in neutrophils [18]. A few, as yet poorly defined
modulators have also been reported including the capacity of a modified, largely
tissue-bound form of C-reactive protein (CRP) and serum from trauma or septic
patients to inhibit neutrophil apoptosis [19, 20] and peritoneal dialysis membranes
and dialysate fluid to enhance neutrophil apoptosis [21]. Of more physiological rel-
evance, a distinct population of circulating neutrophils that are CD54high, CXC che-
mokine receptor 1low has been detected recently, which are typical of cells that have
undergone reverse endothelial transmigration, i.e. cells that have migrated from an
inflamed site back into the systemic circulation; these cells appear to be relatively
apoptosis resistant and in the presence of systemic inflammation can account for up
to 2% of circulating neutrophils [22].
In general, the number of stimuli that induce granulocyte survival outnumber
those that promote apoptosis, and stimuli that induce very transient cellular activa-
tion (e.g. the bacterial tripeptide fMLP or IL-8 in the neutrophil) appear to have
a far more marginal effect on apoptosis compared to more sustained inputs (e.g.
20
Table 1 - Regulation of eosinophil apoptosis
Apoptosis Proposed mechanism Ref(s)

delaying factors
Cytokines IL-3, IL-5, Receptors composed of unique alpha sub-unit and [96]
GM-CSF common beta sub-unit (`c) [97]
Ligand binding triggers recruitment of tyrosine [98]
kinases lyn, syk, Jak2 and SHPTP-2
IL-9 Unknown, may be up-regulation of IL-5R [99]
expression
IL-13 Enhances synthesis/release of IL-3 and [100]
GM-CSF to act in autocrine fashion
TNF-_ Partly by p38 MAP kinase activation [101]
Hormones Leptin May block mitochondrial release of cytochrome c [102]
Bacterial LPS Enhanced autocrine GM-CSF production [103]
endotoxin
Interferons IFN-a Mediated by Jak2 [104]
Integrins `2 Integrin Enhanced paracrine/autocrine synthesis of cyto- [105]
kines, e.g. IL-5/GM-CSF through ICAM-1
and `2 integrin signalling
Fibronectin, Integrin-mediated interaction resulting in [106]
laminin generation of survival cytokines [107]
Galectin Ecalectin Unknown, but independent of IL-3, IL-5 and [108]
GM-CSF
Prostaglandins PGE2 Increasing cytosolic cAMP [109]
EP2R subtype expressed on eosinophils
Cysteinyl LTB4, LTC4, CysLTs produced by eosinophils, mast cells & [110]
leukotrienes LTD4 Th2 lymphocytes
Mediated by GM-CSF which increases CysLT
production
Pharmacological Rolipram Increasing cytosolic cAMP levels [111]
agents
CD antigens CD40 Cross-linking CD40 enhances GM-CSF release [112]
Gaseous Nitric Inhibits apoptosis by cyclic GMP driven process [113]
compounds oxide
Death receptors Fas/APO-1/ Cross-linking with ligand (CD95L, FasL, APO-1L) [114]
CD95
CD69 Ligation of CD69 in GM-CSF cultured eosinophils [115]
21
Table 1 - continued
Apoptosis Proposed mechanism Ref(s)

delaying factors
Cytokines TGF-` Inhibits IL-3-, IL-5-, GM-CSF- and IFN-a- [116]

mediated survival
IL-4 Inhibits IL-3-, IL-5- and GM-CSF-medi- [117]
ated survival
Interleukins IL-10 Inhibits LPS-induced GM-CSF release [103]
Pharmacological Glucocorticoids, e.g. Unknown, may inhibit of production [118]
agents dexamethasone of cytokines by other cells
Theophylline Inhibits IL-5-mediated survival [115]
Sodium Inhibit survival-enhancing action of [119]
channel-blockers, e.g. IL-5 and IFN-a
lidocaine
Ketotifen Inhibit survival-enhancing action of IL-5 [120]
Potassium channel- Inhibit cytokine-induced survival [121]
blockers, e.g. sulfonyl-
ureas
Immunosuppressants, Inhibit survival-enhancing action [120]
e.g. Cyclosporin A of IL-5
Gliotoxin Inhibition of NF-gB [77]
Orazipone Inhibit survival-enhancing action of IL-5 [122]
Antibody Siglec-8 Production of ROS and mitochondrial [123]
cross-linking cleavage and caspase cleavage
GM-CSF, LPS, hypoxia). It is also evident that most agents that induce functional
granulocyte priming, that is, enhance the magnitude of subsequent agonist-stimu-
lated respiratory burst or degranulation responses, also prolong neutrophil survival,
suggesting a functional and/or mechanistic link between these processes.
Stimuli for inducing apoptotic cell death in granulocytes
The extracellular agents that promote neutrophil apoptosis divide into four main
groups, particulate (e.g. E. coli, oil red micro-particles), death receptor ligands
(including TNF-_, TNF-related apoptosis-inducing ligand or TRAIL and Fas-L)
[23, 24], toxins (e.g. Staphylococcus aureus Panton-Valentine leukocidin, which is
22
a pore-forming toxin that targets neutrophil mitochondrial membranes) [25] and

pharmacological agents (e.g. protein synthesis inhibitors, phorbol esters, zVAD-
fmk, curcumin, sodium salicylate, etc.) [18, 26].
The former group underwrites the importance of phagocytosis-induced cell
death (PICD) as being an important mechanism in driving neutrophil apoptosis and
thereby inducing effective elimination of both the inciting particle and the phago-
cyte. Neutrophil apoptosis following phagocytosis of bacteria was first described
for E. coli but subsequently for many other bacteria also [27, 28]; PICD can also
be induced by latex beads, requires reactive oxygen species (ROS) production [28]
and is regulated by differential expression of a large number of apoptosis regulators
[29].
An important distinction needs to be made between the effects of cell adhesion
and the interaction of neutrophils with extracellular matrix components such as
fibronectin, which induce a `2-integrin-dependent survival response, and the phago-
cytic uptake of particles, including bacteria, which, while still `2-integrin dependent,
triggers accelerated granulocyte apoptosis [30]. Likewise, the ability of TNF-_ to
induce neutrophil and eosinophil apoptosis is highly dependent on the state of basal
priming, the duration of agonist stimulation, and whether the cells are adherent or
in suspension [31, 32]. These experiments underline the context-specific nature of
many of the pro-apoptotic responses observed in granulocytes and help clarify some
of the seemingly contradictory data that exist in the literature.
In most studies, however, TNF-_ and FasL have both been shown to induce a
modest increase in the rate of neutrophil apoptosis at early times, although notably
TNF-_ has an overall survival effect if incubations are continued for longer periods
(> 12 h) [31, 33]. Similar findings have also been reported for eosinophils, although
the pro-apoptotic effect of TNF-_ and FasL in these cells appears to be more con-
ditional and modest [34]. Of interest, the early pro-apoptotic effect of TNF-_ in
neutrophils is dependent on co-ligation of both the TNF-RI and TNF-RII, a finding
that has now been reported in other cell types and appears to relate to the capacity
of TNF-RII, which has a higher affinity for TNF-_, to participate in ligand pass-
ing. TNF-_ can also prime neutrophils for apoptotic induction by agents such as
GM-CSF, LPS or the `2-integrin-activating antibody VIM12 [35]. Moreover, the
pro-apoptotic efficacy of TNF-_ in neutrophils varies between individuals and this
appears to relate to the level of cells surface CD13 or aminopeptidase N expression
[36].
Another death ligand, TRAIL (TNF-related apoptosis-inducing ligand), can also
regulate neutrophil apoptosis [24]. Neutrophils express TRAIL-receptor 2 (TRAIL-
R2) and TRAIL-R3 at both mRNA and protein level with the latter thought to act
as a decoy receptor for TRAIL [24]; the same study was unable to detect TRAIL-R1
or TRAIL-R4. Despite evidence from this and other studies that neutrophils can
produce TRAIL, and that ligation of the TRAIL-R2 with a leucine zipper-tagged
form of TRAIL induces apoptosis, no evidence could be found for TRAIL in auto-
23
crine regulation of constitutive neutrophil apoptosis. TRAIL is, however, expressed

on other immune cells such as T cells and macrophages and TRAIL-induced neutro-
phil apoptosis, mediated by autologous T cells, is a likely mechanism of neutropenia
in systemic lupus erythematosus [37].
A similar complexity also exists regarding Fas/FasL interactions in the neutro-
phil. Hence, early reports suggested that human neutrophils expressed both FasL
and its cognate receptor Fas [38]; while the latter is certainly true, independent
examination has failed to identify FasL expression in neutrophils at either mRNA or
protein level [23, 39]. As with His-tagged monomeric TRAIL, certain preparations
of recombinant soluble FasL also appear unable to induce neutrophil apoptosis
[40], whereas that produced by opsonised-zymozan stimulated or apoptotic neu-
trophil-fed macrophages is clearly pro-apoptotic [39]. These latter findings rather
discount Fas/FasL-mediated fratricide as being important in neutrophils but support
the capacity for FasL generated by other cells to induce neutrophil apoptosis.
Intracellular mechanisms mediating granulocyte apoptosis
Granulocyte apoptosis can be initiated by activation of either the extrinsic or intrin-

sic pathways of cell death [41]. The extrinsic pathway is mediated through ligation
of cell-surface death receptors, as described above, leading to formation of the DISC
(death-inducing signalling complex) and cleavage of caspase-8 [42]. The intrinsic or
stress pathway, typically initiated following oxidant damage, cytotoxic agents or
UV radiation, leads to mitochondrial outer membrane permeabilisation (MOMP),
release of small inter-membrane mitochondrial proteins, notably cytochrome C and
apoptosis-inducing factor, and activation of both caspase-9-dependent and cas-
pase-independent pathways [43, 44]. These two pathways can interact to enhance
apoptosis: for example, the pro-apoptotic Bcl-2 protein Bid mediates cross-talk by
actions of a Bid caspase-cleavage product on MOMP [45]. Both pathways culminate
in a common executioner pathway, characterised by activation of caspase-3 and
cleavage of DNA and chromatin [46].
Given the paucity of functional mitochondria in neutrophils [47], it has been
debated whether the intrinsic, mitochondrial death pathway is important in
granulocytes. There is now good evidence, however, for mitochondrial initiation of
apoptosis [48, 49] and that neutrophils can achieve activation of the apoptosome
and caspase-9 processing [50]. Mitochondrial membrane permeabilisation in neu-
trophils, as in other cell types, requires Bax translocation to the mitochondria prior
to caspase-3 activation [51].
Caspases play key roles in the induction of granulocyte apoptosis, as in most
cell types. Neutrophils express many of the known caspases, including the cytokine-
processing caspases-1, -4 and -5 [50, 52, 53], the upstream caspases-8, -9 and -10
[50, 54, 55] and the executioner caspases-3, -6 and -7 [50, 56]. Caspase-1 has an
24
important role in cytokine processing, particularly of pro-IL-1`, as a component

of the inflammasome [53], but has an additional role as an upstream regulator of
neutrophil apoptosis, since spontaneous apoptosis is delayed in caspase-1-deficient
neutrophils [52]. Caspase-8 is activated following death receptor ligation [57], while
caspase-10, in contrast, is activated in spontaneous but not death receptor-mediated
neutrophil apoptosis [55]. Caspase-8 may also have a role in spontaneous neutro-
phil apoptosis, which is independent of death receptor ligation, with ROS causing
aggregation of CD95 in lipid rafts to form a functional DISC and thus activate
caspase-8 [58]. A role for caspase-8 in spontaneous or Fas-mediated eosinophil
apoptosis is less clear [59]. Despite the very low levels of cytochrome C detected in
neutrophils, there is good evidence for caspase-9 processing and apoptosome forma-
tion in neutrophils in both spontaneous and staurosporine-induced apoptosis [50].
Similarly, caspase-9 activation is a feature of eosinophil apoptosis [60].
The broader question of how susceptible neutrophils and eosinophils are to
oxidant-mediated induction of apoptosis is more complex and the subject of some
debate [61]. Certainly, the application of agents such as sodium arsenite and high
concentrations of H2O2 and NO are capable of inducing granulocyte apoptosis
and antioxidants can protect against various forms of PICD; likewise, neutrophils
from patients with chronic granulomatous disease who lack a functional NADPH
oxidase have been variably reported to display delayed spontaneous and Fas-medi-
ated apoptosis [62] and TNF-_ fails to kill neutrophils under anoxic conditions
[61]. In contrast, however, full-scale activation of the very substantial respiratory
burst machinery in neutrophils using sequential incubation of cells with PAF fol-
lowed by fMLP, or the addition of more physiologically relevant concentrations of
pro-oxidants, has no effect on apoptotic thresholds. Likewise early TNF-_-induced
neutrophil apoptosis, again reported to be oxidant-mediated, is preserved in the
presence of a panel of antioxidants including trolox, superoxide dismutase, catalase,
reduced glutathione and N-acetyl-l-cysteine [61]. Actinomycin A, which enhances
mitochondrial ROS generation and augments TNF-_-induced apoptosis in several
other cell types, fails to enhance the pro-apoptotic efficacy of TNF-_ in human neu-
trophils. Together, these data imply that neutrophils, as a physiologically important
source of ROS are, in fact, relatively protected from the pro-apoptotic efficacy of
these toxic intermediates.
Intracellular acidification is an early feature of neutrophil apoptosis and stimu-
lation of the vacuolar (H+) ATPase in cell membranes inhibits both apoptosis and
intracellular acidification [63]. During phagocytosis of bacteria, intracellular alka-
linisation can delay neutrophil apoptosis, while ingestion of larger numbers of bac-
teria leads to intracellular acidification and increased apoptosis [64].
Caspase-3 activation leads to cleavage of specific protein targets that result in
cell death; some of these are generic such as actin [65], lamin B and fodrin [66]
while others are potentially cell-type specific. In neutrophils, caspase-3 activation
cleaves and activates protein kinase C (PKC)-b during spontaneous apoptosis [67],
25
whereas in eosinophils, but not neutrophils, the mammalian sterile 20-like 1 and 2
(Mst1/Mst2) kinases are caspase substrates [68]. Proteases other than caspases have
also been implicated in inducing granulocyte apoptosis: calpains have been shown
to degrade XIAP but, in view of the low levels of XIAP found in neutrophils, the
importance of this is uncertain [69]. Calpain-1 also plays a direct role in neutrophil
apoptosis by enhancing the cleavage of Bax to an 18-kDa form that no longer inter-
acts with Bcl-xL, thus freeing up this Bax isoform to induce MOMP and caspase-3
activation [70].
Intracellular mechanisms that delay granulocyte apoptosis
The principal second-messenger cascades implicated in neutrophil and eosinophil

survival are: (i) NF-gB [71], (ii) phosphoinositide 3-kinase (PI3K)/Akt [72], (iii)
protein kinase-b (PKCb), (iv) adenylyl cyclase/cAMP/PKA [73, 74], (v) p42/44 and
p38 MAPK [75, 76] and (vi) PHD/HIF-1_ pathways [16]. These have been well
reviewed elsewhere and have major similarities to many other cell types. Hence
observations largely by Rossi and co-workers have demonstrated that prostaglan-
din E1 (PGE1), PGE2, adenosine (working through the adenosine A2a receptor) and
cAMP and its analogues are powerful suppressors of neutrophil (and eosinophil)
apoptosis, preventing both loss of mitochondrial membrane potential and caspase
activation via a protein kinase A-independent mechanism [73, 74, 77].
Likewise, the activation of one of the multiple PI3K isoforms expressed in myeloid
cells [78] results in the conversion of phosphatidylinositol 4,5-bisphosphate to the
lipid second messenger phosphatidylinositol 3,4,5-trisphosphate, which induces the
phosphorylation and activation of Akt/protein kinase B. This targets a number of
anti-apoptotic pathways including the BH3 only molecule Bad, Forkhead, caspase-9
and IKK_, although only the former has been confirmed in granulocytes. Activated
Akt can also phosphorylate and release Hsp27.
The NF-gB pathway has a well-defined pro-survival role in granulocytes
and inhibitors such as glitoxin are powerful inducers of apoptosis and enhance
TNF-_-killing; the enhanced survival of neutrophils under hypoxic conditions
may also be mediated by a HIF-1_-mediated transcriptional up-regulation of
this pathway. Of note, the late survival effect of TNF-_ appears to be driven by
NF-gB-mediated release of IL-8, which then operates in an autocrine/paracrine
manner to induce cell survival [79]. PKCb has also been reported to be impor-
tant in mediating the survival effect of TNF-_ in adherent neutrophils through a
positive regulation of RIP and TRAF2 assembly on TNF-RI (thereby enhancing
NF-gB activation) and inhibition of TRADD association (thereby blocking the
formation of the cytosolic FADD, TRADD2, RIP, caspase-8 complex II, which
induces apoptosis) [32, 80].
26
Other major targets that mediate neutrophil longevity include Mcl-1 and A1
that, unlike Bcl-2, are both expressed to a significant degree in neutrophils [8184].
Mcl-1, a member of the Bcl-2 family, is of particular interest given its short half-life
of 23 h and its up-regulation by neutrophil-survival factors including GM-CSF,
IL-1, TNF-_ and IL-15; it is regulated at both a transcriptional and proteosomal
level and is a target for caspase-mediated cleavage. Moreover, gene expression pro-
filing in neutrophils treated with GM-CSF showed additional up-regulation of the
apoptosis inhibitor 5, Bcl-2-like 1, BNIP2, CFLAR, serum/glucocorticoid-regulated
kinase (SGK), and TNF-_-induced protein 8 [85]. Inhibitors of apoptosis proteins
(IAPs) are inhibitors of activated caspases and provide a further regulatory step in
apoptosis pathways [86]. Their role in granulocyte apoptosis is, however, uncertain
since several, including cIAP1, XIAP and survivin, are expressed at very low levels;
however, G-CSF may up-regulate cIAP2 and this could contribute to its pro-survival
effect in neutrophils. cIAP2 is also known to be over-expressed in chronic neutro-
philic leukaemia [87].
Consequences of granulocyte apoptosis
The requirement for a highly effective route of physiological granulocyte apopto-

sis and clearance is exemplified by pneumococcal pneumonia, where bacterial clear-
ance is followed by complete resolution of the neutrophilic inflammatory infiltrate
[88]. Since, as described previously, certain extracellular bacterial factors typically
delay neutrophil apoptosis, whereas phagocytosis of bacteria induces apoptosis, the
regulation of neutrophil apoptosis during infections is complex. These opposing
effects can be integrated by a model of biphasic susceptibility, in which apoptosis is
delayed until bacterial killing (associated with internalisation and ROS generation)
is well advanced [28]. This model would allow for containment of infection and
limit inflammatory tissue injury.
These physiological host responses can, however, be subverted by pathogens
as part of a strategy for immune evasion, as shown in a number of models of mac-
rophage death [89]. Premature neutrophil apoptosis impairs bacterial killing and, if
clearance mechanisms are overwhelmed and secondary necrosis occurs, could cause
bystander tissue injury. Pseudomonas aeruginosa employs this strategy both in vitro
and in vivo via the production of the phenazine metabolite pyocyanin [90, 91] and
Streptococcus pyogenes induces apoptosis by altering the intrinsic programme of
apoptosis at a transcriptional level [92]. Other organisms, notably Staphylococcus
aureus, induce neutrophil necrosis [28], although the S. aureus Panton-Valentin leu-
kocidin induces neutrophil apoptosis via activation of the mitochondrial pathway
[25]. Other inducers of neutrophil necrosis include Burkholderia cenocepacia and
the E. coli hemolysin [9395].
27
Conclusions
Until very recently neutrophils and eosinophils have been stereotyped as largely
short-lived and transcriptionally inert cells capable only of releasing set amounts
of pre-formed mediators. In fact, these cells are remarkably versatile (and certainly
synthetically active) and are involved in a number of biological processes aside from
the orchestration and resolution of inflammation, including the facilitation of the
specific immune response (e.g. through antigen processing), the regulation of angio-
genesis, and modulation of tumour cell fate among others. While critical for host
defence, the capacity for granulocytes to induce significant bystander organ damage
dictates the need for powerful and safe clearance mechanisms for these cells, and
constitutive and pathogen-induced apoptosis, coupled with efficient phagocytic
recognition and disposal appears to afford one such mechanism. While many of the
events that regulate inflammatory cell apoptosis are now understood, part of the
future challenge is to establish how, where and when we can intervene to facilitate
these processes in vivo and to determine the interplay with other non-apoptotic
clearance mechanisms, for example those involved in the removal of circulating
granulocytes by the spleen and bone marrow.
Acknowledgements
The work in the authors laboratories is funded by the MRC, The Wellcome Trust,
Asthma UK and the British Lung Foundation.
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37
Innate immune mechanisms in the resolution of inflammation
Andrew Devitt1 and Christopher D. Gregory2
1
School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK;
2
MRC Centre for Inflammation Research, The Queens Medical Research Institute,
47 Little France Crescent, Edinburgh, EH16 4TJ, UK
Introduction
The inflammatory response is a highly orchestrated and tightly controlled mecha-

nism of defence characterised by infiltration of granulocytes and mononuclear cells.
For this response to be beneficial it must deal with its initial inflammatory stimulus
and then subside to allow the tissue to return to its pre-inflamed state. Such resolu-
tion requires cessation of inflammatory cell recruitment and, importantly, deletion
of recruited cells in a safe and controlled fashion. Failure to delete cells appropri-
ately may permit effete cells to undergo necrosis (lysis due to loss of plasma mem-
brane integrity) with the generation of inflammatory and autoimmune consequences
associated with leakage of intracellular contents [1].
Apoptosis constitutes a vital mechanism by which damaged, infected or unwant-
ed cells (e.g. effete inflammatory cells) are removed from the body via a process that
culminates in the efficient phagocytic removal of dying cells under carefully con-
trolled conditions. This ultimate clearance process is used for both the removal of
physiological cell death associated with normal tissue homeostasis and pathological
cell death associated with inflammation and infection. Thus, deletion of cells in a
resolving inflammatory situation is mediated through apoptosis and rapid phago-
cytosis of cell corpses by recruited and resident phagocytes. Importantly, especially
in the context of innate immune system involvement in resolution of inflammation,
this deletion and clearance must occur in a manner that itself does not generate or
promote inflammatory responses.
The mechanisms mediating non-phlogistic clearance of apoptotic cells are still
poorly understood though the identities of many putative molecular players are
known and include, perhaps counter-intuitively, components of the innate immune
system. In this chapter we focus upon these molecules in the phagocytic clearance
of apoptotic cells that is central to a resolving inflammatory response.

The innate immune system
The innate immune system is a powerful (if often under-rated) system that consti-
tutes an important early defence against pathogens. It comprises a series of factors
that fall into groups including physical barriers to infection (e.g. skin), cellular
factors (e.g. neutrophils, macrophages and their receptors) and humoral factors
(e.g. complement) with the latter group containing induced innate factors (e.g.
chemokines). In this chapter we seek to review the published role of innate immune
components in the context of apoptotic cell clearance with a focus upon the resolu-
tion of inflammation.
Pattern recognition
The concept of self/non-self recognition is central to immunology how and why do

we respond to some antigens and not others? Janeway [2] proposed that the innate
immune system discriminated non-self (infectious non-self) from self (non-
infectious self) at the point of recognition: the former being foreign and exposed
patterns that are evolutionarily conserved (so-called pathogen-associated molecular
patterns or PAMPs) and such PAMPs were proposed to be recognised by specialised
receptors known as pattern recognition receptors (PRRs). The discovery that com-
ponents of the innate immune system are involved in the recognition and removal
of apoptotic cells (unwanted self) clearly raises questions of this view of immune
recognition. The striking similarities in the recognition of bacteria and apoptotic
cells led to the proposal that apoptotic cells themselves expose similar patterns for
recognition termed apoptotic cell-associated molecular patterns (ACAMPs) [3, 4].
These patterns which, by definition, closely resemble the three-dimensional structure
of PAMPs may then also be recognised by PRRs. While the infectious non-self
model proposed by Janeway does not allow for such similarities in self and non-
self, one alternative theory that may does not rely upon non-self being foreign but
on being injurious or dangerous [5]. This danger hypothesis is supported by the
involvement of innate immune system components in the recognition of apoptotic
cells (non-infectious/non-dangerous self) and underlines a central point in immunol-
ogy: it is not what the immune system recognises but how it responds. Whatever the
underlying mechanisms that permit differential responses to self and non-self, it is
clear that the innate immune system is firmly involved in both.
Clearance of apoptotic cells
Over the past decade, the number of putative molecular players involved in the
clearance of apoptotic cells has expanded significantly and these have been reviewed
40
Figure 1.
A cartoon to illustrate innate immune system components implicated in the clearance of
apoptotic cells by phagocytes.
extensively elsewhere [6, 7]. Those molecules that play an important role in both
innate immunity to pathogens and in the clearance of apoptotic cells are depicted
schematically in Figure 1. These factors comprise cellular receptors and, increas-
ingly, factors that act as opsonins for apoptotic cells, thereby bridging apoptotic
cells to phagocytes [8]. While all receptors on macrophages and neutrophils might
reasonably be considered innate immune molecules, a few candidates from Figure
1 stand-out as classical innate immune molecules being known primarily as anti-
microbial effector molecules and fitting the description of PRRs. It was the dis-
covery of CD14, the prototypic PRR, as an important apoptotic cell receptor that
raised the suggestion that innate immune responses of phagocytes to microbes and
apoptotic cells may share common components.
CD14 The prototype PRR
CD14 is the prototypical innate immune receptor. It was the first cloned in the
late 1980s [9, 10] and identified as a glycosylphosphatidylinositol (GPI-)-anchored
membrane glycoprotein [10, 11]. Its widely acknowledged role in the recognition of
a wide range of molecular species from microbes (most notably LPS) and its media-
41
tion of an inflammatory response identify it clearly as a PRR. However, a large array

of ligands from non-microbial origins have also been identified [12].
CD14 and recognition of apoptotic cells
The identification of a role for CD14 in the recognition and clearance of apoptotic
cells stems from the activity of a murine mAb (61D3) against human monocytes
[13] was first established more than two decades ago. This mAb specifically and
strongly stained human monocytes and, during screening for its ability to modulate
the clearance of apoptotic cells by human macrophages, 61D3 was noted to block
apoptotic cell clearance in vitro [14]. Formal identification of the antigen specified
by 61D3 proved more difficult but finally an expression cloning approach using
Cos cells transiently expressing a cDNA library generated from HL60 cells cloned
the 61D3 cDNA [15]. Sequencing led to a surprising result, the antigen defined
by 61D3 was the LPS receptor, CD14, a receptor renowned for its ability to elicit
strong pro-inflammatory responses [16] and mediate endotoxic shock [17, 18]. At
that time, relatively little was understood about the mechanisms and consequences
of apoptotic cell clearance by macrophages, although one thing was certain it was
a non-phlogistic process. More striking still was the suggestion that mAb 61D3
defined both the apoptotic cell- and LPS-binding site on CD14 [15].
Following early antibody studies implicating CD14 in apoptotic cell clearance,
it has taken a range of in vitro and in vivo studies to elucidate the role of CD14 in
apoptotic cell clearance. Initially, over-expression of CD14 in Cos cells was shown
to promote the ability of those cells to recognise and clear apoptotic cells in vitro
in a manner inhibitable by 61D3 [15]. This role for CD14 was further dissected
through the use of interaction assays between macrophages and apoptotic cells
undertaken at temperatures non-permissive for phagocytosis revealing that, at least
minimally, CD14 is a tethering receptor for apoptotic cells. This is further supported
by the observation that soluble CD14 can bind apoptotic cells, suggesting CD14 is
capable of interacting directly with apoptotic cells [19].
While in vitro studies provide an important and useful step in identifying candidate
molecules implicated in a process, full biological significance is gained through assess-
ment of the consequence of loss of their function in vivo. It is of note that a number
of receptors and molecules implicated in the clearance of apoptotic cells through in
vitro studies have proved rather disappointing when attentions were turned to in vivo
studies in knockout animals [6]. For example the inability of SR-A-deficient mice to
present a phenotype in line with the proposed role for SR-A in apoptotic cell clear-
ance [20] has led to widespread acknowledgement of the possibility of redundancy
in clearance mechanisms. In marked contrast, in vivo studies of the role of CD14
demonstrated a widespread phenotype with raised numbers of persistent apoptotic
cell corpses being detectable in a range of tissues (including thymus, spleen, lung and
42
liver) when CD14 was absent [19]. Further detailed studies indicated that this was
due to defective clearance of apoptotic cells rather than increased cell death, with
CD14/ peritoneal macrophages being less competent to remove administered apop-
totic cells and CD14/ thymic phagocytes unable to cope with high loads of thymic
cell death following dexamethasone administration. These observations indicate that
CD14 plays an important role in apoptotic cell clearance during both physiological
and pathological cell death and argue strongly that its role is non-redundant, at least
in the context of the mouse strain and tissues investigated.
A significant role for CD14 in the resolution of inflammation seems highly
likely in light of its ability to remove apoptotic granulocytes [21] and the results
described above. Studies are currently underway to address the consequence of
CD14 deficiency in a range of inflammatory situations. The precise role for CD14
in the resolution of an inflammatory response is not clear but a key attribute for any
molecule involved in resolution must be to function in such a way as not to induce
or exacerbate inflammation. CD14 is thus suited to a resolving role, through the
tethering and removal of apoptotic cells, as ligation of CD14 by apoptotic cells is
not inflammatory [15, 19].
Phagocyte receptors: Scavenger receptors
CD36, a class B scavenger receptor, was one of the first macrophage receptors to be
implicated in the clearance of apoptotic cells. First identified through mAb inhibi-
tion studies [22] and further dissected through over-expression studies [23], CD36
appears to function in concert with the integrin _v`3 (CD51/CD61: the vitronectin
receptor, implicated earlier in apoptotic cell clearance [24]) with thrombospondin
acting as a molecular bridge between the apoptotic neutrophil and the phagocyte
[22]. The conservation of scavenger receptors (both presence and function) through-
out evolution is highlighted by the discovery of Croquemort in Drosophila. Cro-
quemort, a CD36 superfamily member, mediates clearance of apoptotic cells while
playing little role in the clearance of bacteria perhaps suggesting that evolution
of receptors for the clearance of apoptotic cells, at least during development, was
followed by a role in host defence [3]. Of particular relevance to this chapter, stud-
ies highlighting a role for CD36 in clearance of apoptotic cells were focussed upon
the clearance of apoptotic neutrophilic granulocytes, strongly implicating a role for
these molecules in resolution of inflammation.
Additional scavenger receptors have also been identified as apoptotic cell recep-
tors following in vitro studies, including Lox-1 [25, 26], SR-AI and II (CD204) [27,
28] and SRBI [2932]. However, these studies focussed upon clearance of apoptotic
cells other than inflammatory cells (e.g. thymocytes) and, while surface changes
associated with apoptosis are suggested to be conserved [33], the role of these
molecules in resolution of inflammation requires direct study. Furthermore, the role
43
of scavenger receptors for clearing apoptotic cells in vivo is unclear with knockout
studies failing to report any cell clearance defect [20]. This is most likely due to sig-
nificant redundancy in the function of scavenger receptors, a view that is supported
by the observations that macrophages deficient in CD204 show an up-regulation of
CD36 [34]. Studies to address the effect of multiple receptor knockouts on apop-
totic cell clearance and resolution of inflammation are required to identify the in
vivo roles of potentially redundant receptors.
Humoral factors: Complement
The discovery of a role for complement components, collectins and pentraxins in

the clearance of apoptotic cells emphasised the notion that pattern recognition
is clearly more than recognition of PAMPs. It is now clear that molecules of the
complement cascade that were previously thought of as defence molecules are also
involved in removal of dead and dying cells. Deficiency in C1q is the most powerful
genetic risk factor in the aetiology of the autoimmune disease systemic lupus erythe-
matosus (SLE, a condition linked to the failure of apoptotic cell clearance) and in
mice deficient in C1q a dramatic kidney defect is evident with persistent apoptotic
cells and glomerulonephritis [35]. The role of C1q in clearance of apoptotic cells
with particular reference to inflammation was highlighted through the work of
Taylor et al. [36], which demonstrated that both resident and inflammatory perito-
neal macrophages showed reduced competence for the removal of apoptotic cells
in C1q/ animals a deficit that could be complemented through the addition of
C1q-containing serum.
Conflicting reports (from a range of experimental systems) exist as to the extent
of activation of the complement cascade required for complement-dependent clear-
ance of apoptotic cells with C1q possibly functioning as a simple opsonin without
C3 involvement [36, 37]. Other reports support complement fixation [38] and C3
deposition as the major molecular mechanism underlying complement-dependent
clearance of apoptotic cells [3941]. Deposition of C3b on cell surfaces is rapidly
followed by proteolytic cleavage to form iC3b and apoptotic cell-associated iC3b
has been shown to promote apoptotic cell removal [42]. Despite any perceived
differences in these reports, it is clear that complement components contribute to
apoptotic cell removal and any conflicts between reports relating to complement
activation may well be due to timing variation in the experimental systems. It is rea-
sonable that following C1q opsonisation of apoptotic cells macrophages may bind
and remove apoptotic cells but delay at this point may permit activation of comple-
ment to allow the involvement of other, downstream, complement components in
the process. This progressive involvement of different factors is an attractive and
economical model of apoptotic cell removal where additional factors only become
involved in clearance once others have failed.
44
If complement activation provides enhanced apoptotic cell clearance, the stimu-

lus for activation must be addressed. A possible role for phosphatidylserine, exposed
at the apoptotic cell surface, in the activation of complement (via the classical and
alternative pathways) has been suggested as a mechanism leading to the deposition
of iC3b on the apoptotic cell surface [39]. A more detailed mechanism for activation
of complement by the classical pathway at the apoptotic cell surface has also been
put forward where natural IgM antibodies opsonise the apoptotic cell [41, 40, 43]
and initiate complement activation. Such involvement of IgM (while not essential
for complement-mediated apoptotic cell removal) is thought to be required for the
efficient removal of apoptotic cells in vivo [41] and natural IgM has been shown to
be the major pathway leading to complement activation via the classical pathway. It
is suggested that these natural antibodies opsonise apoptotic cells via the recognition
of oxidised lipids at the apoptotic cell surface [44] or through recognition of altered
phospholipids that arise during apoptosis as a result of the activation of phospho-
lipase A2 during apoptosis [45]. Interestingly, this latter report further proposes
that apoptotic cell opsonisation with IgM also activates non-classical pathways of
activation, albeit to a lesser extent.
It is of note that the serum acute-phase protein CRP (a member of the pentraxin
family also implicated in apoptotic cell clearance, see below) is capable of binding
to C1q and amplifying complement activation. It has been shown that CRP binds
apoptotic cells, promoting activation of the classical pathway of complement activa-
tion and iC3b deposition, while preventing assembly of downstream complement
factors (e.g. membrane attack complex) through the recruitment of factor H, a
complement regulatory protein [46]. All of the effects of CRP are dependent upon
C1q and, in inflammatory situations where CRP levels may increase many fold, this
may provide an important mechanisms for promoting complement activation and
apoptotic cell clearance.
It is clear that there is significant research into the molecular mechanisms under-
lying complement-dependent opsonisation and clearance of apoptotic cells. So what
of the receptors? CR3 (CD11a/CD18; _m`2) and CR4 (_d`2) have been implicated
in the removal of iC3b-opsonised apoptotic cells through the use of antibody inhi-
bition studies [42]. While CR3 and CR4 are attractive candidate iC3b receptors,
a new candidate receptor, CRIg (complement receptor of the immunoglobulin
superfamily), has been identified [47]. While CRIg is highly expressed on Kupffer
cells and functions for removal of complement-opsonised circulating pathogens,
it is notably expressed by a number of tissue resident macrophages (e.g. alveolar,
synovial), although a formal role in the clearance of iC3b-opsonised apoptotic cells
is yet to be addressed.
Phagocyte receptors for C1q have been implicated in apoptotic cell clearance
with calreticulin and CD91 binding the collagenous tails of C1q [37, 48]. Other
receptors have been implicated but to date the involvement in apoptotic cell clear-
ance remains unclear. For example, while CD93 (C1qRp) is involved in modulat-
45
ing phagocytic capacity [49] and contributes to apoptotic cell clearance in vivo, it
appears, at least in mice, not to be required for C1q-dependent clearance [50].
Humoral factors: Collectins
Additional humoral factors of the innate immune system, the collectins, have been
implicated in the clearance of apoptotic cells. Collectins comprise a family of C-type
lectins with complex structures based upon a monomer containing a globular head
region with carbohydrate-recognition domain and a collagenous tail arranged into
trimers and associated multimers [51, 52]. The lung collectin proteins surfactant
protein A and D (SP-A and SP-D) and the circulating serum collectin mannose-bind-
ing lectin (MBL) [37, 48, 53, 54] have been shown to bind apoptotic cells and pro-
mote their clearance. This work has been supported by in vivo analyses to indicate
that a deficiency in SP-D [48] can lead to an incompetence to clear apoptotic cells
instilled into the lungs (while SP-A and C1q deficiency showed little effect) [48].
This work is in line with previous ex vivo studies indicating that SP-D and, to a less-
er extent, SP-A promote apoptotic neutrophil clearance by alveolar macrophages,
whereas MBL and C1q showed no effect [53]. Recent work in vivo, however, has
shown that MBL-defective mice [55] fail to efficiently clear apoptotic cells injected
into the peritoneal cavity and raises the possibility that variations in reports are the
result of different experimental model systems or tissue-specific effects of individual
molecules. This latter report supports the results of earlier work using CD14-defi-
cient mice, which demonstrated for the first time that it is possible to have defective
apoptotic cell clearance (with persistence of corpses) that does not lead inextricably
to inflammation or autoimmunity. This suggests a non-redundant role for MBL in
apoptotic cell clearance where large numbers of apoptotic cells may be required to
be removed rapidly, as may occur at the site of inflammation, and has led to the
suggestion that the mechanisms for tethering apoptotic cells and for induction of the
anti-inflammatory effects of apoptotic cells can be uncoupled [19].
Collectin (and C1q) receptors have been implicated in apoptotic cell clearance.
Calreticulin and CD91 are involved in C1q-, SP-A- and SP-D-dependent clearance
of apoptotic cells in the lung through binding of the collagenous tails of the collectin
family [37, 48]. While calreticulin was proposed to act with CD91 on the surface of
the phagocyte as a common collectin receptor complex [48], it has recently been
suggested that, through apoptosis-dependent redistribution of CD47, calreticulin on
the apoptotic cell surface becomes reorganised such that it can interact with CD91
on the phagocyte (in trans) to promote clearance [56]. Interestingly, SIRP-_ on
phagocytes has been shown to bind SP-A and SP-D via their globular head region
[57], suggesting a possible mechanism for modulating the inflammatory environ-
ment. This binding is the reverse of the orientation previously suggested where the
globular heads bind the apoptotic cell surface and collagenous tails mediate receptor
46
binding. How this might, if at all, play a role in apoptotic cell clearance as a direct
collectin receptor has not been addressed. CD14 has also been suggested to function
in the binding of SP-A, SP-D [58, 59] and mannose-binding protein [60]. The role of
collectins in modulating the inflammatory environment may be the major function
of these molecules in the clearance of apoptotic cells [37, 57, 61].
Humoral factors: Pentraxins
The pentraxins are another family of soluble innate immune factors that may be up-
regulated during the acute-phase response to infection and tissue damage. As their
name suggests, they have a pentameric structure and are represented in short forms
(e.g. CRP, SAP) and long forms (e.g. PTX3). These pentraxin members have been
shown to bind apoptotic cells and, in the case of CRP, promote clearance through
involvement of complement (see above [46]). SAP also binds to apoptotic cells,
albeit late apoptotic cells, via phosphatidylethanolamine in blebs [62] and mediates
their uptake by macrophages [63]. The long pentraxin PTX3, however, is somewhat
different; it appears to bind apoptotic cells (lymphocytes and granulocytes) and, to a
lesser extent, necrotic cells and prevents uptake by dendritic cells [64, 65]. This was
suggested as a mechanism by which autoantigens are sequestered from antigen-pre-
senting cells for the control of potential autoimmune reactions. This work has been
followed up with similar results addressing uptake by macrophages and has been
suggested as a causative factor in the appearance of persistent apoptotic neutrophils
at the site of vasculitis [66]. Interestingly, the pentraxins, at least in part, appear to
bind to similar sites on apoptotic cells by virtue of the ability of CRP and SAP to
block binding of labelled PTX3.
CRP, SAP and PTX3 are all capable of binding C1q to activate the classical
complement cascade and, as such, the role for CRP in activating complement at the
apoptotic cell surface (above) may be a family-wide characteristic [6769]. How
then does PTX3 differ in its effects upon apoptotic cell clearance? One possibility is
that PTX3 binds and sequesters soluble C1q to limit its action in binding apoptotic
cells and activating C3 [65]. The significant increase in circulating CRP levels during
inflammation [70] makes this an attractive candidate that is pro-resolution from the
onset of inflammation as has been suggested for other factors [71].
Multiple receptors for CRP were suggested from analyses that indicated the pres-
ence of a low-affinity receptor for CRP (identified as FcaRI [72]) and a high-affinity
receptor for CRP (identified as FcaRII [73]). Further analyses using mice deficient
in individual or combinations of FcaR were used to identify the receptors used for
binding of pentraxin-opsonised apoptotic cells [74] and highlighted a requirement
for FcR a chain. Interestingly, this report showed that only SAP (not CRP, the
major induced acute-phase protein in humans) opsonised and promoted clearance
of apoptotic neutrophils by human macrophages despite both SAP and CRP medi-
47
ating clearance of apoptotic Jurkat cells by the murine macrophage-like cell line
J774. However, in this study, heat-inactivated serum was used and likely therefore
removed the ability of complement to play a part in mediating CRP function. The
pentraxins do appear, however, to use FcaRI and/or FcaRIII for the clearance of
apoptotic cells.
Induced innate immune responses
Although the innate immune response lacks the education and memory of the
acquired immune system, the ability to induce certain components ad hoc pro-
vides an important level of control and refinement to the innate immune system.
Chemokines are an important example of such induced innate immune responses
and underlie the recruitment of inflammatory cells to sites of infection or of tissue
damage from circulation. Such responses have been implicated in the recruitment of
phagocytes to the site of cell death [6, 75] via release of apoptotic blebs [76], S19
ribosomal protein homodimers or lysophosphatidylcholine (LPC) from apoptotic
cells [77]. The generation of LPC, like the generation of epitopes for the binding of
natural IgM is dependent upon the cleavage of surface phospholipids during apop-
tosis [45, 77]. The large number of cell deaths by apoptosis at an acutely inflamed
site makes it highly unlikely that resident phagocytes (professional or amateur)
would be able to cope without recruited help. The extent to which apoptotic cell-
derived chemoattractants play a role in recruitment of phagocytes to sites of inflam-
mation is still unknown and, as yet, no chemokines have been implicated in the
attraction of phagocytes to apoptotic cells.
Pyre prevention
As pattern recognition molecules of the innate immune system (e.g. CD14 and
MBL) are implicated strongly in pathogen recognition and defence via inflamma-
tion, it is intriguing that they also mediate removal of apoptotic cells (a non-phlo-
gistic process). The basis of such differing responses is a current challenge in this
area of research and studies are underway to identify molecular mechanisms that
underlie differing responses to PRR ligation by PAMPs and ACAMPs.
A simple explanation of such a dichotomy may be that ACAMPs (as three-
dimensional structural analogues of PAMPs) do not actually exist and PRRs are
ligated in a different and non-inflammatory manner by apoptotic cell-associated
molecules (e.g. via ligation of different portions of the receptor). Indeed, formal
identification of an ACAMP is yet to be reported; however, epitope mapping using
anti-CD14 mAbs shows close similarity in the LPS and apoptotic cell binding sites
on CD14 [15].
48
The infectious non-self model of recognition proposed by Janeway (suggest-

ing that the innate immune system discriminates self from infectious non-self via
recognition of PAMPs by PRRs) would be difficult to reconcile with any formal
identification of an ACAMP and evidence of strong structural identity between
PAMPs and ACAMPs. Such results would, however, be consistent with an alterna-
tive hypothesis of immune recognition, the Danger Model where the nature of the
receptor is not the critical factor in deciding any resultant response but the presence
or absence of danger signals [5]. In the case of apoptotic cell clearance, there would
be a predicted lack of such danger permitting inflammatory receptors, such as
CD14, to mediate quiet removal of unwanted cell corpses. However, in the event of
failed clearance of apoptotic cells at the site of inflammation, resultant necrotic cells
could provide danger signals (e.g. HMGB-1) and subsequent immune stimulation
[78, 79]. An addition to this is the idea (irrespective of whether ligation of PRRs
is inherently inflammatory) that the reason for the quiet nature of apoptotic cell
removal is in fact the presence of calming influences. Such immunomodulatory
influences (e.g. the release of TGF-`1 and IL-10) have been noted from phagocytes
clearing apoptotic cells and such immunosuppression appears powerful, even in
the face of potent pro-inflammatory stimuli such as LPS [80, 81]. Furthermore,
while such cytokine production promotes an anti-inflammatory environment, it
also tailors phagocytes. IL-10 is known to direct macrophages towards an alter-
natively activated phenotype, so-called M2 [82], which are notable for their
preferential clearance of early apoptotic cells in a manner that is dependent upon
CD14-mediated binding [83]. Taken together, these strands promote the idea that
the appearance of apoptotic cells at a site (e.g. inflamed tissue) would promote an
immunosuppressive milieu that is highly supportive of phagocytes for the clearance
of apoptotic cells in a manner dependent upon component molecules of the innate
immune system.
The basis of any response to phagocytosis using PRRs is most likely defined in
the constitution of the proposed phagocytic synapse [7, 84] that is assembled in
response to ligation of pathogens or apoptotic cells. The net balance of responses to
ligation of a range of receptors and signalling pathways will likely dictate the out-
come. CD14, a GPI-anchored glycoprotein that lacks signalling capacity through a
transmembrane domain, is an interesting case model. For responses to LPS, CD14
functions in association with TLR4 (to elicit inflammatory signals), whereas it seems
likely that responses to apoptotic cells utilise other co-receptors (or alternatively
CD14 acts as a simple tethering molecule). The basis of differing responses to CD14
ligation by LPS or apoptotic cells is yet to be fully characterised, but the discovery
of TLR4 as an important molecule in responses to LPS may have partly answered
the question. TLRs appear not to be involved in recognition of apoptotic cells and
indeed may negatively regulate the degradation of engulfed apoptotic cells [85].
Furthermore, phagocytosis of bacteria but not apoptotic cells was inhibited by the
absence of TLR signalling, supporting a lack of involvement of TLRs in apoptotic
49
cell clearance [86]. An alternative signalling partner for CD14 for AC clearance may
yet be identified and candidate molecules include the `2 integrins [87].
Summary
Removal of effete inflammatory cells in an immunologically quiet, controlled fash-

ion is vital to efficient resolution of inflammation. The ultimate step in resolution is
the recognition and removal of apoptotic cells by phagocytes via a process involving
a range of components of the innate immune system functioning in non-inflam-
matory/anti-inflammatory mode. The involvement of PRR in both inflammatory
defence and anti-inflammatory removal of apoptotic cells is a key issue for study
in the field of apoptotic cell research and has led to suggestions that PRRs evolved
initially for the removal of cell deaths during development [3, 5] and later evolved
a role in defence following PRR subversion by pathogens. As such, it may now be
surprising to identify innate immune components that are not involved in clearance
of apoptotic cells.
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Immunol Today 17: 209212
56
Cell surface molecular changes associated with apoptosis
Ian Dransfield1, Sandra Franz2, Kim Wilkinson1, Aisleen McColl1, Martin Herrmann2
and Simon P. Hart1
1
MRC Centre for Inflammation Research, University of Edinburgh Medical School, Teviot
Place, Edinburgh EH8 9AG, UK; 2Institute for Immunology, FAU Erlangen-Nuremberg, Glck-
strasse 4a, 91054 Erlangen, Germany
Cell surface receptors
Regulation of cellular responses to micro-environmental stimuli is achieved through

cell surface receptors, which may assemble into complexes that permit the bi-direc-
tional transmission of information across the plasma membrane. It has been esti-
mated that circulating lymphocytes may express several hundred different surface
receptors [1], which may confer specificity for an equivalent number of extracellular
cues. The expression and function of these critical portals of communication are
thus extremely tightly regulated to ensure appropriate cellular responses, including
altered adhesion and migration, activation and cellular proliferation. In this chapter
we consider the changes in cell surface receptor profiles that are associated with pro-
grammed cell death or apoptosis, discussing the implications in terms of their influ-
ence upon cellular responses and upon processes that impact upon disease patho-
genesis. Many studies of the process of apoptosis have been performed in vitro,
using model systems in which cell death is initiated following chemical or radiation
(e.g. ultraviolet) insult. Alternatively, apoptosis may be induced by ligation of death
receptors leading to activation of the caspase cascade within the cell. Whether the
changes observed in these in vitro systems mimic physiological death within tissues
is uncertain, but for the purposes of this review, we assume that many of the changes
that are initiated also occur on cells as they undergo apoptosis in situ.
Apoptosis
As discussed elsewhere in this volume, apoptosis is a physiological form of cell

death that initiates a defined series of biochemical changes within the cell leading
to the destruction of proteins, DNA and carbohydrates through the activation of
a variety of proteases, nucleases and glycosidases [2, 3]. These changes result in
the disruption of the cytoskeleton [4], altering intracellular transport [5, 6] and,

Ian Dransfield et al.
crucially for the molecular changes that occur on the plasma membrane, movement
of vesicles within the cell [6]. It is now clear that, while these characteristics of
apoptosis are broadly true, there may be some variation when apoptosis is specifi-
cally accelerated following interactions of cells with pathogens including viruses,
bacteria and mycobacteria. Indeed, there are pathogen-specific alterations in gene
expression profiles associated with apoptosis [7], which may alter surface molecu-
lar profiles.
Importantly, apoptosis is usually associated with the rapid removal of cells from
tissues either by neighbouring cells or by professional phagocytic cells [8]. In this
way the potentially harmful intracellular contents of apoptotic cells are prevented
from being released and are disposed of within the cell responsible for clearing
it. This clearance process represents an important mechanism for the removal of
large numbers of cells that undergo programmed cell death each day. Furthermore,
it is now widely accepted that removal of dying cells is actually more than just a
silent clearance pathway, leading to alteration of cellular behaviour in the phago-
cytic cell that are often considered anti-inflammatory through the suppression of
pro-inflammatory cytokine production and induction of IL-10 and TGF-` release
[911]. A large array of phagocyte receptors have been reported to contribute to
the recognition process [12]. In addition, it has been reported that disablement of
cell detachment may contribute to apoptotic cell uptake by phagocytes [13]. It is
likely that the responses of phagocytes to apoptotic cell uptake will be determined
by the repertoire of phagocyte receptors that are engaged during the binding and
subsequent internalisation of apoptotic cells. Crucially, in the absence of phagocytic
removal, cells that have undergone apoptosis are likely to progress to late apoptosis
and ultimately to secondary necrosis. The balance between apoptotic, late apoptotic
and necrotic cell death within tissues is therefore likely to impact on responses of
cells within tissues and development of immune responses [14].
Membrane alterations associated with apoptosis
One of the best-characterized surface molecular alterations associated with apop-

tosis in many different cell types is the exposure of the anionic phospholipid, phos-
phatidylserine (PS), on the outer leaflet of the plasma membrane [15].
On viable cells, anionic phospholipids are maintained on the inner leaflet of
the plasma membrane through the action of specific phospholipid translocases.
Exposure of PS upon the apoptotic cell membrane appears to represent a critical
determinant of their immunosuppressive and anti-inflammatory effects of apoptotic
cells upon neighbouring cell types. A number of specific adaptor proteins, includ-
ing Gas6, `2-GPI, and MFG-E8 have been shown to bind to PS [16, 17] and,
despite the problems with the definitive identification of the PS receptor [18, 19],
it remains possible that phagocytes are capable of directly recognising PS. Interest-
58
ingly, reported inhibition of phagocytosis of apoptotic cells by liposomes contain-

ing PS is seldom complete, implying involvement of other receptor pathways in the
recognition process.
The neutrophil Surface alterations associated with apoptosis
In considering changes in the plasma membrane molecules that accompany apop-

tosis, the neutrophil is discussed here as a model cell type. The neutrophil granu-
locyte is the archaetypal inflammatory leukocyte that is the major subset (> 70%
of circulating leukocytes) present in the peripheral blood and is the first cell type
recruited in innate defences against invading pathogens [20]. In response to infec-
tion or tissue injury, neutrophils are rapidly (within minutes) recruited into tissues,
binding and transmigrating across activated endothelial cells [21]. Specific recruit-
ment of neutrophils is directed by the adhesion and chemokine receptors that neu-
trophil express. Once present within inflammatory sites, neutrophils are efficient
phagocytes, capable of recognising a wide variety of pathogens via different opso-
nins. Neutrophils express a number of receptors for complement components and
immunoglobulins that are able to bind to pathogens, targeting them for phagocytic
destruction. Phagocytosed pathogens are subjected to attack from an extensive bat-
tery of destructive enzymes capable of breaking down protein, carbohydrate and
lipids together with reactive oxygen species, which further compromises pathogen
survival [22]. Following clearance of pathogens, neutrophils are thought to be
removed from inflammatory sites through the induction of programmed cell death
or apoptosis, which marks them for phagocytic removal [23]. The numbers of neu-
trophils present at inflammatory sites is thus determined by the balance between
pro- and anti-apoptotic stimuli present in the local microenvironment [24].
The profile of membrane receptors that neutrophils express can be rapidly
altered in response to external stimuli, either by mobilisation of intracellular azu-
rophilic and specific granule compartments or through the rapid and specific
proteolytic shedding of the ecto-domain of specific receptors [2527]. Regulation
of expression of receptors allows the generation of distinct receptor repertoires
that directly influence neutrophil functional status. Neutrophils constitutively
undergo apoptosis when cultured in vitro, a process that can be regulated by dif-
ferent exogenous factors, including growth factors (e.g. GM-CSF), cytokines (e.g.
TNF-_) and inflammatory mediators (e.g. prostaglandin E2). Neutrophil apoptosis
may also be regulated by ligation of death receptors (e.g. Fas, TNFR and TRAILR)
[28] and other interactions with other cell types [29]. Interestingly, interactions of
neutrophils with different pathogens may also influence engagement of apoptotic
programmes [30, 31], providing a mechanism for ensuring that highly phagocytic
neutrophils containing pathogenic organisms are functionally silenced. Membrane
receptor alterations observed when neutrophils undergo apoptosis may therefore
59
Figure 1.
Schematic representation of cell surface molecular changes associated with neutrophil apop-
tosis.
differ depending on the initiating apoptotic stimulus (see Fig. 1 for a schematic
representation of changes). As discussed previously, one important consequence of
the surface molecular alterations associated with the apoptotic programme is that
phagocytes are able to specifically recognise and phagocytose apoptotic neutrophils
[32], targeting them for clearance from resolving inflammatory lesions.
Apoptosis-induced surface receptor alterations
In a series of studies relating to the effects of long-term treatment of neutrophils with

cytokines, it was noticed that there were marked alterations to expression of the gly-
cophosphatidylinositol-anchored IgG receptor, FcaRIII (CD16) [33, 34]. Two distinct
subpopulations of in vitro cultured neutrophils could be defined in terms of levels of
60
CD16 expression, correlating with the proportion of apoptotic neutrophils present.

This observation implied that there may be specific alterations in membrane recep-
tor expression that accompany the programme of morphological changes associated
with apoptosis. Levels of receptor expression at the plasma membrane reflect the
balance between production of new proteins from the endoplasmic reticulum, recep-
tor shedding, together with receptor internalisation and recycling. Disruption of the
endoplasmic reticulim (ER)-Golgi-plasma membrane transport represents an early
event in the apoptotic process [5, 6], with the potential to profoundly influence the
levels of expression of plasma membrane receptors. One possibility was that shut-
down of the secretory pathway in apoptotic neutrophils led to the reduced expres-
sion of many surface receptors. However, this was found not to be the case [35].
Importantly, examination of the other GPI-linked molecules indicated that the nature
of the membrane anchor was not responsible for the profound loss (>90% of surface
receptors) of FcaRIII, as similar changes were not seen for CD58 for example.
Further analysis of other receptors expressed on neutrophils cultured in vitro
revealed that there was also a marked down-regulation of a restricted profile of
receptors associated with neutrophil apoptosis [36]. Interestingly, these molecules
[CD62L, CD44, CD43 and TNFRI (CD120a)] have the potential to be shed via the
action of metalloproteases during neutrophil activation. Dual fluorescence labelling
with annexin V to mark PS exposure indicated that there was a catastrophic loss
of these receptors that correlated with PS exposure, as opposed to a gradual loss of
receptors over time. One possibility was that the intense membrane re-organisation
(blebbing) that correlates with exposure of PS during early apoptosis, is accompa-
nied by the activation of surface-associated metalloproteases, resulting in specific
receptor cleavage. This change, together with a shutdown in transport of newly syn-
thesised receptors to the cell surface may account for the marked down-regulation
of these target molecules observed. Our analysis also revealed that many different
surface receptors exhibit a smaller reduction in surface expression (approximately
30% of the levels found on non-apoptotic neutrophils) [35]. Given that this down-
regulation occurs for apparently unrelated molecules, this observation may simply
reflect the loss of plasma membrane and reduction in cell volume that occurs as a
result of membrane blebbing events accompanying apoptosis.
Early studies of the membrane alterations associated with apoptosis suggested
that there may be net changes in the cell surface charge, possibly due to altered
glycosylation patterns [37]. One possibility is that there may be altered activity of
carbohydrate processing enzymes, e.g. sialidases [38], that accompany apoptosis. In
addition, it is also possible that post-translational processing of proteins in the Golgi
is inhibited prior to shutdown of Golgi-plasma membrane transport, allowing incom-
pletely processed carbohydrate moieties to be present on proteins that are expressed
on the plasma membrane. A panel of labelled lectins, each with a relative specificity
for particular carbohydrate structures, were used to analyse the glycosylation status
of the plasma membrane of neutrophils during apoptosis. The lectins derived from
61
Griffonia simplificolia II (GSL II), Narcissus pseudonarcissus (NPn), and Ulex euro-
paeus I (UEA I) increased their binding to surfaces of apoptotic neutrophils [39]. The
exposure of PS and the loss of cellular volume both preceded the increased lectin
binding. Interestingly, these lectins recognize sugar structures (N-acetylglucosamine,
polymannose, and fucose, respectively) predominantly found as terminal residues of
immature glycoproteins during their processing in ER and Golgi. In non-apoptotic
neutrophils the majority of these sugars are located intracellulary. In late apoptotic
stages they get exposed at the cell surfaces, suggesting that the plasma membranes of
apoptotic neutrophils contain incompletely processed proteins.
The terminal residues of oligosaccharides of mature glycoproteins are typically
formed by sialic acid residues. During neutrophil apoptosis mature glycostructures
are lost from cell surfaces as detected by decreased binding of sialic acid recog-
nising lectins derived from Maackia amurensis and Sambucus nigra [35] (Franz,
unpublished data). Increased binding of the galactose binding lectin, peanut agglu-
tinin, was not observed, implying that en masse desialylation caused by activated
sialidases did not represent a major carbohydrate modification event accompanying
apoptosis. More likely, plasma membranes containing sialic acid glycostructures get
lost during the apoptotic blebbing process.
In a recent study we analysed the plasma membrane composition of late apoptot-
ic neutrophils that are reduced in size but have still maintained their plasma mem-
brane impermeability for propidium iodide (Franz, unpublished data). We found the
ER-resident protein calnexin to be exposed in the membranes of late apoptotic cells.
Furthermore, the glycolipid GM1, which is lost from the surfaces of ageing neutro-
phils in the early stages of apoptosis [40], gets re-exposed from internal stores. The
ER-derived proteins and lipids appear at the cell surface with the same time course
as the immature glycoprotein epitopes, detected by GSL II, NPn, and UEA I. These
findings indicate that internal membranes at least partially derived form the ER get
translocated to the surface of late apoptotic neutrophils. Thereby, preformed inter-
nal target structures get access to the cell surface.
This mechanism of membrane exchange may help to explain how apoptotic
neutrophils rapidly alter their glycocalyx and receptor availability. An apoptotic
cell soon shuts down its power production and must, therefore, deal parsimoniously
with its ATP reservoir. From this point of view, the exposure of preformed internal
structures that are sequestered inside viable cells is an economical method for the
concomitant generation of several phagocyte recognition structures on surfaces of
apoptotic neutrophils.
Receptor inactivation
One of the key membrane changes associated with neutrophil apoptosis is the func-
tional uncoupling of signalling receptors, acting to isolate the apoptotic cell from
62
stimuli that normally trigger production of superoxide and degranulation [41]. The
apoptosis-associated increase in activity of caspases and calpains is likely to disrupt
the recruitment and subsequent assembly of signalling complexes that are required
to translate receptor occupancy into a functional response [42]. Furthermore, dis-
ruption of cytoskeletal integrity [4, 42, 43] through proteolysis of actin or other
actin-binding proteins such as gelsolin, ezrin, fodrin or band 4.1 would be likely
to inhibit neutrophil adhesion, migration and degranulation. For apoptotic neutro-
phils, we reported specific loss of the capacity for `2 integrins to bind to ligand,
even though levels of surface integrin expression remained similar on the surface of
apoptotic neutrophils [36]. It is well established that `2 integrins exhibit regulated
ligand binding function, through a process known as inside-out signalling [44].
Another possibility is that loss of integrin ligand binding activity reflects
changes in integrin organisation within the membrane (avidity), together with loss
of affinity regulation. However, examination of binding of antibody NKI-L16 to
apoptotic cells revealed similar levels of expression on apoptotic and non-apoptotic
cells (Dransfield and Figdor, unpublished data). Since NKI-L16 binding has been
suggested to be linked with avidity regulation of LFA-1, this observation suggests
that altered affinity regulation is the principal reason for loss of integrin activity on
apoptotic neutrophils. One possibility is that there is dysregulation of key signalling
pathways following apoptosis. Intriguingly, an active conformation of `2 integrins
could not be forced even in the presence of the divalent cation Mn2+ [36], implying
that the membrane lipid and cytoskeletal alterations associated with apoptosis may
further restrict integrin activity and thereby ensure a lack of neutrophil response to
environmental signals. The effects of apoptosis on plasma membrane organisation,
including assembly and organisation of lipid rafts has not been well studied. In
neutrophils, spontaneous apoptosis may be a consequence of recruitment of death-
inducing signalling complex (DISC) components (FADD, pro-caspase-8 and -10 and
c-FLIP) to lipid rafts, since disruption of raft organisation with nystatin delayed
apoptosis [45]. However, Sherriff and co-workers [40] demonstrated that binding
of the ganglioside GM-1 (thought to be a marker of lipid rafts) was specifically
lost from apoptotic cells. This event appeared to be a very early change, preceding
or accompanying exposure of PS, and thus might represent an early marker of cell
death. Whether these changes influence the function of other receptors is not clear.
One surface receptor that has been proposed to play an important role in the
discrimination of viable and apoptotic cells is the immunoglobulin superfamily,
molecule platelet-cell adhesion molecule-1 (PECAM-1) or CD31 [46]. This 130-kDa
glycoprotein is expressed by endothelial cells, platelets, monocytes, neutrophils,
nave CD4+ cells and memory CD8+ cells. It has been reported that homotypic
interaction of CD31 on neutrophils and CD31 on phagocytes is capable of medi-
ating intercellular adhesion. However, whereas CD31 provides a signal leading to
the active detachment of viable neutrophils, tethering of apoptotic neutrophils via
CD31 may lead to subsequent engulfment [13]. For endothelial cells, CD31 was
63
found to be cleaved during apoptosis into a soluble 100-kDa fragment composed of

the extracellular domain, which was shed into the culture medium, in addition to a
28-kDa truncated form, comprising a small extracellular region, the transmembrane
section and the cytoplasmic domain. In preliminary studies, changes in expression of
CD31 associated with apoptosis in neutrophils was found to be somewhat variable,
but in some experiments was found to be reduced by approximately 50% (Drans-
field, unpublished data) raising the possibility that proteolytic loss of CD31 expres-
sion during apoptosis may be a more general phenomenon. The truncated form was
shown to differentially recruit `-catenin, a-catenin and SHP-2 when compared with
full-length CD31, providing a potential mechanism for differential signalling lead-
ing to a failure to undergo detachment [47]. One possibility is that the truncated
form of CD31 may assume a conformation similar to that of the phosphorylated
form, and thus differentially recruit signalling molecules during apoptosis and thus
CD31 ligation on apoptotic cells is unable to generate signals that promote cell
detachment.
Neutrophils also express CD47, the ligand for the immunoglobulin superfamily
member SIRP_ [48]. For viable neutrophils, this interaction has been proposed to
give rise to dont eat me signals and when this interaction was blocked by CD47
Fab fragments, viable cells were engulfed [49]. In terms of membrane distribu-
tion, CD47 was found to be spatially segregated from calreticulin and PS, putative
counter-receptors for phagocyte recognition molecules, suggesting that regulatory
molecules may act independently of putative recognition receptors to control phago-
cytic function.
Other changes associated with late apoptosis
We have characterised a monoclonal antibody (BOB78) that binds to a subset of

apoptotic neutrophils (annexin V positive) that exhibit distinct forward/side scatter
properties [35]. We have found that the antigen recognised by BOB78 was normally
expressed intracellularly in neutrophils and have described that this BOB78-posi-
tive population represents late apoptotic cells, with a distinctive morphological
appearance, yet distinct from propidium iodide positive, necrotic cells. Preliminary
data indicate that the BOB78 antigen may be a member of the heat-shock protein
family (Ross, unpublished data), possibly implying that these chaperone proteins
become stranded at the cell surface during the later stages of apoptosis. Heat-
shock proteins have been reported to be specifically expressed on the surface of
neutrophils and other cells following stress or pathogen-induced cell death [5052].
This finding may have particular relevance to the promotion of antigen presentation,
as heat-shock proteins represent a potent stimulus of dendritic cell maturation.
For neutrophils, progression from early apoptosis to this late apoptotic phe-
notype appears to be a relatively slow process. In contrast, many other cell types
64
rapidly acquire the late apoptotic phenotype following induction of apoptosis.

Interestingly, late apoptotic neutrophils have been shown to bind a number of
plasma proteins that are importance in homeostatic regulation, e.g. binding of the
matricellular protein thrombospondin [35], the pentraxin C-reactive protein [53],
serum amyloid P [54] and also the complement component C1q [55]. Binding of
these molecules does not appear to represent non-specific protein binding capacity
since other proteins (e.g. labelled bovine serum albumin) do not bind to these cells.
One implication is that certain antigens may become accessible during this phase of
apoptosis and thus potentially alter phagocyte recognition of these cells.
Of particular interest with respect to neutrophil apoptosis is the binding of
anti-neutrophil cytoplasmic autoantibodies (ANCAs) to apoptotic neutrophils.
These antibodies can be shown to have two major patterns of binding, reacting
with cytoplasmic or perinuclear antigens. Electron microscopic analysis suggested
that translocation of intracellular granules to the plasma membrane occurs late
during the apoptotic process, exposing granule proteins such as myeloperoxidase
and proteinase 3 [56]. Although the exposure of these antigens was reported to be
independent of prior priming and activation of the neutrophils, other studies have
suggested that ANCA fail to bind to apoptotic cells unless mobilised to the surface
by isolation procedures [57]. As shown for the BOB78-positive cells, ANCA-posi-
tive neutrophils exhibit extensive nuclear degradation, although they remain able
to exclude propidium iodide. It is possible that other major autoantigens such as
nucleosomal DNA and small ribonucleoproteins, SS-A/Ro and SS-B/La become
accessible during the later stages of apoptosis, prior to loss of membrane integ-
rity.
The opsonisation of cells with complement components also deserves consider-
ation. C1q has been demonstrated to bind to blebs on apoptotic cells [58] and have
a role in clearance of apoptotic cells both in vitro (apoptotic thymocytes) and in vivo
associated with the development of glomerulonephritis in C1q-deficient animals
[59, 60]; together with the well-defined association between C1q deficiency and
incidence of systemic lupus erythematosus, the possibility is raised that defective
apoptotic cell clearance predisposes affected individuals to development of autoim-
mune conditions. However, studies in other animals lacking phagocytic receptors,
e.g. CD14 [61], have suggested that this association is not absolute. Studies from
Kim and co-workers [62] suggested that C1q deposition on apoptotic cells was
mediated in part through the binding of IgM antibodies to the cell surface during
apoptosis. Binding of IgM could be increased by hydrolysis of membrane phos-
pholipids with phospholipase A2, suggesting that lysophosphatidylcholine becomes
exposed during the later stages of apoptotic cell death.
Apoptotic cells have also been reported to become opsonised with C3 compo-
nents [63]. Recognition of complement-opsonised apoptotic cells is then mediated
by `2 integrins CD11b/CD18 and CD11c/CD18. However, opsonisation with
some complement components may only occur later during the apoptotic pro-
65
cess [64]. Most cells express surface receptors that protect against complement
activation, termed complement regulators [65]. CD46 and CD55 act to regulate
C3 convertase activity and CD59 controls the assembly of the membrane attack
complex. Interestingly, the expression of CD55 and CD59 are down-regulated on
apoptotic neutrophils, potentially making these cells vulnerable to complement-
mediated attack [66]. In addition, deposition of C3 components may target these
cells for phagocytic clearance via complement receptors, including CD11b/CD18
and CD35.
It is clear that if these proteins that have been implicated in the recognition and
subsequent phagocytic clearance of apoptotic cells are present at inflammatory sites,
there may be profound consequences in terms of how these cells are subsequently
cleared. From the published data relating to the potential for opsonisation of apop-
totic cells, it seems likely that their clearance will differ both in terms of phagocyte
recognition pathways engaged and also in the efficiency of internalisation. Mecha-
nisms for clearing late apoptotic cells may represent a backup pathway for ensuring
that failure to clear early apoptotic cells does not lead to release of intracellular
contents [12]. Surprisingly, given the potential importance of apoptotic cell removal
in so many diverse processes, there have been few studies that have compared
molecular mechanisms and functional consequences of phagocyte clearance of cells
at different stages of the apoptotic process.
Apoptosis-enabled receptors?
There have been few reports of gain of function of cell surface receptors follow-
ing induction of apoptosis. Interestingly, Moffatt and colleagues [67] reported
that the binding profile of ICAM-3 was altered following apoptosis. Thus,
while ICAM-3 on viable cells was able to bind to the counter-receptor LFA-1,
on apoptotic cells this capacity was lost. Instead, ICAM-3 was suggested to be
able to bind to CD14. Recently, we have described a novel mechanism whereby
apoptotic neutrophils become opsonised by immune complexes [68]. This finding
arose from the characterisation of a monoclonal antibody that exhibited a unique
binding profile for neutrophils. After extensive characterisation, we found that
this murine IgG1 antibody rapidly formed immune complexes in the presence of
the antigen (the foetal calf serum protein fetuin) and bound to apoptotic neutro-
phils via an interaction of the Fc portion to FcaRII. Surprisingly, antibody-antigen
complexes did not bind, or bound weakly to freshly isolated or cytokine/che-
mokine-activated neutrophils, despite abundant expression of FcaRII on these
cells. We believe that this alteration in ligand binding activity is the first example
of a molecule that shows reduced expression on apoptotic cells, yet exhibits
enhanced function. It is possible that other, as-yet-unidentified, surface receptors
may behave in a similar manner. The molecular mechanism(s) responsible for
66
this effect remains to be defined and although the significance of opsonisation of

apoptotic neutrophils by immune complexes during inflammation is not known,
it is likely that subsequent phagocytic clearance will be affected [69].
Apoptosis and antigen presentation
The surface molecular alterations associated with apoptosis may profoundly influ-
ence generation of effective immune responses to endogenous or tumour cell-asso-
ciated antigens. Many different peptide epitopes derived from antigens expressed
by tumours can be recognised by cytotoxic T lymphocytes in the context of MHC
class I molecules [70]. However, endogenous expression of most tumour antigens
may normally induce tolerance, with antigens containing cryptic epitopes capable of
eliciting effective immune responses. It has been established that ex vivo immunisa-
tion of antigen-presenting cells (including dendritic cells and Langerhans cells) with
tumour-derived material could provide effective anti-tumour therapy [7173], par-
ticularly following acquisition of antigens from apoptotic cells [51]. Generation of
antigen-presenting cells with high levels of expression of co-stimulatory molecules
can also be influenced by the mode of cell death [50, 52, 74]. Thus, the mode of
cell death likely represents a critical factor that determines the antigen-presentation
capacity and thus the generation of effective immune responses.
Summary
In conclusion, since the process of programmed cell death was first described in
1972 [75], there has been tremendous progress in defining the underlying regu-
latory mechanisms and the consequences in terms of gene expression patterns,
functional activity and membrane receptor alterations. However, the issues relat-
ing to heterogeneity of the apoptotic cell phenotype discussed here have profound
implications for future studies of phagocyte recognition, uptake and, crucially,
phagocyte responses following phagocytosis of apoptotic cells. Delayed apoptotic
cell clearance within tissues could potentially drive the progression to late apoptosis
and secondary necrosis. Efficient clearance of apoptotic cells by macrophages may
inhibit induction of specific immune responses, a finding of considerable impor-
tance in terms of potential tumour immunotherapy. The presence of late apoptotic
or necrotic cells would be predicted to promote maturation of antigen-presenting
cells to express co-stimulatory molecules that would maximally activate lymphocyte
responses. In addition, masking of the PS on the apoptotic cell membrane would be
predicted to interfere with suppression of pro-inflammatory signals and also with
the generation of anti-inflammatory signals. Indeed, production of IL-1 and TNF-_
was augmented and TGF-` release was suppressed in macrophages challenged with
67
annexin V-coated irradiated tumour cells in vitro when compared with untreated
irradiated cells [76], potentially overcoming tolerogenic responses to apoptotic cell
uptake. Definition of the precise role of membrane receptor alterations associated
with apoptosis upon phagocyte recognition of apoptotic cells and their influence
upon subsequent phagocyte responses remains an important goal. Understanding
the contribution that these changes make to development of inflammatory and auto-
immune diseases represents a considerable challenge for future studies.
Acknowledgements
This work was supported by the Medical Research Council (Clinician Scientist
award to S.P.H.), the Arthritis Research Campaign (grant R0622) and the Wellcome
Trust. We would especially like to thank Prof. Christopher Haslett, Prof. John Savill
and Dr. Simon Brown for constructive discussion.
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73
Fate of macrophages once having ingested apoptotic cells:
Lymphatic clearance or in situ apoptosis?
Centre for Respiratory Research, University College London, Rayne Institute,

London WC1E 6JJ, UK
Introduction
Neutrophil and macrophage kinetics at the inflamed site differ markedly [1, 2].
Unlike neutrophils, many organs and tissues have a population of resident mac-
rophages, hence these cells have a different baseline at the outset of inflammation.
Resident macrophages are a key population in the initiation of local inflammation
[3]. Neutrophils influx rapidly early in the acute inflammatory event, while resident
tissue macrophages may actually decline in numbers due to a process known as the
macrophage disappearance reaction (MDR) [4]. Like neutrophils, inflammatory
monocytes migrate in from the blood stream, although this lags somewhat behind
the insurgence of neutrophils. These monocytes mature locally into inflammatory
macrophages, although their activation state may alter over the course of the inflam-
matory process [5, 6]. Neutrophil numbers peak earlier than macrophages. Their
decline can be due to necrosis, apoptosis and subsequent phagocytosis, or progress-
ing to secondary necrosis if phagocytosis of apoptotic cells fails [7]. Neutrophils
may be able to efflux away from the inflamed site, for example back into the blood
stream, or, with pulmonary inflammation for example, they can migrate into the air-
way lumen [810]. It appears, however, that their main fate is to undergo apoptosis
locally as shown in a number of models and in vivo settings [7, 11]. In normally
resolving inflammation, macrophages phagocytose the apoptotic neutrophils and
their numbers then decline allowing the tissue to return to normal structure and
function [1214]. This chapter examines macrophage clearance in the resolution of
inflammation.
Macrophages are powerful scavengers and are the major cell involved in phago-
cytosis of apoptotic neutrophils. The fate of macrophages once they have ingested
apoptotic neutrophils is open to much debate. There is good evidence that the mac-
rophage, unlike both the neutrophil and the un-activated monocyte, is a long-lived
cell [15, 16]. Despite working in a highly toxic environment, macrophages need
to be robust cells and retain viability and functionality until they have facilitated
clearance of the inciting pathogen(s). Macrophages are resistant to many apoptotic

stimuli including Fas and tumour necrosis factor (TNF)-_ death receptor ligation,
ionising radiation, and multiple anti-neoplastic or cytotoxic agents [1719]. This
suggests that local apoptosis may not be the immediate fate for these cells and
this is supported by several investigators who have shown that macrophages from
inflamed sites can be found in the draining lymph nodes [2022]. Experimental
evidence, using the inflamed peritoneum as a model, demonstrated remarkably
little local apoptosis or subsequent phagocytosis and confirmed emigration into the
draining lymphatics was the major route for macrophage clearance [21, 23]. The
situation is not so clear cut, however, as it is well established that macrophages,
despite their inherent resistance to apoptosis, can undergo apoptotic cell death and
a number of mechanisms are recognized to drive this process [24, 25]. Indeed mac-
rophage apoptosis has been shown to occur in the presence of a range of infections
such as that with Shigella [26]. More recently, we have learned that the perceived
resistance of macrophages to apoptosis is not fixed and that these cells can, in
certain circumstances, gain susceptibility to apoptosis in a potentially beneficial
way for the hosts innate immune response [27]. Hence, both local apoptosis and
emigration appear to be valid modes for macrophage clearance with the resolution
of inflammation.
Macrophage apoptosis
Macrophage resistance to apoptosis
Macrophages produce a number of powerful effector molecules to kill pathogens;

these include reactive oxygen and nitrogen species and histotoxic enzymes. To sur-
vive in such a local toxic environment macrophages have been shown to up-regulate
enzymes including catalase and manganese and copper superoxide dismutase and
buthionine sulfoximine which help in buffering intracellular redox systems provid-
ing a powerful mechanism through which cell survival is increased [2830]. Along
with protection through enhanced protection against free radical damage, there is
also modulation of apoptosis through changes in a variety of intracellular pathways
including activation of JNK but mainly by expression of the pro- and anti-apop-
totic molecules of the Bcl-2 family [31, 32]. Raised Bcl-2 expression is associated
with resistance to apoptosis, while Bax is associated with enhanced apoptosis and
these have been found to have clinical relevance for example in tuberculous disease
where caseating granumoma have increased macrophage apoptosis with a relative
predominance of Bax over Bcl-2 [30, 32]. The phosphatidylinositol 3-kinase (PI3K)
pathway appears to be important for macrophage survival as mice with abnor-
mal regulation of this pathway have altered regulation of macrophage apoptosis,
reduced viral clearance and increased inflammation [33, 34]. Akt-1 is a PI3K-regu-
lated serine/threonine kinase that regulates survival through myeloid cell leukaemia
76
Fate of macrophages once having ingested apoptotic cells: Lymphatic clearance or in situ apoptosis?
sequence (Mcl)-1, a Bcl-2 family member, rather than through caspases, or NF-gB.
Mcl-1 is up-regulated during macrophage differentiation and protects cells from
apoptosis from a range of toxins including irradiation, drugs or through the with-
drawal of growth factors. Importantly Mcl-1 has a relatively short half-life [27].
Macrophage apoptosis and infections
Despite this resistance to apoptosis, it is clear that a number of pathogens can

lead to macrophage apoptotic cell death. This was first shown for Shigella; how-
ever, the number of pathogens shown to lead to macrophage apoptosis is now
large and includes Salmonella enterica serovar typhi, Bacillus anthracis, Yersinia,
Mycobacterium tuberculosis, Streptococcus pneumoniae, Trichomonas vaginalis
and Pneumocystis pneumonia infection [3543]. The timing with which such infec-
tions lead to apoptosis is not clear. In certain circumstances macrophage apoptosis
appears to be a response to an overwhelming infection with a virulent pathogen,
suggesting the macrophage defences are simply overwhelmed. In these situations,
macrophage apoptosis is usually part of the pathogenic process and the host does
badly, e.g. if caspase-9 is inhibited, alveolar macrophage apoptosis is prevented in a
rodent pneumocystis pneumonia model and survival is dramatically enhanced [43].
Similarly, the pathogenic Shigella strain induces macrophage cell death and has a
high mortality, while the non-pathogenic strain is not associated with cell death or
pathogenicity [35].
In other circumstances, apoptosis may follow as a consequence of bacterial
phagocytosis. For example, macrophages infected with M. tuberculosis at low copy
number are stimulated to undergo apoptosis in a TNF-dependent fashion and this
appears to be a host-protective process as it leads to reduced mycobacterial viability
[40]. Of interest, however, when infected with a high copy number, the pathogen
induces macrophage apoptosis, now in a TNF-independent fashion that does not
reduce viability of the mycobacterium and allows for its release and dissemination.
Indeed, it appears that there may be a relationship between the intracellular live
bacterial load and the degree of induction of macrophage apoptosis for other bac-
teria including the Pneumococcus [41]. As with mycobacterial-induced macrophage
apoptosis, pneumococcal induced macrophage apoptosis is linked with bacterial
killing. Marriott et al. [44] have shown that the phagocytosis of pneumococci leads
to nitric oxide production in microvesicles adjacent to the lysosomes. This nitric
oxide contributes to intracellular killing of the pneumococci but is also associated,
later with increased apoptotic death of the macrophage itself. Interestingly, the
pro-apoptotic effect of exogenous nitric oxide alone is far lower, suggesting other
factors including bacterial load are crucial to this process. In the absence of nitric
oxide, pneumococcal infection leads the macrophages to undergo necrosis. These
experiments were performed in vitro; 20 h after infection up to a quarter of the
77
macrophages had evidence of apoptotic features. It is not clear if all the macro-
phages phagocytose pneumococci and, if so, are some relatively protected from cell
death or is apoptosis related to a population more involved in phagocytosis? Also of
interest is the in vivo fate of those macrophages undergoing apoptosis; are they then
phagocytosed by other macrophages and how are these then cleared? This raises the
possibility that different macrophage populations exist with different propensities
for bacterial clearance/killing vs for cell clearance and inflammatory resolution.
This concept is not new: for example, alveolar and peritoneal macrophages have
been shown to have different phagocytic ability for apoptotic cells. However, this
may relate to the site more than the cell type as monocytes recruited to the lung
also phagocytose apoptotic cells poorly [45, 46]. Macrophages can also exist in
different activation states, depending on the local activation signals [5, 47], and the
possibility of activation state altering apoptotic and emigration potential is, as yet,
unexplored.
Macrophage apoptosis and non-infective challenges
Non-infective causes of macrophage apoptosis are also well described, e.g. hydrogen
peroxide induces cell death that can be ameliorated with nitric oxide [29]. Nitric
oxide is also well known as a trigger for macrophage apoptosis [48]. Inflammation,
as noted above, leads to inducible nitric oxide synthase (iNOS) synthesis and hence
high levels of nitric oxide locally. Macrophages elaborate anti-inflammatory prosta-
glandins such as the cyclopentone 15dPGJ2, which could act to inhibit the synthesis
of nitric oxide. Interestingly, however, although treatment with 15dPGJ2 inhibits
the expression of iNOS, it results in an increase in the percentage of apoptotic cells,
probably through preferential formation of peroxynitrite, in these conditions [49].
Serum deprivation can lead to apoptosis in most cells; this is also apparent for
macrophages though at a much lower level. This has recently been shown to be
mediated by interferon secretion [50]. Other mechanisms shown to induce mac-
rophage apoptosis include engagement of Fas ligand [51]. There is now good evi-
dence that macrophages can undergo apoptosis locally at the inflamed site even in
sterile inflammation. Gilroy et al. [52] used an acute pleurisy model in which they
demonstrated an early neutrophil predominance, which declined through apoptosis
to be replaced by monocytes-derived inflammatory macrophages. In this model,
inflammatory resolution was associated with programmed cell death in the mac-
rophages and in both cases apoptosis was mediated by cyclooxygenase 2-derived
15deoxyDelta12-14PGJ2, expressed during the resolution phase. In atheroma
models, cholesterol and oxidized LDL have also been shown to drive apoptosis.
In addition, in atheromatous plaques, overexpression of TIMP-2 leads to a nearly
50% reduction in plaque and this was associated with a reduction in macrophage
apoptosis [53].
78
Differential regulation of macrophage resistance to apoptosis
As discussed above, pathogens can promote macrophage apoptosis. They may also
inhibit apoptosis, e.g. Tunbridge et al. [54] showed that Neisseria meningitides,
which is known to inhibit apoptosis in other cells, prevents macrophage apoptosis
via genes encoding nitric oxide detoxification and a porin. This may contribute to
innate immunity. In a similar vein, Gross et al. [55] demonstrated that Brucella
infection led to overexpression of the A1 gene, a member of the Bcl-2 family and
prevented apoptosis. This ability of an intracellular pathogen to modulate apopto-
sis in the hosts cells suggests this may be an advantageous strategy for Brucella to
avoid its elimination.
Interestingly, Mcl-1 offers a unique control point for macrophage sensitivity to
apoptosis. A number of Mcl-1 splice variants exist and Marriott et al. [27] have
shown that Mcl-1 is up-regulated in the early phase of inflammation, while mac-
rophage functionality is well maintained, with the cells being viable and able to
phagocytose bacteria. Later Mcl-1 is down-regulated, corresponding to emergence
of a smaller splice variant and evidence of mitochondrial membrane permeabilisa-
tion and induction of apoptosis. This change has been shown to be of functional
importance in a rodent model where overexpression of Mcl-1 resulted in delayed
mitochondrial membrane permeability and reduced bacterial clearance both in vitro
and in vivo. This suggests that the switch of Mcl-1 splice variants with associated
enhanced susceptibility to apoptosis may, in certain circumstances, promote resolu-
tion. Such a change in sensitivity to apoptosis is not without precedent as similar
findings have been observed for Bcl-2 and a shortened cleavage fragment Bcl-2/
Delta34 that lacks the ability to heterodimerise with Bax and hence the cleavage
fragment lacks the anti-apoptotic effect of Bcl-2 [56]. Mogga et al. [57] also suggest
that macrophage apoptosis is related to Bcl-2 expression and that overexpression
of Bcl-2 can reduce macrophage apoptosis and may be associated with intracellular
survival of tubercle bacilli.
Macrophage emigration
Emigration is the other major mechanisms for macrophage exit. Our early work
on this was stimulated by the concept that macrophages were indeed long-lived
cells and thus the idea that local apoptosis was the route for clearance was ques-
tioned. We used in vivo fluorescence labelling to track macrophages during the
resolution of sterile inflammatory challenges. These were either short (starch:
24 days), medium (thioglycolate: 510 days) or longer term models (heat-killed
Corynebacterium parvum: 2 weeks or more). Interestingly, we found that with
resolving peritonitis, fluorescence-labelled macrophages increasingly accumulated
in the draining lymph nodes as resolution proceeded, suggesting emigration was a
79
feature of resolution. These experiments could not account for the possibility that
macrophages were dying locally and being phagocytosed, and the labelled cells we
were finding in the lymph nodes had in fact gained the label by ingesting apoptotic
labelled macrophages. To examine this, red fluorescently labelled inflammatory
macrophages from H-2k/d mice were transferred into the peritoneal cavity of H-2k
mice at the start of the resolution of thioglycolate peritonitis. The number of trans-
ferred macrophages within the peritoneum declined rapidly over the next 4 days.
Dual-colour flow cytometry permitted discrimination among donor cells, recipient
cells, and donor cells that had been phagocytosed by recipient macrophages and
this showed that there was little evidence of any significant local phagocytosis
of transferred macrophages [21]. Importantly labelled, non-phagocytosed mac-
rophages were detected with increasing frequency in the draining lymph nodes,
but not in a variety of other tissues. These data suggest that inflammatory macro-
phages normally emigrate rapidly from the peritoneal cavity during the resolution
of inflammation. In conjunction with this, we examined the clearance of resident
peritoneal macrophages and found that they too were capable of emigrating to the
local lymph nodes but, unlike the rapid emigration of inflammatory macrophages,
the resident cells could persist in the non-inflamed peritoneum for weeks. This
major difference in kinetics suggested that the process of emigration was regulated.
To investigate this further we compared the clearance of live and formalin-fixed
adoptively transferred macrophages and found that clearance of the formalin-fixed
cells was slower than that of the live cells and that the formalin-fixed cells were
only found in the draining lymph nodes after they had been phagocytosed by live
macrophages. This confirmed that emigration was an active process. Our work
was in line with that of others, e.g. Rosen and Gordon [58], also using adoptive
transfer of fluorescent macrophages, showing that they were able to migrate to
specialized lymphoid organs. Reviewing the lymphatic drainage system of the peri-
toneum we see that the greater omental lymphoid organ and the subdiaphragmatic
surface of the diaphragm are the main sites for the origin of the draining lymphat-
ics. This drainage occurs through stomata called milky spots. The peritoneum is
lined with flattened mesothelial cells; however, at the milky spots these cells gain a
cuboid morphology with microvilli. They surround a small opening that connects
the peritoneal cavity with an underlying lymph vessel. Along with a lymph duct
there is a prominent vascular supply. In addition, leukocytes are commonly found
adherent to the overlying mesothelial cells. These structures appear to contribute
to the influx of leukocytes into the peritoneum during the onset of inflammation.
The small 58-mm opening can also allow macrophage emigration, although this
would require these cells to adhere and squeeze through. When we looked at ex
vivo tissue from resolving thioglycolate peritonitis we could clearly see fluores-
cently labelled macrophages adhering to milky spots and, looking at lymphatics in
the diaphragm, could then see labelled macrophages in draining lymphatic vessels
tracking away to the draining nodes Figure 1.
80
Figure 1.
Fluorescence-labelled macrophages in the draining parathymic lymph nodes.
To address the question of how this macrophage emigration is regulated, we

focused on the adhesion interaction between macrophages and the mesothelial cells.
Using ex vivo tissue we were able to demonstrate that macrophages did indeed
adhere to the mesothelial lining of the peritoneum and that this adhesion was cal-
cium and magnesium dependent, such that more than 70% of adhesion could be
abolished in the absence of these divalent cations. Despite the powerful importance
of the `2 integrins to macrophage function, inhibiting these molecules alone made
no difference to macrophage-mesothelial cells interactions. Using RGD peptides,
however, we could demonstrate a significant inhibition of adhesion and this was
borne out by using blocking antibodies to very late antigen-4 (VLA-4) and VLA-5.
When the `1 integrins were blocked, the addition of blocking `2 integrin antibodies
exposed a further inhibitory effect on macrophage-mesothelial cell adhesion. The
relevance of this ex vivo work was confirmed when blocking `1 integrins or RGD
peptides was shown to significantly delay macrophage clearance from the inflamed
peritoneum and reduce macrophage accumulation at the parathymic lymph nodes
Figure 2 [23].
In a similar set of investigations, Cao et al. [59] confirmed that inflammatory
macrophages do not die locally but migrate across the peritoneum into the lym-
81
Figure 2.
Protocol for adoptive transfer of fluorescence-labelled macrophages, ensuring donor mac-
rophages are at the same stage in the inflammatory process as the recipient mouse and are
labelled and coated in blocking antibody.
phatics to the regional lymph nodes. They again showed that activation increased
the rate of emigration; however, their work demonstrated that upon further acti-
vation it was the `2 integrin, Mac-1, that was a critical regulator of emigration.
This is in keeping with our finding of an additional `2 integrin activity when `1
integrins were blocked. Moreover, unlike the findings published by Hotchkiss
et al. [60] where the introduction of apoptotic cells prior to the induction of
sepsis was harmful, it has recently been shown that the rate of inflammatory
macrophage clearance from the inflamed site with resolution can be enhanced by
phagocytosis of apoptotic cells [61]. This enhanced emigration again occurred
though `1 integrin-mediated mechanisms [62]. It must be emphasized that local
apoptosis and emigration are not necessarily mutually exclusive. We do not know
what happens to those macrophages that undergo apoptosis after ingesting bac-
teria; are they then engulfed by other macrophage populations that then emigrate
themselves?
This leads us to recognize a situation in which macrophages could undergo
apoptosis early in the inflammatory process through a toxicity-driven process that
82
typically is associated with increased morbidity, while, in the presence of other intra-
cellular pathogens such as M. tuberculosis, the macrophage could undergo altruistic
apoptosis to facilitate pathogen clearance [63]. Generally, however, the macrophage
aims to resist being driven into apoptosis and instead to facilitate pathogen clear-
ance and then inflammatory resolution. Having phagocytosed apoptotic neutrophils
they can then leave by emigration.
Macrophage disappearance reaction
Another situation in which macrophages have been shown to enter the lymphatics is
via the MDR. This is a response of peritoneal macrophages (and probably other res-
ident macrophage populations) to a variety of stimuli in which many of the resident
cells disappear and are not able to be lavaged or recovered from the peritoneum [4].
Using fluorescent labelling, Melnicoff et al. [1] showed that some of these cells are
recoverable after a period of days, suggesting either firm adherence to the surround-
ing parietal peritoneum or a process of recirculation. Many cells do not reappear.
We have examined this process in more detail and see that peritoneal macrophages
adhere to the greater omental lymphoid organ and subdiaphragmatic surface of the
peritoneum, sites representing the highest concentrations of milky spots. Moreover,
the instillation and ingestion of apoptotic cells is a powerful stimulus for the very
rapid disappearance of both apoptotic cells and resident macrophages that are
found co-localised in great numbers adherent to the greater omental lymphoid
organ in particular. Instillation of live cells is not associated with ingestion or with
an MDR. A good number of these adherent macrophages are found then to migrate
into the draining lymphatics. This work supports the concept that ingestion of
apoptotic cells is a key signal for macrophage adherence and emigration into local
lymphatics and efflux to the draining lymph nodes.
Site-specific clearance
Most work examining macrophage emigration has been done in the peritoneum.
Other serosal-lined cavities such as the pleura and pericardium also have milky
spots and resident macrophages and probably have similar mechanisms for clear-
ance. Both peritoneal and pleural macrophages have also been shown to undergo
local apoptosis but this has been in response to a pathogenic stimulus rather than
to ingestion of apoptotic cells. Macrophages are known to emigrate to the drain-
ing lymphatics from solid organ inflammation; this has certainly been shown for
the kidney and the lung [20, 22]. Again, the impact of apoptotic cell ingestion on
this process is not clear. Indeed even Kupffer cells, long believed to be tissue-fixed
macrophages, have been shown to migrate using high resolution video microscopy,
83
although this is more restricted migration along sinusoid walls with or against blood
flow rather than into the lymphatics [64]. There is also increasing evidence that
macrophages actually contribute to lymphangiogenesis in inflammation and this
may further increase cell clearance and reduce the consequences of inflammation
[65, 66].
Summary of emigration
Hence, in summary, the emigration process is regulated by adhesion molecules

and, more importantly, by the ingestion of apoptotic cells. This occurs both for
inflammatory macrophages with the resolution of inflammation and for resident
macrophages as part of the MDR. Such an emigration process has much to com-
mend it teleologically. It makes sense not to have macrophage clearance before
resolution of the neutrophillic phase of inflammation as these cells are required to
clear the burden of polymorphonuclear leukocytes. Moreover, it is also sensible for
the process of engulfment of apoptotic cells to drive the clearance of the engulfing
macrophage. This phagocytosis signals that the cell has performed its key final
local role of cell clearance and it is now time for these macrophages to be removed,
themselves, from the inflamed site. It would appear logical for the macrophage to
travel to the draining lymph nodes where it could then interact with the lympho-
cytes there; impacting on the immune response. Macrophages are not the only cells
that migrate to the draining lymph nodes. Lymphocytes can be cleared by both
apoptosis and emigration and mature lymphocytes participate in a similarly com-
plex pattern of trafficking, passing from sites of inflammation into the lymph ducts
to the regional lymph nodes. This T cell emigration is under specific chemokine
control as CCR7 is required for T cell exit [67]. Interestingly, this same chemokine
is induced on dendritic cells after interaction with apoptotic cells [68]. Dendritic
cells also undertake such a journey as, early on in the inflammatory response,
they rapidly emigrate to the draining lymph nodes where they present antigen and
promote clonal lymphocyte proliferation and an immune response to the inciting
antigen. Apoptotic cell ingestion can even modulate maturation of dendritic cells
[69, 70]. Importantly, it has been demonstrated that macrophages actually have an
immune down-modulating effect on lymphocyte proliferation, suggesting that their
arrival in the lymph nodes could further act to silence the immune/inflammatory
response [71]. Despite this logical set of processes, however, we have seen that the
picture is not so clear-cut as macrophage apoptosis is also well described. Much
work also needs to be done to understand these processes. If emigration is the main
mechanism for clearance after ingestion of apoptotic cells, what signals lead to
altered adhesion? Are these processes similar for both solid organs and for serosal
cavity inflammation? Is regulation of this process only at the level of adhesion or
are there chemokine/cytokine signals at play that either wane allowing clearance
84
or act as lymphatic-specific chemotactic signals? What is the importance of the

hyaluronan receptor LYVE-1 and lymphatic trafficking, in addition to its role in
lymphangiogenesis [72]?
Phagocytosis and clearance
Many factors can alter macrophageneutrophil interaction and phagocytosis. Clear-

ance of apoptotic cells depends on effective phagocytosis and this can be defective,
for example in the NOD mouse, with consequent potential effects on autoimmune
disorders [73]. Other factors are now increasingly recognized as important in this
phagocytic interaction, such as the role of the lipoxins [74]. The study by Amano
et al. [75] suggests that altering neutrophil clearance has an effect on resolution of
inflammation, in this case on lung injury. They demonstrated that levels of mono-
cyte chemoattractant protein (MCP-1) were increased before macrophage inges-
tion of neutrophils, and the administration of blocking-MCP-1 antibodies reduced
macrophage clearance of neutrophils and potentiated lung tissue injury. Conversely,
the administration of MCP-1 itself increased neutrophil ingestion by macrophages
and reduced lung tissue injury. One wonders if the noted effect of cigarette smoke
on reducing macrophage phagocytosis of apoptotic cells contributes to the persist-
ing inflammation seen in the lungs of many smokers [76]. Data from Asada et
al. [77] suggest that peroxisome proliferator-activated receptor gamma (PPARa),
expressed by alveolar macrophages, plays an anti-inflammatory role through inhib-
iting cytokine production, increasing CD36 expression and enhancing phagocytosis
of apoptotic neutrophils, although the relevance directly to inflammation in vivo
and macrophage clearance of apoptotic cells has not been established. Also the
local chemical environment can directly alter the ability of macrophages to ingest
apoptotic cells, for example the presence of steroids augments this while interferon-
gamma prevents this augmentation. [78, 79]. Finally, the ingestion of apoptotic cells
may down-regulate any future ability of macrophages to ingest other apoptotic cells
[80].
This provides the macrophage with a complex life cycle where, as a monocyte,
it is rescued from early apoptotic death by the onset of inflammation. These cells,
which may be derived from a distinct subpopulation of monocytes to those that
spawn resident macrophages, migrate into the inflamed site and adopt a pro-inflam-
matory phenotype where, along with the resident macrophages, they are vital in the
phagocytosis and killing of pathogens and the orchestration of the pro-inflamma-
tory response through their cytokine and chemokine elaboration. Induction of iNOS
and nitric oxide are critically important in this, certainly for live bacterial challenges.
Indeed, such pathogenic insults may overwhelm the macrophages defences, despite
their inherent resistance to apoptosis and longevity, and lead to death locally, either
by necrosis or by apoptosis. With the waning of the inflammatory response, macro-
85
phages adopt different roles; they can, depending on regulation of their own sensi-
tivity, undergo apoptosis locally. They may also undergo alternative activation and
contribute to wound healing and, as we have seen, clear apoptotic cells which then
drives their emigration to the draining lymph nodes. Here they may present antigen
and thus further regulate the immune response and may act to suppress this.
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91
Novel lipid mediators in resolution and their aspirin triggered
epimers: Lipoxins, resolvins, and protectins
Charles N. Serhan
Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology,

Perioperative and Pain Medicine, Brigham and Womens Hospital and Department of Oral
Medicine, Infection, and Immunity, Harvard School of Dental Medicine and Harvard Medical
School, 75 Francis Street, Boston, MA 02115, USA
Introduction
In The Doctors Dilemma (1906) by the popular writer George Bernard Shaw, the
character Sir Bloomfield Bonnington stated:
Drugs can only repress symptoms: they cannot eradicate disease. The true
remedy for all diseases is Natures remedy. Nature and Science are at one, Sir
Patrick, believe me; though you were taught differently. Nature has provided,
in the white corpuscles as you call them in the phagocytes as we call them
a natural means of devouring and destroying all disease germs. There is at
bottom only one genuinely scientific treatment for all diseases, and that is to
stimulate the phagocytes. Stimulate the phagocytes.
In view of our current appreciation of resolution of inflammation, reviewed in this

chapter, these lines from The Doctors Dilemma take on new meaning, namely,
resolution as an active biochemical and cellular process in controlled molecular
terms by specialised mediators that function in resolution and can give rise to new
approaches to therapeutics. Efforts taken in the authors laboratory focus on gain-
ing a molecular understanding of the natural means by which the body controls
phagocytes, both positive and negative physiological signals. In this context, the
eicosanoids generated from arachidonic acid are mediators of special interest, given
their prominent and well-appreciated role(s) as pro-inflammatory mediators [1].
Our interest in endogenous control mechanisms in inflammation led us to recognise
the lipoxins (LXs) as the first local mediators possessing both anti-inflammatory
and pro-resolving actions (recently reviewed in [2]). The connection and role of
omega-3 polyunsaturated fatty acids (t-3 PUFA) was unknown as precursors to
novel mediators operative in resolution. These first pathways in resolution are now
described and overviewed here.
The essential roles of dietary t-3 PUFA were uncovered in the late 1920s [3] and
their importance observed in many studies to date [47]. Inflammation has emerged

Charles N. Serhan
Figure 1A.
Lipid-derived mediators in programmed resolution of acute inflammation.
Precursors of lipid mediators. Arachidonic acid is the precursor to eicosanoids that have
distinct roles as proinflammatory mediators. The prostaglandins (PGs) and leukotrienes (LTs)
each play specific actions pivotal to the progression of inflammation. Arachidonic acid-
derived epoxyeicosatetraenoic acids (EETs) produced via P450 [93, 94] and t-3 polyunsatu-
rated fatty acids (PUFA) P450 epoxides may also play roles [68, 95]. Cell-cell interactions,
exemplified by platelets-leukocytes within blood vessels and/or polymorphonuclear leuko-
cytes (PMN)-mucosal interactions, enhance generation of lipoxins that serve as endogenous
anti-inflammatory mediators self-limiting the course of inflammation [26]. The essential t-3
fatty acids eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6) are
converted to two novel families of lipid mediators, resolvins (Rv) and protectins, that play
pivotal roles in promoting resolution. Resolvin E series are generated from EPA, e.g. RvE1,
and resolvins of the D series, e.g. RvD1, are generated from DHA as well as the protectins
such as neuroprotectin D1 (see text for details).
today as a central component contributing to many prevalent diseases in Western

civilisation that were previously not known to involve inflammation, including
Alzheimers disease, cardiovascular disease [8] and cancer [9]. These now add to
the list of diseases associated with uncontrolled inflammation such as arthritis and
periodontal disease [10, 11]. In an effort to determine a link between t-3 fatty acids
and endogenous anti-inflammation, the author and colleagues identified previously
unknown pathways for enzymatic oxygenated products that were biosynthesised
from the two main t-3 PUFA, eicosapentaenoic acid (EPA) and docosahexae-
noic acid (C22:6, DHA). The isolated compounds proved to possess potent actions
94
Novel lipid mediators in resolution and their aspirin triggered epimers: Lipoxins, resolvins, and protectins
Figure 1B.
Aspirin triggering of lipid mediators. Aspirin impacts the formation of lipoxins and resolvin
by acetylating cyclooxygenase 2 (COX-2), for example in human vascular endothelial cells
that stereoselectively can generate in the case of RvE1 biosynthesis to generate 18R-HPEPE
that is picked up via transcellular cell-cell interactions by leukocytes and converted in a
lipoxygenase-like mechanism to RvE1. The complete stereochemistry of RvE1 and one of its
receptors were established ([46] and see Fig. 2). Of interest, biosynthesis of RvE1 can also
be initiated by P450-like enzymes in microbes [18]. Aspirin also influences the biosynthe-
sis of D-series resolvins. Aspirin catalytically switches COX-2 to a 17R-lipoxygenase-like
mechanism that generates 17R-containing series of resolvin D and protectins, for example,
neuroprotectin D1/protectin D1 (see text).
within resolving inflammatory exudates. Hence, they were coined resolvins (Rv) or
resolution phase interaction products, and protectins. In this chapter, an overview
of these new compounds and pathways that carry potent biological actions is given
as well as the impact of aspirin in these biosynthetic routes.
Specialised lipid mediators in the resolution of inflammation
Given that many, if not most, of the mediators generated from arachidonic acid
are pro-inflammatory [1, 12], it was unexpected to find that specific products, i.e.
LXs, produced from arachidonic acid during cell-cell interactions via transcellular
biosynthesis (Fig. 1) carry potent anti-inflammatory and pro-resolving actions [2].
The resolvins of the E series and those derived from DHA, resolvins of the D series,
95
Charles N. Serhan
as well as bioactive members from DHA that carry conjugated triene structures
(docosatrienes) termed protectins, are also both anti-inflammatory [13, 14] and
neuroprotective mediators [15, 16]. When generated from neural tissues, these DHA
products are denoted neuroprotectins (NPD1) [16] and, in other tissues, protectins
[17]. It is important to point out that other compounds identified earlier from t-3
fatty acids are prostaglandin (PG)- and leukotriene (LT)-like structures (i.e., PGE3
or LT5 series), but were less potent and/or devoid of bioactivity in inflammation.
This sharply contrasts the resolvins and protectins that each evoke potent actions
in vivo [1318]. The LXs, resolvins, and protectins are three structurally distinct
families of chemical mediators that are separated in their biosynthesis in space and
time in experimental inflammation and resolution. Specific members of each family
carry potent agonist properties in endogenous anti-inflammation and pro-resolv-
ing circuits in vivo in animal models (see Tabs 1 and 2 and below). Whether they
counter and can stop the progression to chronic diseases in humans is of interest,
as deficiencies in these resolving pathways might underlie disease pathology not
previously recognised.
The resolution program: An active biochemical progression

at the tissue level
The signs of inflammation (oedema, redness, heat) were known to early civilisations.
Egyptian hieroglyphics, Greek and Chinese ancient medical texts taught of the flame
of inflammation, mistakenly, as a disease unto itself [19]. In 1794, Scottish surgeon
John Hunter wrote that Inflammation in itself is not to be considered as a disease,
but as a salutary operation consequent to some violence or some disease [19]. This
insight is directly traced to our current appreciation of inflammation as a life-saving
reaction, yet this vital process is linked to many widespread diseases not previously
known to involve inflammation [2022]. Sir Henry Dale of England [23] and U. von
Euler of Sweden [24] each focused their seminal investigations on the roles of chemi-
cal mediators as short-range signals or autacoids in regulating cellular and tissue
responses in neural systems. The role of chemical mediators in inflammation became
apparent with the isolation of the prostaglandins, named by von Euler, and later the
discovery of the leukotrienes [1]. Although the contributions of phagocytes in host
defence were described in the early studies of another Nobel laureate, Mechnikov
[25], the lipid mediators biosynthesised by phagocytes, including neutrophils, mono-
cytes and macrophages, and their contributions to homeostasis are still evolving and
an overview of current information is given here (see Figs 2 and 3). Given the intricate
and specialised roles of these individual leukocyte types in the progression, duration
and termination of inflammatory responses, it is not surprising that phagocytes can
produce specific chemical mediators to signal activation of the programmed return
to homeostasis as well as dampen the amplification of inflammation.
96
Table 1 - LX and ATL actions in animal models of disease
Dermal inflammation
Inhibit neutrophil recruitment into ear skin Takano et al. [79]
Prevent vascular permeability Schottelius et al. [80]
Ischaemia-reperfusion (I/R) injury

Attenuate hind-limb I/R-induced lung injury Chiang et al. [37]
Detachment of adherent leukocytes in mesenteric I/R Scalia et al. [81]
Protective in ischaemic acute renal failure Leonard et al. [82]
Peritonitis
Block neutrophil recruitment and vascular leakage Bannenberg et al. [44]
Promote phagocytosis of neutrophil by macrophage Godson et al. [41]
Colitis (inflammatory bowel disease)

Attenuate proinflammatory gene expression and reduce severity Fiorucci et al. [83];
of colitis Arita et al. [49]
Inhibit weight loss, inflammation and immune dysfunction Gewirtz et al. [84]
Glomerulonephritis
Reduce leukocyte rolling and adherence Munger et al. [85]
Decrease neutrophil recruitment Leonard et al. [82]
Asthma
Block airway hyper-responsiveness and pulmonary inflammation Levy et al. [86]
Cystic fibrosis
Decrease neutrophilic inflammation, pulmonary bacterial burden Karp et al. [87]
and disease severity.
Angiogenesis
Reduce angiogenic phenotype: endothelial cell proliferation Fierro et al. [58]
and migration
Periodontitis (oral inflammation and bone loss)*

Reduce microbe-initiated, neutrophil-mediated tissue damage Serhan et al. [61]
and bone destruction
Eye
Accelerate cornea re-epithelialisation, limit neovascularisation, Cotran et al. [88];
and promote host defense in the eye Gronert et al. [77]
Bone marrow transplant

Protect against graft-vs-host diseases (GvHD) Devchand et al. [89]
*The cited animal models were carried out with mice or rats, except for periodontitis, carried
out in rabbits.
97
Charles N. Serhan
Table 2 - Resolvins and protectins: Actions of animal disease models.
Mediator Action Reference

Resolvin E1 Reduces PMN infiltration, murine skin air Serhan et al. [18];
pouch inflammation, peritonitis Serhan et al. [13];
Arita et al. [46];
Bannenberg et al. [44]
Gastrointestinal protection in TNBS colitis Arita et al. [49]
Protects in periodontitis, stops inflammation Hasturk et al. [76]
and bone loss
Resolvin D1 Reduces PMN infiltration, murine skin air Serhan et al. [13]
pouch inflammation
Reduces peritonitis Hong et al. [14]
Reduces cytokine expression in microglial cells
Protects in renal ischaemic injury Duffield et al., [90]
Protectin D1/ Reduces stroke damage Marcheselli et al. [15]
neuroprotectin D1 Reduces PMN infiltration Hong et al. [14]
Protects from retinal injury Mukherjee et al. [16]
Regulates Th2 cells, apoptosis and raft Ariel et al. [91]
formation
Shortens resolution interval in murine Bannenberg et al. [44]
peritonitis; regulates cytokines and chemokines
Protectin D1/ Reduces PMN infiltration; reduces peritonitis Serhan et al. [17]
neuroprotectin D1 Diminished production in human Alzheimers Lukiw et al. [92]
disease and promotes neural cell survival
Promotes corneal epithelial cell wound healing Gronert et al. [77]
There are many local, short-acting chemical mediators that are pro-inflamma-
tory mediators that are initially produced while granulocytes approach invading
microbes to be neutralised [26]. From the early studies of Borgeat and Samuelsson
[27], it is clear that the peripheral blood neutrophils, when encountering materials
to devour after they exit the post-capillary venules, release mediators derived from
arachidonic acid via both cyclooxygenase (COX) and lipoxygenases (LOs) that are,
for the most part, pro-inflammatory [1]. Consider, for example, pro-inflammatory
LTB4, which is a potent chemoattractant involved in the recruitment of additional
neutrophils and leukocytes to the initial area of insult. Lipid-derived chemoattrac-
tants like LTB4 work in concert spatially and temporally with peptide chemoattrac-
tants, chemokines and cytokines (for reviews see [28, 29]). Together, the appearance
of these mediators is enhanced in many inflammatory diseases, and hence they are
targets of many pharmaceutical companies that quest to control inflammation.
98
Figure 2.
RvE1 Biosynthesis from EPA.
Left panel: TNF-_-stimulated PMN infiltration spontaneously resolves during this phase,
indicated by ?. RvE1 was identified [18]. Right panel: The biosynthesis of RvE1 in human
cells was established. For example, hypoxic human endothelial cells expressing COX-2
treated with aspirin transform EPA. The mechanism involves abstracting hydrogen at carbon
16 in EPA to give R insertion of molecular oxygen, yielding 18R-hydroperoxy-EPE that is
reduced to 18R-HEPE. They can be further converted via sequential actions of human leuko-
cyte 5-lipoxygenase leading to formation of the trihydroxy bioactive product RvE1 [13]. The
complete stereochemistry of RvE1 is 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-EPA and one
of its receptors identified as a G protein-coupled receptor [46].
Because there are a very large number of pro-inflammatory mediators gov-

erning the vitality of the amplification phase from initial host-defence response
to microbes, surgical trauma, and/or injury from within [10], searching for key
regulators to control the excessive trafficking is a paramount task. The neutrophils
dual mask and that of the effector response of neutrophils are well known [28]. As
primary defenders, neutrophils are able to congregate swiftly in large numbers, yet
99
Charles N. Serhan
Figure 3.
Protectin and D series resolvin biosynthesis.
DHA is precursor to the lipoxygenase product 17S-H(p)DHA that is converted to a 16(17)-
epoxide converted to the 10,17-dihydroxy bioactive product [14] denoted earlier as 10,
17S-docosatriene (DT) [15] and recently coined neuroprotectin D1/protectin D1 based on its
potent actions in vivo [15, 16]. The complete stereochemistry was recently established [17].
Aspirin-triggered epimers: The 17R series resolvins are produced from DHA in the presence
of aspirin. Human endothelial cells expressing COX-2 treated with aspirin transform DHA to
17R-HpDHA. Also, recombinant COX-2 treated with aspirin converts DHA to 17R-HpDHA.
Human PMN convert 17R-HDHA to two compounds via 5-lipoxygenation; each is rapidly
transformed into two epoxide intermediates. One of these is a 7(8)-epoxide [13] and the
other a 4(5)-epoxide. These two novel epoxide intermediates can be enzymatically opened
to bioactive products denoted 17R series ATRvD1 through ATRvD6 [13]. The total organic
synthesis of RvD was recently reported [96].
they can inadvertently spill noxious agents, intended to kill or neutralise invaders.
These anti-microbial agents released or spilled from phagocytes can in turn evoke
tissue damage and inflammation. The uncovering of novel endogenous mediators of
anti-inflammation that control or dampen inflammation to keep it self limited and
100
Figure 4.
Temporal-differential analyses of resolution, mediator lipidomics, and proteomics.
Schematic outline of the approach to identifying pathways, mediators, proteins, and genes
critical to resolution.
promote resolution (summarised in Figs 24) raised awareness of the potential for
new therapeutic approaches to inflammatory diseases that target these active path-
ways [30, 31] and potentially the molecular basis underlying deficiencies in essential
t-3 PUFA and related pathways.
Temporal progression _ signals t in resolution:

Arachidonic acid to t-3-derived mediators
We initiated a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-

based informatics approach for systematic studies of lipid mediators within the
course of a spontaneously resolving inflammatory response (Fig. 2 and [32]). Dur-
101
Charles N. Serhan
ing the initial phase, PGs such as PGE2 are generated [33] that are involved in the
early steps in the control of blood flow and vessel dilation needed for leukocytes to
undergo firm adhesion and diapedesis [34]. The required traffic from the post-capil-
lary lumen to the interstitial space is a process that is, in part, governed by LTB4.
Programmed within this initial phase, there is also the activation of signalling path-
ways for the normal self-limiting or termination at local contained sites of inflam-
mation [13, 18, 33]. Signalling pathways [35] lead to PGE2 and PGD2, which in turn
actively switch on production at the transcriptional level of enzymes required for
the generation of LXs [33], as well as the novel families of lipid mediators, resolvins
and protectins (Figs 2 and 3) generated from t-3 PUFA that signal for resolution in
this phase of the tissue response.
The LXs are now widely appreciated for their ability to actively promote reso-
lution by regulating the entry of new neutrophils to sites of inflammation [36] and
organs of reperfusion injury [37]; they reduce vascular permeability and oedema
[38, 39], while also stimulating the nonphlogistic infiltration of monocytes [40]
that appear to be required for wound healing, and stimulate macrophages to
uptake apoptotic neutrophils [41]. This temporal switch in lipid mediator class
within the family of eicosanoids from pro- to anti-inflammatory eicosanoids (e.g.
the progression of PG and LT to LX) is an active process and also underscores
the ability of leukocytes to trigger the self-limited response of acute inflammation
[33]. This switch in lipid mediator classes of arachidonate-derived eicosanoids also
appears to be linked to a change in the phenotype and the internal cellular clock
of individual neutrophils within the site of inflammation, for example, within
pustules or contained sites of inflammatory exudates. Once a neutrophil para-
chutes into an evolving exudate, it can, by interacting with cells in its immediate
surroundings (i.e., other leukocytes, blood-borne cell types, platelets, endothelia,
mucosal epithelia [42] and/or interstitial cells, fibroblasts), via transcellular bio-
synthesis switch its lipid mediator profile from LT to LX biosynthesis [33]. This
initiation of the termination sequence in the early steps of the phagocytes response
appears to be a circuit that is utilised at the extracellular mediator level as well as
the intracellular signalling events via NF-gB [43]. It follows then that the begin-
ning events in inflammation governed by arachidonic acid-derived mediators
(alpha) signal the end (omega) or termination that utilises t-3 PUFA precursors to
biosynthesise new mediators in resolution.
Spontaneous resolution: Identification of resolvins and protectins
During the course of acute inflammatory response, mediators not only switch
classes but also substrates to form novel families of chemical mediators [44]. The
air pouch, initially studied in rats by Willoughby and colleagues [45], undergoes
spontaneous resolution. In Figure 2, the systematic analysis of this phase using a
102
lipidomic/lipid mediator informatics approach employing LC-MS/MS revealed that,

during the time course of spontaneous resolution, novel lipid mediators were gener-
ated that were not previously known. These new resolution phase mediators utilise
t-3 PUFA (DHA and EPA) as substrates and are agonists for promoting resolution
by stopping the further recruitment of leukocytes. The first of these are resolvins of
the E series that are biosynthesised from the precursor EPA [13, 18, 46].
The t-3 PUFA were widely known to play an essential role in maintaining
healthy organs [3]. Many reports in the clinical literature emphasise the importance
of t-3 supplementation in correcting disease-mediated events. However, until the
GISSI studies [4, 47, 48], this investigator remained cautioned on the impact of
t-3s in many diseases. Indeed, the role(s) and mechanisms of t-3 EPA and DHA
at the molecular level were the subject of much debate. The major mechanism of
action for t-3s was thought to block formation of pro-inflammatory mediators
via substrate competition [4]. In sharp contrast, during the course of inflammation
and its resolution, we found that t-3 PUFAs are utilised to generate novel lipid
mediators that are agonists in anti-inflammation. Systematic analysis undertaken
to define the indices of resolution using a combined trafficking proteomic and
lipidomic approach (Fig. 4) revealed that representatives for each family of lipid
mediators (LXA4, RvE1 and NPD1/PD1) act at different steps or points within the
resolution indices recently defined [44] and given in Figure 5. Each mediator is anti-
inflammatory when given in vivo, but more importantly can promote resolution by
shortening the time interval (Ri) or time for catabasis and return of involved tissue
to homeostasis [44].
Systematic temporal-spatial analysis of murine peritonitis demonstrated a pro-
gram of events in resolution and that cellular trafficking and clearance is regulated
in an active fashion by lipid-derived mediators [44] as well as protein-derived medi-
ators, such as annexins [29, 41]. LXA4 and annexin-1 share the ability to regulate
the uptake of apoptotic neutrophils by macrophages (Fig. 1B). The identification of
the resolvin RvE1 receptor and its presence on leukocytes and dendritic cells (DCs),
as well as the key role of these cell types in inflammatory responses, underscores
the communication between early initial events in lipid-mediated biosynthesis and
their linking to cellular trafficking [46], as well as governance for inflammation as
in models of inflammatory bowel disease [49, 50].
Mapping of the resolution phase of acute inflammatory responses appears to be
organ specific as to the temporal relationship(s) between lipid mediator classes (cf.
[33, 44, 51]). Well-established and widely used drugs impact resolution [29, 35].
In this context, aspirin, which is well known for its ability to inhibit formation of
lipid mediators, such as PGs [52], actually triggers the generation of epimeric forms
of arachidonic acid [53], as well as the newly identified EPA and DHA-derived
mediators (Figs 13). These endogenous mediators and their aspirin-triggered (AT)
epimer forms demonstrate potent anti-inflammatory and pro-resolving actions (see
below and Tabs 1 and 2). The epimeric forms of LXs, for example, also denoted AT
103
Charles N. Serhan
Figure 5.
Resolution indices.
Resolution indices are a defined set of parameters enabling laboratory assessment of key
events in resolution. These are the precise impact of inhibitors, drugs, and novel endogenous
mediators.
15-epi-LXs, share their actions in vitro and in vivo, as appears to be the case with
EPA-derived resolvins and DHA-derived resolvins of the D series (17R-contain-
ing resolvins/protectins) (Tab. 2). There is also clear evidence that glucocorticoids
enhance the uptake and the limitation of apoptotic neutrophils [29, 41, 54], a key
step in the clearance or expedition of the return of the tissue to homeostasis. Hence,
the process of catabasis appears to be genetically programmed at the level of chemi-
cal mediators including lipid mediator and protein mediator levels to direct tissue
level events [44]. Their role is likely governing both intracellular and extracellular
signalling events that are involved in dampening inflammation and promoting its
resolution. Since aspirin and glucocorticoids both impact resolution and share, in
the case of LX, a common site of action (the LXA4 receptor) [29], it is possible that
other widely used common drugs can impact resolution pathways.
104
AT epimers: Jump-starting resolution
Cell-cell interactions in the vasculature are impinged upon by aspirin [53, 55].
Aspirin inhibits thromboxane production by platelets and prostacyclin biosynthesis
in vascular endothelial cells [56]. During polymorphonuclear leukocyte (PMN)-
endothelial and/or PMN-epithelial interactions, aspirin triggers the biosynthesis of
15-epi-LXs (AT LX; ATL) [53]. Both LX and ATL and their respective stable ana-
logues are potent regulators of transendothelial/transepithelial migration of PMN
across these cells and endothelial cell proliferation, in vitro and in vivo ([5759]
and Tab. 1). Also, transgenic mice overexpressing the human LXA4 receptor with
a myeloid-specific promoter display reduced PMN infiltration in peritonitis and
heightened sensitivity to LXA4 and ATL [60]. Transgenic rabbits overexpressing 15-
LO type I generate enhanced levels of LXs, have an enhanced anti-inflammatory sta-
tus, and are protected from the inflammatory bone loss of periodontal disease [61].
Taken together, the results of these studies heightened our awareness that PMN, in
addition to their host defence position and releasing mediators such as the classic
eicosanoids, prostanoids, and leukotrienes [52, 62], can also produce novel protec-
tive lipid mediators that actively counter-regulate inflammation (Figs 2 and 3).
In view of the compelling results from the GISSI study showing improvements
in > 11 000 cardiovascular patients [47, 48]: namely, reduction in sudden death by
~ 45% by taking ~1 g t-3 per day, we recently addressed a potential role of t-3
PUFA. Inspection of their protocol indicated that all patients were also taking daily
aspirin that remained unaccounted for in their published analysis. Despite very large
doses (mg to grams daily), an abundant literature with t-3 PUFA points to potential
beneficial actions in many human diseases, including periodontal [63], anti-inflam-
matory, and anti-tumour actions [4, 64]. Each of the three major human LO activi-
ties (5-LO, 12-LO, 15-LO) can convert DHA to various monohydroxy-containing
products; however, at the time their in vivo functions were neither apparent nor did
they display bioactivity [65, 66].
Aspirin is present as an active ingredient in more than 60 over-the-counter reme-
dies, making it a difficult substance to rigorously control for in some human studies.
In view of the aforementioned findings, the questions became apparent, namely, what
is the molecular basis for t-3s protective action, and are there potential overlap(s)
in their actions at the molecular and cellular levels? To address this in an experimen-
tal setting, we used murine dorsal skin pouches (Fig. 2). This model of inflammation
is known to spontaneously resolve in rats [45]. We adapted this for mice to include
both genetics and to set up lipidomics employing LC-UV-MS/MS-based analyses
geared to evaluating whether potential novel lipid mediators are indeed generated
during the resolution phase of inflammation [13, 18]. In this pouch, representing
an experimentally contained local inflammation, after ~ 4 h, PMN numbers began
to drop within the exudates. Exudates were taken at timed intervals, focusing on
the period of spontaneous resolution, and lipid mediator profiles were analysed
105
Charles N. Serhan
using LC-UV-MS/MS. We constructed lipid mediator libraries with physical proper-

ties (i.e., MS and MS/MS spectra, elution times, UV spectra, etc.) for matching and
to assess whether known and/or potential novel lipid mediators were present within
the exudates, and we are presently expanding these libraries and the software for
their matching [32]. If novel lipid mediators were encountered, their structures were
elucidated by carrying out retrograde analysis for both biogenic enzymatic synthesis
and total organic synthesis. This approach permitted assessment of structure-activ-
ity relationships as well as the scale-up required to confirm the bioactions of novel
compounds identified [13, 17, 18].
18R E series resolvins and 17R D series resolvins
Resolving exudates in mice contain 18R-HEPE as well as several related bioactive

compounds [18]. These novel compounds are produced from EPA by at least one
biosynthetic pathway operative in human cells. This pathway is illustrated in Figure
2; blood vessel-derived vascular endothelial cells treated with aspirin convert EPA to
18R-hydro(peroxy)-EPE that is reduced to 18R-HEPE. This conversion is enhanced
by hypoxia. The 18R-HEPE is released from endothelium and rapidly converted by
activated human PMN in their proximity, for example by adherent PMN that trans-
form 18R-HEPE to a 5(6) epoxide-containing intermediate that is further transformed
to the bioactive 5,12,18R-trihydroxy-EPE. This bioactive mediator termed a resolvin,
specifically RvE1 because it was identified in the resolution phase in mice, appeared
as cell-cell interaction/transcellular biosynthetic products with isolated human cells,
and importantly proved to be a potent regulator of PMN and inflammation (Figs 3
and 4). Without aspirin treatment, 18R-HEPE can be produced via P450-like mecha-
nisms as well as by intact microbes [67, 68]. Total organic synthesis of RvE1 as well
as related isomers was carried out and its complete stereochemical assignment was
established as 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-EPA [46].
In the resolving inflammatory exudates from mice given aspirin and DHA, we
also identified novel 17R-hydroxy-DHA (17R-HDHA) and several related bioactive
compounds (Fig. 3). Human microvascular endothelial cells, also aspirin-treated
in a hypoxia chamber, biosynthesise 17R-HDHA. In addition, DHA is converted
by human recombinant COX-2 [13, 14] to 13-hydroxy-DHA. With aspirin, this
switches to 17R-oxygenation to give epimeric AT forms, also in brain [13, 14], of
both resolvins (RvD1 through RvD4) and protectins (Fig. 3).
17S D series resolvins
Using lipid mediator-informatics and LC-MS/MS-based analyses, we learned that

without aspirin or added DHA the endogenous DHA (enriched in neural tissues)
106
was converted in vivo to a 17S series of resolvins (RvD1 through RvD4) as well
as docosatrienes (such as NPD1/PD1) [14, 15]. As in most structural elucidation
experiments, added substrates were used to confirm biosynthesis, and to isolate
quantities of the novel active principle for bioassay. In this case, given the large
doses in humans, experimental animals, and in vitro cell culture studies needed to
observe effects in t-3 supplement studies reported in the literature (see [47] and
references within), we anticipated that EPA and DHA needed to be added in these
studies, which proved not to be the case. Normal mouse tissue and isolated human
neural cells contain DHA that is available upon activation to produce NPD1/PD1
and RvDs in vivo [1316, 18] possessing potent actions (Tab. 2).
With microglial cells that liberate cytokines in the brain, the D class resolvins
block TNF-_-induced IL-1` transcripts and are potent regulators of PMN infiltra-
tion in brain, skin, and peritonitis in vivo [14, 15]. Of the protectin family, the
NPD1/PD1 pathway (Fig. 3), proved a potent regulator of PMN influx in exudates
at sites where it is formed from endogenous precursors [13, 14], limiting stroke
brain injury [15] and retinal pigmented cellular damage [16]. Other dihydroxy-
docosanoids were less active in these bioassay settings [14, 16].
Direct comparisons between the E versus the D resolvins (17R and 17S epimer
series) for their ability to regulate PMN in vivo were carried out [13, 14, 61]. Both
the D and E classes of resolvins are potent regulators of PMN infiltration. The RvD
class 17R series, triggered by aspirin, and the 17S series give essentially similar
results (DHA-derived tri-hydroxy resolvins), indicating that the S to R switch does
not diminish their bioactions. When injected i.v. at 100 ng/mouse, they both gave
~ 50% inhibition, and the RvE1 gave ~75 80% inhibition. In comparison, indo-
methacin at 100 ng/mouse (or ~3 +g/kg) gave roughly 25% inhibition [13, 14].
Pro-resolving biosynthetic pathways: Microbes in the mix
The main bioactive resolvins and protectins as representative members are shown in
Figures 2 and 3. The formation of these compounds may involve enzymes that are
also known to convert arachidonic acid as substrate. It is possible that, in view of
the many LOs identified to date with unknown function(s) and/or specific PUFAs as
substrates [69, 70], strategically positioned enzymes may be specifically involved in
pathways that produce these novel compounds. In general, LOs are defined by their
ability to convert PUFAs that contain cis,cis-1,4-pentadiene subunits to hydroper-
oxy-containing products that can serve as intermediates. A well-known substrate in
human tissues is arachidonic acid; the main LOs convert arachidonic acid to the cor-
responding 5S-, 12S-, or 15S-hydroperoxyeicosatetraenoic acids. These enzymes are
known as the arachidonate:oxygen 5-oxidoreductase (5-LO), arachidonate:oxygen
12-oxidoreductase (12-LO), and arachidonate:oxygen 15-oxidoreductase (15-LO).
The release and availability of substrate are critical to indicating the preferred sub-
107
Charles N. Serhan
strate of a given LO, which is best appreciated in the case of 5-LO and LT biosyn-
thesis. With the identification of LO via molecular cloning, many additional LOs
are known, but their preferred substrates and functions in vivo are not established.
These include, for example, 12R-LO, 15-LO-type 2, soluble 15-LO (LoxA), and 8S-
LO. Each LO was catalogued according to its position of molecular insertion into
arachidonate [7174]. It follows that specific hydrolase(s), synthase(s), and related
enzymes specialised to handle DHA and EPA-derived intermediates are likely to be
involved in these pathways. Of interest, fish, which are abundant in t-3 PUFA, actu-
ally biosynthesise these compounds, demonstrating that the resolvins and protectins
are highly conserved structures [75].
Resolvins and protectins in disease
Resolvins and protectins have potent agonist actions that are of interest in man-
aging human disease. RvE1 was identified in human plasma [46]. At nanomolar
levels, RvE1 dramatically reduced dermal inflammation, peritonitis, DC migra-
tion and IL-12 production (Tab. 2). We screened GPCRs and identified one,
denoted earlier as the orphan G protein-coupled receptor ChemR23, that medi-
ates RvE1 signal to attenuate NF-gB. Specific binding of RvE1 to this receptor
was confirmed using synthetic 3H-labeled RvE1 that was prepared and isolated
to confirm the specific interactions of RvE1 with ChemR23. Treatment of DCs
with small-interfering RNA specific for ChemR23 sharply reduced RvE1 regula-
tion of IL-12.
RvE1, as a synthetic anti-inflammatory lipid mediator, reduces leukocyte infil-
tration in several mouse disease models as well as in a rabbit model of periodontal
disease (Tab. 1). Administration of synthetic RvE1 blocks PMN infiltration in
periodontal disease [76] and protects against the development of 2,4,6-trinitro-
benzene sulphonic acid (TNBS)-induced colitis [49]. The beneficial action of RvE1
was quantified by increased survival rates, sustained body weight, improvement of
histological scores, reduced serum anti-TNBS IgG, decreased leukocyte infiltration
and pro-inflammatory gene expression including IL-12 p40, TNF-_, and inducible
nitric oxide synthase. Thus, RvE1 counter-regulates in vivo leukocyte-mediated
tissue injury and pro-inflammatory gene expression [44]. These findings show a
novel endogenous mechanism that may underlie the beneficial actions of t-3 EPA
and provide new approaches for the treatment of gastrointestinal mucosal and oral
inflammation.
PD1, NPD1 when generated by neural cells, was found to possess potent bioac-
tions both in vivo and in vitro [35]. The complete stereochemistry of PD1 (10,17S-
docosatriene), i.e. chirality of the carbon 10 alcohol and geometry of the conjugated
triene required for bioactivity, remained to be established and was recently assigned.
PD1 generated by human neutrophils during murine peritonitis and by neural tis-
108
sues was separated from related natural isomers and then subjected to LC-MS/MS
and gas chromatography-MS-based analyses [17]. Comparison with six 10,17-
dihydroxydocosatrienes prepared by total organic and biogenic synthesis showed
that PD1, identified earlier from human cells (Tab. 2), carries potent bioactivity; its
complete stereochemistry is 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-
hexaenoic acid.
Additional isomers identified in these studies include 615-trans-PD1 (iso-
mer III), 10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid
(isomer IV), and a double dioxygenation product 10S,17S-dihydroxy-docosa-
4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid that was also present in murine exu-
dates. 18O2 labelling showed that 10S,17S-diHDHA (isomer I) carried 18O in the
10-position alcohol, indicating sequential lipoxygenation. This biosynthetic route
is in sharp contrast to PD1 formation, which proceeds via an epoxide intermedi-
ate in situ and leads to the potent bioactive mediator. Synthetic PD1 at 10 nM
attenuated (~ 50%) human neutrophil transmigration and its 615-trans-PD1 was
essentially inactive. In addition, PD1 proved to be a potent regulator of PMN
infiltration (~ 40% at 1 ng/mouse) in peritonitis. The rank order at 110 ng dose
was PD1 5 PD1 methyl ester >> 615-trans PD1 > 10S,17S-diHDHA (isomer I). Of
interest to potential treatment roles for these new compounds, PD1 also reduced
PMN infiltration after initiation (2 h) of inflammation and was additive with RvE1.
These results establish that PD1 is a potent stereoselective anti-inflammatory mol-
ecule [13, 14, 17]. Moreover, results of studies with Bazan and colleagues in neural
tissues (Tab. 2) and recently other laboratories [77] demonstrate and confirm that
PD1 displays potent protective actions as well as wound healing capacity. Table 3
indicates the abbreviations and stereochemistry of some lipid mediators and related
isomers used in this chapter.
Concluding remarks
The resolvins and protectins are new families comprised of distinct chemical series
of t-3 PUFA-derived mediators, each with unique structures and apparent comple-
mentary anti-inflammatory actions. These families of compounds, resolvins and
protectins, are also generated when aspirin is given in mammalian systems in their
respective epimeric forms, as we established earlier with the LXs and their AT-15-
epi-LXs [2]. Since both aspirin and glucocorticoids impact resolution and share, in
the case of LX, a common site of action (the LXA4 receptor), it is possible that other
widely used common drugs can impact resolution pathways. The resolvins and pro-
tectins each dampen inflammation and PMN-mediated injury from within, which
are key culprits in many widely occurring human diseases. The results of our stud-
ies to date underscore their role(s) in resolution as well as catabasis, and spotlight
potential therapeutics for this new arena of immunomodulation and protection. It
109
Charles N. Serhan
Table 3 - Abbreviations and stereochemistry for some lipids and isomers used in this chapter
5S,15S-diHETE 5S,15S-dihydroxy-6E,8Z,11Z,13E-eicosatetranoic acid

7S, 17S-diHDHA 7S,17S-dihydroxy-docosa-4Z,8E,10Z,13Z,15E,19Z-hexaenoic acid
(resolvin D5)
10S-HDHA 10S-hydroxy-docosa-hexaenoic acid
10S,17S- 10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid
docosatriene (the dioxygenation product)
10,17-docosatriene 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15E,19Z-hexaenoic acid;
isomers 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid;
10S,17R-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid;
10S,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid
17S-HDHA 17S-hydroxy-docosa-4Z,7Z,10Z,13Z,15E,19Z-hexaenoic acid
17S-H(p)DHA 17S-hydroxy(peroxy)-docosa-4Z,7Z,10Z,13Z,15E,19Z-hexaenoic acid
PD1 Protectin D1/neuroprotectin D1, 10R,17S-dihydroxy-docosa-
4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid
RvE1 Resolvin E1, 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosa-
pentaenoic acid
is likely that the resolvins, protectins and their AT-related forms may play roles in
specific tissues and organs. It is likely that the resolvins and protectins are conserved
in evolution since they are made by fish from t-3, possibly to serve as self-protec-
tive and host-protective chemical mediators. In view of the essential roles of DHA
and EPA in human biology and medicine uncovered to date [78], the physiologi-
cal relevance of the resolvins and protectins is likely to extend beyond our current
understanding [1316, 18].
Acknowledgments
We thank Mary Halm Small for assistance in preparing the manuscript. Studies
in the authors laboratory were supported in part by National Institutes of Health
grants GM38675, DK074448 and P50-DE016191.
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117
Beyond inflammation: Lipoxins; resolution of inflammation
and regulation of fibrosis
UCD School of Medicine and Medical Science, UCD Conway Institute, University College
Dublin, Ireland
Introduction
It is increasingly apparent that effective host defence involves biphasic production of

mediators. An initial acute response involves leukocyte activation and recruitment, a
second phase is characterised by the production of mediators regulating phagocytic
clearance of apoptotic cells and the active suppression of the initial inflammatory
response [110]. Eicosanoid production in inflammation tightly regulates these
processes. During the initial phase, proinflammatory mediators including leukot-
riene (LT) B4, the cysteinyl LTs and prostaglandins (PG) evoke potent chemotactic
responses of leukocytes whose activation is coupled to the production of proinflam-
matory (Th1-derived cytokines) at sites of inflammation [11]. To facilitate resolu-
tion, a second phase of lipid mediators may be produced favouring agents with
pro-resolution activities, including lipoxins (LXs) and the more recently described
resolvins and protectins [5, 1220].
Lipoxins: the originals of the species
The term lipoxin (LX) is an acronym for lipoxygenase interaction products, which
describes the provenance of these lipid mediators. LXs were first described by
Serhan et al [21] and initial observations stressed their role as anti-inflammatory
mediators inhibiting polymorphonuclear neutrophil (PMN) chemotaxis, adhe-
sion and transmigration across endothelia and epithelia [2225]. LXs are highly
conserved in the course of evolution since leukocytes from fish species are able
to generate LXs from endogenous sources of substrate [26]. 5S,6R,15S-trihy-
droxy-7,9,13-trans-11-cis-eicosatetraenoicacid (LXA4) and its positional isomer
5S,14R,15S-trihy-droxy-6,10,12-trans-8-cis-eicosatetraenoic acid (LXB4) are the
principal species formed in mammals [27, 28]. LXs are typically formed by tran-
scellular metabolism initiated by sequential oxygenation of arachidonic acid by

both 5-lipoxygenase (LO) and 12-LO or 15 and 5-LO [13, 6, 7, 9]. In a cyto-
kine-primed milieu, aspirin acetylation of cyclooxygenase-2 (COX-2) switches the
catalytic activity of the enzyme to an R-LO with the formation of 15R hydroxye-
icosatetraenoic acid that is rapidly converted by 5-LO in activated PMN to 15-epi-
LX (aspirin-triggered LXs, ATL) that share many of the bioactions of the native
LXs [2, 3, 2932]. Aspirin-acetylated COX-2 is also involved in the production of
novel mediators from t-3 polyunsaturated fatty acid, called resolvins ([2, 5, 14,
15, 19, 20] and reviewed elsewhere in this volume).
LXs act locally and are rapidly inactivated by dehydrogenation at C15 and pos-
sibly by t-oxidation at C20 [13, 5, 6, 9]. When compared to the native LXs, the
ATLs display longer biologic half-life [13, 33]. To circumvent the metabolic inac-
tivation of native compounds, LX and ATL analogues were designed with specific
modifications of the native structures of LXA4 and LXB4, such as the addition of
methyl groups on C-15 and C-5 of LXA4 and LXB4, respectively, or with a phe-
noxy group bonded to C-16 replacing the t-end of the molecule [3335]. Recently,
a second generation of LX stable analogues, 3-oxa-LX analogues showing potent
actions in vivo were designed [36]. The availability of LX analogues active via oral,
topical, and systemic routes will facilitate studies on the functions and therapeutic
applications of LX in vivo [3638].
LXs are generated in vivo within an inflammatory milieu. A reduction in LX
production has been demonstrated in human diseases such as airway inflammation
[39, 40], cystic fibrosis [41], glomerulonephritis [42], in patients with chronic liver
disease [43] and in chronic myelocytic leukaemia due to the lack of 12-LO activity
in platelets [44]. In contrast, LXA4 production is up-regulated in localised juvenile
periodontitis [45] mild asthma [46], following atherosclerotic plaque rupture [47]
and with nasal polyps [48]. Aspirin-intolerant asthmatics display lower biosynthetic
capacity for these potentially protective lipid mediators relative to aspirin-tolerant
asthmatics or healthy subjects [46].
LX formation has been demonstrated in an immune complex model of glo-
merulonephritis [42], in pleural exudates upon allergen challenge in rats [49] and
in ischemic lungs [50]. Decreased LXA4 biosynthesis is associated with exaggerated
neutrophil infiltration in nephrotoxic serum nephritis in P-selectin knockout mice
and administration of wild-type platelets, which express P-selectin, restore LX gen-
eration [51]. LXA4 levels generated during microbial infection with Toxoplasma
gondii in a murine model are remarkably increased during the acute phase and stay
high during chronic disease [52, 53]. ATL have been detected in vivo, e.g. in an
aspirin-dependent manner in murine peritonitis [54], in dorsal air-pouches [55], in
rat kidney [42] and in liver [56]. ATL is formed in rat stomach after aspirin adminis-
tration, indicating that ATL production is one of the mechanisms of gastric adapta-
tion to aspirin [57]. Administration of low doses of aspirin to healthy subjects was
shown to significantly increase plasma levels of ATL with a concomitant inhibition
of thromboxane biosynthesis [58] with a positive correlation between age and ATL
120
Beyond inflammation: Lipoxins; resolution of inflammation and regulation of fibrosis
for women, but not for men [59], supporting in part the reported gender-dependent
therapeutics of aspirin [60].
LXA4 receptor: A receptor for pleiotropic ligands
LXA4 has been shown to bind with high affinity (subnanomolar) to specific cell
surface receptors. Although the limited availability of a high specific activity LXA4
radioligand has slowed progress towards defining the pharmacological charac-
teristics of LXA4 binding, it is now accepted that LXA4 binds to at least one G
protein-coupled receptor (GPCR), which has been cloned, characterised and des-
ignated as ALXR [3, 6, 7]. ALXR, belonging to the cluster of chemoattractant
peptide receptors, is expressed in neutrophils [61], monocytes [35], activated T
cells [62], basolateral membrane of gastrointestinal epithelial cells [63], synovial
fibroblasts [64], bronchial epithelial cells [65] and mesangial cells [66]. ALXR was
originally identified as a low-affinity N-formyl-methyonyl-leucyl-phenylalanine
(fMLP) receptor-like 1 (FPRL-1) and there is considerable evidence that ALX can
bind pleiotropic ligands, i.e. both lipid and peptide such as MHC binding peptide
(a potent necrotactic peptide derived from NADH dehydrogenase subunit 1 from
mitochondria) [67], anti-microbial peptides (e.g. LL37 and temporin A) [68, 69],
truncated chemotactic peptides (e.g. CKbeta8-1) [70], a urokinase type plasmino-
gen activator receptor (uPar) fragment [71], and the HIV envelope peptides [72,
73]. ALX can bind also prion protein [74], serum amyloid A [75], amyloid `42 [76]
and the glucocorticoid-inducible protein annexin 1 [55]. Annexin 1 and annexin
1 mimetics (shorter peptide from the N-terminal region of the protein), such as
peptide Ac226 showed anti-inflammatory actions in many experimental models
of inflammation [7780] (also reviewed in the chapter by Perretti and Flower). The
binding of lipids and small peptide to the receptor occurs with different affinities
and/or distinct interaction sites, facilitating activation of distinct signalling path-
ways that depends on the cell type and system [81].
Bioactions of LXs
LXs have been shown to modulate specific actions in cells of both myeloid and non-
myeloid origin typically consistent with the distribution of the ALXR [13, 6, 7, 9].
LXs, ATLs and stable synthetic LX analogues inhibit PMN and eosinophil chemo-
taxis [22, 23] as well as PMN adhesion to and transmigration across endothelial
cells and intestinal epithelia [24, 25, 82]. Both LXs and ATL antagonise many of the
effects of proinflammatory LTs including PMN-endothelial cell adhesion mediated
by CD11/CD18 expression [25], endothelial PMN adhesion dependent on endothe-
lial P-selectin [83] and integrin clustering and mobility on PMN [84].
121
LXs and ATL have been shown to play a key role in regulating cytokine-che-
mokine axes directly modulating the cytokine composition in the inflammatory
environment. In activated human synovial fibroblasts, LXs inhibit the synthesis
of inflammatory cytokines and matrix metalloproteinases, while stimulating tis-
sue inhibitor of metalloproteinase expression [64]. LXs and LX analogues inhibit
interleukin-8 (IL-8) release from tumour necrosis factor-_ (TNF-_)-primed colonic
cell lines [85], human colon ex vivo [86], and intestinal epithelia in response to
challenge with Salmonella typhimurium [87]. Interestingly, ALXR is preferentially
expressed on the basolateral surface of intestinal epithelia; therefore, LX generation
at the paracellular space via neutrophil-epithelial interactions can rapidly act on
the receptor to down-regulate intestinal inflammation [63]. Additional evidence for
the involvement of LXs in regulatory cytokine loops is demonstrated by the inhibi-
tion of TNF-_-stimulated IL-1` expression and superoxide production in LX- and
ATL-treated PMNs, effects mediated in part by suppression of NF-gB activity in the
nucleus [88, 89]. Indeed, modulation of NF-gB via activation of a specific GPCR
has been shown to underlie the anti-inflammatory bioactions of the anti-inflamma-
tory t-3-derived resolvin E1 [90].
LXs are potent inhibitors of mesangial cell proliferation in response to mitogens
such as LTD4 [66], platelet-derived growth factor (PDGF) and epidermal growth
factor, with a mechanism that involves elaborate cross-talk between AXLR and
receptor tyrosine kinases [66, 91, 92]. In addition to modifying proliferation, LXA4
can counteract PDGF-induced gene expression in mesangial cells [93]. Noteworthy,
amongst the genes whose expression was modified by LXA4 were PDGF-induced
profibrotic genes, suggesting that LXA4 might protect the tubulointerstitium from
the deleterious effects of the activated glomerulus. Consistent with this hypothesis,
supernatants derived from mesangial cells treated with PDGF caused a morpho-
logical change in murine renal tubular cells, with loss of epithelial tight junction
marker E-cadherin and gain of _-smooth muscle actin, whereas LXA4 pre-treatment
diminished these effects, suggesting that LXA4 has a potential anti-fibrotic activ-
ity, preventing epithelial mesenchymal transformation implicated in fibrosis [93].
LXs and ATLs have also been found to inhibit vascular endothelial growth factor
(VEGF)-induced endothelial cell proliferation and migration via inhibition of actin
polymerisation and assembly of focal adhesions [94, 95], and to inhibit prolifera-
tion of human lung fibroblasts by connective tissue growth factor [96].
In vivo models of disease
The stable analogues of LXs and ATL have been a useful tool to evaluate the role
of LXs in different experimental animal models. Stable analogues of LXs and ATL
have shown efficacy in a variety of models of dermal inflammation [32, 36, 97],
periodontitis, an inflammatory disease characterised by leukocyte-mediated bone
122
loss and inflammation caused by specific gram-negative microorganisms [98]. Ana-

logues of LXs and ATLs inhibit PMN recruitment to Phorphyromonas gingivalis in
a murine model [99].
The impact of LX on airway responsiveness and inflammation was recently
investigated in vivo showing that an LXA4 stable analogue attenuates both airway
hyperreactivity and inflammation in a murine model of asthma inhibiting the gen-
eration of proinflammatory mediators [100]. The dysregulated proinflammatory
environment of the cystic fibrotic airway, characterised by up-regulated IL-8 pro-
duction and persistent, destructive neutrophilic inflammation, is consistent with a
deficiency in LX-mediated anti-inflammatory activity [41]. In a mouse model of the
chronic airway inflammation and infection associated with cystic fibrosis, an ATL
stable analogue suppresses neutrophilic inflammation, decreases pulmonary bacte-
rial burden and attenuates disease severity [41].
ATLs are protective in intestinal inflammation in a mouse model of dextran sodi-
um sulphate-induced colitis [101] and have a role in defence against opportunistic
infections inducing transcriptional activation of bactericidal/permeability-increasing
protein by epithelial cells of the mucosa to produce a protective protein that inhibits
endotoxin signalling [102]. LXs are potential therapeutics in ischaemic acute renal
failure, showing a protective action in an experimental murine model of acute renal
failure in vivo [103, 104]. Administration of the ATL, prior to ischaemia, resulted
in significant functional and morphological protection, and attenuated chemokine
and cytokine responses and modification of the expression of many differentially
expressed pathogenic mediators, including cytokines, growth factors, adhesion mol-
ecules, and proteases [103, 104].
Resolution of inflammation and phagocytosis of apoptotic cells
Resolution of inflammation is a dynamically regulated process that may provide

several opportunities for therapeutic intervention [5, 10]. In this regard, as previ-
ously discussed, the switch in the production from pro-inflammatory mediators to
LXs as well as resolvins and protectins is critical [5, 1220]. As a consequence, the
recruitment of PMNs ends and they can undergo apoptosis. This temporal switch
in lipid class is, therefore, an active process able to trigger a self-limiting response
to acute inflammation [4, 5, 8].
During inflammation the removal of apoptotic cells is an important step in
sparing tissue from exposure to the noxious and immunogenic contents of necrotic
cells and is a prerequisite to restore normal tissue function and plays a critical role
in the resolution of inflammation [4, 5, 8, 105107]. Clearance of apoptotic cells
is mediated by professional phagocytes such as macrophages (M\) and immature
dendritic cells and by a variety of non-professional phagocytes that engulf apoptotic
cells albeit with less efficiency [105109]. In contrast to phagocytosis of bacteria
123
and opsonised particles, the engulfment of apoptotic cells is associated with the
release of anti-inflammatory mediators, such as TGF-`, IL-10 and PGE2 and with
inhibition of the secretion of pro-inflammatory mediators, such as TNF-_, as dem-
onstrated by in vitro and in vivo studies [110112], and triggers secretion of VEGF,
which is critical for repair of endothelial and epithelial injury [113].
Apoptosis induces cell surface changes that are important for recognition and
engulfment of cells by phagocytes [4, 5, 8, 105107]. In addition, opsonisation of
apoptotic cells by components of the innate immune system such as complement
factors facilitates and modulates the clearance of apoptotic cells by classical phago-
cytic receptors [114]. Other potential opsonins such as the collectins, pentraxins and
anticoagulant proteins may be involved in the opsonisation of apoptotic cells and
they have the ability to bind not only to intact apoptotic cells, but also to micropar-
ticles that are released from the cell during apoptosis [114, 115]. Phagocytes show
significant redundancy in recognition strategies and are able to use many receptors
at the same time, reflecting multiple phases in the interaction between apoptotic cells
and phagocytes. Some receptors may simply play a role in tethering of phagocyte
to apoptotic cells without generating a signal, whereas others may activate a signal
pathway leading to cytoskeleton rearrangements and engulfment [8, 105]. Crucial
regulators of actin-based cytoskeleton rearrangement as a consequence of apoptotic
cell recognition include Rho GTPases (Rho, Rac and cdc42), and phosphatidylino-
sitol 3-kinase (PI3K) that play a role in the extension of pseudopodia and in the
formation and the maturation of the actual phagosome [116]. Finally, the ingested
particle enters the lysosomal system in the phagocyte where it is degraded. Defin-
ing the ligands on apoptotic cells and the corresponding receptors on phagocytes is
likely to lead to the development of novel anti-inflammatory pro-resolution drugs.
Among the multiple changes on the surface of the apoptotic cells that facilitate
their recognition, the best characterised is the loss of phospholipid asymmetry
and subsequent exposure of phosphatidylserine (PS) [117, 118]. While necessary,
PS exposure is not sufficient to complete clearance of apoptotic cells [119, 120],
suggesting that other recognition factors might be expressed on apoptotic cells to
facilitate their uptake. Recently, annexin 1 was found to co-localise with PS in apop-
totic cells and was associated with the efficient tethering and internalisation [121].
Annexin 1 is exported from the cytosol to the plasma membrane of apoptotic cells
by a mechanism dependent on caspase activation and is required for the clustering
of overexpressed PS receptor around apoptotic cells [121].
Since the identification of the M\ vitronectin receptor (_v`3) as the first recep-
tor to recognise and engulf apoptotic cells [122], numerous molecules involved in
phagocytosis of apoptotic cells, belonging to many different receptor families, were
characterised including PS receptor, the scavenger receptors, lectins, the receptor
tyrosine kinase Mer, the lipopolysaccharide receptor CD14, which binds to the Ig-
superfamily member adhesion molecule ICAM-3, members of the collectin family
and their receptors CD91 and calreticulin (see reviews [8, 105107, 123]).
124
The modulation of phagocytic capacity for apoptotic cell clearance represents

a potential therapeutic target in the control of inflammatory disease since defects
in clearance of apoptotic cells have been closely associated with several chronic
inflammatory conditions including systemic lupus erythematosus (SLE) in which
autoantibodies against a number of self antigens derived from apoptotic cells are
developed [124, 125]. Further evidence for the importance of efficient mechanisms
for apoptotic cell clearance in vivo is supported by the observation that autoimmune
responses can be provoked in mice when key molecules for apoptotic cell recogni-
tion and uptake, such as complement protein C1q, the tyrosine kinase receptor gene
Mer and milk fat globule factor 8, are missing or mutated [126128]. Interestingly,
an increased number of apoptotic cells have been demonstrated in the airways of
patients with cystic fibrosis and non-cystic fibrosis bronchiectasis, probably as a
consequence of elastase-mediated cleavage of PS receptor on phagocytes [129].
Endogenous regulators of phagocytosis of apoptotic cells
Given that recognition and engulfment of apoptotic cells is an important process

in the resolution of inflammation, a positive regulation of the capacity of M or
non-professional phagocytes for phagocytosis of dying cells represents a potential
therapeutic target in the control of inflammatory disease. In recent years, the role
of endogenous anti-inflammatory mediators in the modulation of these processes
has emerged. Phagocytosis of apoptotic cells is stimulated by glucocorticoids, which
promote cytoskeleton rearrangement [130, 131]. Recently, a potential role of lipid
mediators has emerged by the observation that apoptotic cells release, through
caspase-3-mediated activation of phospholipase A2, phosphatidylcholine (LPC) that
can specifically attract phagocytes to sites of resolution [132]. The release of chemo-
tactic factors by dying cells could actively participate in their own removal through
cell recruitment. Indeed, research in our laboratory has shown that apoptotic cells
may release both chemotactic and pro-phagocytic factors associated with actin rear-
rangement and release of TGF-` (unpublished observations). The identification of
such factor(s) has recently been achieved [132a].
LXs and phagocytosis
As previously discussed native LXs and ATL are well-described braking signals in
inflammation [13, 6, 7, 9]. In contrast to inhibiting PMN function, LXs are potent
activators of monocytes, stimulating their chemotaxis and adherence without caus-
ing degranulation or release of reactive species [35]. This observation suggested that
LXs might be involved in the recruitment of monocytes to sites of wound healing or
clearance. We have shown that native LX, ATL, and stable synthetic LX analogues
125
Figure 1.
Fluorescence micrograph of lipoxin (LX)-stimulated macrophage (M\) ingesting an apoptotic
polymorphonuclear leukocyte (PMN).
Differentiated THP-1 or human monocytes derived M\ were treated with LXA4 (1 nM for
15 min) before co-incubation with aged PMNs for 30 min. Cells were fixed with paraformal-
dehyde. (A) THP-1 stained with ALXR antibody (C1508, kind gift from Dr. J. F. Parkinson,
Berlex, CA, USA), followed by Alexa Fluor 568 goat anti-rabbit antibody (Molecular Probes,
Eugene, OR). Cells were stained with Oregon Green phalloidin (Molecular Probe) to visual-
ize actin. Images were obtained using a Zeiss LSM 510 META scanning confocal microscope.
(B) Localisation of actin was determined in M\ using Oregon Green phalloidin and nuclei
were stained with Hoechst 33258. Images were visualised by fluorescence microscopy using
an 100 oil objective.
promote the resolution of inflammation stimulating non-phlogistic phagocytosis of

apoptotic PMNs (Fig. 1) and lymphocytes by M\ in vitro and in vivo in a murine
model of thioglycolate-induced peritonitis [133135]. Consistent with a role for LX
promoting the resolution of inflammation are the observations that LX-stimulated
phagocytosis is associated with increased TGF-`1 release from M\ and a suppression
of IL-8 and monocytes chemotactic protein-1 (MCP-1) release [133, 134]. The effect
of LXs on phagocytosis of apoptotic PMNs by M\ can be blocked by antibodies
to several M\ surface proteins known to contribute to the recognition of apoptotic
leukocytes such CD36, _v`3 and CD11b/CD18 and it is mediated by protein kinase
C and PI3K [133, 134]. A modulatory role for cAMP is suggested by the observa-
tion that LX-induced phagocytosis is inhibited by a cell permeant cAMP analogue
and mimicked by a PKA inhibitor [133]. Furthermore, LXs might prime M\ for
chemotaxis and phagocytosis inducing changes in the ultrastructure and reorgani-
sation of actin in human monocytes and M\ (Fig. 2), resulting in the promotion
126
Figure 2.
LXA4, its stable analogue 15-(R/S)-methyl-LXA4 and Ac2-26 induce actin reorganisation in
human monocyte-derived M\.
M\ were exposed to vehicle, LXA4 (1 nM), 15-(R/S)-methyl-LXA4 (10 pM) and Ac2-26
(32 +M) for 15 min at 37C. Cells were fixed with paraformaldehyde and localisation of actin
was determined using Oregon Green phalloidin and visualised by fluorescence microscopy
using a 100 oil objective.
of cytoplasmic extensions and in the formation of pseudopodia with a mechanism

that is dependent on activation of the GTPases RhoA and Rac [136]. In addition,
LXA4-stimulated phagocytosis is associated with decreased phosphorylation and
redistribution of MYH9, a non-muscle myosin H chain II isoform A, involved in
127
cytoskeleton rearrangement and polarisation of M\ with activated cdc42 localised

toward the leading edge and MYH9 at the cell posterior [135]. LXA4 stimulates the
phosphorylation of polarity-organisation molecules such as Akt, protein kinase Cc
and glycogen synthase kinase-3`, suggesting that the effect of LXA4 on cell polarisa-
tion is a key early event in LXA-induced phagocytosis [135].
Bone marrow derived-M\ (BMDM) are uncommitted phagocytes that can
develop phenotypically distinct properties after cytokine programming. Interferon-a
(IFN-a)/TNF-_ stimulated-BMDM are characterised by sustained nitric oxide (NO)
production and a diminished phagocytic capacity, whereas exposure of BMDM to
TNF-_ stimulates phagocytosis of apoptotic PMNs and is not associated with NO
release, characteristic of a reparative phenotype. Intriguingly, exposure of TNF-_-
programmed M\ to LX further enhances their ability to phagocytose apoptotic
PMN, whereas LX rescues the compromised phagocytic activity of IFN-a/TNF-_-
primed BMDM [134].
The beneficial effect of LXs on phagocytosis of apoptotic cells may contribute
to broaden the potential role of LXs in the treatment of diseases in which impaired
apoptotic cell clearance has been demonstrated, for example in cystic fibrosis [129].
In addition, the ability of the ALXR to interact with amyloid [76, 77] suggests that
it may have a role in modulating the accumulation of extracellular amyloid, a key
feature of neurodegenerative disorders. In addition, the promotion of resolution by
apoptotic cell clearance and the previously mentioned modulation of growth factor
receptor signalling [9193] suggest a potential anti-fibrotic role for LXs.
Agonists of LX receptor and phagocytosis
As previously discussed ALXR can bind pleiotropic ligands, i.e. both lipid and
peptide [68]. The release of N-formylated peptides from mitochondria of damaged
cells is a signal for PMN chemotaxis [68]. Interestingly, the peptide mimetics MHC-
binding peptide (MHC bp, MYFINILTL) derived from NADPH dehydrogenase and
a synthetic rogue peptide MMK-1 (LESIFRSLLFRVM) stimulate M phagocytosis
of apoptotic cells in association with TGF-` release via the ALXR [134].
A role for endogenous annexin in phagocytosis of apoptotic cells has been
recently hypothesised through the observation that annexin 1 is exported from the
cytosol to the plasma membrane of apoptotic cells [121]. In addition, phagocytosis
of apoptotic lymphocytes by M\ was inhibited by pre-treatment of either target
cells or phagocytes with an antibody to annexin 1, suggesting that annexin serves
as both ligand and receptor in promoting phagocytosis [137]. The N-terminal
peptide of annexin 1, Ac2-26 promotes phagocytosis of apoptotic PMNs through
a mechanism involving the ALXR [138]. This effect is coupled to TGF-`1 release
and to changes in F-actin reorganisation in M\ (Fig. 2) and MYH9 dephosphoryla-
tion and redistribution [135, 138]. Interestingly, the endogenous annexin 1 released
128
by dexamethasone-treated M\ presents a pro-phagocytic effect of apoptotic cells

by M\ and induces actin rearrangement [138], suggesting a physiological role of
endogenous annexin 1 and expanding the therapeutic potential of ALXR agonists.
Conclusions
The uptake of apoptotic cells by professional phagocytes such as M\ is an impor-

tant step in the resolution of inflammation that may be harnessed for therapeutic
gain. However, more studies are necessary to define a clear link between impaired
phagocytosis of apoptotic cells and inflammatory diseases. The potential pro-
resolution activities of lipid mediators such as LXs and alternative LX receptor
agonists may be of benefit in this context. Additionally, the growing appreciation
of the role of non-professional phagocytes in disposal of apoptotic cells coupled
to the diversity of cell types responsive to LX suggest that this may be a more
widespread phenomenon than previously thought. The powerful anti-inflamma-
tory and pro-resolution actions of endogenous and aspirin-triggered LXs coupled
to their efficacy in vivo suggest these agents possess therapeutic potential for use
in human disease.
Acknowledgements
Work in the Authors laboratory is supported by The Health Research Board
Ireland, Science Foundation Ireland and The Wellcome Trust and funded under
the Programme for Research in Third Level Institutions by the Higher Education
Authority and EU FP 6 EICOSANOX Program LSHM-CT-2004-005033.
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Anti-inflammatory glucocorticoids and annexin 1
William Harvey Research Institute, Charterhouse Square, London EC1M 6BQ,

United Kingdom
Glucocorticoids: From endogenous anti-inflammation to drug exploitation
It is now evident that several endogenous anti-inflammatory pathways are activated

in parallel with the host inflammatory response to maintain a homeostatic control.
From this idea has arisen the concept of anti-inflammation, a term used to describe
the balance that exists between pro-inflammatory and anti-inflammatory media-
tors/pathways that operate in concert to initiate, maintain and finally resolve the
inflammatory reaction.
Leukocyte extravasation is a hallmark of the inflammatory reaction mounted by
the host to counteract xenobiotic invasion and infections, thus a good understand-
ing of its functioning is likely to be a fruitful option for identifying new targets for
innovative drug discovery [1]. Pro-inflammatory mediators operate in an interre-
lated and complex manner to co-ordinate the onset and resolution of this process.
This list includes chemoattractants, cytokines, adhesion molecules and proteolytic
enzymes to name just a few [2, 3]. The crucial role of inflammation in host defence
is demonstrated by the poor life expectancy of patients having a genetic deficiency
in integrin expression, and who are thus unable to mount a rapid and effective
leukocyte extravasation process [4]. Therefore, inflammation is a protective process
overall and its perfect functioning is crucial for health. Several anti-inflammatory
mediators and mechanisms (e.g. apoptosis [5]) operate within the host to promote
and control the resolution phase that is characterised by a reduction in leukocyte
egress from the blood vessels into the extra-vascular tissue [6]. If one or more of
these pathways are altered or ineffective, this could lead to an inflammatory pathol-
ogy, such as asthma, rheumatoid arthritis or inflammatory bowel disease. There
are several examples of anti-inflammatory agonists including low molecular weight
molecules, ranging from adenosine to lipoxins, as well as more complex substances,
from galectins to melanocortins [610].
Within this large group of anti-inflammatory mediators, it is the glucocorticoids
(GC; cortisol in man and corticosterone in rodents) that were the first to be identi-

fied and to be fully exploited for drug development. Current synthetic derivatives
of these hormones are the most effective drugs for an array of therapeutic applica-
tions.
Historical view
In 1949, a hormone Compound E, prepared from the adrenal cortex was found
to posses potent anti-arthritic properties [11]. It was a seminal discovery that led to
the further purification of GC and eventually to their extensive clinical application
in the treatment of several chronic inflammatory pathologies. However, at the time,
Henchs observations came as a big surprise. The prevailing ethos of the day was
that the mobilisation of GC, which occurred during injury represented part of the
general adaptation syndrome a term coined by Selye to describe the co-ordi-
nated response of an organism to injury or stress. In this context, it was believed
that these hormones probably contributed to the inflammatory response. Henchs
observations therefore ran completely counter to the expectations of the day.
Nevertheless, the results were dramatic and reproducible. Within only a year or
so Hench had been awarded the Nobel Prize for Physiology or Medicine (jointly
with chemists Kendall and Reichstein) and the use of GC had been extended to
many other conditions including skin disorders, asthma and allergies. As a historical
note, it is interesting that the first compound that Hench administered (Compound
E) was actually cortisone the inactive 11-ketone metabolite of the endogenous
hormone hydrocortisone, which we now know to have negligible binding affinity
at the GC receptor. However, the interconversion of cortisone and cortisol (hydro-
cortisone) can occur in some tissues in the body through the action of the 11`-
hydroxy steroid dehydrogenase enzyme, providing an explanation of this apparent
anomaly.
To begin with, preparations of GC were prepared semi-synthetically using, natu-
rally occurring plant steroid precursors. Initially, the supply of these hard-to-obtain
starting materials severely restricted the use of the drug to a few hospital clinics
(almost all within the US). It was a situation that put a strain on international rela-
tions at the time as news of the potential of this drug spread throughout the world.
However, within a few years, the total synthesis of GC initiated a new era in drug
discovery and enabled the pharmaceutical industry to commence a serious search
for analogues. By this time (mid 1950s) the side effects incurred by prolonged GC
usage were well recognised and the immediate aim of the drug development pro-
grams were to minimise these (this has been a Leitmotiv that recurs throughout the
entire gamut of GC research). The 1960s saw a veritable explosion in the number
of GC analogues available many of which were vastly more potent than the native
hydrocortisone molecule. Unfortunately, all shared virtually the same profile of side
effects.
142
In the late 70s, GC actions were being re-interpreted and the theory that these
hormones were important in preventing over-shooting of the host inflammatory
reaction was put forward in a landmark review by Munck and co-workers [12].
Thus, the following scheme was proposed; during an inflammatory response,
adrenocorticotropic hormone (ACTH) released from the anterior pituitary gland
acting on the adrenal gland promotes the release of GC, which down-regulates the
inflammatory response [13], ensuring that it does not damage the host. In agree-
ment with this idea, adrenalectomised animals exhibit an exacerbated inflammatory
response [14] and even normally mild inflammatory provocations may have lethal
consequences [15]. There is a circadian rhythm in ACTH secretion with a maximal
pulse of GC release occurs early in the morning [16]; superimposed on this, there
is an ultradian rhythm that results in a pulsatile release of these adrenal hormones
throughout the 24-h cycle [17]. Interestingly, alterations of this important physi-
ological loop early in life (e.g. by mean of stress or exposure to lipopolysaccharide)
affect subsequent responses of the host to inflammatory insults, as demonstrated in
many experimental systems [13, 18].
In parallel with this, largely endocrinological approach, substantial progress
was being made in understanding the molecular basis of GC action. The discovery
of intracellular binding proteins for steroid hormones, including the sex steroids as
well as the GC, led to the gradual realisation that the action of these hormones was
mediated by a sophisticated intracellular receptor system, which eventually culmi-
nated in changes in gene transcription.
The notion that the GC also exerted their anti-inflammatory effects in this way,
was supported by studies in the late 1970s when several groups were able to show
that the anti-inflammatory properties of these hormone/drugs could be abolished in
the presence of GC receptor antagonists or inhibitors of de novo protein or RNA
synthesis in several experimental models of inflammation. Since that time, this idea
has remained a corner stone of GC pharmacology, although, interestingly, increas-
ing attention is now being paid to the idea that the liganded receptor itself may
exert some cytosolic signalling action independent of its effect within the nuclear
compartment (as addressed below).
Genomic and non-genomic effects
The development of synthetic GC derivatives therefore represents the first and most
successful example of the exploitation of an endogenous anti-inflammatory mediator
for therapeutic purposes. The widespread clinical utility of GC is due to their mul-
tiple mechanisms of action, which poses a problem to drug discovery programmes
oriented towards single targets. At physiological doses GC regulate key metabolic
enzymes and up-regulate specific cytokine receptors thereby assuring the correct
physiological functions of the body [12, 19]; at higher doses, in the therapeutic
143
range, GC inhibit cytokine release, block the innate immune response and modulate
adaptive immunity [19, 20]. Most of the inflammatory actions occur within the
microcirculation of inflamed vascular beds [21]. At the molecular level, these effects
are again brought about by multiple mechanisms (see [22] and below).
GC produce profound effects on gene expression: a figure that is often quoted
is that approximately 1% of total genome transcription can be influenced by these
drugs. As these GC are lipophilic, they rapidly cross plasma membranes to reach
their specific cytoplasmic receptors, termed glucocorticoid receptors or GR (Fig.
1). In the conventional view, binding to the receptor is followed by disassociation
from bound proteins that form a complex with GR in the inactive status. There
are two potential outcomes. On one hand, the GR homodimer complex travels to
the nucleus where it binds to specific positive or negative glucocorticoid-response
elements (GRE) [22] that are present in the promoter region of target genes, to
increase or decrease gene transcription (Fig. 1). In the second option, a monomeric
GC-GR complex remains in the cytosol where it can bind to transcription factors,
preventing their activation, the end-point of which is blockade of gene expression.
Examples of transcription factors susceptible to GC inhibition include nuclear
factor (NF)-gB, and the complex c-jun/c-fos (activated protein 1, or AP-1). This
second option can be effected in different ways (a detailed analysis is beyond the
scope of the present chapter). Briefly, GC can induce (presumably by interacting
through a positive GRE) the synthesis of inhibitors (as in the case of NF-gB); or
they can bind directly to a transcription factor or finally, interfere with transcription
factor binding to responsive gene [22, 23]. Recent evidence indicates a crucial role
for GR acetylation in determining its ability to suppress transcription factor-related
mechanisms [24]. It is unclear how and which of these modulatory mechanisms
operate in each specific cell type.
A different line of research has also revealed the existence of rapid receptor-
dependent non-genomic effects exerted by GC, possibly through direct protein-pro-
tein interactions [25]. Indeed, GC can produce several actions unlikely to require
modifications in gene activity. One example is linked to the externalisation of
annexin 1, which occurs within the first 510 min post-GC exposure, and may dif-
fer among different GC [26, 27]. Figure 2 is a schematic of this important area of
GC biology, as recently recognised in the field [22].
Also of interest is the observation that there are two forms of the GC receptor
termed GR_ and GR`. Both forms are produced by alternative splicing from the
same gene. GR_ is the principal receptor for GC. It is a protein of 777 amino acids
that is expressed in most cells in the body. GR`, which has a C-terminal truncation
(742 amino acids) does not function as a receptor and its intracellular role is cur-
rently unclear, although there have been some interesting hypotheses. GR_ follows
the same general pattern as other members of the nuclear receptor family to which it
belongs: that is to say, there is a unique ligand binding domain (C terminus) as well
144
Figure 1.
Schematic representation of the glucocorticoid receptor cycle.
Following glucocorticoid (GC) binding to glucocorticoid receptor (GR), associated proteins
(e.g. heat-shock protein, hsp) are removed from the complex formed by GR in the inactive
status. Dimers may then form, with GR phosphorylation (P group) and the complex can travel
to the nucleus; here it can bind to specific positive or negative glucocorticoid-response ele-
ments (GRE) that are present in the promoter region of several genes, to increase or decrease
gene transcription. Alternatively, a monomeric GC-GR complex remains in the cytosol and
here it can bind to transcription factors, preventing them from becoming activated and so are
trapped in an inactive status: the end-point is blockade of gene expression.
145
Figure 2.
Glucocorticoid modulation of annexin 1 expression.
GC exert both genomic and non-genomic control on annexin 1 synthesis and post-trans-
lational modifications. Rapid (515 min) non-genomic effects are linked to protein phos-
phorylation and externalisation (as demonstrated in a pituitary cell line); it is not yet clear if
phosphorylated annexin 1 binds with a different affinity to its receptor. More delayed (>1 h)
effects are genomic and require de novo protein synthesis. See text for more details.
as DNA binding, nuclear localisation, transcriptional activation and HSP binding

motifs, many of which are common to all members of the nuclear receptor family.
Finally, there are continuing reports of membrane receptors for GC, although
it is unclear if these are identical to intracellular GR, which could better explain
146
the rapid non-genomic actions of GC, especially on selected cell types such as the
monocyte [28]. A similar unpredicted story may also be unravelling in platelets. We
have recently reported the rapid anti-aggregating effect of prednisolone on human
platelets: this effect occurred in less than 5 min and was specific to the GC, with
prednisolone but not dexamethasone being active. In addition, the classical GR acti-
vation loop described predominantly in monocytes and lymphocytes, and schemati-
cally shown in Figure 1, does not seem to operate in platelets, since prednisolone
interaction with GR causes selective dissociation of heat-shock proteins [29].
The molecular mechanisms briefly touched upon here are responsible for GC
modulatory effects on the production and/or release of several pivotal mediators
of inflammation (e.g. cytokines and cytokine receptors, adhesion molecules, eico-
sanoids, interleukin-10, galectin-1 and annexin 1) and form the basis for the GC
therapeutic application to control inflammatory pathologies.
Anti-inflammatory actions
It is often believed that GC affect most if not all facets of the host response to
infection and xenobiotic attack. This is probably not entirely true, although it is
evident that these lipophilic compounds can influence many cell targets and host
responses. With respect to inflammation, many events occurring in the microcircula-
tion are altered by GC, including blood flow, oedema formation and cell trafficking.
Changes in endothelial cell permeability are likely responsible for the anti-oedema
effects [30, 31], although alterations of endothelial cell lifespan contribute to the
hypertensive effects evident upon long-term administration [32].
GC inhibit the production and/or function of short-lived as well as many
long-lived inflammatory mediators, ranging from platelet-activating factor and
arachidonic acid metabolites, to cytokines and chemokines. Effects upon cytokine
synthesis and action are more subtle since a distinction must be made between the
anti-inflammatory/pharmacological doses of GC and the low physiological concen-
trations required for homeostatic regulation. Low doses of GC augment cytokine
receptor expression on specific cell targets, thereby exerting a permissive effect on
specific physiological actions, the clearest example here being liver maturation and
response to cytokines [19]. At high(er) doses GC exert dual effects on cytokines,
with clear inhibition of pro-inflammatory cytokines synthesis and release, and an
increase of anti-inflammatory cytokines, such as interleukin-10 or interleukin-1
receptor antagonist. An exception to this scheme is GC induction of the inflamma-
tory mediator macrophage-inhibitory factor (MIF), which acts as a functional GC
antagonist [33].
The complexity of GC mechanisms of action is responsible for their clinical effi-
cacy, but also their wide spectrum of side effects. Actions on transcription factors
are schematically summarised in this chapter; more recently, GC effects on rapid
147
signalling events have also been discovered, including their ability to induce the
expression of mitogen-activate protein kinase phosphatase-1 thereby favouring de-
phosphorylation, hence inactivation, of mitogen-activated protein kinases [34]. This
effect has been so far elucidated in osteoblasts and in synovial-derived fibroblasts.
To summarise this section, GC inhibit the synthesis, release or function of several
pro-inflammatory mediators, one exception being MIF. Conversely, they up-regulate
several protective, anti-inflammatory and homeostatic mediators, one of which is
the protein called annexin 1. Other examples would include interleukin-10 and the
anti-inflammatory protein galectin-1 [35], as well as intracellular phosphatases or
transcription factor inhibitors.
Annexin 1: one of the GC mediators
The search for secondary mediators of anti-inflammatory GC was launched many

years ago, and several candidates have been proposed as major players in this
respect, some which we have discussed above. It is possible that some of these
mediators may be functionally linked, as is the case for annexin 1 and eosinophil
cell surface integrin function [36], and this may vary with the cell type under inves-
tigation.
Historical view
Originally identified as a GC-inducible 37-kDa protein and termed lipocortin [37],

annexin 1 inhibited phospholipase A2 activity and hence prostaglandin generation
from perfused lungs and activated macrophages [38]. The protein was cloned in
1986 [39] and subsequently shown to inhibit the acute inflammatory response in
the rat paw [40].
The attempt to identify pharmacophore regions within the 346-amino acid
sequence of annexin 1, led to the study of peptido-mimetics derived from the N-
terminal region, which retained anti-inflammatory activity in models insensitive to
inhibitors of lipid metabolism [41, 42]. These pharmacological studies opened the
way to further work highlighting the novel effects of this protein on several blood
cell types. Furthermore, passive immunisation strategies [43] and the development
of annexin 1 null mice [44] have allowed a better definition of the role of the endog-
enous protein in several cellular functions, including phagocytosis, extravasation
and mediator generation [45]. Over the past decade, our own studies have focussed
upon the mobilisation of PMN and the function that this protein plays with respect
to the process of neutrophil recruitment, and we now refer to the annexin 1 sys-
tem as an endogenous biochemical process that operates in the context of the
adherent and migrating neutrophil [46].
148
The annexin 1 system
In resting conditions, annexin 1 is abundantly expressed in white blood cells, includ-

ing blood neutrophils and monocytes [47, 48], as well as in tissue dwelling cells such
as macrophages and epithelial cells [49]. Human neutrophil adhesion to endothe-
lial monolayers in vitro mobilises large amounts of the protein on the cell surface
[50]. This process is explained by the presence of the protein in secretory gelatinase
granules in resting cells [51], as demonstrated also with a proteomic approach
[52]. Controlled exocytosis [53] brings the protein onto the cell surface. Indeed,
the entire annexin 1 system is activated in the context of neutrophil adhesion to
the endothelium, externalising protein and receptor (as discussed below) and also
activating a process of catabolism, leading us to postulate the existence of a specific
lipocortinase [6]. Figure 3 highlights the salient events occurring upon activation
of the annexin 1 system. Of note here and supporting further discussion, are the
data indicating gelatinase granule-dependent up-regulation on the cell surface of
activating neutrophils of a specific receptor, termed FPRL-1 [54].
How does annexin 1 control neutrophil function once on the cell surface? Using
specific rabbit sera, neutrophil-derived annexin 1 could be immunoprecipitated
with a specific seven transmembrane G protein-coupled receptor, termed formyl-
peptide receptor-like 1 or FPRL-1 [55]. This receptor is also utilised by lipoxin
A4 and its stable aspirin-related analogues; therefore several anti-inflammatory
mediators, including short-lived lipids, activate this specific leukocyte receptor
[55, 56]. The biochemical interaction between annexin 1 and FPRL-1 was sup-
ported by binding experiments using transfected cells or primary neutrophils. In
addition, heterologous competition in the binding assays was observed when using
lipoxin A4 or a annexin 1 mimetic, the bioactive peptide Ac2-26 [57], as a ligand
[55]. More recently these data have been refined with observations indicating that
in in vitro systems the short N-terminal-derived peptide Ac2-26 binds both FPRL-
1 and the structurally related receptor FPR, whereas the full protein maintains
a degree of selectivity towards FPRL-1 [58]. In particular, peptide Ac2-26 and
other peptides derived from the same region of annexin 1 bind and activate all
three receptors of the family, FPR, FPRL-1 and the monocyte/dendritic restricted
FPRL-2 [59, 60].
Initial analysis of post-receptor signalling revealed an annexin 1/FPRL-1-depen-
dent selective activation of mitogen-activated protein kinase, with a positive effect
on extracellular regulated kinase [58]. At higher concentrations calcium fluxes
are also produced [61], although disparate reports on the downstream effects on
neutrophil adhesion molecules have been published [58, 62, 63]. Thus, to sum-
marise this section, it is interesting to note the possibility that distinct receptors and
downstream signalling mechanisms could be activated by annexin 1 and its shorter
peptides, although it is clear that in in vivo settings they both elicit anti-inflamma-
tory responses [46, 64].
149
Figure 3.
The annexin 1 system. Human neutrophils (used here as prototypes) display multiple pools
of annexin 1.
Intracellularly the protein is either in the cytosol or in gelatinase granules. Upon cell adhe-
sion, controlled exocytosis brings annexin 1 onto the cell surface, where it binds in a calcium-
dependent fashion to its receptor, FPRL-1. Cell adhesion may also up-regulate the receptor.
Annexin 1 activation of its receptor causes extracellular regulated kinase phosphorylation,
calcium fluxes and actively promotes cell detachment. Thus, activation of the annexin 1
system acts a fine-tuning break control mechanism in the complex cascade of events leading
to neutrophil extravasation.
Both annexin 1 and lipoxin A4 (a ligand with high selectivity towards FPRL-1,
with no effect on FPR and FPRL-2) share similar effects upon leukocyte adhe-
sion molecule expression, with up-regulation of L-selectin and down-regulation of
CD11b [58, 65], indicative of an inactive (or refractory?) status of the neutrophil.
150
Data with annexin 1 null neutrophils prepared from the mouse blood corroborate
this view [66].
Figure 3 highlights the major event regulating activation of the annexin 1 sys-
tem in the context of an adherent neutrophil. Both annexin 1 and FPRL-1, basally
expressed on the cell surface of resting neutrophils, can be markedly externalised via
a process of controlled exocytosis with membrane export of granule-contained pro-
teins [53, 67]. It is unclear if the gelatinase granule pool of annexin 1 interacts with
the receptor when still inside the leukocyte cytosol. It is more likely that once on the
extracellular surface where it is exposed to > 1 mM calcium, annexin 1 acquires the
active conformation [6870] necessary for interacting with FPRL-1. Downstream
signalling events control the extent of cell extravasation, promoting detachment
[71] and hence reducing the rate and the degree of adherent neutrophils entering
into diapedesis and travelling into the sub-endothelial matrix.
As a final note for this section, in our working model, the hypothesis that
externalised annexin 1 would act in an autocrine/paracrine manner on the adher-
ent leukocyte has often been put forward [72]; however, endothelial cells have also
been shown to express lipoxin A4 receptors [73], along with specific binding sites
for annexin 1 [74], and have been reported to re-uptake annexin 1, possibly in its
cleaved isoform, from emigrating leukocytes [75].
We have analysed here the annexin 1 system in the context of the neutrophil. It is
also clear that annexin 1 can affect several other cell types and systems, as recently
reviewed [64, 76, 77], although it is likely that some of the granulocyte mechanisms
might also be applicable to other cell targets too.
New perspectives
Whereas most of the studies on annexin 1 and its bioactive peptides have been
focussed upon the innate immune response, and its cellular players, much less
attention has been devoted to their potential involvement and role on adaptive
immunity. GC are known to display profound effects on thymus development,
thymocyte differentiation and subsequent T cell lineage commitment. For instance,
GC favour a Th2 response, which could be beneficial in certain immune-mediated
pathologies (e.g. inflammatory bowel disease and rheumatoid arthritis) but not in
asthma. Recent work indicates a prolonged (> 4 week) amplification of the Th2
response following brief exposure of mice to budesonide [78]. While on a shorter
time scale, this is reminiscent of the effects of early neonatal exposure to GC, or
alterations of circulating corticosterone levels by means of low endotoxin expo-
sure, on subsequent susceptibility to inflammation in adulthood [18]. Therefore,
GC affect several complex aspects of the adaptive immune response including
immune tolerance; however, the role that endogenous annexin 1 plays has yet to
be determined.
151
Some studies have indicated that annexin 1 could mediate part of the anti-pro-
liferative actions of GC on peripheral blood mononuclear cell proliferation [79].
In those studies a mixed cell culture was used, making it difficult to determine if
annexin 1 acts directly on T cell functions or indirectly through an action on con-
taminating monocytes. At high concentrations, peptide Ac2-26 inhibits both phy-
tohaemoagglutinin- and antigen-induced T cellular proliferation [80]. In any case,
much more work is required to dissect the actions and roles of endogenous and
exogenous annexin 1 on pivotal players of adaptive immunity and to determine if
FPRL-1 or other receptor types mediate these effects.
Another aspect to highlight is the potential of the microarray approach. Recent
studies using global unbiased approaches have identified annexin 1 as a key media-
tor whose expression was modified in pathology, or pathological models [81, 82].
We suggest that the trans-activating effects of GC, i.e. their ability to induce gene
expression upon receptor binding, has been generally overlooked, in favour of their
trans-repressive actions [83, 84], which is essentially focused on the inhibition of
specific transcription factor functions upon GC application. We believe that the
complex homeostatic properties of GC, partly reviewed here, underline multiple
positive effects on gene programming and the ensuing cellular phenotype. Initial
analysis in T cells seems to confirm this novel opinion [85].
Conclusion
We have highlighted the allure of the anti-inflammation approach, i.e. the interest
raised by investigating endogenous pathways operating in the host to counter-regu-
late the inflammatory reaction, assuring rapid resolution and restoration of homeo-
stasis. In particular, we have focused on the archetypal anti-inflammatory media-
tors, the GC, whose exploitation has been of immense impact on clinical practice
and therapy management. We propose that more mediators under this umbrella of
anti-inflammation should be studied, confident that this will lead to the develop-
ment of better anti-inflammatory drugs. As an example, we have illustrated the case
of annexin 1, a mediator strictly but not solely related to GC. Understanding the
molecular mechanisms switched on by annexin 1 activation of its receptor, and the
events modulated in target cells, will be of great help in developing innovative ways
to control inflammatory pathologies.
Acknowledgements
Work carried out in the Authors lab and mentioned in this review is predomi-
nantly funded by the Arthritis Research Campaign UK (15755), the Wellcome
Trust UK, the British Heart Foundation and the William Harvey Research Foun-
dation.
152
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158
The resolution of airway inflammation in asthma and chronic
obstructive pulmonary disease
School of Medicine, Institute of Medical Sciences, University of Aberdeen, Foresterhill,

Aberdeen, United Kingdom
Introduction
Asthma is now one of the most common chronic diseases in westernised countries
and is characterised by reversible airway obstruction, bronchial hyperresponsive-
ness and airway inflammation. Key pathological features include: infiltration of
the airways by activated lymphocytes and eosinophils; damage to, and loss of, the
bronchial epithelium; mast cell degranulation; mucous gland hyperplasia; and col-
lagen deposition in the epithelial sub-basement membrane area. Asthma pathology
is associated with the release of myriad pro-inflammatory substances including lipid
mediators, inflammatory peptides, chemokines, cytokines, and growth factors. In
addition to infiltrating leukocytes, structural cells in the airways, including smooth
muscle cells, endothelial cells, fibroblasts and airway epithelial cells, are all impor-
tant sources of asthma-causing or -enhancing mediators [1]. This complex scenario
means that potential targets for therapeutic intervention are many and varied and
the task of successful therapy a challenging one.
For many years, anti-inflammatory therapy in asthma has been largely reliant on
glucocorticoids (GCs) particularly in their inhaled form and their use is associ-
ated with a striking reduction in the numbers of activated eosinophils, mast cells
and T cells in vivo. However, although GCs can be efficacious, they are also rela-
tively non-specific in their actions and may not be of benefit to patients with severe
asthma who experience virally induced exacerbations of their disease. Their use also
raises concerns regarding side effects and compliance, particularly in children and
adolescents. Furthermore, even in cases of good compliance, patients with moderate
and severe asthma may experience significant residual symptoms including exacer-
bations of their disease that in some cases can be life-threatening [2]. Moreover, a
small proportion of patients with asthma fails to respond to GCs even at high doses.
Although relatively uncommon, steroid resistance in asthmatic patients places a
burden on scarce resources and presents considerable management problems, as
few alternative therapies are available [3, 4]. Consequently, there is much interest in
developing more specific and effective asthma treatments.

Chronic obstructive pulmonary disease (COPD) is a debilitating inflamma-

tory disease of the lungs and a major and increasing global health problem. In the
United Kingdom alone it accounts for more than 30 000 deaths a year and is an
increasing cause of chronic disability. The pathogenesis underlying COPD involves a
progressive chronic inflammatory process affecting the peripheral airways and lung
parenchyma, leading to airflow limitation that is not fully reversible [5]. Airflow
limitation is usually progressive and is associated with an abnormal inflammatory
response of the lungs to noxious particles or gases, primarily caused by cigarette
smoking. Inflammation in COPD results in airway remodelling, extracellular matrix
(ECM) destruction and fibrosis of the small airways. There is a significant inflam-
matory infiltration of the bronchial mucosa and the alveolar space by CD8+ T cells
together with increased numbers of neutrophils and macrophages in the airways
and increased airway epithelial cell death [6]. However, understanding of the basic
pathogenesis of the disease is somewhat limited and there are no effective treat-
ments. Indeed, in contrast to its beneficial effects on the inflammatory changes in
asthma, inhaled corticosteroid therapy is unable to slow the relentless progression
of COPD [7]. Currently available therapies for COPD are grossly ineffective, mak-
ing research and development in this area vital [8]. The mechanisms underlying the
inability of steroids to attenuate airway inflammation in COPD appear to involve
inactivation of histone deacetylase [9]. This hypothesis has been reviewed in detail
elsewhere [10] and is therefore not discussed further here.
Novel glucocorticoids
Corticosteroids have powerful anti-inflammatory effects by virtue of their ability

to inhibit pro-inflammatory cell recruitment and to down-regulate the production
of pro-inflammatory cytokines. Presently, anti-inflammatory therapy in asthma is
largely reliant on corticosteroids, particularly in their inhaled form, and their use is
associated with a striking reduction in the numbers of activated eosinophils, mast
cells and T cells in vivo, most likely through the induction of apoptosis in these cells
and/or reduced levels of pro-inflammatory cytokines and chemokines that promote
their survival. For the majority of patients, corticosteroids are effective at suppress-
ing airway inflammation and the associated re-modelling of the airways that leads
to progressive and irreversible loss of lung function. The mechanisms by which cur-
rently available corticosteroids inhibit inflammatory processes in asthma have been
described in detail elsewhere (for a review see [11]). A major goal for novel inhaled
GCs might be to achieve selectivity using compounds requiring bioactivation in the
lung and thus avoid unwanted systemic activity. An example of this approach is
ciclesonide, an inhaled non-halogenated GC with little or no oral bioavailability.
Ciclesonide remains inactive until cleaved by esterases present in the airway, where
its active metabolite, desisobutyryl-ciclesonide, binds GC receptors at a 100-fold
160
The resolution of airway inflammation in asthma and chronic obstructive pulmonary disease
higher binding efficiency. In healthy volunteer subjects, a placebo-controlled, ran-

domised, double-blind, change-over equivalence trial demonstrated that daily doses
of ciclesonide (800 g for 7 days) were found to have no clinically relevant effects on
the hypothalamic-pituitary-adrenal axis; this was independent of the time of admin-
istration indicating that it should prove to be well tolerated [12]. A number of clini-
cal trials have been published recently that suggest ciclesonide may be an effective
therapy for asthmatic patients. For example, ciclesonide has been shown to be clini-
cally effective in patients with bronchial asthma whose airway responsiveness was
assessed using adenosine-5-monophosphate challenge [13]. In a double-blind, pla-
cebo-controlled, randomised, crossover study, ciclesonide treatment (800 +g twice
daily via a Cyclohaler) also protected against early and late-phase bronchoconstric-
tion following an antigen challenge in asthmatic patients [14]. A more recent study
utilised a multi-centre, double-blind, randomised, placebo-controlled, 3-period
crossover study comparing 7 days of treatment with inhaled ciclesonide at 50 mg
and 100 mg in 22 subjects with stable allergic asthma. Low-dose ciclesonide pro-
tected against allergen-induced late and early asthmatic reactions, including changes
in FEV1, and significantly reduced levels of serum eosinophil cationic protein and
sputum eosinophils at 24 h post challenge [15]. Another double-blind, randomised,
parallel group, placebo-controlled study performed over 12 weeks found that cicle-
sonide effectively maintained asthma control in adults with persistent asthma [16].
These findings suggest that inhaled ciclesonide will be an effective asthma medica-
tion and attenuates the inflammatory changes in the asthmatic airways. However,
before ciclesonide can be fully evaluated for the treatment of asthma, we require
data from longer-term studies comparing ciclesonide against other inhaled cortico-
steroids in patients with natural day-to-day asthmatic symptoms.
Most of the anti-inflammatory actions of corticosteroids can be accounted for
by inhibition of transcription factors such as activator protein-1 (AP-1), nuclear
factor-gB (NF-gB) and nuclear factor of activated T cells (NF-AT) that regulate
inflammatory gene expression (transrepression). However, the systemic side effects
of corticosteroids are mediated largely through DNA binding (transactivation) [17]
(Fig. 1). It may therefore be possible to dissociate the anti-inflammatory effects from
unwanted adverse effects, thus improving the risk-benefit profile. Several dissoci-
ated corticosteroids have been synthesised, for example RU 24858, that are able
to selectively cause transrepression to a greater extent than transactivation in cells
in vitro and in gene reporter systems [18]. To date, this dissociation has not been
confirmed in whole animal studies.
Mediator antagonists
As many mediators contribute to the pathophysiology of asthma the development

of specific antagonists directed at these substances represents an attractive target
161
Figure 1.
Dissociation of anti-inflammatory effects from side effects of corticosteroids [17]. GR: Glu-
cocorticoid receptor.
for inflammation resolution. However, it is unlikely that a single antagonist will

have a major clinical effect compared with non-specific agents such as corticoste-
roids. Indeed, strategies to block a single mediator, such as platelet-activating factor
antagonists, thromboxane inhibitors and bradykinin antagonists, have all proved to
be disappointing [19]. However, some specific inhibitors, notably cysteinyl leukotri-
ene (LT) antagonists, have had promising clinical effects. Since COPD is character-
ised by neutrophilic inflammation, attention has focused on mediators involved in
recruitment and activation of neutrophils or on reactive oxygen species.
The LTs are derived from arachidonic acid via the 5-lipoxygenase (LO) path-
way (Fig. 2). The cysteinyl LTs (LTC4, LTD4, LTE4) are highly potent mediators of
inflammation, and induce bronchoconstriction. LTB4 has minimal bronchoconstric-
tor effects but is a potent chemoattractant and activator of neutrophils [20]. The
cysteinyl LT receptor antagonists were the first new class of anti-asthma drugs to
be introduced in the last 30 years and are now an established part of the asthma
162
Figure 2.
The 5-lipoxygenase (LO) pathway and sites of action of leukotriene (LT)-modifying drugs.
armamentarium. Overall, they are less effective than inhaled corticosteroids but
some patients show a striking improvement and a corticosteroid-sparing effect has
been demonstrated [21]. Inhibition of 5-LO inhibits the formation of LTB4 and cys-
teinyl LTs. Many 5-LO inhibitors have been developed but only one, Zileuton, has
been marketed and that only in the USA. It is limited by its requirement for frequent
administration and a 5% incidence of liver function test abnormalities. The limited
data available suggest that the anti-asthmatic effects of 5-LO inhibitors and LT
receptor antagonists are indistinguishable [22]. LTB4 may be an important media-
tor in nocturnal asthma, severe status asthmaticus and sudden-onset fatal asthma in
which airway neutrophilia predominates rather than eosinophilia [2326]. It is also
likely to be an important pro-inflammatory mediator in COPD [27]. A number of
antagonists for LTB4 have been developed; however, trials to date in patients with
COPD with either LTB4 synthesis inhibitors or antagonists have proved disappoint-
ing [28, 29].
163
Phosphodiesterase inhibitors
Phosphodiesterases (PDE) break down cyclic nucleotides, particularly cAMP which

has a range of biological actions on the respiratory system, including airway smooth
muscle relaxation, suppression of the actions of immune and inflammatory cells and
neuromodulation [30]. Eleven families of enzymes have been identified with the
levels of different isozymes varying among tissues and cell types. Theophylline, long
used as an asthma treatment, is a non-selective PDE inhibitor whose therapeutic
utility is limited by its marked gastrointestinal, cardiovascular and central nervous
system (CNS) side effects. PDE4 is the predominant family of PDE in inflammatory
cells, including mast cells, eosinophils, CD8+ T lymphocytes, macrophages and
structural cells such as sensory nerves and epithelial cells [31]. This has suggested
that PDE4 inhibitors would be useful as an anti-inflammatory treatment in asthma,
particularly as there is some evidence for overexpression of PDE4 in cells of atopic
patients. Many pharmaceutical companies have developed PDE4 inhibitors, several
of which are now in phase III clinical trials including cilomilast (ariflo) and roflu-
milast [19]. Both have been shown to produce clinically relevant activity in patients
with asthma. In a 6-week, double-blind study in patients with mild to moderate
asthma who were receiving concomitant treatment with inhaled corticosteroids and
as-required salbutamol, cilomilast improved trough pulmonary function test param-
eters from week 1 in addition to having a first-dose bronchodilator effect. Sus-
tained improvement in pulmonary function associated with a reduction in asthma
symptoms has been reported in a 12-month study with cilomilast, suggesting toler-
ance does not occur. PDE4 inhibitors do have an improved side-effect profile over
non-selective compounds, particularly with regard to cardiovascular and CNS side
effects. Drugs with dual PDE3/4 inhibition are being developed with the expectation
that they will have more pronounced anti-inflammatory properties, as PDE3 is pres-
ent in macrophages and airway smooth muscle [19, 32]. PDE inhibitors may also
have therapeutic utility in COPD, as compounds such as cilomilast and roflumilast
are known to be active in animal models of neutrophilic inflammation [33, 34], with
cilomilast producing beneficial clinical effects in patients with COPD [35].
Protease inhibitors
There is compelling evidence for an imbalance between proteases that digest elastin
and other structural proteins and antiproteases that protect against this in COPD
[36]. This suggests that either inhibiting these proteolytic enzymes or increasing
anti-proteases may be beneficial in COPD, as well as having a potential role in
addressing airway remodelling in asthma. In addition to its major extracellular
proteolytic activity, neutrophil elastase, a neutral serine protease, potently stimu-
lates mucus secretion and induces interleukin (IL)-8 release from epithelial cells and
164
therefore may perpetuate the inflammatory state [37]. Both peptide and non-peptide
inhibitors of neutrophil elastase (e.g. ICI 200355 and ONO-5046, respectively)
have been developed and have high potency, inhibiting neutrophil elastase-induced
lung injury in experimental animals and inhibiting neutrophil elastase-induced
mucus secretion in vitro [38]. Other proteolytic enzymes may need to be inhibited
together with neutrophil elastase, e.g. cathepsin G and proteinase 3, also released
by neutrophils, and cathepsins B, L and S, released from macrophages. However, to
date targeting of these mediators using selective inhibitors in COPD has not led to
clinical improvement.
Immunotherapy
Asthma is a manifestation of an imbalance in cytokine and signalling pathways

that mediate inflammatory and structural changes within the lung [39]. Emerging
treatments include strategies to alter the cytokine/chemokine balance or to skew the
cytokine profile away from T helper (Th) 2 responses and towards Th1 responses.
Immunotherapy may potentially attenuate symptoms by disease modification
through the induction of tolerance to common environmental allergens rather than
by suppressing inflammation. A major advantage is the potential for a positive effect
to remain for several years after the end of the treatment period. The use of allergen-
specific immunotherapy is not a new approach to asthma therapy [40] and, until
relatively recently, the crude nature of the allergen extracts available meant that its
use was limited by unwanted side effects such as anaphylaxis. Strategies to over-
come these problems include the use of hypoallergenic isoforms, recombinant aller-
gens or DNA vaccines [41]. For example, in mice with chronic airway inflammation
maintained by repeated ovalbumin inhalation, mucosal administration of CpG
DNA oligonucleotides significantly reversed airway hyperreactivity (AHR) together
with both acute and chronic markers of inflammation [42]. Another approach is the
use of short, synthetic, allergen-derived peptides that induce T cell tolerance but are
unable to cross-link IgE on mast cells or basophils and induce anaphylaxis. The main
effect seems to be a shift from a Th2 to Th1 profile and also induction of regulatory
cytokines such as IL-10 (see below) and transforming growth factor (TGF)-`. A
recent study using patients with asthmatic reactions to cats demonstrated that treat-
ment with a desensitising vaccine based on many short, overlapping, HLA-binding,
T cell peptides derived from Fel d 1 inhibited both early- and late-phase reactions
to a subsequent whole allergen challenge. Changes in immunological parameters
included modulation of the proliferation of blood mononuclear cells together with
their production of IL-4, IL-10, and IL-13, and of interferon (IFN)-a [43]. A more
recent study from the same group demonstrated that treatment of cat-allergic asth-
matic subjects with Fel d 1-derived T cell peptides significantly improved clinically
relevant outcome measurements, including reductions in late asthmatic reactions to
165
inhaled whole cat dander and significant improvements in asthma-related quality of

life [44]. Although the results from these studies are encouraging, we require larger,
dose-ranging studies before firm conclusions on clinical efficacy of peptide allergen
therapy can be made. Although it is conceivable that targeting T cells may induce
Th1-type autoimmune pathology in humans, to date there is no evidence that this
occurs. In addition, non-allergic mechanisms are also likely to be important in the
pathogenesis of asthma; thus it is possible that novel treatments targeted at the
allergy fraction of the phenotype may not be as efficacious as hoped.
Pro-inflammatory cytokines
Multiple cytokines have been implicated in the pathophysiology of asthma and

COPD [45]. There are several possible approaches to resolving airway inflammation
by inhibiting specific pro-inflammatory cytokines. These include drugs that inhibit
cytokine synthesis, for example GCs, cyclosporin A and tacrolimus, humanised
blocking antibodies to cytokines or their receptors, soluble receptors that mop up
secreted cytokines, receptor antagonists or drugs that block the signal transduction
pathways activated by cytokines. Alternatively, anti-inflammatory cytokines may
have therapeutic potential in asthma and COPD [46].
A number of anti-cytokine therapies have focussed on monoclonal antibody
(mAb) ablation of the effects of IL-4, IL-13 or IL-5. Much asthmatic inflammation
is thought to be a consequence of the inappropriate accumulation of eosinophils
and the subsequent release of their potent pro-inflammatory arsenal that includes
such diverse elements as granule-derived basic proteins, mediators, cytokines and
chemokines [47]. IL-5 is crucial to the development and release of eosinophils from
the bone marrow, their enhanced adhesion to endothelial cells lining the post-capil-
lary venules, and their persistence, activation and secretory activity in the tissues.
Ameliorating the effects of IL-5 may therefore have a beneficial effect on the eosino-
phil-driven inflammation underlying much of asthma pathogenesis. Several animal
models of asthma, including primates, have provided good evidence that inhibiting
the effects of IL-5 using specific mAb inhibited eosinophilic inflammation and AHR
[48]. Given its central role in regulating eosinophil development and function, IL-5
was therefore chosen as a potentially attractive target to prevent or blunt eosinophil-
mediated inflammation in patients with asthma. However, several clinical trials have
reported disappointing clinical outcomes following treatment of asthmatic patients
with a humanised anti-IL-5 mAb. The first study was designed to validate the safety
of the anti-IL-5 mAb mepolizumab [49] but was criticised for lack of power [50]
and the validity of patient selection [51]. A later placebo-controlled study [52]
found that treatment of mild asthmatic patients with mepolizumab abolished circu-
lating eosinophils and reduced airway and bone marrow eosinophils but reported
no significant improvement of clinical measures of asthma. Critically, lung biopsy
166
samples from the treatment group contained intact tissue eosinophils together with
large quantities of eosinophil granule proteins, findings that likely explain the lack
of clinical benefit following mepolizumab treatment. Similar findings were reported
with the anti-IL-5 mAb SCH55700 in patients with severe asthma that had not been
controlled by inhaled corticosteroid use. These authors reported profound reduc-
tions in circulating eosinophils, but no significant improvement in either asthma
symptoms or lung function [53]. An alternative to humanised anti-IL-5 mAb is the
use of molecular modelling of the IL-5 receptor _-chain to develop specific recep-
tor antagonists. Recently such a compound (YM-90709) has been shown to be a
relatively selective inhibitor of the IL-5R [54].
It has been suggested by some that the disappointing results with humanised
anti-IL-5 mAb cast doubt on the role of the eosinophil in asthma. However, there
is a vast literature that demonstrates that eosinophils are important pro-inflamma-
tory cells in asthma pathogenesis. Moreover, asthma is a complex heterogeneous
condition and eosinophils are likely to be more important in some forms of asthma
than others. Two recently developed eosinophil-deficient mouse models, although
yielding differing results, have provided strong support for eosinophil involvement
in asthma. One study found that eosinophils were required for both airway hyper-
responsiveness and mucus accumulation [55], while the other demonstrated a criti-
cal role for the cell in airway remodelling [56]. Indeed, eosinophils are also thought
to contribute to the pathophysiology of airway remodelling primarily through the
release of substances, particularly TGF-`, involved in ECM deposition leading to
sub-epithelial membrane thickening [57]. This view is further reinforced by a study
showing that TGF-`2, secreted primarily by tissue eosinophils, is the predominant
isoform in severe asthma and is associated with augmented profibrotic responses
[58]. These are important findings and reinforce a recent study demonstrating that
treatment of asthmatics with mepolizumab again specifically decreased airway
eosinophil numbers. Importantly, compared with placebo, mepolizumab significant-
ly reduced the expression of the ECM proteins tenascin, lumican, and procollagen
III in the bronchial mucosal reticular basement membrane. In addition, anti-IL-5
treatment was associated with a significant reduction in both airway eosinophils
expressing mRNA for TGF-`1 and the concentration of TGF-`1 in bronchoalveolar
lavage (BAL) fluid [59]. The authors concluded that eosinophils may contribute to
tissue remodelling processes in asthma by regulating the deposition of ECM proteins
and mepolizumab may prove useful in preventing this. However, to fully establish
the roles of eosinophils and IL-5 in asthma, longer-term studies aimed at eliminat-
ing tissue eosinophils are required. Furthermore, the studies with anti-IL-5 mAb
emphasise that eosinophil accumulation is not solely dependent on IL-5 [60], a view
supported by an elegant study in which eosinophils were recruited to the lungs of IL-
5/ mice following infection with paramyxovirus [61]. These studies emphasise the
desirability of research aimed at the development of more effective anti-eosinophil
strategies for asthma treatment.
167
Another cytokine important in eosinophil accumulation is IL-4 and, together with

its close relative IL-13, it is important in IgE synthesis by B cells. Both cytokines sig-
nal through a shared surface receptor, IL-4R_, which then activates the transcription
factor STAT-6 [62]. Studies with soluble IL-4R given in a nebulised form demonstrat-
ed that the fall in lung function induced by withdrawal of inhaled corticosteroids was
prevented in patients with moderately severe asthma [63]. However, despite these
promising findings subsequent trials have not been as successful and consequently
this treatment is no longer being developed. Other approaches for blocking the IL-4
receptor include administration of antibodies against the receptor and mutant IL-4
proteins. Interrupting downstream IL-4 receptor signalling by targeting transcription
factors such as STAT-6, GATA-3, or FOG-1 might also be possible [19].
IL-13 has been found in BAL following allergen provocation of asthmatic sub-
jects, which strongly correlated with the increase in eosinophil numbers [64] and
mRNA expression for IL-13 was detected in bronchial biopsies from both allergic
and non-allergic asthmatic subjects [65]. In animal models, IL-13 mimics many of
the pro-inflammatory changes associated with asthma [66]. It is therefore another
potential therapeutic target for the resolution of airway inflammation. Two recep-
tors for IL-13 have been described, IL-13R_1 and IL-13R_2. The latter exists in
soluble form and has a high affinity for IL-13 and can thus mop up secreted
IL-13; in mice IL-13R_2 blocked the actions of IL-13, including IgE production,
pulmonary eosinophilia and AHR [67]. A humanised IL-13R_2 is now in clinical
development as a novel therapy for asthma. Another mouse-based study reported
that intratracheal administration of human IL-13 induced leukocyte infiltration
in the lung, AHR, and goblet cell metaplasia with allergic eosinophilic inflamma-
tion in the oesophagus. An anti-human IL-13 IgG4 mAb (CAT-354) significantly
reduced many of these parameters. In contrast, another study using mice sensitised
by intranasal application of ovalbumin as a model of asthma/allergy found that
the inhibition of the IL-4/IL-13 system efficiently prevented the development of the
asthmatic phenotype, including goblet cell metaplasia and airway responsiveness to
methacholine, but had little effect on established asthma [68]. However, findings
to date suggest that the therapeutic effects of IL-4/IL-13 inhibitors in patients with
allergic asthma are difficult to predict.
Another Th2 cytokine, IL-9, and its receptor are found in asthmatic airways
in increased levels [69]. IL-9 has several pro-inflammatory effects on eosinophils,
including enhancement of eosinophil IL-5 receptor expression, differentiation in the
bone marrow and prolonged survival through inhibition of apoptosis [70]. Trans-
gene expression of IL-9 in the lungs of mice resulted in lymphocytic and eosinophilic
infiltration of the lung, airway epithelial hypertrophy with mucus production, mast
cell hyperplasia and production of IL-4, IL-5 and IL-13 [71]. Blocking humanised
antibodies to IL-9 are currently in development [72].
TNF-_ is expressed in asthmatic airways and levels are elevated in induced
sputum from patients with COPD [73]. TNF-_ may play a key role in amplifying
168
airway inflammation through activation of transcription factors such as NF-gB and

AP-1. Humanised anti-TNF mAb (infliximab) and soluble TNF receptor blockers
(etanercept) have been developed and shown to be effective in other inflammatory
diseases such as Crohns disease [74] or rheumatoid arthritis [75]; these may also
be effective in asthma and COPD. Small molecule inhibitors of TNF-_ converting
enzyme (TACE) have also been synthesised and these too may be attractive thera-
peutic targets for asthma and COPD [76, 77].
In summary, current evidence seems to suggest that targeting single T cell pro-
inflammatory cytokines in asthma or COPD would not be an effective therapeutic
approach.
Anti-inflammatory cytokines
An alternative approach might involve the use of cytokines with proven anti-inflam-
matory potential. These include IL-10, IL-12 and IFN-a. IL-10 would appear to
have the most promise as its anti-inflammatory effects include switching of B cells
from IgE to IgG4 production and inhibition of the production of cytokines includ-
ing TNF-_, GM-CSF or IL-5, together with inflammatory enzymes such as induc-
ible nitric oxide synthase that are overexpressed in asthma. Furthermore, there is
evidence that macrophages isolated from asthmatic patients have a defect in IL-10
production [78, 79]. Moreover, a sub-set of regulatory T cells also produce IL-10
and there is some evidence that their function might be impaired in allergic and
asthmatic disease [80]. Thus, novel therapeutic regimens for asthma might promote
regulatory T cell generation. However, although IL-10 has proved to be effective in
controlling inflammatory bowel disease and psoriasis [81], there are as yet no studies
demonstrating its usefulness or otherwise in asthma. In ovalbumin-sensitised mice
with AHR and eosinophilic inflammation following antigen challenge, a significant
and sustained increase in IL-10-producing CD4+ T cells was observed, mainly of
the CD45RBlow subset. Anti-IL-10 antibody treatment before ovalbumin challenge
had no effect on eosinophilic inflammation but significantly inhibited AHR. In con-
trast, anti-IL-10 antibody treatment just prior to the last ovalbumin challenge sig-
nificantly attenuated the resolution of eosinophilic inflammation without affecting
airway responsiveness 2 weeks after challenge. Thus, in this animal model, IL-10 is
associated with AHR in early inflammatory responses, while it is associated with the
later resolution of airway inflammation [82]. Sputum levels of IL-10 are reduced in
COPD [83] and it also decreases the expression of matrix metalloproteinases, while
increasing the expression of tissue inhibitors of matrix metalloproteinases (TIMPs)
from macrophages, suggesting a potential beneficial role in COPD [84].
IFN-_ promotes differentiation of Th1 cells in vitro, and may influence cytokine
production by regulatory T cells, in particular increasing the expression of IL-10. A
small-scale study [85] looked at the effect of IFN-_ treatment on ten patients with
169
corticosteroid-resistant asthma, including three patients with Churg-Strauss syn-

drome. Treatment was associated with an early clinical improvement in all patients
as assessed by pulmonary function tests and tapering of their corticosteroid treat-
ment. In terms of inflammatory parameters, IFN-_ treatment was associated with
decreased leukocyte numbers, increased CD4+ T cells, increased differentiation of
Th1 cells together with increased expression of IL-10 in peripheral blood mono-
nuclear cells. The authors suggested that the potential mechanisms of action might
include the establishment of an adequate Th1-Th2 balance and the induction of the
anti-inflammatory IL-10 gene. Thus, those patients with steroid-resistant asthma or
severe chronic disease might benefit from IFN-_ therapy, although these findings
have yet to be confirmed in larger placebo-controlled studies.
IgE inhibitors
IgE plays a central role in the pathogenesis of diseases associated with immediate
hypersensitivity reactions, including allergic asthma. Its actions depend on its bind-
ing to high-affinity (FcRI) receptors on mast cells and basophils and to low-affin-
ity (FcRII) receptors on macrophages, dendritic cells and B lymphocytes. Allergen
molecules cross-link adjacent Fab components of IgE on the cell surface, activating
intracellular signal transduction. In mast cells, this leads to the release of preformed
mediators and the rapid synthesis and release of other mediators responsible for
bronchoconstriction and airway inflammation. Therefore, blocking the action of
IgE using blocking antibodies that do not result in cell activation is an attractive
approach.
Omalizumab (rhuMab-E25) is a humanised mAb directed to the FcRI bind-
ing domain of human IgE. It inhibited early-phase and late-phase allergen-induced
asthmatic reactions [86, 87], reduces serum free IgE concentrations to less than
5% of baseline and has now progressed through clinical development [88]. A large
Phase II trial studied fortnightly intravenous administration of omalizumab for 20
weeks in 317 patients [89], while two Phase III trials, including over 500 patients
each, studied omalizumab given subcutaneously every 24 weeks for 12 months
[90, 91]. Ayres and colleagues [92] examined the effects of omalizumab in patients
with moderate-to-severe allergic asthma whose symptoms were poorly controlled
by high doses of inhaled GC. Omalizumab was administered for 12 months and
benefited these patients as shown by a 50% reduction in their asthma deteriora-
tion-related incidents. Another recent study reported that omalizumab treatment of
subjects with both persistent rhinitis and difficult to treat asthma resulted in signifi-
cantly reduced asthma exacerbations and improved quality of life in those patients
receiving anti-IgE therapy over the 28-week study period [93]. Omalizumab has
also been shown to be beneficial as an add-on therapy in patients who have inad-
equately controlled severe persistent asthma [94]. Consistent findings from these
170
trials showed that omalizumab is an effective therapy for patients with symptomatic
moderate-severe allergic asthma despite treatment with corticosteroids and rescue
medication. It reduced the frequency of exacerbations and improved symptom con-
trol while allowing a reduction in the use of corticosteroids and `2-agonists. It also
improved patient quality of life and produced a significant improvement in lung
function as measured by PEFR and FEV1. Omalizumab appears to be well toler-
ated with few side effects reported in these studies and development of circulating
antibodies against omalizumab was not reported. Although more long-term studies
are needed to fully elucidate the benefit and safety of anti-IgE therapy in asthma,
its niche may be in the treatment of patients with severe asthma who are dependent
on oral corticosteroids.
Targeting inflammatory cell accumulation in the lung
Eosinophils: Accumulation
A considerable body of research has accumulated over many years with the purpose
of furthering our understanding of the complex and inter-related events that con-
trol pro-inflammatory leukocyte accumulation in the asthmatic lung. As mentioned
above, eosinophils are key effector cells in the inflammation underlying asthma
pathogenesis [47]. Their accumulation in the asthmatic lung is complex involving
their maturation in and release from the bone marrow, adhesion to and transmigra-
tion through the post-capillary endothelium, followed by their chemotaxis to and
activation/degranulation at inflammatory foci [95]. In normal individuals eosino-
phils are rarely found in the lung and are confined to the tissues surrounding the
gut where they are thought to contribute to immune protection against helminthic
parasitic worms [47]. Eosinophil numbers in the asthmatic lung correlate with
disease severity and their accumulation thus suggests that pathways exist for selec-
tion of eosinophils over other leukocytes. Selective eosinophil adhesion appears
to be dependent on the very late antigen (VLA)-4 (_4`1)/VCAM-1 pathway [96].
Moreover, both IL-4 or IL-13 stimulation of endothelial cells selectively up-regu-
lates VCAM-1 in the absence of expression of E-selectin or ICAM-1, and IL-4 also
enhances eosinophil transmigration through endothelium in a VLA-4/VCAM-1-
dependent manner [97, 98]. Thus, blocking the _4`1integrin may provide a suitable
target for preventing eosinophil accumulation in the lung. A humanised anti-_4`1
mAb (natalizumab) has proved beneficial to patients with multiple sclerosis [99] and
small peptide antagonists of the integrins _4`1 and _4`7 (TR14035 and BIO1211)
have had positive effects in animal models of asthma [100]. These are currently in
Phase II trials for efficacy in asthma [101].
However, the potential side effects of targeting adhesion pathways as therapeu-
tic avenues should always be considered as demonstrated by the recently reported
171
series of fatal CNS infections occurring during systemic treatment with natalizumab
[102].
Chemokines are chemoattractant cytokine molecules and are crucial in eosino-
phil recruitment. RANTES, MCP-3, MCP-4 and eotaxin act on a common receptor
CCR3 (cysteine-cysteine chemokine receptor-3) that is expressed predominantly on
eosinophils. CCR3 is therefore a promising and selective target to blunt or prevent
eosinophil entry into the lung. Modified chemokines such as met-RANTES are
potent CCR3 antagonists that inhibit CCR3 receptor signalling and consequent
eosinophil migration. CCR3 is the receptor not only for eotaxin 1, but also eotaxin
2 and 3 and several other asthma-relevant chemokines. Furthermore, CCR3 repre-
sents a very attractive therapeutic target as it is expressed not only on eosinophils
but also on basophils [103], mast cell subpopulations [104], activated Th2 cells
[105], and airway epithelial cells [106], all of which make significant contributions
to asthmatic inflammation. Although the bronchial epithelium consists of struc-
tural non-migratory cells, expression of the CCR3 receptor may represent an auto-
regulatory feedback mechanism to monitor chemokine production. Furthermore,
eotaxin produced by the epithelium may be sequestered by the CCR3 receptor and
presented to infiltrating cells thereby enhancing their activation, a phenomenon
observed with IL-8 and its receptor. Hence, CCR3 is closely associated with asthma
and allergy and blockade of this receptor may have pronounced beneficial effects
in these diseases [107]. The N-(ureidoalkyl)-benzpiperidines have been identified as
potent CCR3 antagonists, inhibiting eosinophil chemotaxis and calcium mobilisa-
tion in the micro- to nanomolar concentration range [108]. There is evidence from
animal models that IL-5 and eotaxin may work in a synergistic fashion to promote
the release of mature eosinophils from the bone marrow [109]. Thus, it might be
that combination therapies of CCR3 antagonist and humanised anti-IL-5 mAb may
prove an effective approach to limit or prevent eosinophil toxicity in the asthmatic
lung.
Eosinophils: Apoptosis
Another approach that has received much attention of late is the development of
strategies to encourage eosinophil removal from the asthmatic lung via apoptosis
induction and their subsequent recognition and removal by phagocytic cells. Apop-
tosis or programmed cell death is a central and essential process in the resolution
of inflammation. Our clinical study [110] and those of others [111, 112] provide
evidence that apoptosis induction in eosinophils and their subsequent phagocytic
removal is a rational avenue for development of novel therapies for asthma. The
load of lung eosinophils in asthmatic disease is likely to be related to a balance in
the tissue microenvironment between pro- and anti-apoptotic signals. Eosinophil
persistence in the airways is enhanced by the presence of several asthma relevant
172
cytokines that prolong eosinophil survival by inhibition of apoptosis. The role of

IL-3, IL-5, GM-CSF in this regard is well established [113], while more recently
eosinophil viability-enhancing effects have been reported for IL-9 [114], IL-13 [115]
and IL-15 [116]. Furthermore, eosinophil contact with the proteins of the ECM are
likely to contribute to their persistence. For example, integrin-mediated eosinophil
adhesion to fibronectin [117, 118] results in the autocrine production of viability-
enhancing cytokines GM-CSF, IL-3 and IL-5. TNF-_-mediated eosinophil survival
as a result of autocrine GM-CSF production appears dependent on activation of
NF-gB [119] and inhibition of NF-gB activation plays a critical role in eosinophil
responsiveness and apoptosis [120]. It is therefore of interest that a recent report
provided evidence of persistent activation of NF-gB pathways in the mononuclear
cells of patients with severe uncontrolled asthma [121]. Activation of NF-gB path-
ways are associated with pro-inflammatory cytokine and chemokine production,
thus they may represent a plausible therapeutic target for asthma.
A number of signals have been described that accelerate apoptosis in human
eosinophils. GCs induce apoptosis in peripheral blood eosinophils [122] and also
in tissue eosinophils resident in nasal polyp tissue sections [123], suggesting that
eosinophil apoptosis induction by GCs might be relevant to their anti-inflamma-
tory effects in asthma. IL-12 induces apoptosis in human eosinophils, findings that
likely explains its ability to decrease tissue eosinophilia in murine models of allergic
inflammation [124]. Furthermore, the level of IL-12 mRNA expression in the air-
ways of asthmatic subjects is significantly lower than that in non-asthmatic controls
and levels significantly increased following treatment with GCs [125]. IL-12 would
therefore appear to be a good candidate for the treatment of asthma. However,
whereas a clinical study demonstrated that treatment of asthmatic patients with
rhIL-12 reduced sputum and blood eosinophil numbers, no significant effect was
observed on either AHR or the late-asthmatic response to an inhaled antigen chal-
lenge [126].
The desire to understand how the process of apoptosis can be harnessed in
the quest for novel asthma therapy has led to much interest in furthering our
understanding of the triggers and intracellular mechanisms controlling apoptosis
induction in human eosinophils. For example, several studies have demonstrated
that ligation of membrane receptors including Fas (CD95) [127, 128] CD69 [129],
siglec-8 [130] and CD45 [131] induce eosinophil apoptosis. Interestingly, eosino-
phil expression of the latter receptor is elevated in patients with asthma compared
with normal controls [132]. The intracellular signalling mechanisms by which
GCs induce apoptosis in human eosinophils include the involvement of caspases
and release of mitochondrial cytochrome C [133]. Caspases are key regulators of
apoptosis in diverse human cells. Previous work with eosinophils derived from
both healthy and asymptomatic allergic individuals has demonstrated an involve-
ment of caspase-3 and -8 in GC-induced apoptosis [134]. In contrast, others have
reported that dexamethasone-induced apoptosis failed to induce specific caspase-3
173
and -8 activity in eosinophils compared with spontaneous apoptosis [135]. Our own
findings demonstrate that different caspase pathways are involved in controlling
receptor-ligation-mediated apoptosis induction in human eosinophils [136]. While
caspases are key regulators of apoptosis in diverse human cells, oxidant-induced
mitochondrial injury associated with translocation of the pro-apoptotic protein Bax
to the mitochondria has been shown to be pivotal in eosinophil apoptosis. This
effect was mediated by GC-induced prolonged activation of c-Jun NH2-terminal
kinase that was, in turn, inhibited by GM-CSF [137].
Taken together, these observations indicate potential avenues for the develop-
ment of novel therapeutic approaches to target eosinophil-induced inflammation
in asthma, particularly in those patients who exhibit steroid resistance. Other fac-
tors important in the control of apoptosis and caspase activation in many cellular
systems include the Bcl-2 family of proteins [138]. Bcl-2 and Bcl-xL inhibit cell
death, whereas other members such as Bax and Bcl-xs promote apoptosis. There
are several reports demonstrating constitutive expression of Bcl-2 [139, 140] or
Bax and Bcl-x [141] by human eosinophils, whereas a decrease in Bcl-xL messenger
RNA and protein levels was found to be associated with eosinophil apoptosis [142].
These studies are all increasing our knowledge of the complex mechanisms that
regulate eosinophil survival or apoptosis-induction in the asthmatic lung.
Eosinophils: Phagocytic disposal and luminal entry
While much attention has rightly been paid to the study of the mechanisms by
which pro-inflammatory cells such as the eosinophil can be induced to become
apoptotic, it must be remembered that removal of cellular corpses by phagocytosis
is as vital a process as apoptosis itself [143]. Failure to do so will result in disinte-
gration of the apoptotic cell via a process termed secondary necrosis and the sub-
sequent uncontrolled leakage of the dying cells contents, resulting in a propagated
inflammatory response. Indeed, defects in apoptosis and/or subsequent phagocytic
clearance of pro-inflammatory cells are increasingly recognised in chronic inflam-
matory diseases [144]. While the macrophage is considered to be one of the most
important cells involved in apoptotic cell removal, including that of apoptotic
eosinophils [145], many lines of evidence suggest an important role for non-pro-
fessional phagocytes such as dendritic cells, fibroblasts or hepatocytes in the rec-
ognition and removal of apoptotic cells [146]. Our own work has established that
both primary cultures of human small airway epithelial cells (AEC) [147] and the
alveolar epithelial cell line A549 [148] phagocytose apoptotic, but not freshly iso-
lated, eosinophils (Fig. 3). Recognition and phagocytosis of apoptotic eosinophils
was a specific event under the control of integrin, lectin and phosphatidylserine
membrane receptors. Importantly, we also demonstrated that the corticosteroid
dexamethasone increased both the percentage of AEC engulfing apoptotic eosino-
174
Figure 3.
A representative scanning electron micrograph (original with close-up underneath) obtained
using a Philips 505 Scanning electron microscope of phagocytosis of apoptotic eosinophils
by a small airway bronchial epithelial cell.
Two partially phagocytosed eosinophils are clearly visible by their globular surface features,
while another is almost completely engulfed by an encroaching smooth, dark small airway
epithelial cells (AEC) membrane (white arrowheads). The membrane advances further to
cover an adjacent eosinophil and projections of AEC membrane (black arrow) clearly extend
around the apoptotic eosinophil. (Original magnification 3200) [148].
175
phils and, in particular, in the number of apoptotic eosinophils ingested by each

epithelial cell. These findings add a new dimension to the anti-inflammatory effects
of corticosteroids. We also demonstrated that actin rearrangement is involved in
the phagocytosis of apoptotic eosinophils by AEC and that the phagocytic capac-
ity of cytokine-stimulated small and large AEC was approximately half that of
human monocyte-derived macrophages. Importantly we found that AEC do not
recognise or ingest apoptotic neutrophils. This latter observation does suggest that
clearance of apoptotic neutrophils by the bronchial epithelium does not represent
a potential anti-inflammatory pathway in COPD [149]. What is clear is that given
the extent of the lung epithelium and that LPS-dependent phagocytosis of apoptotic
cells by alveolar macrophages is greatly impaired in patients with chronic asthma
[150], bronchial epithelial cells may prove to be vital in the clearance of apoptotic
eosinophils.
Luminal entry by viable eosinophils has been expounded as the major mecha-
nism for their clearance [151153]. Several papers from the same group have cham-
pioned eosinophil efflux into the airway lumen in the absence of tissue apoptosis.
This mechanism does not preclude granulocyte apoptosis in the airway lumen and
subsequent clearance by macrophages [145] or AEC [147149] as numerous studies
have demonstrated apoptosis and phagocytic removal in vivo [154156]. Indeed,
it has been suggested that phagocytosis of apoptotic cells during quiescent periods
is an important process with transepithelial egress providing a necessary second-
ary means of removal during acute inflammation [157]. What is clear is that the
complexities of granulocyte interactions with other cells, ECM and cytokines mean
that although reductionist in vitro experiments provide key insights into specific
granulocyte interactions, they fail to provide information on the ultimate fate of
granulocytes in a particular in vivo situation.
Mast cells
Mast cells might also prove to be an attractive target for novel asthma therapy as
their infiltration of airway smooth muscle has recently been found to be associ-
ated with the disordered airway function found in asthma [158]. In this elegant
study, Brightling and colleagues compared lung biopsies from patients with asthma,
eosinophilic bronchitis and normal controls and found the number of mast cells was
significantly higher in the airway smooth muscle of the subjects with asthma than
either the normal subjects or patients with eosinophilic bronchitis, a condition that
is similar to asthma and therefore provides an appropriate control. More recently,
the same group has demonstrated localisation of IL-4 and IL-13 within mast cells
resident in smooth muscle in biopsies from asthmatic subjects [159]. Thus, thera-
pies that target mast cells or their mediators such as tryptase or prostaglandin D2
might prove fruitful. There is accumulating evidence that prostaglandin D2 plays an
176
important role in asthma [160]. Importantly, prostaglandin D2 is a specific eosino-

phil chemoattractant that exerts its effects via a novel adenylyl cyclase-coupled
receptor for prostaglandin D2 (DP receptor) with comparable potency to that of
eotaxin [161].
Neutrophils
The role of neutrophils in asthma is often overlooked, although they play an impor-
tant pro-inflammatory role, particularly in exacerbations and more severe forms of
the disease [162]. The latter are associated with increased sputum neutrophils [25],
although it could be argued that the observed neutrophilia is at least partially a con-
sequence of corticosteroid treatment favouring neutrophil survival while decreasing
the relative proportion of eosinophils via apoptosis induction (see below). However,
in patients with severe asthma who exhibit resistance to corticosteroid therapy, neu-
trophil persistence may at least partially explain the continued airway inflammation
despite effective reduction in eosinophil numbers.
Selectins are responsible for the early adhesive events between leukocytes and
the endothelial cells lining the post-capillary venules. TBC1269 is a synthetic com-
puter-designed pan selectin antagonist targeted against all three identified selectins
[163]. In a sheep model of allergy, inhaled TBC1269 potently inhibited allergic
airway responses, lavage levels of histamine and tissue kallikrein and neutrophilic
inflammation [164]. In patients with asthma, a single intravenous dose of TBC1269
had only a minor effect on sputum eosinophils or inhaled allergen-induced late
asthmatic reactions [165]. In contrast, inhaled TBC1269 significantly reduced late-
phase asthmatic reactions by approximately 50% compared with placebo in mild
asthmatic subjects [166]. Thus, the inhaled route of TBC1269 may offer advantages
over systemic delivery in terms of both efficacy and safety. Since selectins are also
vital in early adhesive neutrophil interactions with the endothelium TBC1269 may
also prove an effective therapy for COPD.
COPD progression is characterised by increased small airway wall and luminal
neutrophilia and neutrophilic inflammation correlates with both the clinical sever-
ity of COPD and increased mucus production. As well as being present in cigarette
smoke, oxidants are produced endogenously by activated inflammatory cells,
including neutrophils and alveolar macrophages. Oxidative stress and protease-
mediated inflammation in COPD results in the release of a host of pro-inflamma-
tory mediators that include chemokines, cytokines, elastase, and metalloproteases
[167, 168]. This suggests that antioxidants may be beneficial in COPD therapy.
N-Acetylcysteine (NAC) provides cysteine for enhanced production of glutathione
and has antioxidant effects in vitro and in vivo. In clinical studies, NAC reduces the
number of COPD exacerbations and appeared to reduce the rate of FEV1 decline
over a 2-year period in an uncontrolled study. It is likely that more effective antioxi-
177
dants will be developed for future clinical use. For example, cyclic nitrone spin-trap
antioxidants are much more potent and inhibit intracellular reactive oxygen species
formation by forming stable compounds [169].
IL-8 represents a potent chemoattractant for neutrophils and airway concen-
trations of this chemokine are markedly elevated in COPD, particularly during
exacerbations [170]. IL-8 represents a significant part of the chemotactic activity of
airway secretions [171]. Humanised anti-IL-8 mAb have been developed and have
shown promise in a number of chronic inflammatory conditions [172]. A pilot trial
in patients with COPD over a 3-month period demonstrated that anti-IL-8 mAb
treatment reduced dyspnea with no significant changes in lung function or exercise
capacity reported. No direct markers of airway inflammation were reported in this
study but it does suggest that more comprehensive large-scale studies with IL-8 mAb
in COPD are warranted. IL-8 attracts neutrophils via a high-affinity G protein-
coupled receptor CXCR1 and a common receptor CXCR2. A non-peptide inhibitor
of CXCR2 (SB225002) has been developed that blocks the chemotactic response of
neutrophils to IL-8 and other CXC chemokines [173]. Chemokines involved in the
recruitment of activated macrophages present further therapeutic targets in both
asthma and COPD.
As mentioned above, apoptosis induction and subsequent phagocytic removal
of phagocytic corpses are important mechanisms in the resolution of airway
inflammation in asthma and COPD. Given that current therapies for COPD are
inadequate and many new strategies have proved disappointing, this may provide
an avenue for development of new therapies for COPD. Indirect evidence that
targeting apoptosis/phagocytosis in COPD is a rational approach is provided by
one study that demonstrated defects in recognition of apoptotic epithelial cells
by alveolar macrophages from patients with COPD [174]. Another avenue that
might prove fruitful would be the dissection of the mechanisms responsible for the
promotion of apoptosis in eosinophils by corticosteroids, while these drugs inhibit
this process in neutrophils. Preliminary findings suggest that GC-mediated delay of
neutrophil apoptosis may be reversed by inhibition of protein synthesis and block-
ade of NF-gB [175]. In addition, antagonism of LTB4 receptors and inhibition of
LT synthesis resulted in reversal of LPS-, GM-CSF- and dexamethasone-induced
neutrophil survival [176]. The infected and inflamed tissue of the lung in COPD
results in a relatively low oxygen tension; since hypoxia can delay neutrophil
apoptosis these conditions may prolong their pro-inflammatory potential [177]. It
may also be possible to selectively induce neutrophil apoptosis in COPD by target-
ing cell regulatory molecules such as phosphatidylinositol and protein kinase C-b
[178]. These findings suggest that reduction of neutrophil survival represents a
potentially important anti-inflammatory mechanism. Overall, phagocytic removal
of apoptotic pro-inflammatory cells represents a major mechanism for inflamma-
tion resolution in many conditions including asthma and COPD [179] and is an
important and growing area of research.
178
Conclusion
Current therapies for asthma and COPD are clearly unsatisfactory. In asthma, sig-
nificant numbers of patients respond poorly or not at all to corticosteroids, which
in turn can elicit significant side-effects. Importantly, current therapy only treats
symptoms and does not effect a cure. Thus even those patients with asthma who
respond well to inhaled corticosteroid therapy must continue to use their medication
for life. Current treatments in COPD are also far from effective. What is clear is that
in both conditions airway inflammation is the most important target for the devel-
opment of more effective and specific therapy. However, the complicated nature of
the processes underlying airway inflammation and the heterogeneity of the asthma
and COPD phenotypes strongly suggest that a great deal more work is needed if
more effective treatments are to become available.
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191
Resolution of glomerular inflammation
MRC Centre for Inflammation Research, University of Edinburgh, Queens Medical Research
Centre, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom
Introduction
A key role of the kidney is to eliminate many of the waste products generated by
cellular metabolism and to maintain biochemical and acid base homeostasis of the
organism. Kidneys contain spherical microvascular capillary networks called glom-
eruli that filter the plasma through a highly specialised filtration barrier (Fig. 1).
Glomeruli are composed of three main cell types: mesangial cells, endothelial cells
and glomerular epithelial cells (podocytes). The contractile mesangial cells are locat-
ed within the centre of the glomerulus and support the delicate glomerular capillary
network. The outermost podocytes are key components of the glomerular filtration
barrier. The glomerular filtrate passes into Bowmans space and the composition
of this fluid is altered by tubular epithelial cells as it passes along the lumen of the
nephrons. A more detailed description of renal physiology is outside the scope of
the chapter but it should be noted that end stage renal failure is uniformly fatal if
patients do not undergo dialysis or receive a functioning kidney transplant. It is
therefore apparent that strategies that promote the resolution of glomerular inflam-
mation would be predicted to be of great therapeutic benefit.
Glomerular inflammation is a feature of various forms of glomerular injury with
many types of glomerulonephritis (GN) resulting in the development of chronic
renal failure. Severe acute or persistent chronic GN often results in glomerulosclero-
sis (complete glomerular scarring), renal tubular atrophy, microvascular rarefaction
and interstitial scarring with an accompanying loss of renal function. Many types
of GN exhibit a remitting and relapsing course and may be responsive to various
therapies. The future challenge is to understand the mechanisms involved in such
reparative processes and utilise them for patient benefit by devising novel therapies
to promote the resolution of inflammatory glomerular injury including the reversal
of glomerular scarring and restoration of a normal glomerular architecture. In this
chapter we briefly cover the various features of glomerular injury including various
experimental models of GN that have provided significant insights into glomerular
inflammation and healing. We then consider the requirements for the resolution of

Figure 1.
Glomerulus structure. The glomerulus is a highly specialised network of capillaries and is fed
by an afferent arteriole. The filtration barrier consists of the endothelium, glomerular base-
ment membrane and the podocytes. The fenestrated endothelial cells lie on the basement
membrane and are supported by mesangial cells and mesangial matrix. On the other side of
the basement membrane lie the foot processes of podocytes. The glomerular filtrate that is
produced passes into the space between the layers of Bowmans capsule and passes into the
proximal tubule. Blood leaving the glomerular capillaries does so via the efferent arteriole,
which then supplies the peritubular capillary network.
glomerular injury and finally discuss potential strategies to harness natural repair
mechanisms.
It is important to recognise at the outset that, although the inexorable progres-
sion of renal disease is seen in many conditions, glomerular inflammation may
resolve completely. For example, post-streptococcal GN may follow a throat or skin
194
infection and classically exhibits the histological appearance of diffuse proliferative

GN with infiltration by macrophages (M) and neutrophils (PMN). Despite the
severity of the histological lesion, post-streptococcal GN typically resolves over 12
weeks with complete restoration of normal glomerular structure and function. This
emphasises that natural endogenous mechanisms exist that promote the complete
resolution of glomerular inflammation and repair of the structurally damaged glom-
erulus. There are also anecdotal reports of patients with conditions such as lupus
GN who exhibit a stabilisation or improvement of renal function following treat-
ment, suggestive of the partial resolution of glomerular disease. Also, some diabetic
patients who received a pancreas transplant many years previously develop regres-
sion of some of the features of diabetic nephropathy [1]. Other forms of glomerular
disease such as IgA nephropathy exhibit a chronic clinical course. Despite this, an
acute exacerbation of disease associated with macroscopic haematuria and a decline
in renal function is usually followed by at least a partial spontaneous resolution of
inflammation. Severe forms of acute GN such as anti-neutrophil cytoplasmic anti-
body (ANCA)-positive vasculitis associated with crescent formation may resolve
with specific anti-inflammatory immunosuppressive treatment.
Experimental animal models of glomerular disease such as nephrotoxic nephri-
tis (NTN) induced by the administration to heterologous antibody directed at
glomerular components have been very useful in the study of the pathogenesis of
glomerular inflammation and the progression of glomerular injury to glomeru-
losclerosis. Also, the Thy 1.1 model of GN in the rat, which exhibits features of
human mesangioproliferative GN, is of particular interest [2]. Disease is induced
by the administration of antibodies to the Thy 1.1 antigen expressed on rat mesan-
gial cells. The model is characterised by an initial profound loss of mesangial cells
(mesangiolysis) that is followed by the development of mesangial hypercellularity
and accumulation of excess glomerular extracellular matrix (ECM). Over time,
however, this model exhibits complete resolution including the restoration of
glomerular normocellularity and degradation of excess ECM. These and other
animal models have facilitated the analysis of chemical and cellular mediators
involved in both the initiation and progression of glomerular disease and illustrated
the potential to manipulate the inflammatory system to promote resolution. This
has led to two main approaches to ameliorate glomerular inflammation; firstly the
inhibition of pro-inflammatory mediators and cells and secondly the augmentation
of anti-inflammatory effectors.
Initiation of glomerular inflammation
Glomerular inflammation can be initiated by a wide range of insults (Tab. 1) and it

should be noted that the severity of the features of inflammation is often predictive
of the rate of progression.
195
Table 1 - Mechanisms of glomerular injury.
Glomerular disease Mechanism of injury Animal model
ANCA-positive ANCA bind to and activates ANCA-induced glomerulo-

vasculitis neutrophils and monocytes and leads nephritis
to endothelial cell death
Anti-GBM disease IgG antibodies bind to the _3 chain Nephrotoxic nephritis
of the NC1 domain of type IV
collagen
SLE Immune complex deposition leads to Certain mice strains spontane-
diffuse proliferative inflammation ously develop lupus including
glomerulonephritis
Membranous Immune complex deposition along Heymann nephritis, antibodies
nephropathy the glomerular basement membrane. against megalin appear central
Can occur in hepatitis B, SLE and
malignancy
Post-streptococcal Cross-reactivity to glomerular anti- Infection of mice with group A
glomerulonephritis gens induced by streptococcal infec- streptococcus
tion with immune complex deposition
Hepatitis C-induced Glomerular immune complex Thymic stromal lymphopoietin
cryoglobulinemia deposition transgenic mice develop mixed
cryoglobulinemia
IgA nephropathy Increased IgA production with abnor- Animal IgA has different charac-
mal glycosylation leads to teristics thus models limited
glomerular IgA deposition
ANCA, anti-neutrophil cytoplasmic antibody; GBM, glomerular basement membrane; SLE,

systemic lupus erythematosus
Cellular mediators of glomerular inflammation
Irrespective of the initial insult, glomerular inflammation is characterised by infiltra-

tion by inflammatory cells including PMNs, M and lymphocytes [3].
Macrophages
Many studies indicate that M are key inflammatory cells. For example, the admin-
istration of an anti-M serum reduced injury in experimental NTN in rats [4].
196
Cyclophosphamide treatment induced a protective leukopenia in rat NTN with

injury being restored by intravenous administration of a M cell line [5]. In our
recent work, we used a murine conditional M ablation model [6] to investigate
the role of M in progressive murine NTN. M depletion performed between days
15 and 20 reduced glomerular crescent formation, tubular cell apoptosis, interstitial
fibrosis, proteinuria and improved renal function thereby reinforcing a major injuri-
ous role for infiltrating M [7].
Lymphocytes
Although the antigenic targets of the immune response in many forms of GN are
unknown, lymphocytes have a central role both in providing T cell help to activate
cells, including M via IFN-a production and B lymphocytes leading to production
of antibody. The development of severe crescentic GN in murine NTN is driven
by a Th1-type immune response, while the Th2 immune response favours anti-
body production and glomerular immune deposits [8]. Thus, lymphocyte-derived
cytokines activate cells involved in glomerular inflammation and provide help in
peripheral lymphoid organs to drive the injurious immune response.
Neutrophils
Neutrophil influx is seen in many acute models of glomerular injury especially in

experimental models such as NTN [9]. In human disease neutrophils can be identi-
fied in ANCA-positive vasculitis, post-streptococcal GN and lupus nephritis [3].
In later stages of disease, neutrophil numbers reduce but evidence implicates both
neutrophil production of reactive oxygen species and proteases in acute glomerular
injury [10, 11]. Particular interest has focused on effect of ANCA antibodies on
neutrophils. Both proteinase 3 and myeloperoxidase-ANCA are able to activate
neutrophils by binding to cell surface expressed antigens and via binding of Fc-
gamma receptors leading to both superoxide generation and cytokine release [12,
13]. Neutrophils activated in this way have the potential to kill endothelial cells
and further propagate injury. ANCA antibodies also activate monocytes in a similar
mechanism.
Chemical mediators of glomerular inflammation
Animal experiments have defined a number of key mediators involved in driving

glomerular inflammation.
197
TNF-_ and IL-1`
The administration of IL-1` or TNF-_ to rats with NTN augments disease, while
passive immunisation against either of these cytokines ameliorates glomerular
inflammation [14, 15]. Treatment of NTN in rats with IL-1 receptor antagonist (IL-
1ra) is similarly beneficial [16]. The administration of soluble TNF receptor inhib-
ited glomerular inflammation in WKY rats with crescentic GN when administered
both before the onset of disease and after the initiation of disease (the latter being
a requirement of any putative therapeutic intervention) [17]. The administration of
anti-TNF-_ antibodies in the same model after disease onset was also able to pre-
serve renal function and reduce crescent formation and scarring [18]. In a rat model
of ANCA-associated vasculitis, treatment with anti-TNF-_ antibodies after disease
onset reduced inflammatory crescent formation, proteinuria and the development
of lung haemorrhage [19].
Production of IL-1 and TNF-_ is a prominent feature of GN in human biop-
sies in patients with ANCA-associated Wegeners granulomatosis or microscopic
polyarteritis [20], lupus nephritis [21, 22] and IgA nephropathy [23]. In addition,
expression of these cytokines is associated with a worse clinical outcome [24]. The
use of anti-TNF-_ strategies is well established in rheumatoid arthritis and inflam-
matory bowel disease. Small-scale studies have shown that infliximab, a neutralising
anti-TNF-_ antibody, was able to attenuate inflammation in patients with ANCA-
associated vasculitis, including patients with glomerular involvement [25]. Thus,
inhibition of pro-inflammatory cytokines can reduce glomerular inflammation and
permit remission of the disease process.
IFN-a and IL-12
Most forms of glomerular inflammation involve an adaptive immune response

requiring antigen presentation and help from T cells. Despite the uncertainty of
the antigenic targets, there is no doubt that cytokines of the adaptive immune
system are important. The development of crescentic GN in NTN was found in
Th1-biased strains of mice (C57BL/6) and was less evident in Th2-biased strains
(BALB/c) [26]. IFN-a stimulates T cell proliferation and activates M, while IL-12
promotes both T cell proliferation and IFN-a production. Mice deficient in IFN-a
develop less severe GN, with fewer crescents and inflammatory cell infiltrates [27]
and treatment with anti-IFN-a antibody reduces injury [28]. A similar role has
been identified for IL-12 with IL-12 knockout mice developing less severe GN,
while injury is inhibited by IL-12-blocking antibodies [29, 30]. Deficiency of IFN-a
or IL-12 in lupus prone mice inhibits the development of GN with mice exhibiting
reduced T cell and M accumulation [31, 32]. It should be noted, however, that
although glomerular inflammation was retarded it continued to progress which
198
raises the important concept that pro-inflammatory cytokines may have roles in
both progression and resolution.
Nitric oxide
Glomeruli isolated from rats with NTN generate large amounts of nitric oxide (NO)
[33] with M being the principal source of NO. Similarly in Heymanns nephritis,
a model of membranous GN, macrophages have been shown to generate NO [34].
The exact contribution of NO to glomerular injury remains controversial. For
example, decreasing NO production by L-arginine depletion in rat NTN exacer-
bated proteinuria [35], while administration of the synthetic inducible nitric oxide
synthase (iNOS) inhibitor in WKY rat NTN reduced crescent formation [36]. NO
production peaks at the point of maximum mesangiolysis in Thy 1.1 GN [37] and
is derived from iNOS-positive infiltrating inflammatory cells. NO, together with
TNF-_, is involved in M-dependent mesangial cell apoptosis in vitro [38] and is
also a key mediator of M-dependent apoptosis of murine tubular epithelial cells
[39]. Thus, although NO appears to be a key mediator of cytotoxicity in acute
glomerular inflammation, it may also facilitate death of effete cells as part of the
resolution process (see later).
Glomerular localisation of inflammatory cells
The classical paradigm is that inflammatory cells initially adhere loosely to the
endothelium via selectins. Activation of cell surface integrins occurs during leuko-
cyte rolling along the endothelium and this leads to firm binding to endothelial cell
adhesion molecules, such as intercellular adhesion molecule (ICAM) and vascular
cell adhesion molecule (VCAM). The monocytes then cross the endothelium and
basement membrane following a chemotactic gradient to enter the inflamed site.
However, different vascular beds often utilise distinct adhesion molecules depend-
ing on both the nature and timing of inflammation [40, 41]. For example, in NTN
there is limited evidence for a role of selectins in leukocyte adhesion in the rat [42]
and P-selectin deficiency in mice leads to augmented inflammatory injury in NTN
due to the loss of circulating P-selectin [43].
Integrin-mediated adhesion is involved in PMN and monocyte localisation
to inflamed glomeruli. Blockade of the VLA-4-VCAM interaction with anti-
VLA4 antibodies administered 2 weeks after disease onset significantly improved
renal function and reduced scarring. Interestingly, this effect was associated with
increased glomerular T cell and M infiltration [44, 45], implying that adhesion
molecule blockade either alters specific profibrotic cellular interactions or modu-
lates disease progression, possibly by altering inflammatory cell trafficking outside
199
of the glomeruli. Blockade of the CD11a-ICAM-1 interaction reduced M infiltra-

tion and glomerular injury in a lupus nephritis model [46]. In addition, deficiency of
ICAM-1 and CD11b/CD18 integrin is protective in NTN [47] and immune complex
GN [48], respectively.
There is also a role for chemokines in adhesion. For example, GRO-_ can be
immobilised on cell surface proteoglycans, which are up-regulated on inflamed
endothelium and mediate adherence under conditions of flow [49]. Direct blockade
of GRO-_ receptor on the M surface reduced M localisation to inflamed glom-
eruli in rats with NTN [50]. Similarly, inhibition of fractalkine, a chemokine with
a transmembrane domain facilitating cell surface expression that mediates adhe-
sion, reduces M infiltration and injury in NTN. Similarly, administration of an
antagonistic truncated form of fractalkine reduces glomerular M infiltration and
glomerular injury in lupus prone mice [51].
Thus, modulating adhesion may inhibit inflammation, although exactly which
functional effects are most important is as yet unclear. In particular, it is unclear
whether blockade of adhesion can either promote influx of leukocytes that favour
resolution or encourage the egress of pro-inflammatory cells. Similarly blockade
of adhesion may alter the trafficking of inflammatory leukocytes elsewhere within
the immune system and this may modulate the evolution of the inflammatory
response.
Glomerular injury is often inexorably progressive but, as outlined previously,

complete resolution may occur (Fig. 2). What is required for the resolution of
glomerular injury to take place? In many ways, the requirements for the resolution
of glomerular inflammation are the opposite of those involved in the initiation
and progression of disease and include the following: (1) cessation of the insult
that initiated disease; (2) restoration of a normal number of resident glomerular
cells; (3) removal of injurious glomerular leukocytes; (4) clearance of apoptotic cell
corpses; (5) reversion of resident glomerular cells to a quiescent phenotype; (6)
reprogramming of infiltrating glomerular macrophages to a reparative phenotype;
(7) modulation of the glomerular cytokine milieu; and (8) down-regulation of inju-
rious immune responses.
Cessation of the insult that initiated disease
Such factors may include successful treatment of hypertension, improved dia-

betic control (e.g. pancreas transplant) or the successful induction of a remission in
immunological diseases such as ANCA-associated vasculitis. Glomerular inflamma-
200
Figure 2.
Glomerular inflammation. A wide range of disease processes lead to acute glomerular inflam-
mation. In anti-neutrophil cytoplasmic antibody (ANCA)-positive vasculitis there is a char-
acteristic crescent of inflammatory cells that have passed across the endothelium and glo-
merular basement membrane to surround the glomerulus. In systemic lupus erythematosus
(SLE), deposition of immune complexes leads to diffuse infiltration of inflammatory cells and
proliferation of mesangial and endothelial cells. In both of these diseases the trend is towards
progressive inflammation and subsequent scarring (bold line), leading to sclerosed glomeruli
with obliteration of the capillary network and replacement by matrix proteins. However, it is
possible for inflammation to resolve and once again establish a normal glomerular architec-
ture and function (grey line) (images at 200 magnification).
tion may be associated with bacterial or viral infection, such as hepatitis, and the
eradication of such infections may also facilitate glomerular healing.
Restoration of a normal number of resident glomerular cells
Unwanted cells that are surplus to requirements are typically deleted by undergoing
apoptosis. Apoptosis was first described in 1972 [52] and is now recognised as play-
201
ing a key role in the initiation, progression and resolution of GN [53]. All glomeru-
lar cells may undergo apoptosis and may either desquamate into the urinary space
or the capillary lumen or be ingested by adjacent cells or infiltrating M. There is
little glomerular cell apoptosis evident in normal human kidneys but it is mark-
edly increased in disease states such as acute post-infectious GN, IgA nephropathy
or lupus nephritis. Apoptosis is very much a double-edged sword. For example,
apoptosis may facilitate beneficial glomerular remodelling and the clearance of
infiltrating leukocytes in acute post-infectious GN but may also result in excessive
loss of resident glomerular cells leading to hypocellular scarring in chronic diseases
such as IgA nephropathy or lupus nephritis. The resident glomerular cells comprise
mesangial cells, endothelial cells and glomerular epithelial cells (podocytes) and all
of these cells may undergo apoptosis during disease. Injured glomeruli may exhibit
either glomerular hypercellularity or hypocellularity and both are associated with
glomerular scarring that is followed by eventual glomerulosclerosis and loss of
function.
Glomerular hypercellularity
Mesangial cells
Human mesangioproliferative GN exhibits increased numbers of glomerular mesan-
gial cells and, with appropriate treatment, may resolve thereby indicating that
mesangial hypercellularity may be successfully remodelled. Mesangial hypercellular-
ity has been modelled experimentally in the rat by the administration of anti-Thy
1.1 antibodies that directly target mesangial cells. This model is characterised by
an initial wave of mesangial cell death followed by the development of mesangial
hypercellularity that resolves completely. Initial investigations focused upon the
mechanisms driving mesangial cell proliferation with a key role found for the potent
mitogen platelet-derived growth factor (PDGF). PDGF-BB is the predominant iso-
form present during glomerular injury and infiltrating M and platelets produce
PDGF-B. Expression of PDGF-B is found in many human glomerular pathologies
including IgA nephropathy, lupus nephritis and crescentic vasculitis [54]. The treat-
ment of rats with PDGF-BB induces mesangial cell proliferation [55], while inhibi-
tion of PDGF-B in the Thy 1.1 GN model attenuated mesangial hypercellularity and
ECM deposition [56].
Seminal work by Baker et al. [57], however, indicated that the resolving phase
of the Thy 1.1 GN model is characterised by a significant tenfold increase in the
level of mesangial cell apoptosis with kinetic analysis indicating that this is the
major mechanism responsible for the restoration of a normal complement of
mesangial cells. Although a somewhat simplistic concept, it was suggested that
the balance between cell survival factors and pro-apoptotic death factors may
be a critical regulator of mesangial cell apoptosis during disease and glomerular
healing.
202
Survival factors. A significant feature of the Thy1.1 model is that the peak of mesan-
gial cell proliferation and apoptosis coincide, suggesting that survival factors may
regulate total cell number as proliferating cells are more susceptible to apoptosis if
survival factors are limited. Thus, competition for survival factors at the peak of
mesangial hypercellularity may lead to the deletion of surplus mesangial cells by
undergoing apoptosis. Insulin growth factor-1 (IGF-1), IGF-2 and basic fibroblast
growth factor (bFGF) protect mesangial cells from apoptosis induced by serum
starvation, while transforming growth factor-`1 (TGF-`1), epidermal growth fac-
tor (EGF) or PDGF do not, reinforcing the fact that survival factors are cell lineage
specific. It is important to note that in vitro work indicates that survival factors do
not protect mesangial cells from all pro-apoptotic stimuli as IGF-1, IGF-2 or bFGF
do not inhibit Fas-mediated apoptosis.
The transcription factor nuclear factor-kappaB (NF-gB) promotes mesangial
cell survival and inhibition of NF-gB activity sensitises mesangial cells to apoptosis
induced by TNF-_ [58], which is employed by inflammatory M to induce mesan-
gial cell death in vitro [38]. It is of interest, however, that methylprednisolone inhib-
its mesangial cell NF-gB activity and ameliorates hypercellularity in Thy 1.1 GN
[59]. Patients with mesangioproliferative GN may be treated with corticosteroids
but there are no data to suggest whether this action plays any role in the potentially
beneficial effect of corticosteroids.
Extracellular matrix. ECM modulates mesangial cell behaviour and phenotype with
appropriate ECM such as collagen IV and laminin imparting `1 integrin-mediated
survival signals [60]. In contrast, the ECM found in glomerular scarring, such as
collagen I and fibronectin, is non-protective. Thus, although there are no supportive
data, the degradation of ECM in hypercellular glomeruli during the remodelling
phase of glomerular repair would be predicted to promote mesangial cell apoptosis
and a restoration of normal cell numbers. In contrast, glomerular scarring may
increase the vulnerability of mesangial cells to pro-apoptotic stimuli and facilitate
further mesangial cell loss.
Pro-apoptotic stimuli. Many factors may induce mesangial cell apoptosis including
complement, immune complexes, reactive oxygen species, cytokines, Fas ligation,
etc. There is evidence to support the involvement of such pro-apoptotic factors in
disease initiation and progression but there are no data to indicate whether such
death effectors are utilised to delete mesangial cells from hypercellular glomeruli.
Inflammatory M. M play an important role in GN [61] and may induce mesangial

cell apoptosis in vitro with co-culture experiments indicating involvement of NO, a
lesser role for TNF-_ and no demonstrable involvement of FasL [38]. Interestingly,
M-derived NO also inhibits mesangial cell proliferation and it can be appreciated
that inhibition of proliferation and induction of apoptosis may be ideal to combat
203
mesangial proliferative nephritis. Although monocyte/M depletion has been per-

formed in the Thy1.1 model [62] and does diminish matrix deposition, the effects
upon the mesangial cell numbers during the resolution phase were not studied.
Although the role of NO in human disease is somewhat controversial, there is a
significant correlation between the numbers of cells expressing iNOS and glomeru-
lar cell apoptosis in lupus nephritis renal biopsies [63]. It should be noted that M-
derived NO may be used for the beneficial or detrimental deletion of mesangial cells
from hypercellular or normo/hypocellular glomeruli, respectively. A key concept,
however, is that cytotoxic mediators and mechanisms that are involved in disease
processes may also be involved in tissue repair and remodelling.
Podocytes
Podocytes adhere to the external surface of the glomerular tuft and are critically
important in the maintenance of the normal glomerular filtration barrier. Podocytes
also produce VEGF and this is important for the integrity of the glomerular endo-
thelial cells [64]. There is no doubt that podocyte apoptosis and subsequent loss is
a key factor in the development of glomerulosclerosis following injury [65].
Studies using immortalised podocyte cell lines have indicated that multiple fac-
tors may induce podocyte death, e.g. reactive oxygen species, complement, angio-
tensin II, mechanical strain, endothelin and TGF-`. Podocyte apoptosis may be
amenable to modulation as hepatocyte growth factor (HGF) is protective in vitro,
while retinoids reduce podocyte injury and apoptosis induced by puromycin in vivo
and in vitro [65]. Also, endothelin 1 antagonists inhibit spontaneous age-dependent
glomerulosclerosis in rats and protect podocytes from puromycin-induced apoptosis
in vitro [66]. The down-regulation of endogenous survival factors would be pre-
dicted to predispose podocytes to undergoing apoptosis and it is therefore of interest
that the down-regulation of podocyte Bcl-2 expression is associated with a worse
outcome in patients with chronic IgA nephropathy [67].
In the majority of disease states, podocytes appear to lack the capacity to under-
go cell proliferation and restore coverage of the podocyte-depleted glomerular
surface and stimulation of such restorative cell cycle progression may be of thera-
peutic benefit. Since a reduction in podocyte number is found in many glomerular
pathologies, it is not surprising that apoptosis of podocytes would not be expected
to be beneficial and indeed there are no data to indicate that apoptosis of podocytes
is key to glomerular repair.
Endothelial cells
Endothelial cells may undergo apoptosis and this is important in both renal inflam-
mation and healing. For example, active endothelial injury is present in patients
with active ANCA-associated small-vessel vasculitis as active disease is associated
204
with increased numbers of circulating necrotic endothelial cells with numbers fall-
ing with effective treatment [68]. Endothelial cell survival factors include VEGF,
angiopoietin-1, integrin-mediated adhesion, endothelial NOS-derived NO and shear
stress and, like other glomerular cells, endothelial cell apoptosis may be controlled
by the balance between pro-apoptotic stimuli and survival signals. Although there is
no direct evidence that inflammatory M actively induce microvascular endothelial
cell apoptosis during renal inflammation, M infiltration co-localises with reduced
podocyte VEGF expression and glomerular capillary loss in the rat remnant kidney
model [69]. The downregulation of such a key survival signal suggests that M may
lower the threshold at which endothelial cells may undergo apoptosis and it is
therefore pertinent that VEGF administration is protective in this model. Prevention
of endothelial injury/apoptosis during acute disease would be predicted to reduce
the detrimental microvascular rarefaction implicated in disease progression. Lastly,
glucocorticoids that are commonly used to treat patients with acute GN inhibit
TNF-_- and LPS-induced apoptosis of glomerular endothelial cells in vitro [70],
although in vivo data are lacking.
Accumulating evidence indicates that the injured glomerular capillary network may
undergo reparative angiogenesis [71] that is driven by up-regulation of VEGF, FGF
and VEGF receptor expression. Furthermore, the administration of exogenous VEGF
to rats treated with anti-Thy 1.1 antibody and Habu-snake venom promoted the repair
of injured glomerular capillaries and accelerated the resolution process [72].
Glomerular hypocellularity
A reduced number of mesangial cells, endothelial cells or podocytes is detrimental
for glomerular health. There are various putative mechanisms to combat the inexo-
rable development of hypocellular glomerular scarring. For example, there may be
an alteration of the balance between proliferation and apoptosis of resident cells
(i.e. increased proliferation and reduced apoptosis) to promote restorative net cell
accumulation. This is likely to be an important mechanism of action of various
growth factors that exhibit mitogenic and pro-survival effects.
Also, there is evidence that there may be repopulation of the hypocellular glom-
erulus by migration of cells to the mesangium from outside the glomerulus. Elegant
work by Hugo et al. [73] examined the repopulation of mesangial cells during the
early phase of Thy 1.1 GN in the rat. Although the majority (> 90%) of mesangial
cells are killed in the early stage of this model, there were residual Thy 1.1-positive
cells present within the juxtaglomerular apparatus (JGA) just outside the glomeru-
lus. Careful radiolabelling and morphometric studies demonstrated the migration
of these Thy 1.1-positive cells into the hypocellular glomerulus to repopulate the
denuded mesangium and restore cell numbers.
Recently, there has been great interest in the potential for repopulating injured
and scarred sites by stem cells derived from the kidney or bone marrow [74]. Stem
205
cells offer a very appealing prospect for treatment as they may differentiate into any
of the resident cells of the glomerulus. Studies have induced disease in female mice
that have received a bone marrow transplant (BMT) from syngeneic male so that
BM-derived cells can be identified by the presence of the Y chromosome detected by
in situ hybridisation. BMT can also be performed using BM-derived cells from mice
transgenic for the enhanced green fluorescent protein gene or mice expressing the
bacterial LacZ gene. Investigators have also studied male patients who have received
allografts from female donors. Initial reports suggested that BM-derived cells made
a significant contribution towards renal regeneration. However, more recent studies
of murine renal ischaemia-reperfusion injury using rigorous deconvolution confocal
microscopy indicate that the contribution of BM-derived cells to tubular epithelial
regeneration is negligible [75], while the contribution to peritubular capillary repair
is minor [76]. The authors suggest that the imaging methods previously used may
have overestimated the involvement of BM-derived stem cells. Studies examining
glomerular injury are limited but recent work suggest that some mesangial cells
and podocytes may be derived from BM-derived cells in a murine model of Alports
syndrome and that these cells contribute to repair of the basement membrane [77].
Stem cells may also reside within the kidney [78, 79] and the role played by this
population during renal repair is unclear at present.
Removal of injurious glomerular leukocytes
Injured glomeruli typically exhibit a leukocytic infiltrate and these must be removed
before complete resolution can take place.
Reduced glomerular expression of chemokines and pro-inflammatory

cytokines together with reduced adhesion molecule expression by
glomerular endothelial cells
The resolution of GN is associated with reduced chemokine and chemokine recep-
tor expression [80]. Interestingly, in a number of experimental settings, inhibition
of chemokines can lead to augmented glomerular injury suggesting that, as with
adhesion molecules, chemokines and their receptors are involved in regulation of the
immune response outside the glomerulus. As yet, the role of chemokines in direct-
ing the egress of glomerular leukocytes as injury resolves has not been addressed.
Removal of infiltrating leukocytes

There are limited possibilities for leukocyte removal. Leukocytes may die by under-
going apoptosis within the kidney or emigrate via lymphatic vessels.
206
Removal of infiltrating PMNs

Infiltrating PMNs may initiate and worsen tissue injury and the efficient removal
of PMNs is thus important. PMNs constitutively undergo apoptosis and are readily
ingested by M at inflamed sites. Previous studies of tracking radiolabelled PMNs
in a rat model of immune complex GN [81] indicated that approximately one fifth
of infiltrating glomerular PMNs underwent apoptosis and were cleared by phagocy-
tosis. The majority of PMNs, however, left the glomerular capillary network prior to
undergoing apoptosis and underwent clearance elsewhere in the reticuloendothelial
system. The proportion of PMNs that die in situ within the glomerulus may, how-
ever, vary according to the nature of the glomerular inflammation. It is important to
note, however, that PMN apoptosis undoubtedly occurs in human GN [82].
Removal of infiltrating M and lymphocytes

This has been little studied in the kidney and the limited data available suggest
that infiltrating M may undergo apoptosis within the inflamed kidney or exit to
draining lymph nodes [83]. This may be slightly different to M fate in non-renal
inflammation as studies of peritoneal inflammation indicates that inflammatory
M do not undergo apoptosis in situ but exit the peritoneum and traffic to draining
lymph nodes via the lymphatic system [84]. M trafficking during and following
glomerular inflammation merits further study. Similarly, little is known regarding
the fate of infiltrating lymphocytes, although it would be logical to assume that they
traffic to draining lymph nodes.
Clearance of apoptotic cell corpses
The ingestion of apoptotic PMNs by both M and mesangial cells is evident in NTN
in the rat. Primary human mesangial cells phagocytose apoptotic cells in vitro and
this may represent an important back-up to professional M clearance. The inges-
tion of apoptotic cells such as PMNs by M is a powerful biological stimulus and
the administration of apoptotic cells to inflamed sites has been shown to promote
the resolution of inflammation [85]. It is therefore also likely to be important in the
resolution of GN.
It is also noteworthy that various factors involved in the initiation of inflamma-
tion appear to play a role in subsequent tissue repair. For example, pro-inflamma-
tory cytokines such as IL-1 and TNF-_ increase the capacity of M to phagocytose
apoptotic cells thereby facilitating the clearance of apoptotic cells and promoting
the generation of reparative M. Glucocorticoids exert a similar effect upon M,
although it is unclear whether this action is important in their beneficial action upon
inflammatory GN.
207
Defective apoptotic cell clearance and glomerular inflammation and repair

There are myriad receptors and bridging molecules involved in the recognition and
uptake of apoptotic cells by M [86] and mice deficient in many of these molecules
have been engineered. There are also strong links between the defective phagocy-
tosis of apoptotic cells, autoimmunity and hence GN. C1q, the first component of
complement bridges apoptotic cells to M, and C1q-knockout mice exhibit defec-
tive apoptotic cell clearance, autoimmune GN and develop more severe glomerular
injury following the induction of NTN [87]. It is very pertinent that the majority
of human patients with C1q deficiency develop SLE, while M from patients with
SLE exhibit decreased capacity to phagocytose apoptotic cells [88]. Defective apop-
totic cell uptake may result in inadequate deactivation of infiltrating inflammatory
M and promote ongoing tissue injury. Also, defective or inadequate apoptotic cell
uptake may place mesangial cells at risk of ongoing M cytotoxicity since in vitro
data indicate that uptake of apoptotic cells diminishes M induction of mesangial
cell apoptosis by TNF-_ [38]. Although, defective phagocytosis of apoptotic cells
may be involved in the development and progression of GN, there are also impor-
tant implications for the resolution of GN since defective apoptotic cell ingestion
would be predicted to impair the generation of reparative M and this may impede
glomerular remodelling. However, the effect of such a phagocytic defect upon the
resolution of glomerular injury has not been studied.
Reversion of resident glomerular cells to a quiescent phenotype
TGF-` is the major pro-fibrotic cytokine in chronic renal inflammation. Increased

TGF-` expression is found in all human glomerular disease exhibiting chronic scar-
ring, especially the progressive interstitial fibrosis that is the common end stage of
all forms of chronic renal injury. TGF-` induces the epithelial to mesenchymal tran-
sition (EMT), a process characterised by tubular epithelial cells becoming E-cad-
herin-negative and smooth muscle actin-positive myofibroblasts that are believed to
promote interstitial scarring. TGF-` also promotes ECM synthesis and deposition.
Early studies in GN established that inhibition of TGF-` by decorin, a natural TGF-
` antagonist, or anti-TGF-` antibodies reduced glomerular and interstitial scarring
[89].
During GN, the glomerular mesangial cells similarly adopt a myofibroblast phe-
notype and are characterised by de novo expression of smooth muscle actin. Such
cells are regarded as activated and generate and deposit ECM. It is thus apparent
that such cells must either be deleted or revert to a quiescent phenotype during the
resolution process. Although there are few data available for mesangial cells, inter-
est has focused on bone morphogenic protein-7 (BMP-7), a member of the TGF-`
superfamily that has been shown to antgonise TGF-`-induced EMT [90]. Indeed, the
phenotypic changes of tubular cells undergoing EMT appear to be plastic such that
208
TGF-`1 may induce tubular cell EMT, while BMP-7 can reverse tubular cell EMT
in vitro [91]. Furthermore, the administration of BMP-7 to mice with NTN was
able to preserve renal function and reduce mortality, even when treatment was com-
menced up to 4 weeks after disease onset [92]. This impressive reversal of chronic
tubulointerstitial injury in vivo was associated with a reduction in fibrosis and in
vivo evidence of EMT. Similar beneficial effects were seen in models of murine lupus
with reduced formation of inflammatory glomerular crescents, decreased collagen I
deposition and increased MMP2 and 9 expression [93]. Thus, BMP-7 shows prom-
ise as an agent that can reverse progressive inflammatory scarring.
Reprogramming of infiltrating glomerular macrophages to a reparative

phenotype
In the early stages of experimental GN M appear to have the properties of clas-

sically activated M, producing NO and being unresponsive to anti-inflammatory
cytokines such as IL-4 and TGF-` [94]. It is likely that generation of NO in this
context plays an important role in injury as M-derived NO may be cytotoxic to
both mesangial and tubular cells, leading to their apoptosis [38, 39]. M are emi-
nently capable of ingesting apoptotic cells and, as outlined previously, the removal
of apoptotic cells is a key stage in resolution. Uptake of apoptotic PMNs by M
leads to further release of anti-inflammatory and reparative cytokines such as TGF-
`1. Thus, there is the concept of early M influx promoting injury, but if the driving
insult is withdrawn M will play a key role in dampening down further injury.
Modulation of the cytokine milieu
Models of crescentic glomerular inflammation are dominated by a Th1-biased

immune response driven by pro-inflammatory cytokines such as IFN-a, TNF-_ and
IL-12 generated primarily by infiltrating leukocytes but also by resident glomerular
cells [95, 96]. In contrast, IL-4 and IL-13 are key cytokines favouring the develop-
ment of Th2 immune responses. IL-4-knockout mice develop more aggressive NTN
[26, 97], whereas IL-13-knockout mice develop enhanced antibody deposition and
comparable glomerular inflammation compared to wild-type mice. Administration
of IL-4 ameliorates acute crescentic GN even if treatment is delayed until after the
onset of injury [98100], a requirement for effectiveness in human disease. Although
IL-4 administration after disease onset did not alter M infiltration, it did reduce
the state of M activation with attenuated iNOS and ED3 expression.
IL-10 has an important role in attenuating cell-mediated glomerular inflamma-
tion. IL-10-knockout mice develop more severe glomerular inflammation [101] and
IL-10 administration is protective when administered before or after disease onset
209
[99]. In certain lupus prone mouse strains deficiency of IL-10 leads to more severe
GN with IL-10 administration being protective [102]. Adenoviral transduction of
the liver to express IL-10 markedly improved renal function in WKY rats with
NTN and was associated with reduced M infiltration and activation [103]. Long-
term administration of IL-10 to rats achieved by administering adeno-associated
virus expressing IL-10 [104] resulted in persistent elevation of IL-10 levels. These
animals subsequently underwent a 5/6 nephrectomy and exhibited improved renal
function compared to controls associated with reduced M and T cell infiltration.
This implies that IL-10 can benefit both immune and non-immune-mediated renal
injury.
It is thus likely that the resolution of glomerular inflammation will require a shift
in the cytokine milieu from that dominated by pro-inflammatory mediators to one
comprising key cytokines such as IL-10, that down-regulate injury and inflamma-
tory responses and set the scene for effective tissue repair.
Down-regulation of injurious immune responses
Immune-mediated glomerular inflammation is directed at specific antigens, although

the targets in human disease are generally unclear. This response requires antigen
presentation by dendritic cells or other antigen-presenting cells (APCs) to T cells.
This involves the interaction between MHC class II molecules on the APC and its
cognate T cell receptor (TCR) with full activation requiring activation of co-stimu-
latory molecules. The interaction between CD80/86 on the APC with CD28 on the
T cell with TCR engagement leads to T cell activation and promotion of antigen-
specific immunity. This interaction can be prevented by CTLA4-Ig, which binds to
CD80/86, and administration of CTLA4-Ig to WKY rats with experimental GN
reduced albuminuria and glomerular M and T cell infiltration [105]. Similarly,
the administration of CTLA4-Ig to mice with NTN preserved renal function and
reduced proteinuria [106]. More recent data suggest that CD80 and CD86 may
have differential roles in glomerular inflammation. CD80-deficient mice are pro-
tected from the development of anti-GBM GN with reduced M and T cell accu-
mulation, whereas CD86-deficient mice developed more severe inflammation with
reduced IL-4 and increased IFN-a expression [107]. Another member of the CD28
family is inducible costimulator (ICOS) expressed on antigen-primed T cells and
has been shown to have a role in driving humoral immune responses. In mice with
lupus nephritis, expression of ICOS on T cells increased with disease progression
and antibody blockade after disease onset reduced glomerular inflammation and
improved survival [108]. Thus, reducing antigen-primed T cell activation can slow
progression and promote resolution.
The resolution process requires regulation of T and B cell responses outside
the kidney and increasing interest has focused on the role of T regulatory (Treg)
210
cells that have important roles in the maintenance of peripheral tolerance. These
cells produce IL-10 and TGF-` and are characterised by expression of CD25 and
Foxp3 [109]. In experimental anti-glomerular basement membrane GN, transfer
of CD4+CD25+ T cells before the induction of disease markedly attenuated inflam-
mation with reduced glomerular M and T cell infiltration and less proteinuria
compared to control administered CD25 cells [110]. The administered CD25+
cells predominantly localised to secondary lymphoid organs and this mirrors results
found in other inflammatory diseases including inflammatory colitis, arthritis and
diabetes.
There is some evidence in human GN that resolution of disease is associated with
the generation of Treg cells. In Goodpastures disease mononuclear cells isolated
from patients at disease presentation proliferate in response to peptides derived
from the Goodpastures antigen (NC1 domain of the alpha 3 chain of type IV col-
lagen) and preferentially produced IFN-a. In contrast, T cells isolated after the reso-
lution of active disease produced IL-10 in response to the peptides and suppressed
proliferation. Thus, the generation of Treg cells may have a crucial role in switching
off autoimmune glomerular disease.
Old and new therapies to combat glomerular injury and

promote resolution
Although challenging, the manipulation of apoptosis in GN may be of therapeutic

benefit. The situation is complicated in that one may wish to induce apoptosis of
infiltrating leukocytes but protect resident renal cells from undergoing apoptosis; it
may be possible to use a combination of pro-apoptotic agents and survival factors
to achieve such effects or target drugs to particular cell types. It should be noted
that, since infiltrating leukocytes may induce glomerular cell apoptosis, currently
employed anti-inflammatory treatments such as corticosteroids or cytotoxic drug
treatment that act to diminish renal leukocyte infiltration and the levels of pro-
apoptotic cytokines would be predicted to reduce apoptosis of resident renal cells.
Also, corticosteroids may inhibit endothelial cell apoptosis and promote M inges-
tion of apoptotic cells, thus facilitating tissue repair.
The scarred, fibrotic kidney is a hostile microenvironment and provides a
reduced supply of ECM-derived survival signals that promotes a chronic low level
of cell drop out by apoptosis. Thus, anti-fibrotic treatments such as angiotensin-
converting enzyme inhibitors should promote renal cell survival. Renal ischaemia
and associated hypoxia is detrimental and promotes cell loss and further fibrosis.
The administration of VEGF ameliorates various experimental models of renal dis-
ease and suggests that inhibition of endothelial cell apoptosis as well as stimulation
of reparative angiogenesis may be a useful strategy as the improved tissue blood
flow and oxygenation would promote survival of non-endothelial renal cells.
211
For many years the treatment of acute GN has mainly involved non-specific
immunosuppression with steroids and cytotoxic agents. However, as our under-
standing of the inflammatory response has evolved, more specific biological agents
have been developed and brought to the clinical arena. Anti-TNF-_ therapies
have been used and more recently there has been interest in cell-targeted therapy.
Rituximab is an anti-CD20 monoclonal antibody developed for treatment of B cell
lymphomas. CD20 is expressed exclusively on human B cells and rituximab depletes
circulating B cells for 69 months. In lupus nephritis, treatment with rituximab in
refractory GN shows early promise [111]. Similarly, rituximab can lead to disease
remission in ANCA-positive vasculitis. The precise mechanism of the effect of B cell
depletion on glomerular inflammation is unclear. Reduced autoantibody production
is the most obvious effect, although disease may remit without a change in autoan-
tibody levels. Thus, B cells may have other roles at the tissue level including antigen
presentation that may be equally important.
An alternative approach to promote resolution has been to prevent cell division.
Cell proliferation is a near constant feature of GN involving leukocytes, mesangial
cells, epithelial cells and endothelium, with the cells responding to a diverse range
of signals. The cyclin-dependent kinases (CDK), a family of serine/threonine protein
kinases, are key regulatory cell cycle proteins. CDK-2 is required for progression
of S phase and specific inhibitors have been developed with initial interest being
the treatment of malignant disease. Roscovitine is a CDK-2 inhibitor and has been
shown to reduce renal cell proliferation in Heymann nephritis [112], a model of
membranous nephropathy in humans. Similarly, roscovitine treatment of rats with
Thy 1 GN reduced endothelial and mesangial cell proliferation in a manner similar
to the chemotherapeutic agent mycophenolate mofetil (MMF). In addition, mice
genetically deficient in the endogenous CDK inhibitor p21 develop increased glo-
merular proliferation and worse renal function compared to wild-type mice in a
murine model of mesangioproliferative GN [113]. Further work will establish the
utility of cell cycle inhibition in GN. An important caveat of this approach is that,
although increased cell proliferation is prominent and detrimental in acute GN, it
is also a feature of tissue repair and such anti-proliferative treatment may hinder
concurrent reparative processes.
Cell-based therapy has also generated interest as a means of promoting resolu-
tion. M are present in acute and chronic renal injury and play a role in the ini-
tiation, progression and resolution of inflammation. M are ideally suited to alter
inflammatory disease due to their preferential localisation to inflamed tissue and
their suitability for ex vivo manipulation involving genetic, cytokine or chemical
manipulation. The majority of work has focused on adenoviral transduction of M
ex vivo [114]. Systemic injection of M transfected with recombinant adenovirus to
express IL-1 receptor antagonist (IL-1ra) reduced the severity of glomerular inflam-
mation in mice with NTN and reduced interstitial M infiltration in a model of
unilateral ureteric obstruction [115, 116]. Injection of NR8383 cells (a rat alveolar
212
M cell line) expressing IL-4 localised efficiently to inflamed glomeruli of rats with
NTN following direct injection into the left renal artery [117]. These cells produced
the IL-4 locally and reduced M infiltration, histological markers of glomerular
inflammation and proteinuria for up to 7 days [117]. In the same model injection
of primary cultures of rat BMDM transduced to express IL-10 similarly produced a
marked reduction in albuminuria and M activation, as demonstrated by reduced
MHC class II and ED3 expression [118]. The impact of IL-10-expressing cells was
more pronounced than IL-4 with the most significant differences in injury seen 7
days after a single injection of cells. Interestingly, in these experiments, despite the
fact that there is no evidence of systemic cytokine production, the localised glomeru-
lar production alters the function of bystander infiltrating M and resident glomeru-
lar cells including mesangial cells, endothelial cells and podocytes. These experiments
demonstrate that the administration of a small number of M, with altered function,
is able to produce a sustained reorientation of the inflammatory response.
Conclusions and future prospects
Until recently the emphasis regarding resolution focused upon the inhibition of
various pro-inflammatory mediators such as TNF-_, chemokines or adhesion mol-
ecules. This is reflected in many of our current therapies for inflammatory GN,
which reduce the pro-inflammatory behaviour of leukocytes, inhibit cell division
or target pro-inflammatory mediators such as TNF-_. There is no doubt that the
process of renal healing is an active and tightly coordinated series of events and,
although the exact mechanisms underlying the resolution of glomerular inflamma-
tion and associated glomerular remodelling are still incompletely understood, we
are now beginning to dissect key processes and players. Further research will yield
insights that will lead to novel future therapies, but we will also need to characterise
various biomarkers of tissue repair to enable such therapies to be monitored and
administered effectively. It is also probable that such therapies are likely to require
the simultaneous modulation of multiple biological processes such as cell prolifera-
tion, angiogenesis, and matrix remodelling.
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Resolution of mucosal inflammation
John L. Wallace1 and Philip M. Sherman2
1
Inflammation Research Network, University of Calgary, 3330 Hospital Drive NW,
Calgary, Alberta, T2N 4N1, Canada; 2Hospital for Sick Children and University of Toronto,
555 University Avenue, Toronto, Ontario, M5G 1X8, Canada
Introduction
Inflammation of the mucosal lining of the gastrointestinal tract is not only common,
it is often described as normal. This is particularly the case in the intestine, where
a single layer of epithelial cells separates the vascular and immune systems from
billions of microbes. Of course, uncontrolled inflammation is also associated with a
number of gastrointestinal disorders, some of which are quite common. Many of the
current therapies for disease such as inflammatory bowel disease (IBD) are aimed
at bringing the inflammatory response under control, by inhibiting production or
action of pro-inflammatory mediators, so that repair of tissue injury can proceed. In
recent years, there has been increasing interest in the notion that better understand-
ing the endogenous mechanisms for resolution of inflammation will provide impor-
tant clues for the design of more effective therapies for inflammatory diseases.
Oddly, it was the introduction of a new type of nonsteroidal anti-inflamma-
tory drug (NSAID) that triggered a burst of research into endogenous anti-inflam-
matory drugs. Cyclooxygenase (COX) is an essential enzyme for the synthesis of
prostaglandins (PGs), some of which (e.g., PGE2) have long been recognised as
contributors to the oedema and pain associated with inflammation. Suppression
of COX activity by NSAIDs is widely accepted as the main mechanism underlying
the anti-inflammatory activity of this class of drugs [1]. In the stomach, however,
PGs are important mediators of several components of mucosal defence [2], so the
inhibition of their synthesis by NSAIDs predisposes the stomach to ulceration. A
second isoform of COX was identified in 1991 [3], and was found to be expressed
in particularly high levels at sites of inflammation [4]. Selective COX-2 inhibitors
were quickly developed with the notion that they would inhibit inflammatory PG
synthesis (thereby reducing oedema and pain), but not gastric PG synthesis (thereby
not causing ulceration).
Unfortunately, the expression of COX-1 and COX-2 did not turn out to be quite
as clearly divided as originally proposed. Selective COX-2 inhibitors were found
to cause gastrointestinal injury [5, 6], as well as exhibiting toxicity similar to, or

even worse than, conventional NSAIDs in the renal and cardiovascular systems [7].
Nevertheless, the advent of the selective COX-2 inhibitor did permit researchers to
begin to determine the contribution of this enzyme to inflammation, which resulted
in some important discoveries.
This chapter focuses on resolution of inflammation in the gastrointestinal tract
and, in particular, on the chemical mediators that contribute to this process. Better
understanding the mechanisms of resolution of inflammation will contribute to the
development of new therapies for a variety of inflammatory disorders of the diges-
tive system.
COX-2 in the resolution of inflammation
Several studies published in the mid-to-late 1990s provided important evidence that
COX-2 makes an important contribution to the resolution of inflammation. Reuter
et al. [8] examined the contribution of COX-2 to the resolution of colitis in rats.
It has previously been demonstrated that NSAIDs exacerbated colitis in this model
[9]. Administration of selective COX-2 inhibitors significantly reduced colonic PG
synthesis, and caused a marked worsening of the colonic damage, while infiltration
of granulocytes into the mucosa was increased [8]. Most importantly, with contin-
ued administration of selective COX-2 inhibitors for a week, the colitis worsened to
the point where perforation and death occurred in most animals. Subsequent studies
demonstrated that COX-2-derived PGD2 synthesis occurs soon after the induction
of colitis in rats, and that this acts as an early stop signal on granulocyte infiltra-
tion into the colon [10].
Another study examined the role of COX-2 in paw oedema induced in mice
via injection of carrageenan [11]. Whereas the inflammation subsided within 48
h in wild-type mice, it was still evident in COX-2-deficient mice 7 days later. In
a study of carrageenan-induced pleurisy in rats, COX-2 expression was increased
most significantly during the phase when inflammation was resolving (i.e. when
leukocyte numbers in the pleural cavity were declining) [12]. When a selective
COX-2 inhibitor was administered, the resolution of inflammation was blocked.
However, the reduction of pleural leukocyte numbers was restored by adminis-
tration of a prostaglandin that was produced via COX-2, namely 15-deoxy-612-
14
-PGJ2 (subsequently abbreviated as 15dPGJ2). This prostanoid is a hydration
product of PGD2.
Studies from Serhans group have convincingly demonstrated the crucial role of
lipoxins in the resolution of inflammation [13] (as discussed in the chapter by C. N.
Serhan). Lipoxins can be produced via several pathways, most involving transcel-
lular metabolism of fatty acids. Interestingly, COX-2 can play an important role in
lipoxin formation in one circumstance. Aspirin covalently acetylates both COX-1
and COX-2. While in the case of COX-1 this prevents all metabolism of arachidonic
224
acid to prostanoids, acetylated COX-2 is still capable of metabolising arachidonic

acid to 15-R-HETE. This can be converted, via neutrophil 5-lipoxygenase, to 15-
R-lipoxin A4 (aspirin-triggered lipoxin) [14]. Aspirin-triggered lipoxin accounts
for a significant portion of the anti-inflammatory effects of aspirin. It binds to the
same receptor and exerts virtually identical actions to its epimeric counterpart [15,
16]. These actions include inhibitory effects on neutrophil chemotaxis, adherence,
transmigration and superoxide anion production [1318], and suppression of the
production of IL-8 by intestinal and pulmonary epithelial cells [19, 20].
Gastric inflammation
The discovery of a link between gastric colonisation by Helicobacter pylori and

ulcer disease led to a renewed interest in mechanisms regulating inflammation in the
gastric mucosa. As mentioned above, PGs mediate many components of mucosal
defence and, in general, act to down-regulate inflammatory processes. The predomi-
nant PGs formed by the gastric mucosa are those of the E and I series [21], which
are vasodilators, can suppress leukocyte adherence to the vascular endothelium and
can suppress release of pro-inflammatory mediators (e.g. leukotrienes, platelet-acti-
vating factor) from immunocytes and leukocytes [22]. Many of these pro-inflamma-
tory mediators also contribute to mucosal injury in various circumstances, including
that associated with administration of NSAIDs [22]. The anti-inflammatory and
gastroprotective actions of PGs can also be elicited by other endogenous media-
tors, such as nitric oxide (NO) and hydrogen sulphide (H2S). Both NO and H2S are
vasodilators and inhibitors of leukocyte-endothelial adhesive interactions and exert
anti-inflammatory and gastroprotective effects [23, 24].
COX-1 is the predominant isoform expressed in the healthy gastric mucosa,
but COX-2 is rapidly induced when COX-1 is suppressed [25], when the mucosa
is challenged with an irritant [26] or when the mucosa becomes ischaemic [27].
This up-regulation of COX-2 is a defensive and anti-inflammatory response aimed
at maintenance of mucosal integrity (increased blood flow, reduction of leukocyte
adherence and activation). Suppression of COX-2 activity in these circumstances
leads to elevated leukocyte adherence to the vascular endothelium in the gastroin-
testinal microcirculation and to mucosal injury [28].
The observation that co-administration of aspirin and a selective COX-2 inhibi-
tor results in extensive gastric damage in animals and humans [18, 29] raised the
possibility that a COX-2-derived factor was contributing to the resistance of the
mucosa to injury. As mentioned above, the covalent acetylation of COX-2 by
aspirin can result in the formation of aspirin-triggered lipoxin. Intraperitoneal
administration of very low (~23 nM) doses of LXA4 reduced the severity of aspi-
rin-induced damage in the rat stomach [18]. The potency of LXA4 in this regard is
very similar to that of PGs of the E and I series [30]. This protective effect is likely
225
attributable to the anti-inflammatory properties of LXA4, specifically, inhibition of

aspirin-induced leukocyte adherence [18]. When a COX-2 inhibitor is administered
together with aspirin, the formation of this lipoxin is inhibited, resulting in exten-
sive leukocyte adherence and mucosal injury. These same effects can be observed
when aspirin is co-administered with an antagonist of the receptor (ALX) that is
activated by LXA4 and aspirin-triggered lipoxin [18]. Interestingly, when the gastric
mucosa is inflamed, there is greater expression of COX-2 and a greater contribu-
tion of COX-2-derived products to mucosal defence [31]. Indeed, the presence of
mucosal inflammation results in a reduced severity of damage induced by aspirin,
but this effect is lost if a COX-2 inhibitor is co-administered with aspirin. Aspirin-
triggered lipoxin generation is, not surprisingly, elevated in the inflamed versus the
healthy gastric mucosa [31].
The ALX receptor that is activated by lipoxins can also be activated by another
anti-inflammatory mediator, annexin 1. The most potent anti-inflammatory drugs
are the glucocorticoids. It has been recognised for many years that much of this
activity is attributed to the induction by these drugs of the production of annexin
1 [32]. Annexin 1 inhibits the activity of cytosolic phospholipase A2 and, there-
fore, blocks arachidonic acid metabolism [33]. It also promotes inflammatory cell
apoptosis through caspase-3 activation, and has been suggested to act as a signal
on apoptotic cells to allow them to be recognised and ingested by phagocytes [33].
In cultured macrophages, annexin 1 stimulates IL-10 production which, in turn,
down-regulates expression of inducible nitric oxide synthase and IL-12 [34]. High-
lighting their roles in resolution of inflammation, both annexin 1 and LXA4 cause
disengagement of neutrophils from the vascular endothelium [35]. Neutrophils are
a primary source of annexin 1 and express the ALX receptor [35].
Once again demonstrating the link between endogenous anti-inflammatory
mediators and gastric resistance to injury, annexin 1 has been shown to contribute
to mucosal defence. Acute administration of corticosteroids reduces the severity of
gastric damage in animal models [36, 37]. This protective effect is likely mediated
by annexin 1 since it is abolished by antagonism of the ALX receptor and by an
immunoneutralising antibody [37] (Fig. 1). These protective effects of annexin 1
are likely attributable to its ability to reduce leukocyte adherence to the vascular
endothelium within the gastric microcirculation [37].
Intestinal inflammation
As mentioned previously, the intestine is regarded as being in a state of controlled

inflammation. As a result of the bacterial load present in the lumen of the large
bowel and distal small intestine, mucosal injury is associated with robust inflamma-
tory reactions. Regulation and resolution of these reactions is, therefore, extremely
important to avoid excessive damage to host tissues.
226
Figure 1.
Annexin 1 is an important mediator of gastric mucosal resistance to damage.
Indomethacin causes the formation of extensive hemorrhagic erosions in the rat stomach.
Pretreatment with dexamethasone significantly (*p < 0.05) reduces the severity of gastric
damage. However, when annexin 1 is immunoneutralised, the protective effect of dexa-
methasone is lost. For further details of these studies, see [37].
Studies using complementary models of infectious enterocolitis and models of

IBD have provided substantial information on the chemical mediators involved in
the resolution of mucosal inflammation. In addition to the anti-inflammatory roles
played by PGs and lipoxins, as in the stomach, several cytokines have been identi-
fied as important in resolving intestinal inflammation. For example, IL-10 is well
recognised as a critical down-regulatory factor. The spontaneous enterocolitis that
develops in IL-10-deficient mice exemplifies the importance of this anti-inflamma-
tory cytokine [38].
The endogenous antagonist of the IL-1 receptor (IL-1RA) is another example of
a cytokine produced during inflammatory reactions in the intestine that acts as a
brake. In IBD, there is an imbalance between the production of IL-1` and IL-1RA,
thereby favouring dysregulation of inflammation [39].
The lower incidence of Crohns disease in the developing world provided a
basis for the hypothesis that helminthic infections, by driving a Th2 lymphocyte
predominance, may protect against IBD. Elliott et al. [40] reported that infection of
IL-10-deficient mice with Heligmosomoides polygyrus results in a down-regulation
of the inflammatory response, and reduced production of Th1-type cytokines (IL-
227
Figure 2.
Colonic synthesis of prostaglandin (PG) E2 and D2 in the rat after induction of colitis by
intracolonic administration of trinitrobenzene sulphonic acid (TNBS).
Colitis is most severe during the first week after TNBS administration, and gradually heals
during the ensuing 5 weeks. By week 6, the colon is macroscopically normal and the inflam-
mation has resolved. While PGE2 synthesis returns to basal levels by week 2, there is a
prolonged increase in colonic PGD2 synthesis during the period of resolution. The PGD2 is
produced via cyclooxygenase-2, as indicated by the ability of a selective inhibitor of this
enzyme (rofecoxib) to reduce colonic PGD2 synthesis to basal levels. For further details of
these studies, see [43, 44].
12, IFN-a). They also demonstrated marked up-regulation of an anti-inflammatory

cytokine, IL-13, and suggested that it was this factor responsible for suppressing
pro-inflammatory cytokine production.
As mentioned in the introduction of this chapter, COX-2-derived prostanoids
have been implicated as important anti-inflammatory signals in the context of
colitis. PGD2 is released early after induction of colitis in rats and acts to suppress
granulocyte infiltration into the mucosa [10]. Selective inhibition of COX-2 dur-
ing active colitis results in exacerbation of the inflammation and ulcers [8]. These
drugs have also been reported to exacerbate or reactivate colitis in humans [41,
42]. Interestingly, PGD2 synthesis increases over the period of weeks during which
colitis induced by trinitrobenzene sulphonic acid heals [43, 44] (Fig. 2). This PGD2
228
synthesis occurs via COX-2. Even when the colonic mucosa has returned to a non-
inflamed state, synthesis of PGD2 and expression of COX-2 and PGD synthase
remain markedly elevated [43, 44]. There is evidence that this persistent up-regula-
tion of PGD2 synthesis and COX-2 expression contributes significantly to long-term
changes in colonic function after a bout of colitis, including barrier dysfunction,
epithelial secretory dysfunction, hyper-proliferation and a predisposition to carcino-
gen-induced colon cancer [43, 44]. Suppression of COX-2 activity or antagonism
of one of the receptors for PGD2 (DP1) normalises these changes in function and
structure [44].
As discussed above, annexin 1 is an important factor in the resolution of inflam-
mation, acting through several mechanisms. Vergnolle et al. [45] demonstrated that
annexin 1 expression is markedly increased early in the course of experimental coli-
tis in rats. Of the six annexins studied, only expression of annexin 1 secretion was
increased. Annexin 1 secretion is no longer increased once the colitis has resolved
[45]. The secretion of annexin 1 occurs primarily from neutrophils and macrophages
[46], but in the first 24 h of colitis, annexin 1 secretion is from neutrophils alone.
The expression of annexin 1 by neutrophils is only detectable in neutrophils at the
site of inflammation (i.e. not neutrophils from blood or from the tunica muscularis
of the colon). Annexin 1 secretion is also observed in biopsies taken from patients
with active ulcerative colitis [47].
The ability of lipoxins to suppress pro-inflammatory cytokine production by
intestinal epithelial cells [19] was the impetus for an assessment of the effects of
stable lipoxin analogues in experimental colitis. Several groups have independently
demonstrated an acceleration in the rate of resolution of experiment colitis in rodents
through treatment with synthetic lipoxins [4850]. The lipoxins dose-dependently
accelerate the healing of mucosal injury and reduce the extent of granulocyte and
lymphocyte infiltration into the colonic mucosa.
Enteric bacteria and resolution of mucosal inflammation
Mouse models of chronic IBD demonstrate that the intestinal bacterial microflora,
which is present both in the lumen and adherent to the mucosa of the colon, is
involved in various aspects of the development, prevention, and resolution of
mucosal inflammation. Intestinal injury and mucosal inflammation are substantially
reduced in the same animals when raised under germ-free conditions [51]. Dysbiosis
is a term that has been coined recently to denote imbalances in the complex, normal
inter-relationships that occur among the luminal commensal bacteria residing in the
non-inflamed large intestine and host surface epithelial cells, and the underlying
innate and adaptive immune cells [52]. When the normal balance between prokary-
otes and host cells is disrupted, the net result is the development of intestinal injury.
Restitution of such an imbalance results in the resolution of mucosal inflammation.
229
Probiotics is a term that refers to live organisms that, when delivered to mucosal
surfaces, have a beneficial effect on human and animal health. Increasing evidence,
obtained from both relevant experimental animals and humans with IBD, indicates
that alteration of the luminal flora by employing probiotics can alter mucosal
inflammatory responses of the host [53]. In contrast, enteric pathogens can activate
Fib-mediated pro-inflammatory signal transduction with production of chemokines,
like interleukin-8, that serves as a chemoattractant for neutrophils. Probiotics and
commensal bacteria inhibit the activation of latent, inactive NF-gB in the cytosol
of host epithelial cells [54]. The end result is the prevention of the production and
secretion of chemokines and a reduction in the attendant inflammatory cell infiltrate
in the gut mucosa.
Host cell mediators that have an impact on normal wound healing processes
are influenced by exposure to enteric pathogens. For instance, Salmonella enteritica
serovar Typhimurium activates epithelial cell production of matrix metalloprotein-
ase (MMP)-9 in a mouse model of enterocolitis. Inflammation is muted in MMP-9
knockout mice and wound healing impaired in injured polarised human epithelial
(Caco2) cells exposed to purified MMP-9 [55].
Transforming growth factor (TGF)-` also has an impact on intestinal epithelial
cell responses to enteric pathogens. For example, Howe et al. [56] showed that
TGF-` blocks the drop in transepithelial electrical resistance induced by exposure of
polarised T84 epithelial cells to enterohaemorrhagic Escherichia coli O157:H7. The
protective effects of the anti-inflammatory cytokine are mediated through prevent-
ing bacterial-mediated reductions in the expression of intercellular apical junction
proteins, including zonula occludens-1, occludin, and claudin-2.
Conclusions
Better understanding of the mechanisms that contribute to resolution of mucosal

inflammation is crucial to the development of improved therapies for diseases
such as IBD. Considerable progress has been made in recent years in identifying
endogenous anti-inflammatory molecules and the mechanisms through which they
modulate inflammation. Studies in animal models have provided evidence that
pharmacological promotion of the resolution of inflammation is a viable approach,
such as in the case of lipoxins as a potential therapy for IBD. Research over the past
two decades has demonstrated that many of the chemical signals for resolution of
inflammation are also important factors in mediating mucosal resistance to injury.
The release of these mediators is therefore a defensive response aimed at reducing
mucosal injury and at tempering the inflammatory response, so as to minimise
damage to host tissue by infiltrating granulocytes. Drugs based on some of the key
mediators of resolution of inflammation may therefore be beneficial for any gastro-
intestinal disorder characterised by mucosal injury.
230
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234
Index
actin reorganisation 127 asthma 97, 120, 159, 165, 166

activator protein-1 161 asthma, cat-allergic 165
acute inflammation 112 atherosclerotic plaque rupture 120
adhesion molecule, glomerulonephritis 199, ATL 97, 120, 123
200 autacoids 96
airway epithelial cell 172, 174
airway hyperreactivity 165 basophils 172
airway inflammation 120, 123, 159179 Bax 174
airway remodelling 160 Bcl-2 174
alveolar macrophage 178 Bcl-x 174
ALXR 121, 122, 128 Bcl-xL 174
angiogenesis 97 bone marrow transplant 97
annexin 1 (AnxA1) 124, 128, 148152, 229 bone morphogenic protein-7 208
annexin 1, knockout 148 braking signal 125
annexin 1, peptide 148, 149 bronchial epithelium 159
anti-IL-5 mAb 166
anti-TNF mAb, humanized, see infliximab C1q deficiency 208
antigen (VLA)-4 171 caspase 173, 174
apoptosis 1928, 5768, 7586, 172, 174,176, cathepsin 165
178, 202 CD45 173
apoptotic cell, opsonisation of 124 CD69 173
apoptotic cell clearance 4047, 119, 207, 208 CD95, see Fas
apoptotic cell clearance, complement 44 cell-based therapy 212
apoptotic cell clearance, pattern recognition 40 cell-cell interaction 95, 106
apoptotic cell death 2224 chemoattractant 98
apoptotic cell recognition 124 chronic myeloid leukaemia 120
apoptotic cell-associated molecular pattern chronic obstructive pulmonary disease
(ACAMP) 40 (COPD) 160, 162164
arthritis 94 ciclesonide 160
aspirin 106, 120 cigarette smoke 160
235
Index
cilomilast 164 Fas (CD95) 173

colitis, see inflammatory bowel disease Fel d 1 165
collectin 46 fibroblast 102, 159, 174
corticosteroid 162 fibronectin 173
Crohns disease 227 fibrosis 160
CTLA4-Ig 210 formyl peptide-receptor like-1 (FPRL-1) 121,
cyclin-dependent kinase 212 149151
cyclooxygenase (COX) 98
cyclooxygenase-2 (COX-2), aspirin acetylation gastrointestinal tract 223
of 120 glomerular injury, mechanism of 196
cysteine-cysteine chemokine receptor-3 glomerulonephritis 97, 119, 120, 198200,
(CCR3) 172 203, 204, 209213
CCR3 antagonist 172 glucocorticoid 104, 125, 141148, 151, 152,
cysteinyl leukotriene (LT) antagonist 162 159161
cystic fibrosis 97, 120 glucocorticoid, history 142, 143
cytokine 160, 166170 glucocorticoid, non genomic action 143145
cytokine, anti-inflammatory 169, 170 glucocorticoid, receptor 143147
cytokine, pro-inflammatory 166169 GM-CSF 173
cytoskeleton rearrangement 124 Goodpastures disease 211
granulocyte apoptosis 1928
dendritic cell 103, 174 granulocyte apoptosis, intracellular
dermal inflammation 97 mechanism 26, 27
DHA 107, 108 GTPase 127
DNA vaccine 165
drug discovery, anti-inflammatory 1 Helicobacter pylori 225
hepatocyte 174
eicosanoid 3 human PMN 100
endothelial cell 102, 106, 159, 204, 205 human vascular endothelial cell 99
eosinophil 159, 171176 hypoallergenic isoforms 165
eosinophil apoptosis 21, 22, 172174
eotaxin 172 IgE 165
EPA 107, 108 IgE inhibitor 170, 171
15-epi-LX, see lipoxin, aspirin-triggered IL-1` 198
epithelial cell, apoptotic 178 IL-3 173
epithelial mesenchymal trans- IL-4 165, 168, 176, 209, 213
differentiation 122 IL-5 173
epithelial to mesenchymal transition IL-8 122
(EMT) 208 IL-9 168, 173
E-selectin 171 IL-10 165, 169, 209, 210, 213
E-series, resolvin 95, 96 IL-12 169, 173, 198
etanercept 169 IL-13 165, 168, 173, 176
eye, LX and ATL actions 97 IL-15 173
236
Index
immunotherapy, asthma 165, 166 mitochondrial outer membrane permeabilisation

inducible costimulator (ICOS) 210 (MOMP) 24
inflammatory bowel disease (IBD) 97, 123, monocyte 126, 127
223 mononuclear cell 173
infliximab 169 mouse tissue 107
interferon (IFN)-_ 169 mucosal epithelia 102
interferon (IFN)-a 165, 198 mycophenolate mofetil (MMF) 212
interleukin-8 (IL-8) 122 MYH9 128
interstitial cell 102
intracellular acidification 25 natalizumab 171
ischaemia-reperfusion (I/R) injury 97 neutrophil 59, 60, 75, 160, 165, 176, 177, 197
ischaemic acute renal failure 123 neutrophil apoptosis 19, 20, 22, 172, 176
neutrophil elastase 165
knockouts 148 NF-gB 9, 26, 161, 173
nitric oxide 199
LC-UV-MS/MS 105, 106 nonsteroidal anti-inflammatory drug
leukocyte 99, 103 (NSAID) 223
lipidomic approach 101, 103 nuclear factor of activated T cells (NF-AT) 161
lipoxin (LX) 97, 119123, 125, 225
lipoxin, aspirin-triggered 120 omalizumab 170
lipoxin A4 122, 149, 151 omega-3 polyunsaturated fatty acid
lipoxygenase (LO) 98, 108 (t-3 PUFA) 93
LPC 125 oxidative stress 177
LTB 178
LTB4 162, 163 P450 106
luminal entry 176 pathogen-associated molecular pattern
lymphocyte 197 (PAMP) 40
pattern recognition receptor (PRR) 40
macrophage 4, 7586, 123, 160, 174, 196, PRR, prototype CD14 4143
197, 203, 204 PD1 108
macrophage M\, glomerulonephritis 203, 204 pentraxin 47
macrophage apoptosis 7679 peptide, anti-inflammatory 142, 149
macrophage emigration 7985 periodontitis 94, 97, 120
mast cell 159, 172, 176 peripheral blood neutrophils 98
MCP-3 172 peritonitis 97
MCP-4 172 phagocyte 93, 123125
Mechnikov 96 phagocytic cell 172
mediator, anti-inflammatory 8 phagocytic clearance 174
mediator antagonist 161163 phagocytosis 59, 85
mepolizumab 166 phosphatidylcholine 125
mesangial cell proliferation 122 phosphodiesterase inhibitor 164
mitochondrial cytochrome C 173 phospholipid asymmetry 124
237
Index
platelet 102 scavenger receptor 43

platelet-derived growth factor (PDGF) 202 secondary necrosis 174
PMN, human 100 selectin 171, 177
PMN recruitment 123 siglec-8 173
podocyte 204 smooth muscle cell 159
probiotics 230 STAT-6 168
programmed cell death, see neutrophil apoptosis stem cell, glomerular 206
prostaglandin (PG) 7, 176 surface receptor 57, 6064
protease inhibitor 164
protease-mediated inflammation 177 T regulatory (Treg) cell 210
protectin 96, 108, 119 TBC1269 177
proteinase 3 165 Th2 cell 172
proteomic approach 101, 103 tissue inhibitor of matrix metalloproteinase
t-3 PUFA 93 (TIMP) 169
TNF-_ 122, 168, 173, 198
RANTES 172 TNF-related apoptosis-inducing ligand
receptor tyrosine kinase 122 (TRAIL) 23, 24
recombinant allergen 165 transcellular biosynthesis 102, 106
resolution indices 104 transforming growth factor (TGF)-` 165, 208
resolvin 95, 96, 107, 108, 119, 120
rituximab 212 VCAM-1 171
RU 24858 161 VEGF 205, 211
RvE1 106, 108
238
The PIR-Series
Progress in Inflammation Research
Homepage: http://www.birkhauser.ch
Up-to-date information on the latest developments in the pathology, mechanisms and the-
rapy of inflammatory disease are provided in this monograph series. Areas covered include
vascular responses, skin inflammation, pain, neuroinflammation, arthritis cartilage and bone,
airways inflammation and asthma, allergy, cytokines and inflammatory mediators, cell signal-
ling, and recent advances in drug therapy. Each volume is edited by acknowledged experts
providing succinct overviews on specific topics intended to inform and explain. The series is
of interest to academic and industrial biomedical researchers, drug development personnel
and rheumatologists, allergists, pathologists, dermatologists and other clinicians requiring
regular scientific updates.
Available volumes:
T Cells in Arthritis, P. Miossec, W. van den Berg, G. Firestein (Editors), 1998
Chemokines and Skin, E. Kownatzki, J. Norgauer (Editors), 1998
Medicinal Fatty Acids, J. Kremer (Editor), 1998
Inducible Enzymes in the Inflammatory Response,
D.A. Willoughby, A. Tomlinson (Editors), 1999
Cytokines in Severe Sepsis and Septic Shock, H. Redl, G. Schlag (Editors), 1999
Fatty Acids and Inflammatory Skin Diseases, J.-M. Schrder (Editor), 1999
Immunomodulatory Agents from Plants, H. Wagner (Editor), 1999
Cytokines and Pain, L. Watkins, S. Maier (Editors), 1999
In Vivo Models of Inflammation, D. Morgan, L. Marshall (Editors), 1999
Pain and Neurogenic Inflammation, S.D. Brain, P. Moore (Editors), 1999
Anti-Inflammatory Drugs in Asthma, A.P. Sampson, M.K. Church (Editors), 1999
Novel Inhibitors of Leukotrienes, G. Folco, B. Samuelsson, R.C. Murphy (Editors), 1999
Vascular Adhesion Molecules and Inflammation, J.D. Pearson (Editor), 1999
Metalloproteinases as Targets for Anti-Inflammatory Drugs,
K.M.K. Bottomley, D. Bradshaw, J.S. Nixon (Editors), 1999
Free Radicals and Inflammation, P.G. Winyard, D.R. Blake, C.H. Evans (Editors), 1999
Gene Therapy in Inflammatory Diseases, C.H. Evans, P. Robbins (Editors), 2000
New Cytokines as Potential Drugs, S. K. Narula, R. Coffmann (Editors), 2000
High Throughput Screening for Novel Anti-inflammatories, M. Kahn (Editor), 2000
Immunology and Drug Therapy of Atopic Skin Diseases,
C.A.F. Bruijnzeel-Komen, E.F. Knol (Editors), 2000
Novel Cytokine Inhibitors, G.A. Higgs, B. Henderson (Editors), 2000
Inflammatory Processes. Molecular Mechanisms and Therapeutic Opportunities,
L.G. Letts, D.W. Morgan (Editors), 2000
Cellular Mechanisms in Airways Inflammation, C. Page, K. Banner, D. Spina (Editors), 2000
Inflammatory and Infectious Basis of Atherosclerosis, J.L. Mehta (Editor), 2001
Muscarinic Receptors in Airways Diseases, J. Zaagsma, H. Meurs, A.F. Roffel (Editors), 2001
TGF-` and Related Cytokines in Inflammation, S.N. Breit, S. Wahl (Editors), 2001
Nitric Oxide and Inflammation, D. Salvemini, T.R. Billiar, Y. Vodovotz (Editors), 2001
Neuroinflammatory Mechanisms in Alzheimers Disease. Basic and Clinical Research,
J. Rogers (Editor), 2001
Disease-modifying Therapy in Vasculitides,
C.G.M. Kallenberg, J.W. Cohen Tervaert (Editors), 2001
Inflammation and Stroke, G.Z. Feuerstein (Editor), 2001
NMDA Antagonists as Potential Analgesic Drugs,
D.J.S. Sirinathsinghji, R.G. Hill (Editors), 2002
Migraine: A Neuroinflammatory Disease? E.L.H. Spierings, M. Sanchez del Rio (Editors), 2002
Mechanisms and Mediators of Neuropathic pain, A.B. Malmberg, S.R. Chaplan (Editors),
2002
Bone Morphogenetic Proteins. From Laboratory to Clinical Practice,
S. Vukicevic, K.T. Sampath (Editors), 2002
The Hereditary Basis of Allergic Diseases, J. Holloway, S. Holgate (Editors), 2002
Inflammation and Cardiac Diseases, G.Z. Feuerstein, P. Libby, D.L. Mann (Editors), 2003
Mind over Matter Regulation of Peripheral Inflammation by the CNS,
M. Schfer, C. Stein (Editors), 2003
Heat Shock Proteins and Inflammation, W. van Eden (Editor), 2003
Pharmacotherapy of Gastrointestinal Inflammation, A. Guglietta (Editor), 2004
Arachidonate Remodeling and Inflammation, A.N. Fonteh, R.L. Wykle (Editors), 2004
Recent Advances in Pathophysiology of COPD, P.J. Barnes, T.T. Hansel (Editors), 2004
Cytokines and Joint Injury, W.B. van den Berg, P. Miossec (Editors), 2004
Cancer and Inflammation, D.W. Morgan, U. Forssmann, M.T. Nakada (Editors), 2004
Bone Morphogenetic Proteins: Bone Regeneration and Beyond, S. Vukicevic, K.T. Sampath
(Editors), 2004
Antibiotics as Anti-Inflammatory and Immunomodulatory Agents, B.K. Rubin, J. Tamaoki
(Editors), 2005
Antirheumatic Therapy: Actions and Outcomes, R.O. Day, D.E. Furst, P.L.C.M. van Riel,
B. Bresnihan (Editors), 2005
Regulatory T-Cells in Inflammation, L. Taams, A.N. Akbar, M.H.M Wauben (Editors), 2005
Sodium Channels, Pain, and Analgesia, K. Coward, M. Baker (Editors), 2005
Turning up the Heat on Pain: TRPV1 Receptors in Pain and Inflammation, A.B Malmberg,
K.R. Bley (Editors), 2005
The NPY Family of Peptides in Immune Disorders, Inflammation, Angiogenesis and Cancer,
Z. Zukowska, G. Z. Feuerstein (Editors), 2005
Toll-like Receptors in Inflammation, L.A.J. ONeill, E. Brint (Editors), 2005
Complement and Kidney Disease, P. F. Zipfel (Editor), 2006
Chemokine Biology Basic Research and Clinical Application, Volume 1: Immunobiology
of Chemokines, B. Moser, G. L. Letts, K. Neote (Editors), 2006
The Hereditary Basis of Rheumatic Diseases, R. Holmdahl (Editor), 2006
Lymphocyte Trafficking in Health and Disease, R. Badolato, S. Sozzani (Editors), 2006
In Vivo Models of Inflammation, 2nd Edition, Volume I, C.S. Stevenson, L.A. Marshall,
D.W. Morgan (Editors), 2006
In Vivo Models of Inflammation, 2nd Edition, Volume II, C.S. Stevenson, L.A. Marshall,
D.W. Morgan (Editors), 2006
Chemokine Biology Basic Research and Clinical Application. Volume II: Pathophysiology
of Chemokines, K. Neote, G.L. Letts, B. Moser (Editors), 2007
Adhesion Molecules: Function and Inhibition, K. Ley (Editor), 2007
The Immune Synapse as a Novel Target for Therapy, L. Graca (Editor), 2008

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Progress in Inflammation Research

Prof. Michael J. Parnham PhD

G. Z. Feuerstein (Wyeth Research, Collegeville, PA, USA)

New Therapeutic Targets in Rheumatoid Arthritis, P.-P. Tak (Editor), 2008

(Already published titles see last page.)

Library of Congress Control Number: 2007934182

Bibliographic information published by Die Deutsche Bibliothek

ISBN 978-3-7643-7505-8 Birkhuser Verlag AG, Basel Boston Berlin

2008 Birkhuser Verlag AG

List of contributors ................................................................. vii

Derek Gilroy and Toby Lawrence

Moira K.B. Whyte, Christopher Haslett and Edwin R. Chilvers

Andrew Devitt and Christopher D. Gregory

Ian Dransfield, Sandra Franz, Kim Wilkinson, Aisleen McColl,

Geoffrey J. Bellingan and Geoffrey J. Laurent

Paola Maderna and Catherine Godson

Mauro Perretti and Roderick J. Flower

Garry M. Walsh and Catherine M. McDougall

David C. Kluth and Jeremy Hughes

John L. Wallace and Philip M. Sherman

Index ................................................................................ 235

Geoffrey J. Bellingan, Centre for Respiratory Research, University College London,

Edwin R. Chilvers, Respiratory Medicine Division, Department of Medicine, Uni-

Ian Dransfield, MRC Centre for Inflammation Research, University of Edinburgh

Sandra Franz, Institute for Immunology, FAU Erlangen-Nuremberg, Glckstrasse

Christopher D. Gregory, MRC Centre for Inflammation Research, The Queens

Simon P. Hart, MRC Centre for Inflammation Research,University of Edinburgh

Christopher Haslett, MRC Centre for Inflammation Research, Queens Medical

Martin Herrmann, Institute for Immunology, FAU Erlangen-Nuremberg, Glck-

Jeremy Hughes, MRC Centre for Inflammation Research, University of Edinburgh,

David C. Kluth, MRC Centre for Inflammation Research, University of Edinburgh,

Geoffrey J. Laurent, Centre for Respiratory Research, University College London,

Aisleen McColl, MRC Centre for Inflammation Research,University of Edinburgh

Catherine M. McDougall, School of Medicine, Institute of Medical Sciences, Uni-

Charles N. Serhan, Center for Experimental Therapeutics and Reperfusion Injury,

John L. Wallace, Department of Pharmacology and Therapeutics, University of Cal-

Garry M. Walsh, School of Medicine, IMS Building, University of Aberdeen, For-

Moira K. B. Whyte, Academic Unit of Respiratory Medicine, School of Medicine

Kim Wilkinson, MRC Centre for Inflammation Research,University of Edinburgh

October 2007 Adriano G. Rossi

Derek Gilroy1 and Toby Lawrence2

The scope of this chapter

The Resolution of Inflammation, edited by Adriano G. Rossi and Deborah A. Sawatzky

Resolution of acute inflammation

Controlling the early phase of acute inflammation

Inflammation is a reaction of the microcirculation that is characterised by the move-

The transition to resolution

response. Therefore, increasing the rate of apoptosis, as a potentially anti-inflam-

Soluble mediators of resolution: Opportunities for drug discovery

Returning to eicosanoids, prostaglandin (PG) D2, a metabolite of the action of

Signalling pathways that regulate inflammation

The inflammatory response is characterised by coordinated activation of various

Negative regulation of pro-inflammatory signalling

As discussed below, pro-inflammatory signalling pathways have the capacity

Resolution of T cell-driven inflammation

Inflammation has an important role in instructing the adaptive immune response,

of adaptive immune diseases. For instance, in a purified protein derivative-induced

Unblocking the drains!

In conclusion, it is clear the inflammatory response has a number of built-in check-

in these endogenous anti-inflammatory pathways will undoubtedly predispose to

10 Bellingan GJ, Xu P, Cooksley H, Cauldwell H, Shock A, Bottoms S, Haslett C, Mut-

binding proteins in p38 mitogen-activated protein kinase pathway-mediated mRNA

Moira K. B. Whyte1, Christopher Haslett2 and Edwin R. Chilvers3

Neutrophil apoptosis (programmed cell death) is now recognised to play a funda-