The Resolution of Inflammation (Progress in Inflammation Research) (Progress in Inflammation Research)

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. Prilaz baruna Filipovic´a 29 HR-10000 Zagreb Croatia 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. Schultheiß, M. Noutsias (Editors), 2008 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

Birkhäuser Basel · Boston · Berlin

Editors Dr Adriano G. Rossi Dr Deborah A. Sawatzky MRC Centre for Inflammation Research Queen’s Medical Research Institute University of Edinburgh 47 Little France Crescent Edinburgh, EH16 4TJ Scotland United Kingdom

Library of Congress Control Number: 2007934182

Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the internet at http://dnb.ddb.de

ISBN 978-3-7643-7505-8 Birkhäuser Verlag AG, Basel – Boston – Berlin The publisher and editor can give no guarantee for the information on drug dosage and administration contained in this publication. The respective user must check its accuracy by consulting other sources of reference in each individual case. The use of registered names, trademarks etc. in this publication, even if not identified as such, does not imply that they are exempt from the relevant protective laws and regulations or free for general use. This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, re-use of illustrations, recitation, broadcasting, reproduction on microfilms or in other ways, and storage in data banks. For any kind of use, permission of the copyright owner must be obtained. © 2008 Birkhäuser Verlag AG Basel · Boston · Berlin P.O. Box 133, CH-4010 Basel, Switzerland Part of Springer Science+Business Media Printed on acid-free paper produced from chlorine-free pulp. TCF ' Cover design: Markus Etterich, Basel Cover illustration: see page 126. With the friendly permission of Catherine Godson and Paola Maderna. Printed in Germany ISBN 978-3-7643-7505-8 e-ISBN 978-3-7643-7506-5 987654321

www.birkhauser.ch

Contents

List of contributors

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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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Andrew Devitt and Christopher D. Gregory Innate immune mechanisms in the resolution of inflammation

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Ian Dransfield, Sandra Franz, Kim Wilkinson, Aisleen McColl, Martin Herrmann and Simon P. Hart Cell surface molecular changes associated with apoptosis . . . . . . . . . . . . . . . . . . . . . . .

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Geoffrey J. Bellingan and Geoffrey J. Laurent Fate of macrophages once having ingested apoptotic cells: Lymphatic clearance or in situ apoptosis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Charles N. Serhan Novel lipid mediators in resolution and their aspirin triggered epimers: lipoxins, resolvins, and protectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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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

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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

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List of contributors

Geoffrey J. Bellingan, Centre for Respiratory Research, University College London, Rayne Institute, London, WC1E 6JJ, UK; e-mail: [email protected] Edwin R. Chilvers, Respiratory Medicine Division, Department of Medicine, University of Cambridge, School of Clinical Medicine, Addenbrooke’s and Papworth Hospitals, Cambridge, CB2 2QQ, UK, e-mail: [email protected] Andrew Devitt, School of Life & Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK; e-mail: [email protected] Ian Dransfield, MRC Centre for Inflammation Research, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, UK; ¨ e-mail: [email protected] Roderick J. Flower, William Harvey Research Institute, Bart’s and the London, Queen Mary School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK Sandra Franz, Institute for Immunology, FAU Erlangen-Nuremberg, Glückstrasse 4a, 91054 Erlangen, Germany Derek Gilroy, Rayne Institute, Centre for Clinical Pharmacology, University College London, 5 University Street, London WC1 6JJ, UK; e-mail: [email protected] Catherine Godson, UCD School of Medicine and Medical Science, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland; e-mail: [email protected] Christopher D. Gregory, MRC Centre for Inflammation Research, The Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK

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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, Queen’s Medical Research Institute, University of Edinburgh Medical School, Edinburgh, EH16 4TJ, UK; e-mail: [email protected] Martin Herrmann, Institute for Immunology, FAU Erlangen-Nuremberg, Glückstrasse 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: [email protected] 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, Bart’s and The London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK; e-mail: [email protected] Paola Maderna, UCD School of Medicine and Medical Science, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland; e-mail: [email protected] Aisleen McColl, MRC Centre for Inflammation Research,University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, UK Catherine M. McDougall, School of Medicine, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, UK Mauro Perretti, William Harvey Research Institute, Bart’s and the London, Queen Mary School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK; email: [email protected] Charles N. Serhan, Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital and Harvard University, 75 Francis St., Thorn Building for Medical Research, Boston, MA 02115, USA

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Philip M. Sherman, Hospital for Sick Children and University of Toronto, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada John L. Wallace, Department of Pharmacology and Therapeutics, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, Canada; e-mail: [email protected] Garry M. Walsh, School of Medicine, IMS Building, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK; e-mail: [email protected] Moira K. B. Whyte, Academic Unit of Respiratory Medicine, School of Medicine and Biomedical Sciences, University of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2JF, UK; e-mail: [email protected] Kim Wilkinson, MRC Centre for Inflammation Research,University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, UK

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Preface

It was with tremendous enthusiasm that we endeavoured to compile and edit this volume for Progress in Inflammation Research describing novel findings and developments 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 attention had been specifically devoted to elucidating the mechanisms orchestrating the resolution of inflammation, although a variety of mechanisms that limit the inflammatory 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, recognised as being actively involved in resolution, are therapeutically manipulatable for the treatment of inflammatory diseases. Indeed, patients with chronic inflammatory diseases are by necessity treated in order to reduce established and persistent inflammation with the added hope of preventing further progression of the inflammatory 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 however is a notable exception) and augment apoptotic cell clearance by phagocytes. Similarly, aspirin, the most widely used NSAID, is involved in an unorthodox biosynthetic 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

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mediators. In addition, there are sections that describe how existing anti-inflammatory 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 understanding 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

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Adriano G. Rossi Deborah A. Sawatzky

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, Bart’s 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 circulation, classically defined by the initial trafficking of polymorphonuclear granulocytes, 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 capacity 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 proinflammatory 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 Birkhäuser Verlag Basel/Switzerland

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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 augment 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 physiological 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]).

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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 SRA and CD36, both of which have been described to mediate the phagocytosis of apoptotic cells, leading to a down-regulation of macrophage activation [4–6] (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 highlighting 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-resolution 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 activation 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 becoming 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

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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 predisposition to recurrent bacterial and fungal infections and the development of inflammatory 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 lymphocytes 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 contrast 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 movement 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 monocytes 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

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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 lipoxygenase/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/sIL6R 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 formidable 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 potentially 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

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response. Therefore, increasing the rate of apoptosis, as a potentially anti-inflammatory strategy, must be matched by a mechanism that up-regulates macrophage phagocytic clearance capacity [20]. Thus, the removal process might also be susceptible 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-professional 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-chemoattracted macrophages accelerate resolution by enhancing phagocytosis of apoptotic 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 macrophage phenotype [23]. Functional and morphological homogeneity was matched by cell surface phenotype, including specific induction of expression of the haemoglobin 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 cardiopulmonary bypass surgery. In each case, binding of the haemoglobin–haptoglobin 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 24–48 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 (24–48-h phase) of a rat carrageenin-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-

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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 association between C1q deficiency and disease [27]. The phagocytosis of apoptotic cells has been suggested to play an important role in the negative regulation of macrophage activation, apoptotic leukocytes may well fit into the category of endogenous anti-inflammatory mediators, therefore the mechanisms of apoptosis and the clearance 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 proliferators-activated receptor gamma (PPARa), 15d-PGJ2 also exerts its effects through PPARa-dependent as well as -independent mechanisms to suppress pro-inflammatory signalling pathways and the expression of genes that drive the inflammatory response. 15d-PGJ2 also preferentially inhibits monocyte rather than PMN trafficking 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 synthesis of PGD2 and 15d-PGJ2 [29], which, along with the alternative DNA-binding p50–p50 homodimers complexes of NF-gB [30] bring about resolution by inducing 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 differential 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 eicosapentaneoic 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-

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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 members of IL-1 and TNF receptor families and the Toll-like microbial pattern recognition 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 germlineencoded non-self recognition system that is hard-wired to trigger inflammation. However, there is some suggestion that endogenous ligands may trigger TLRs during 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 Toll–IL-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-inflammatory genes. MAPKs also regulate pro-inflammatory gene expression through post-transcriptional mechanisms such as mRNA turnover, mRNA transport and translation [50–52]. 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

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and MAPK pathways include IL-1 and TNF-_, generating a feed-forward mechanism 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 activation [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 negative 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/NFgB 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 inflammatory response when challenged with bacteria, fungal cell wall particles, or even immune complexes [61]. These studies established that, while IKK` catalytic activity 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 accelerated 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.

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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 expression 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-inflammatory 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 activation 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 signalling pathways, genetic studies in mice have shown that even the absence of one negative regulator is sufficient to result in serious inflammatory disorders. Undoubtedly, 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 adaptive 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 lymphocyteor 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

The resolution of acute inflammation

Figure 2 Schematic illustration of the co-ordinated activation of pro-inflammatory signalling pathways 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 proinflammatory 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 factors 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

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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 critical 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 macrophages 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 draining 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 macrophage–mesothelial interactions. This report highlights the importance of adhesion molecules controlling 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 checkpoint controls that limit the duration and magnitude of acute inflammation. Defects

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The resolution of acute inflammation

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 complex 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 resolution of inflammation and identify possible approaches to promote this process in combination with anti-inflammatory therapy. Perhaps the, as-yet-unidentified, antiresolution factors that may prevent the proper resolution of inflammation would represent appropriate targets to achieve this goal.

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Granulocyte apoptosis Moira K. B. Whyte1, Christopher Haslett2 and Edwin R. Chilvers3 1

Academic Unit of Respiratory Medicine, School of Medicine and Biomedical Sciences, University of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2JF, UK; 2MRC Centre for Inflammation Research, Queen’s 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, Addenbrooke’s and Papworth Hospitals, Cambridge, CB2 2QQ, UK

Introduction Neutrophil apoptosis (programmed cell death) is now recognised to play a fundamental role in the physiological resolution of innate immune responses. Early work by Metchnikoff correlated the ingestion of microphages (neutrophils) by macrophages 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 granulocytes 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 triggering; this latter event triggers phagocytic removal by alveolar or inflammatory macrophages, 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 circumstances; 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 exquisitely regulated and involves multiple input signals, including both host-derived and The Resolution of Inflammation, edited by Adriano G. Rossi and Deborah A. Sawatzky © 2008 Birkhäuser Verlag Basel/Switzerland

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pathogen-derived molecules [9, 10]. Our current understanding of these events predicts 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 strategy, 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) [9–13], cell adhesion or interaction with particulate material, including bacteria and bacterial products (e.g. VCAM-1, LPS) [10, 14, 15] and various physicochemical 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 relevance, a distinct population of circulating neutrophils that are CD54high, CXC chemokine 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 activation (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.

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Table 1 - Regulation of eosinophil apoptosis Apoptosis delaying factors Cytokines

Proposed mechanism

Ref(s)

IL-3, IL-5, GM-CSF

Receptors composed of unique alpha sub-unit and [96] 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 expression

[99]

IL-13

Enhances synthesis/release of IL-3 and GM-CSF to act in autocrine fashion

[100]

TNF-_

Partly by p38 MAP kinase activation

[101]

Hormones

Leptin

May block mitochondrial release of cytochrome c

[102]

Bacterial endotoxin

LPS

Enhanced autocrine GM-CSF production

[103]

Interferons

IFN-a

Mediated by Jak2

[104]

Integrins

`2 Integrin

Enhanced paracrine/autocrine synthesis of cytokines, e.g. IL-5/GM-CSF through ICAM-1 and `2 integrin signalling

[105]

Fibronectin, laminin

Integrin-mediated interaction resulting in generation of survival cytokines

[106] [107]

Galectin

Ecalectin

Unknown, but independent of IL-3, IL-5 and GM-CSF

[108]

Prostaglandins

PGE2

Increasing cytosolic cAMP EP2R subtype expressed on eosinophils

[109]

Cysteinyl leukotrienes

LTB4, LTC4, LTD4

CysLTs produced by eosinophils, mast cells & Th2 lymphocytes Mediated by GM-CSF which increases CysLT production

[110]

Pharmacological Rolipram agents

Increasing cytosolic cAMP levels

[111]

CD antigens

CD40

Cross-linking CD40 enhances GM-CSF release

[112]

Gaseous compounds

Nitric oxide

Inhibits apoptosis by cyclic GMP driven process

[113]

Death receptors

Fas/APO-1/ CD95

Cross-linking with ligand (CD95L, FasL, APO-1L)

[114]

CD69

Ligation of CD69 in GM-CSF cultured eosinophils

[115]

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Table 1 - continued Apoptosis delaying factors Cytokines

Interleukins

Proposed mechanism

TGF-`

Inhibits IL-3-, IL-5-, GM-CSF- and IFN-a- [116] mediated survival

IL-4

Inhibits IL-3-, IL-5- and GM-CSF-mediated survival

[117]

IL-10

Inhibits LPS-induced GM-CSF release

[103]

Unknown, may inhibit of production of cytokines by other cells

[118]

Theophylline

Inhibits IL-5-mediated survival

[115]

Sodium channel-blockers, e.g. lidocaine

Inhibit survival-enhancing action of IL-5 and IFN-a

[119]

Ketotifen

Inhibit survival-enhancing action of IL-5

[120]

Potassium channelblockers, e.g. sulfonylureas

Inhibit cytokine-induced survival

[121]

Immunosuppressants, e.g. Cyclosporin A

Inhibit survival-enhancing action of IL-5

[120]

Gliotoxin

Inhibition of NF-gB

[77]

Orazipone

Inhibit survival-enhancing action of IL-5

[122]

Siglec-8

Production of ROS and mitochondrial cleavage and caspase cleavage

[123]

Pharmacological Glucocorticoids, e.g. agents dexamethasone

Antibody cross-linking

Ref(s)

GM-CSF, LPS, hypoxia). It is also evident that most agents that induce functional granulocyte priming, that is, enhance the magnitude of subsequent agonist-stimulated 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

Granulocyte apoptosis

a pore-forming toxin that targets neutrophil mitochondrial membranes) [25] and pharmacological agents (e.g. protein synthesis inhibitors, phorbol esters, zVADfmk, 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 phagocyte. 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 phagocytic 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 conditional 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 passing. 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 (TRAILR2) 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-

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Moira K. B. Whyte et al.

crine regulation of constitutive neutrophil apoptosis. TRAIL is, however, expressed on other immune cells such as T cells and macrophages and TRAIL-induced neutrophil 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 neutrophil. 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 neutrophil-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 intrinsic 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 caspase-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 neutrophils, 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 cytokineprocessing 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

Granulocyte apoptosis

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 neutrophil 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 formation 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-mediated 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 followed 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 neutrophils. 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 stimulation of the vacuolar (H+) ATPase in cell membranes inhibits both apoptosis and intracellular acidification [63]. During phagocytosis of bacteria, intracellular alkalinisation can delay neutrophil apoptosis, while ingestion of larger numbers of bacteria 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],

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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 interacts 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 prostaglandin 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 important 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

Granulocyte apoptosis

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 [81–84]. Mcl-1, a member of the Bcl-2 family, is of particular interest given its short half-life of 2–3 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 profiling 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 neutrophilic leukaemia [87].

Consequences of granulocyte apoptosis The requirement for a highly effective route of “physiological” granulocyte apoptosis and clearance is exemplified by pneumococcal pneumonia, where bacterial clearance 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 macrophage 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 leukocidin induces neutrophil apoptosis via activation of the mitochondrial pathway [25]. Other inducers of neutrophil necrosis include Burkholderia cenocepacia and the E. coli hemolysin [93–95].

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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 angiogenesis, 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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(IFN-gamma) inhibits eosinophil apoptosis by up-regulation of bcl-2 expression. Clin Exp Immunol 107: 198–204 Chihara J, Kakazu T, Higashimoto I, Saito N, Honda K, Sannohe S, Kayaba H, Urayama O (2000) Signaling through the beta2 integrin prolongs eosinophil survival. J Allergy Clin Immunol 106: S99-S103 Tourkin A, Anderson T, LeRoy EC, Hoffman S (1993) Eosinophil adhesion and maturation is modulated by laminin. Cell Adhes Commun 1: 161–176 Anwar AR, Moqbel R, Walsh GM, Kay AB, Wardlaw AJ (1993) Adhesion to fibronectin prolongs eosinophil survival. J Exp Med 177: 839–843 Matsumoto R, Hirashima M, Kita H, Gleich GJ (2002) Biological activities of ecalectin: A novel eosinophil-activating factor. J Immunol 168: 1961–1967 Peacock CD, Misso NL, Watkins DN, Thompson PJ (1999) PGE 2 and dibutyryl cyclic adenosine monophosphate prolong eosinophil survival in vitro. J Allergy Clin Immunol 104: 153–162 Lee E, Robertson T, Smith J, Kilfeather S (2001) Leukotriene receptor antagonists and synthesis inhibitors reverse survival in eosinophils of asthmatic individuals. Am J Respir Crit Care Med 161: 1881–1886 Yasui K, Agematsu K, Shinozaki K, Hokibara S, Nagumo H, Yamada S, Kobayashi N, Komiyama A (2000) Effects of theophylline on human eosinophil functions: Comparative study with neutrophil functions. J Leukoc Biol 68: 194–200 Kim JT, Gleich GJ, Kita H (1997) Roles of CD molecules in survival and activation of human eosinophils. J Immunol 159: 926–933 Beauvais F, Joly F (1999) Effects of nitric oxide on the eosinophil survival in vitro. A role for nitrosyl-heme. FEBS Lett 443: 37–40 Tsuyuki S, Bertrand C, Erard F, Trifilieff A, Tsuyuki J, Wesp M, Anderson G, Coyle AJ (1995) Activation of the Fas receptor on lung eosinophils leads to apoptosis and the resolution of eosinophilic inflammation of the airways. J Clin Invest 96: 2924–2931 Yasui K, Hu B, Nakazawa T, Agematsu K, Komiyama A (1997) Theophylline accelerates human granulocyte apoptosis not via phosphodiesterase inhibition. J Clin Invest 100: 1677–1684 Alam R, Forsythe P, Stafford S, Fukuda Y (1994) Transforming growth factor beta abrogates the effects of hematopoietins on eosinophils and induces their apoptosis. J Exp Med 179: 1041–1045 Wedi B, Raap U, Lewrick H, Kapp A (1998) IL-4-induced apoptosis in peripheral blood eosinophils. J Allergy Clin Immunol 102: 1013–1020 Meagher LC, Cousin JM, Seckl JR, Haslett C (1996) Opposing effects of glucocorticoids on the rate of apoptosis in neutrophilic and eosinophilic granulocytes. J Immunol 156: 4422–4428 Okada S, Hagan JB, Kato M, Bankers-Fulbright JL, Hunt LW, Gleich GJ, Kita H (1998) Lidocaine and its analogues inhibit IL-5-mediated survival and activation of human eosinophils. J Immunol 160: 4010–4017

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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; MRC Centre for Inflammation Research, The Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK 2

Introduction The inflammatory response is a highly orchestrated and tightly controlled mechanism 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 resolution requires cessation of inflammatory cell recruitment and, importantly, deletion of recruited cells in a safe and controlled fashion. Failure to delete cells appropriately may permit effete cells to undergo necrosis (lysis due to loss of plasma membrane 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 unwanted 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 controlled 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 phagocytosis 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 Resolution of Inflammation, edited by Adriano G. Rossi and Deborah A. Sawatzky © 2008 Birkhäuser Verlag Basel/Switzerland

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The innate immune system The innate immune system is a powerful (if often under-rated) system that constitutes 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 resolution 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’ (‘noninfectious 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 components 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 nonself, 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 immunology: 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

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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, increasingly, 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 antimicrobial effector molecules and fitting the description of PRRs. It was the discovery 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-

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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 assessment 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 clearance [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

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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 apoptotic 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 inhibition 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) throughout evolution is highlighted by the discovery of Croquemort in Drosophila. Croquemort, 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, studies 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 receptors following in vitro studies, including Lox-1 [25, 26], SR-AI and II (CD204) [27, 28] and SRBI [29–32]. 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

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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 significant 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 apoptotic 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 erythematosus (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 peritoneal 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 clearance 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 [39–41]. 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 reasonable that following C1q opsonisation of apoptotic cells macrophages may bind and remove apoptotic cells but delay at this point may permit activation of complement 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.

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If complement activation provides enhanced apoptotic cell clearance, the stimulus 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 phospholipase 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 activation 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 underlying 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 inhibition 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 clearance remains unclear. For example, while CD93 (C1qRp) is involved in modulat-

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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-binding lectin (MBL) [37, 48, 53, 54] have been shown to bind apoptotic cells and promote 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 lesser 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-deficient 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 environment. This binding is the reverse of the orientation previously suggested where the globular heads bind the apoptotic cell surface and collagenous tails mediate receptor

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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 upregulated 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-presenting 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 [67–69]. 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 presence 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-

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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 provides 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 apoptosis [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 cellderived chemoattractants play a role in recruitment of phagocytes to sites of inflammation 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 inflammation, it is intriguing that they also mediate removal of apoptotic cells (a non-phlogistic 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 threedimensional 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].

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The “infectious non-self” model of recognition proposed by Janeway (suggesting 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 alternative 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 “alternatively 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 outcome. 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

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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 fashion 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-inflammatory/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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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, Glückstrasse 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-directional transmission of information across the plasma membrane. It has been estimated 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 programmed cell death or apoptosis, discussing the implications in terms of their influence upon cellular responses and upon processes that impact upon disease pathogenesis. 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, The Resolution of Inflammation, edited by Adriano G. Rossi and Deborah A. Sawatzky © 2008 Birkhäuser Verlag Basel/Switzerland

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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 specifically 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 molecular 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 phagocytic cell that are often considered anti-inflammatory through the suppression of pro-inflammatory cytokine production and induction of IL-10 and TGF-` release [9–11]. 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 apoptosis in many different cell types is the exposure of the anionic phospholipid, phosphatidylserine (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, including 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-

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ingly, reported inhibition of phagocytosis of apoptotic cells by liposomes containing 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 apoptosis, the neutrophil is discussed here as a model cell type. The neutrophil granulocyte 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 infection or tissue injury, neutrophils are rapidly (within minutes) recruited into tissues, binding and transmigrating across activated endothelial cells [21]. Specific recruitment of neutrophils is directed by the adhesion and chemokine receptors that neutrophil express. Once present within inflammatory sites, neutrophils are efficient phagocytes, capable of recognising a wide variety of pathogens via different opsonins. 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 battery 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 neutrophils 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 azurophilic and specific granule compartments or through the rapid and specific proteolytic shedding of the ecto-domain of specific receptors [25–27]. 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 different 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

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Figure 1. Schematic representation of cell surface molecular changes associated with neutrophil apoptosis.

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 glycophosphatidylinositol-anchored IgG receptor, FcaRIII (CD16) [33, 34]. Two distinct subpopulations of in vitro cultured neutrophils could be defined in terms of levels of

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CD16 expression, correlating with the proportion of apoptotic neutrophils present. This observation implied that there may be specific alterations in membrane receptor 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, receptor 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 shutdown of the secretory pathway in apoptotic neutrophils led to the reduced expression 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 accompanied by the activation of surface-associated metalloproteases, resulting in specific receptor cleavage. This change, together with a shutdown in transport of newly synthesised 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 downregulation 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 incompletely 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

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Griffonia simplificolia II (GSL II), Narcissus pseudonarcissus (NPn), and Ulex europaeus 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 recognising lectins derived from Maackia amurensis and Sambucus nigra [35] (Franz, unpublished data). Increased binding of the galactose binding lectin, peanut agglutinin, 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 apoptotic neutrophils that are reduced in size but have still maintained their plasma membrane 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 neutrophils 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 internal 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 functional uncoupling of signalling receptors, acting to isolate the apoptotic cell from

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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, disruption 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 neutrophils, 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 deathinducing 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, naïve CD4+ cells and memory CD8+ cells. It has been reported that homotypic interaction of CD31 on neutrophils and CD31 on phagocytes is capable of mediating 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

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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% (Dransfield, unpublished data) raising the possibility that proteolytic loss of CD31 expression 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 leading 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 ‘don’t eat me’ signals and when this interaction was blocked by CD47 Fab’ fragments, viable cells were engulfed [49]. In terms of membrane distribution, 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 phagocytic 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-positive 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. Heatshock proteins have been reported to be specifically expressed on the surface of neutrophils and other cells following stress or pathogen-induced cell death [50–52]. 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 phenotype appears to be a relatively slow process. In contrast, many other cell types

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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-positive 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 integrity. The opsonisation of cells with complement components also deserves consideration. 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 autoimmune 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 phospholipids 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 components [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-

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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 complementmediated 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 apoptotic cells, it seems likely that their clearance will differ both in terms of phagocyte recognition pathways engaged and also in the efficiency of internalisation. Mechanisms 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 following 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 neutrophils via an interaction of the Fc portion to FcaRII. Surprisingly, antibody-antigen complexes did not bind, or bound weakly to freshly isolated or cytokine/chemokine-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

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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 influence generation of effective immune responses to endogenous or tumour cell-associated 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 immunisation of antigen-presenting cells (including dendritic cells and Langerhans cells) with tumour-derived material could provide effective anti-tumour therapy [71–73], particularly 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 regulatory mechanisms and the consequences in terms of gene expression patterns, functional activity and membrane receptor alterations. However, the issues relating 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 importance 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

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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 autoimmune 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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of GM1 surface expression precedes annexin V-phycoerythrin binding of neutrophils undergoing spontaneous apoptosis during in vitro aging. Cytometry A 62: 75–80 Whyte MK, Meagher LC, MacDermot J, Haslett C (1993) Impairment of function in aging neutrophils is associated with apoptosis. J Immunol 150: 5124–5134 Knepper-Nicolai B, Savill J, Brown SB (1998) Constitutive apoptosis in human neutrophils requires synergy between calpains and the proteasome downstream of caspases. J Biol Chem 273: 30530–30536 Kothakota S, Azuma T, Reinhard C, Klippel A, Tang J, Chu K, McGarry TJ, Kirschner MW, Koths K, Kwiatkowski DJ, Williams LT (1997) Caspase-3-generated fragment of gelsolin: Effector of morphological change in apoptosis. Science 278: 294–298 Brown E, Hogg N (1996) Where the outside meets the inside: Integrins as activators and targets of signal transduction cascades. Immunol Lett 54: 189–193 Scheel-Toellner D, Wang K, Singh R, Majeed S, Raza K, Curnow SJ, Salmon M, Lord JM (2002) The death-inducing signalling complex is recruited to lipid rafts in Fasinduced apoptosis. Biochem Biophys Res Commun 297: 876–879 Ilan N, Madri JA (2003) PECAM-1: Old friend, new partners. Curr Opin Cell Biol 15: 515–524 Ilan N, Mohsenin A, Cheung L, Madri JA (2001) PECAM-1 shedding during apoptosis generates a membrane-anchored truncated molecule with unique signaling characteristics. FASEB J 15: 362–372 Vernon-Wilson EF, Kee WJ, Willis AC, Barclay, Simmons DL, Brown MH (2000) CD47 is a ligand for rat macrophage membrane signal regulatory protein SIRP (OX41) and human SIRPalpha 1. Eur J Immunol 30: 2130–2137 Gardai SJ, McPhillips KA, Frasch SC, Janssen WJ, Starefeldt A, Murphy-Ullrich JE, Bratton DL, Oldenborg PA, Michalak M, Henson PM (2005) Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte. Cell 123: 321–334 Gallucci S, Lolkema M, Matzinger P (1999) Natural adjuvants: Endogenous activators of dendritic cells. Nat Med 5: 1249–1255 Sauter B, Albert ML, Francisco L, Larsson M, Somersan S, Bhardwaj N (2000) Consequences of cell death: Exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells. J Exp Med 191: 423–434 Zheng L, He M, Long M, Blomgran R, Stendahl O (2004) Pathogen-induced apoptotic neutrophils express heat shock proteins and elicit activation of human macrophages. J Immunol 173: 6319–6326 Hart SP, Alexander KM, MacCall SM, Dransfield I (2005) C-reactive protein does not opsonize early apoptotic human neutrophils, but binds only membrane-permeable late apoptotic cells and has no effect on their phagocytosis by macrophages. J Inflamm (Lond) 2: 5 Familian A, Zwart B, Huisman HG, Rensink I, Roem D, Hordijk PL, Aarden LA, Hack

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Moffatt OD, Devitt A, Bell ED, Simmons DL, Gregory CD (1999) Macrophage recognition of ICAM-3 on apoptotic leukocytes. J Immunol 162: 6800–6810 Hart SP, Jackson C, Kremmel LM, McNeill MS, Jersmann H, Alexander KM, Ross JA, Dransfield I (2003) Specific binding of an antigen-antibody complex to apoptotic human neutrophils. Am J Pathol 162: 1011–1018 Hart SP, Alexander KM, Dransfield I (2004) Immune complexes bind preferentially to FcgammaRIIA (CD32) on apoptotic neutrophils, leading to augmented phagocytosis by macrophages and release of proinflammatory cytokines. J Immunol 172: 1882–1887 Novellino L, Castelli C, Parmiani G (2005) A listing of human tumor antigens recognized by T cells: March 2004 update. Cancer Immunol Immunother 54: 187–207 Pospisilova D, Borovickova J, Polouckova A, Spisek R, Sediva A, Hrusak O, Stary J, Bartunkova J (2002) Generation of functional dendritic cells for potential use in the treatment of acute lymphoblastic leukemia. Cancer Immunol Immunother 51: 72–78 Goldszmid RS, Idoyaga J, Bravo AI, Steinman R, Mordoh J, Wainstok R (2003) Dendritic cells charged with apoptotic tumor cells induce long-lived protective CD4+ and CD8+ T cell immunity against B16 melanoma. J Immunol 171: 5940–5947 Andersen MH, Becker JC, Straten P (2005) Regulators of apoptosis: suitable targets for immune therapy of cancer. Nat Rev Drug Discov 4: 399–409 Scheffer SR, Nave H, Korangy F, Schlote K, Pabst R, Jaffee EM, Manns MP, Greten TF (2003) Apoptotic, but not necrotic, tumor cell vaccines induce a potent immune response in vivo. Int J Cancer 103: 205–211 Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239–257 Bondanza A, Zimmermann VS, Rovere-Querini P, Turnay J, Dumitriu IE, Stach CM, Voll RE, Gaipl US, Bertling W, Poschl E, Kalden JR, Manfredi AA, Herrmann M (2004) Inhibition of phosphatidylserine recognition heightens the immunogenicity of irradiated lymphoma cells in vivo. J Exp Med 200: 1157–1165

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Fate of macrophages once having ingested apoptotic cells: Lymphatic clearance or in situ apoptosis? Geoffrey J. Bellingan and Geoffrey J. Laurent 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 macrophages, 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 inflammatory process [5, 6]. Neutrophil numbers peak earlier than macrophages. Their decline can be due to necrosis, apoptosis and subsequent phagocytosis, or progressing 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 airway lumen [8–10]. 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 [12–14]. This chapter examines macrophage clearance in the resolution of inflammation. Macrophages are powerful scavengers and are the major cell involved in phagocytosis of apoptotic neutrophils. The fate of macrophages once they have ingested apoptotic neutrophils is open to much debate. There is good evidence that the macrophage, 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 The Resolution of Inflammation, edited by Adriano G. Rossi and Deborah A. Sawatzky © 2008 Birkhäuser Verlag Basel/Switzerland

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stimuli including Fas and tumour necrosis factor (TNF)-_ death receptor ligation, ionising radiation, and multiple anti-neoplastic or cytotoxic agents [17–19]. 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 [20–22]. 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 macrophage 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 survive 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 providing a powerful mechanism through which cell survival is increased [28–30]. 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-apoptotic 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 abnormal regulation of this pathway have altered regulation of macrophage apoptosis, reduced viral clearance and increased inflammation [33, 34]. Akt-1 is a PI3K-regulated serine/threonine kinase that regulates survival through myeloid cell leukaemia

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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 withdrawal 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; however, 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 [35–43]. The timing with which such infections 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 bacteria 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

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macrophages had evidence of apoptotic features. It is not clear if all the macrophages 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 prostaglandins 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 macrophage apoptosis include engagement of Fas ligand [51]. There is now good evidence 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 macrophages 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].

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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 apoptosis in the host‘s 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 macrophage 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 permeabilisation 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 resolution. 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 questioned. We used in vivo fluorescence labelling to track macrophages during the resolution of sterile inflammatory challenges. These were either short (starch: 2–4 days), medium (thioglycolate: 5–10 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

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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 transferred 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 macrophages were detected with increasing frequency in the draining lymph nodes, but not in a variety of other tissues. These data suggest that inflammatory macrophages 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 peritoneum 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 lymphatics. 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 5–8-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 fluorescently 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.

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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 calcium 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-

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Figure 2. Protocol for adoptive transfer of fluorescence-labelled macrophages, ensuring donor macrophages 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 activation 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 bacteria; 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

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typically is associated with increased morbidity, while, in the presence of other intracellular 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 clearance 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 resident 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 surrounding 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 clearance. 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 draining 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,

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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 commend 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 lymphocytes 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 complex 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

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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 macrophage–neutrophil interaction and phagocytosis. Clearance 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 monocyte chemoattractant protein (MCP-1) were increased before macrophage ingestion 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 persisting 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 inhibiting 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 interferongamma 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-inflammatory phenotype where, along with the resident macrophages, they are vital in the phagocytosis and killing of pathogens and the orchestration of the pro-inflammatory 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-

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phages adopt different roles; they can, depending on regulation of their own sensitivity, 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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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 Women’s 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 Doctor’s 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 Nature’s 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 Doctor’s 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 author’s laboratory focus on gaining 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 [4–7]. Inflammation has emerged The Resolution of Inflammation, edited by Adriano G. Rossi and Deborah A. Sawatzky © 2008 Birkhäuser Verlag Basel/Switzerland

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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 acidderived epoxyeicosatetraenoic acids (EETs) produced via P450 [93, 94] and t-3 polyunsaturated fatty acids (PUFA) P450 epoxides may also play roles [68, 95]. Cell-cell interactions, exemplified by platelets-leukocytes within blood vessels and/or polymorphonuclear leukocytes (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 Alzheimer’s 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 docosahexaenoic acid (C22:6, DHA). The isolated compounds proved to possess potent actions

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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 biosynthesis 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,

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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 [13–18]. 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-resolving 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 [20–22]. Sir Henry Dale of England [23] and U. von Euler of Sweden [24] each focused their seminal investigations on the roles of chemical 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, monocytes 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.

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Table 1 - LX and ATL actions in animal models of disease Dermal inflammation Inhibit neutrophil recruitment into ear skin Prevent vascular permeability

Takano et al. [79] Schottelius et al. [80]

Ischaemia-reperfusion (I/R) injury Attenuate hind-limb I/R-induced lung injury Detachment of adherent leukocytes in mesenteric I/R Protective in ischaemic acute renal failure

Chiang et al. [37] Scalia et al. [81] Leonard et al. [82]

Peritonitis Block neutrophil recruitment and vascular leakage Promote phagocytosis of neutrophil by macrophage

Bannenberg et al. [44] Godson et al. [41]

Colitis (inflammatory bowel disease) Attenuate proinflammatory gene expression and reduce severity of colitis Inhibit weight loss, inflammation and immune dysfunction

Fiorucci et al. [83]; Arita et al. [49] Gewirtz et al. [84]

Glomerulonephritis Reduce leukocyte rolling and adherence Decrease neutrophil recruitment

Munger et al. [85] Leonard et al. [82]

Asthma Block airway hyper-responsiveness and pulmonary inflammation

Levy et al. [86]

Cystic fibrosis Decrease neutrophilic inflammation, pulmonary bacterial burden and disease severity.

Karp et al. [87]

Angiogenesis Reduce angiogenic phenotype: endothelial cell proliferation and migration

Fierro et al. [58]

Periodontitis (oral inflammation and bone loss)* Reduce microbe-initiated, neutrophil-mediated tissue damage and bone destruction

Serhan et al. [61]

Eye Accelerate cornea re-epithelialisation, limit neovascularisation, and promote host defense in the eye

Cotran et al. [88]; 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.

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Table 2 - Resolvins and protectins: Actions of animal disease models. Mediator

Action

Reference

Resolvin E1

Reduces PMN infiltration, murine skin air pouch inflammation, peritonitis

Serhan et al. [18]; Serhan et al. [13]; Arita et al. [46]; Bannenberg et al. [44] Arita et al. [49] Hasturk et al. [76]

Gastrointestinal protection in TNBS colitis Protects in periodontitis, stops inflammation and bone loss Resolvin D1

Protectin D1/ neuroprotectin D1

Protectin D1/ neuroprotectin D1

Reduces PMN infiltration, murine skin air pouch inflammation Reduces peritonitis Reduces cytokine expression in microglial cells Protects in renal ischaemic injury

Serhan et al. [13]

Reduces stroke damage Reduces PMN infiltration Protects from retinal injury Regulates Th2 cells, apoptosis and raft formation Shortens resolution interval in murine peritonitis; regulates cytokines and chemokines

Marcheselli et al. [15] Hong et al. [14] Mukherjee et al. [16] Ariel et al. [91]

Reduces PMN infiltration; reduces peritonitis Diminished production in human Alzheimer’s disease and promotes neural cell survival Promotes corneal epithelial cell wound healing

Serhan et al. [17] Lukiw et al. [92]

Hong et al. [14] Duffield et al., [90]

Bannenberg et al. [44]

Gronert et al. [77]

There are many local, short-acting chemical mediators that are pro-inflammatory 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 chemoattractants like LTB4 work in concert spatially and temporally with peptide chemoattractants, 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.

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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 leukocyte 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 governing 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 neutrophil’s 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

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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

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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 2–4) raised awareness of the potential for new therapeutic approaches to inflammatory diseases that target these active pathways [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-

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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-capillary 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 pathways for the normal self-limiting or termination at local contained sites of inflammation [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 resolution 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 parachutes 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 biosynthesis 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 beginning 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

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lipidomic/lipid mediator informatics approach employing LC-MS/MS revealed that, during the time course of spontaneous resolution, novel lipid mediators were generated 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-3’s 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-3’s 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 antiinflammatory 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 program 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 mediators, 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 1–3). 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

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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-containing 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 chemical 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.

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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 analogues are potent regulators of transendothelial/transepithelial migration of PMN across these cells and endothelial cell proliferation, in vitro and in vivo ([57–59] 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 15LO type I generate enhanced levels of LXs, have an enhanced anti-inflammatory status, 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 protective 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-inflammatory, and anti-tumour actions [4, 64]. Each of the three major human LO activities (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 remedies, 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-3’s protective action, and are there potential overlap(s) in their actions at the molecular and cellular levels? To address this in an experimental 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

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using LC-UV-MS/MS. We constructed lipid mediator libraries with physical properties (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-activity 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 transform 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 mechanisms 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)

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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 [4–7] 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 [13–16, 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 infiltration 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 dihydroxydocosanoids 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, indomethacin 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 hydroperoxy-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 corresponding 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-

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strate of a given LO, which is best appreciated in the case of 5-LO and LT biosynthesis. 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 8SLO. Each LO was catalogued according to its position of molecular insertion into arachidonate [71–74]. 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, actually 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 managing human disease. RvE1 was identified in human plasma [46]. At nanomolar levels, RvE1 dramatically reduced dermal inflammation, peritonitis, DC migration and IL-12 production (Tab. 2). We screened GPCRs and identified one, denoted earlier as the orphan G protein-coupled receptor ChemR23, that mediates 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 regulation of IL-12. RvE1, as a synthetic anti-inflammatory lipid mediator, reduces leukocyte infiltration 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-trinitrobenzene 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 bioactions both in vivo and in vitro [3–5]. The complete stereochemistry of PD1 (10,17Sdocosatriene), 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-

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sues was separated from related natural isomers and then subjected to LC-MS/MS and gas chromatography-MS-based analyses [17]. Comparison with six 10,17dihydroxydocosatrienes 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,19Zhexaenoic acid. Additional isomers identified in these studies include 615-trans-PD1 (isomer III), 10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid (isomer IV), and a double dioxygenation product 10S,17S-dihydroxy-docosa4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid that was also present in murine exudates. 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 intermediate 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 1–10 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 molecule [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 complementary 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-15epi-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 protectins each dampen inflammation and PMN-mediated injury from within, which are key culprits in many widely occurring human diseases. The results of our studies 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

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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,17Sdocosatriene

10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid (the dioxygenation product)

10,17-docosatriene isomers

10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15E,19Z-hexaenoic acid; 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-docosa4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid

RvE1

Resolvin E1, 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic 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-protective 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 physiological relevance of the resolvins and protectins is likely to extend beyond our current understanding [13–16, 18].

Acknowledgments We thank Mary Halm Small for assistance in preparing the manuscript. Studies in the author’s laboratory were supported in part by National Institutes of Health grants GM38675, DK074448 and P50-DE016191.

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Beyond inflammation: Lipoxins; resolution of inflammation and regulation of fibrosis Paola Maderna and Catherine Godson 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 [1–10]. Eicosanoid production in inflammation tightly regulates these processes. During the initial phase, proinflammatory mediators including leukotriene (LT) B4, the cysteinyl LTs and prostaglandins (PG) evoke potent chemotactic responses of leukocytes whose activation is coupled to the production of proinflammatory (Th1-derived cytokines) at sites of inflammation [11]. To facilitate resolution, 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, 12–20].

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, adhesion and transmigration across endothelia and epithelia [22–25]. 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-trihydroxy-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 transcellular metabolism initiated by sequential oxygenation of arachidonic acid by The Resolution of Inflammation, edited by Adriano G. Rossi and Deborah A. Sawatzky © 2008 Birkhäuser Verlag Basel/Switzerland

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both 5-lipoxygenase (LO) and 12-LO or 15 and 5-LO [1–3, 6, 7, 9]. In a cytokine-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 hydroxyeicosatetraenoic acid that is rapidly converted by 5-LO in activated PMN to 15-epiLX (aspirin-triggered LXs, ATL) that share many of the bioactions of the native LXs [2, 3, 29–32]. 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 possibly by t-oxidation at C20 [1–3, 5, 6, 9]. When compared to the native LXs, the ATLs display longer biologic half-life [1–3, 33]. To circumvent the metabolic inactivation 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 phenoxy group bonded to C-16 replacing the t-end of the molecule [33–35]. 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 [36–38]. 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 glomerulonephritis [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 generation [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 administration, indicating that ATL production is one of the mechanisms of gastric adaptation 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

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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 characteristics 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 designated 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 plasminogen 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 Ac2–26 showed anti-inflammatory actions in many experimental models of inflammation [77–80] (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 pathways 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 nonmyeloid origin typically consistent with the distribution of the ALXR [1–3, 6, 7, 9]. LXs, ATLs and stable synthetic LX analogues inhibit PMN and eosinophil chemotaxis [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 endothelial P-selectin [83] and integrin clustering and mobility on PMN [84].

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LXs and ATL have been shown to play a key role in regulating cytokine-chemokine 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 tissue 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 inhibition 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-inflammatory 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 morphological 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 activity, 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 proliferation 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

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loss and inflammation caused by specific gram-negative microorganisms [98]. Analogues 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 generation of proinflammatory mediators [100]. The dysregulated proinflammatory environment of the cystic fibrotic airway, characterised by up-regulated IL-8 production 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 bacterial burden and attenuates disease severity [41]. ATLs are protective in intestinal inflammation in a mouse model of dextran sodium 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 molecules, 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 previously discussed, the switch in the production from pro-inflammatory mediators to LXs as well as resolvins and protectins is critical [5, 12–20]. 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, 105–107]. 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 [105–109]. In contrast to phagocytosis of bacteria

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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 demonstrated by in vitro and in vivo studies [110–112], 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, 105–107]. 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 phagocytic 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 microparticles 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 phosphatidylinositol 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. Defining 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 apoptotic 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 receptor 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 Igsuperfamily member adhesion molecule ICAM-3, members of the collectin family and their receptors CD91 and calreticulin (see reviews [8, 105–107, 123]).

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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 recognition and uptake, such as complement protein C1q, the tyrosine kinase receptor gene Mer and milk fat globule factor 8, are missing or mutated [126–128]. 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 chemotactic 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 rearrangement 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 [1–3, 6, 7, 9]. In contrast to inhibiting PMN function, LXs are potent activators of monocytes, stimulating their chemotaxis and adherence without causing 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

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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 paraformaldehyde. (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 visualize 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 [133–135]. 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 observation 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 reorganisation of actin in human monocytes and M\ (Fig. 2), resulting in the promotion

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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 37°C. 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

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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 polarisation 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 [91–93] 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 MHCbinding 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 dephosphorylation and redistribution [135, 138]. Interestingly, the endogenous annexin 1 released

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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 important 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 proresolution 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-inflammatory 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 Author’s 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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phosphatidylserine in recognition of apoptotic cells by phagocytes. Cell Death Differ 5: 551–562 Fadok VA, de Cathelineau A, Daleke DL, Henson PM, Bratton DL (2001) Loss of phospholipid asymmetry and surface exposure of phosphatidylserine is required for phagocytosis of apoptotic cells by macrophages and fibroblasts. J Biol Chem 276: 1071–1077 Anderson HA, Englert R, Gursel I, Shacter E (2002) Oxidative stress inhibits the phagocytosis of apoptotic cells that have externalised phosphatidylserine, Cell Death Differ 9: 616–625 A Devitt, Pierce S, Oldreive C, Shingler WH, Gregory CD (2003) CD14-dependent clearance of apoptotic cells by human macrophages: the role of phosphatidylserine. Cell Death Differ 10: 371–382 Arur S, Uche UE, Rezaul K, Fong M, Scranton V, Cowan AE, Mohler W, Han DK (2003) Annexin I is an endogenous ligand that mediates apoptotic cell engulfment. Dev Cell 4: 587–598 Savill J, Dransfield I, Hogg N, Haslett C (1990) Vitronectin receptor-mediated phagocytosis of cells undergoing apoptosis. Nature 343: 170–173 Fadok VA, Bratton DL, Henson PM (2001) Phagocyte receptors for apoptotic cells: recognition, uptake and consequences. J Clin Invest 108: 957–962 Gaipl US, Voll RE, Sheriff A, Franz S, Kalden JR, Herrmann M (2005) Impaired clearance of dying cells in systemic lupus erythematosus. Autoimmun Rev 4: 189–194 Gaipl US, Kuhn A, Sheriff A, Munoz LE, Franz S, Voll RE, Kalden JR, Herrmann M (2006) Clearance of apoptotic cells in human SLE. Curr Dir Autoimmun 9: 173–187 Botto M, Del’Agnola C, Bygrave AE, Thompson EM, Cook HT, Petry F, Loos M, Pandolfi PP, Walport MJ (2002) Homozygous C1q deficiency causes glomerulo-nephritis associated with multiple apoptotic bodies. Nat Genet 19: 56–59 Cohen PL, Caricchio R, Abraham V, Camenisch TD, Jennette JC, Roubey RA, Earp HS, Matsushima G, Reap EA (2002) Delayed apoptotic cell clearance and lupus-like autoimmunity in mice lacking the c-mer membrane tyrosine kinase. J Exp Med 196: 135–140 Hanayama R, Tanaka M, Miyasaka K, Aozasa K, Koike M, Uchiyama Y, Nagata S (2004) Autoimmune disease and impaired uptake of apoptotic cells in MFG-E8-deficient mice. Science 304: 1147–1150 Vandivier RW, Fadok VA, Hoffmann PR, Bratton DL, Penvari C, Brown KK, Brain JD, Accurso FJ, Henson PM (2002) Elastase-mediated phosphatidylserine receptor cleavage impairs apoptotic cell clearance in cystic fibrosis and bronchiectasis. J Immunol 167: 976–986 Giles KM, Ross K, Rossi AG, Hotchin NA, Haslett C, Dransfield I (2001) Glucocorticoid augmentation of macrophage capacity for phagocytosis of apoptotic cells is associated with reduced p130Cas expression, loss of paxillin/pyk2 phosphorylation and high levels of active Rac. J Immunol 167: 976–986 Liu Y, Cousin JM, Hughes J, Van Damme J, Seckl JR, Haslett C, Dransfield I, Savill J, Rossi AG (1999) Glucocorticoids promote nonphlogistic phagocytosis of apoptotic leukocytes. J Immunol 162: 3639–3646

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132 Lauber K, Bohn E, Krober SM, Xiao YJ, Blumenthal SG, Lindemann RK, Marini P, Wiedig C, Zobywalski A, Baksh S et al (2003) Apoptotic cells induce migration of phagocytes via caspase-3-mediated release of a lipid attraction signal. Cell 113: 717–730 132a Scannell M, Flanagan MD, de Stefani A, Wynne KJ, Cagney G, Godson C, Maderna P (2007) Annexin-1 and peptide derivatives are released by apoptotic cells and stimulate phagocytosis of apoptotic neutrophil by macrophages. J Immunol 178: 4595–4605 133 Godson C, Mitchell S, Harvey K, Petasis NA, Hogg N, Brady HR (2000) Cutting edge: Lipoxins rapidly stimulate non-phlogistic phagocytosis of apoptotic neutrophils by monocyte-derived macrophage. J Immunol 164: 1663–1667 134 Mitchell S, Thomas G, Harvey K, Cottell D, Reville K, Berlasconi G, Petasis NA, Erwig L, Rees AJ, Savill J et al (2002) Lipoxins, aspirin-triggered epi-lipoxins, lipoxin stable analogues, and the resolution of inflammation: Stimulation of macrophage phagocytosis of apoptotic neutrophils in vivo. J Am Soc Nephrol 13: 2497–2507 135 Reville K, Crean JK, Vivers S, Dransfield I, Godson C (2006) Lipoxin A4 redistributes myosin IIA and Cdc42 in macrophages: implications for phagocytosis of apoptotic leukocytes. J Immunol 176: 1878–1888 136 Maderna P, Cottell DC, Berlasconi G, Petasis NA, Brady HR, Godson C (2002) Lipoxins induce actin reorganisation in monocytes and macrophages, but not in neutrophils: Differential involvement of Rho GTPases. Am J Pathol 160: 2275–2283 137 Fan X, Krahling S, Smith D, Williamson P, Schlegel RA (2004) Macrophage surface expression of annexins I and II in the phagocytosis of apoptotic lymphocytes. Mol Biol Cell 15: 2863–2872 138 Maderna P, Yona S, Perretti M, Godson C (2005) Modulation of phagocytosis of apoptotic neutrophils by supernatant from dexamethasone-treated macrophages and annexin-derived peptide Ac2–26. J Immunol 174: 3727–3733

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Anti-inflammatory glucocorticoids and annexin 1 Mauro Perretti and Roderick J. Flower 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 mediators/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 understanding 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 interrelated 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 pathology, 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 [6–10]. 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 identiThe Resolution of Inflammation, edited by Adriano G. Rossi and Deborah A. Sawatzky © 2008 Birkhäuser Verlag Basel/Switzerland

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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 applications.

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, Hench’s 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-ordinated response of an organism to injury or stress. In this context, it was believed that these hormones probably contributed to the inflammatory response. Hench’s 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 (hydrocortisone) 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, naturally 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 relations 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 programs 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.

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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 agreement 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 physiological 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 culminated 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, increasing 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 multiple 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

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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 receptordependent non-genomic effects exerted by GC, possibly through direct protein-protein 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 5–10 min post-GC exposure, and may differ 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 currently 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

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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 elements (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.

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Figure 2. Glucocorticoid modulation of annexin 1 expression. GC exert both genomic and non-genomic control on annexin 1 synthesis and post-translational modifications. Rapid (5–15 min) non-genomic effects are linked to protein phosphorylation 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

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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 activation loop described predominantly in monocytes and lymphocytes, and schematically 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, eicosanoids, 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 microcirculation 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 concentrations 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 inflammatory 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 efficacy, but also their wide spectrum of side effects. Actions on transcription factors are schematically summarised in this chapter; more recently, GC effects on rapid

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signalling events have also been discovered, including their ability to induce the expression of mitogen-activate protein kinase phosphatase-1 thereby favouring dephosphorylation, 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 investigation.

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 Nterminal 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 endogenous 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 system” as an endogenous biochemical process that operates in the context of the adherent and migrating neutrophil [46].

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“The annexin 1 system” In resting conditions, annexin 1 is abundantly expressed in white blood cells, including blood neutrophils and monocytes [47, 48], as well as in tissue dwelling cells such as macrophages and epithelial cells [49]. Human neutrophil adhesion to endothelial 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 formylpeptide 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 supported 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 FPRL1 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-dependent 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 summarise 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-inflammatory responses [46, 64].

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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 adhesion, controlled exocytosis brings annexin 1 onto the cell surface, where it binds in a calciumdependent 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 adhesion 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.

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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 system 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 proteins [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 [68–70] 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 adherent 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 exposure, 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.

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Some studies have indicated that annexin 1 could mediate part of the anti-proliferative 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 contaminating monocytes. At high concentrations, peptide Ac2-26 inhibits both phytohaemoagglutinin- 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 mediator 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-regulate the inflammatory reaction, assuring rapid resolution and restoration of homeostasis. In particular, we have focused on the archetypal anti-inflammatory mediators, 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 development 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 predominantly funded by the Arthritis Research Campaign UK (15755), the Wellcome Trust UK, the British Heart Foundation and the William Harvey Research Foundation.

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The resolution of airway inflammation in asthma and chronic obstructive pulmonary disease Garry M. Walsh and Catherine M. McDougall 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 hyperresponsiveness 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 collagen 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 important 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 associated 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 relatively 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 exacerbations 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. The Resolution of Inflammation, edited by Adriano G. Rossi and Deborah A. Sawatzky © 2008 Birkhäuser Verlag Basel/Switzerland

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Chronic obstructive pulmonary disease (COPD) is a debilitating inflammatory 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 inflammatory 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 treatments. 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, making 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 suppressing airway inflammation and the associated re-modelling of the airways that leads to progressive and irreversible loss of lung function. The mechanisms by which currently 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

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higher binding efficiency. In healthy volunteer subjects, a placebo-controlled, randomised, double-blind, change-over equivalence trial demonstrated that daily doses of ciclesonide (800 μg for 7 days) were found to have no clinically relevant effects on the hypothalamic-pituitary-adrenal axis; this was independent of the time of administration indicating that it should prove to be well tolerated [12]. A number of clinical trials have been published recently that suggest ciclesonide may be an effective therapy for asthmatic patients. For example, ciclesonide has been shown to be clinically effective in patients with bronchial asthma whose airway responsiveness was assessed using adenosine-5’-monophosphate challenge [13]. In a double-blind, placebo-controlled, randomised, crossover study, ciclesonide treatment (800 +g twice daily via a Cyclohaler) also protected against early and late-phase bronchoconstriction 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 protected 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 ciclesonide effectively maintained asthma control in adults with persistent asthma [16]. These findings suggest that inhaled ciclesonide will be an effective asthma medication 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 corticosteroids 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 “dissociated 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

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Figure 1. Dissociation of anti-inflammatory effects from side effects of corticosteroids [17]. GR: Glucocorticoid 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 corticosteroids. 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 leukotriene (LT) antagonists, have had promising clinical effects. Since COPD is characterised 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) pathway (Fig. 2). The cysteinyl LTs (LTC4, LTD4, LTE4) are highly potent mediators of inflammation, and induce bronchoconstriction. LTB4 has minimal bronchoconstrictor 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

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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 cysteinyl 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 mediator in nocturnal asthma, severe status asthmaticus and sudden-onset fatal asthma in which airway neutrophilia predominates rather than eosinophilia [23–26]. 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 disappointing [28, 29].

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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 roflumilast [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 parameters from week 1 in addition to having a first-dose bronchodilator effect. Sustained improvement in pulmonary function associated with a reduction in asthma symptoms has been reported in a 12-month study with cilomilast, suggesting tolerance 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 present 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 stimulates mucus secretion and induces interleukin (IL)-8 release from epithelial cells and

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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 allergenspecific 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 overcome these problems include the use of hypoallergenic isoforms, recombinant allergens 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 treatment 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 asthmatic subjects with Fel d 1-derived T cell peptides significantly improved clinically relevant outcome measurements, including reductions in late asthmatic reactions to

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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-capillary 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 eosinophil-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 eosinophilmediated 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 circulating eosinophils and reduced airway and bone marrow eosinophils but reported no significant improvement of clinical measures of asthma. Critically, lung biopsy

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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 reductions 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 receptor 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-inflammatory 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 hyperresponsiveness and mucus accumulation [55], while the other demonstrated a critical 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 significantly 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 eliminating 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 IL5–/– 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.

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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 signal 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 demonstrated 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 subjects, 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 receptors 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 inflammation 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]. Transgene 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

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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 Crohn’s 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 therapeutic targets for asthma and COPD [76, 77]. In summary, current evidence seems to suggest that targeting single T cell proinflammatory 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-inflammatory 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 including TNF-_, GM-CSF or IL-5, together with inflammatory enzymes such as inducible 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 contrast, anti-IL-10 antibody treatment just prior to the last ovalbumin challenge significantly 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

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corticosteroid-resistant asthma, including three patients with Churg-Strauss syndrome. Treatment was associated with an early clinical improvement in all patients as assessed by pulmonary function tests and tapering of their corticosteroid treatment. 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 mononuclear 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 binding to high-affinity (Fc¡RI) receptors on mast cells and basophils and to low-affinity (Fc¡RII) 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 Fc¡RI binding 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 2–4 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 deterioration-related incidents. Another recent study reported that omalizumab treatment of subjects with both persistent rhinitis and difficult to treat asthma resulted in significantly 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 inadequately controlled severe persistent asthma [94]. Consistent findings from these

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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 control 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 tolerated 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 control 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 transmigration through the post-capillary endothelium, followed by their chemotaxis to and activation/degranulation at inflammatory foci [95]. In normal individuals eosinophils 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 selection 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-regulates 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-1dependent 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 therapeutic avenues should always be considered as demonstrated by the recently reported

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series of fatal CNS infections occurring during systemic treatment with natalizumab [102]. Chemokines are chemoattractant cytokine molecules and are crucial in eosinophil 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 represents 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 structural non-migratory cells, expression of the CCR3 receptor may represent an autoregulatory 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 mobilisation 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. Apoptosis 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

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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 viabilityenhancing 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 pathways 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-inflammatory 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 airways 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 challenge [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, eosinophil 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 involvement 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

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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 development of novel therapeutic approaches to target eosinophil-induced inflammation in asthma, particularly in those patients who exhibit steroid resistance. Other factors 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 disintegration of the apoptotic cell via a process termed secondary necrosis and the subsequent uncontrolled leakage of the dying cell’s 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 inflammatory 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-professional phagocytes such as dendritic cells, fibroblasts or hepatocytes in the recognition 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 isolated, 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-

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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].

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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 capacity 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 mechanism for their clearance [151–153]. Several papers from the same group have championed 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 [147–149] as numerous studies have demonstrated apoptosis and phagocytic removal in vivo [154–156]. Indeed, it has been suggested that phagocytosis of apoptotic cells during quiescent periods is an important process with transepithelial egress providing a necessary secondary 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 associated 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, therapies 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

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important role in asthma [160]. Importantly, prostaglandin D2 is a specific eosinophil 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 important 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 consequence 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, neutrophil 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 computer-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 latephase 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 severity 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 proteasemediated inflammation in COPD results in the release of a host of pro-inflammatory 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-

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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 concentrations 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 proteincoupled 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 blockade 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 targeting 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 inflammation resolution in many conditions including asthma and COPD [179] and is an important and growing area of research.

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Conclusion Current therapies for asthma and COPD are clearly unsatisfactory. In asthma, significant 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 development 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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