Trends in Innate Immunity (Contributions to Microbiology)

Trends in Innate Immunity

Contributions to Microbiology Vol. 15

Series Editors

Axel Schmidt Witten Heiko Herwald Lund

Trends in Innate Immunity Volume Editors

Arne Egesten Lund Axel Schmidt Witten Heiko Herwald Lund 20 figures, 1 in color, and 8 tables, 2008

Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Shanghai · Singapore · Tokyo · Sydney

Contributions to Microbiology formerly ‘Concepts in Immunopathology’ and ‘Contributions to Microbiology and Immunology’

Arne Egesten, MD, PhD

Heiko Herwald, PhD

Section for Respiratory Medicine Department of Clinical Sciences Biomedical Center B14 Lund University Tornavägen 10 SE–221 84 Lund (Sweden)

Division of Infection Medicine Department of Clinical Sciences Biomedical Center B14 Lund University Tornavägen 10 SE–221 84 Lund (Sweden)

Axel Schmidt, MD, PhD Professor of Clinical Microbiology Institute of Microbiology and Virology Private University of Witten/Herdecke Stockumer Strasse 10 DE–58448 Witten (Germany)

Library of Congress Cataloging-in-Publication Data Trends in innate immunity/volume editors, Arne Egesten, Axel Schmidt, Heiko Herwald p. ; cm. – (Contributions to microbiology, ISSN 1420–9519; v. 15) Includes bibliographical references and indexes. ISBN 978–3–8055–8548–4 (hard cover : alk. paper) 1. Natural immunity. I. Egesten, Arne. II. Schmidt, Axel, 1962– III. Herwald, Heiko. IV. Series. [DNLM: 1. Immunity, Natural–physiology. 2. Immune System–physiology. W1 CO778UE v.15 2008/QW 541 T793 2008] QR185.2.T74 2008 616.07⬘9–dc22 2008012887

Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® and PubMed/MEDLINE. Disclaimer. The statements, options and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2008 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free and non-aging paper (ISO 9706) by Reinhardt Druck, Basel ISSN 1420–9519 ISBN 978–3–8055–8548–4

Contents

VII

1 12

21 45

61 78 101 118 147 164

Foreword Egesten, A. (Lund); Schmidt, A. (Witten); Herwald, H. (Lund) From Darwin and Metchnikoff to Burnet and Beyond Cooper, E.L. (Los Angeles, Calif.) General Introduction to Innate Immunity: Dr. Jekyl/Mr. Hyde Quality of the Innate Immune System Zänker, K.S. (Witten) The Innate Immune System of Mammals and Insects Müller, U. (Leipzig); Vogel, P. (Bochum); Alber, G. (Leipzig); Schaub, G.A. (Bochum) Pattern Recognition Receptors and Their Role in Innate Immunity: Focus on Microbial Protein Ligands Areschoug, T. (Lund); Gordon, S. (Oxford) Antimicrobial Peptides in Innate Immune Responses Sørensen, O.E. (Lund); Borregaard, N. (Copenhagen); Cole, A.M. (Orlando, Fla.) Complement: An Efficient Sword of Innate Immunity Rambach, G.; Würzner, R.; Speth, C. (Innsbruck) Antibacterial Chemokines – Actors in Both Innate and Adaptive Immunity Eliasson, M.; Egesten, A. (Lund) The Role of Neutrophils and Monocytes in Innate Immunity Kantari, C. (Paris); Pederzoli-Ribeil, M. (London); Witko-Sarsat, V. (Paris) Innate Immune Functions of the Airway Epithelium Bartlett, J.A.; Fischer, A.J.; McCray Jr, P.B. (Iowa City, Iowa) Oxidative Innate Immune Defenses by Nox/Duox Family NADPH Oxidases Rada, B.; Leto, T.L. (Rockville, Md.)

188

Aging and Impairment of Innate Immunity Nomellini, V. (Maywood, Ill.); Gomez, C.R. (Maywood, Ill./Santiago); Kovacs, E.J. (Maywood, Ill.)

206

Author Index Subject Index

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VI

Contents

Foreword

Interestingly, it was not long ago that the area of innate immunity was delineated as a separate entity of immunity, i.e. in contrast to adaptive immunity. Many host defense mechanisms provided by the innate immune system are essential to keep organisms in a state of health, primarily e.g. against potentially harmful microorganisms or aeroplankton present in their environment. In addition to securing the integrity against pathogenic microorganisms of the milieu, vertebrates will have time to mount a specific adaptive immune response owing to innate immune functions. On the other hand, failure of mechanisms involved in innate immunity can contribute to disequilibria, resulting in both infectious and noninfectious diseases such asthma, allergies, and inflammatory bowel disease. The longstanding evolutionary pressure on innate immune mechanisms, as a consequence of host-parasite interactions, can provide keys to how we can tackle future challenges such as bacterial resistance to conventional antibiotics and other antiinfective agents. Therefore, an improved understanding of innate immunity is important to develop novel strategies to combat a broad range of diseases. Innate immunity is an area of research that is rapidly expanding, and it is rightfully attracting an increasing interest among a broad audience of scientists and clinicians. In the present volume of Contributions to Microbiology, we provide a wide and updated overview on several important mechanisms involved in innate immunity. We have been fortunate to recruit several eminent scientists from around the world, giving overviews in their fields of expertise. It is our belief that this volume will provide interesting and thought-provoking aspects on this ancient and important part of the immune system. Arne Egesten, Lund Axel Schmidt, Witten Heiko Herwald, Lund

Egesten A, Schmidt A, Herwald H (eds): Trends in Innate Immunity. Contrib Microbiol. Basel, Karger, 2008, vol 15, pp 1–11

From Darwin and Metchnikoff to Burnet and Beyond Edwin L. Cooper Laboratory of Comparative Neuroimmunology, Department of Neurobiology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, Calif., USA

Abstract Phagocytosis in unicellular animals represents the most ancient and ubiquitous form of defense against foreign material. Unicellular invertebrates can phagocytose for food and defense. Multicellular invertebrates and vertebrates possess phagocytic cells and have evolved more complex functions attributed to immunodefense cells that specialized into cellular and humoral immune responses. Thus all animals possess: innate, natural, nonspecific (no memory) nonanticipatory, nonclonal, germline (hard wired) host defense functions. In addition, all vertebrates possess: adaptive, induced, specific (memory), anticipatory, clonal, somatic (flexible) immune responses. A similar situation exists with respect to components of the signaling system, immunity and development. With multicellularity, clearly numerous immune response characteristics are not possible in unicellular forms or even those that straddle the divide between unicellularity and multicellularity, beginning with colonial/social protozoans. Still, it is instructive to elucidate a hierarchy of animals based upon immunologic characteristics and how they parallel other physiological traits. Evidence is presented that the most primitive of invertebrates prior to the evolution of multicellular organisms possess varying degrees of complexity at the molecular level of those hallmarks that now characterize the immune system. Copyright © 2008 S. Karger AG, Basel

It seems like a long stretch to leap two centuries to the 21st and still praise the prescience of the 19th century Metchnikoff [1]. Still, even more farfetched and almost unimaginable is the source of and reason for our enormous acclamations. The answer is not complicated but it is all encompassing. Multiple accolades are derived from observations on simpler invertebrate animals and hardly would we ever think of them as crucial to understanding more in depth about the breadth of immune competence [2–7]. Invertebrates are often considered as queer (praying mantis), pests (mosquitoes, parasites, flies), edible (crabs, shrimp, oysters, lobsters), medicinal (certain fly larvae, leeches, earthworms, bees), pollinators (bees) essential for soil and therefore agricultural (earthworms). Perhaps still further from 20th century immunologists’ intellectual grasp was the realization that such a simple event as phagocytosis as it was first observed in an invertebrate could exert such a profound influence on how the immune system really

Table 1. Early evidence of self nonself recognition in unicellular species correlated with recent evidence of signaling systems Animal group (Protozoa)

Assay for self non-self recognition

Results of assay(s)

Signaling system and/or adhesion protein families

Investigator

Amoeba (Amoeba proteus) Amoeba discoides

Transplantation Allogeneic nuclei Xenogenic nuclei

90% clones 0% clones 0% clones

–

Hawkins, 1964 [see chapter 5 in ref. 3]

Social amoebae (Dictyostelium discoideum) slime molds

S cells Phagocytosis of bacteria

–

TIR domain proteins

Chen et al., 2007

Choanoflagellates (Unicellular colonial)

–

–

C-type lectins Tyrosine kinase signaling components

King et al., 2003

Ciliata Stentor coeruleus Stentor polymorphus

Lack of Chimera formation

Ejection of symbiotic Chlorella

–

Tartar, 1970 [see chapter 5 in ref. 3]

works or our understanding of how to fine tune it to improve the quality of life of humans. We can now explore easily how potential external threats to life by continuously mutating microbes are first perceived, recognized, and the resulting signals interpreted and presumably survival from infection insured – or the blocking of cancer development an internal threat averted. This chapter will focus primarily on unicellular (Protozoa) and examples of multicellular animals (Sponges, Cnidarians); more complex invertebrates are excluded. Three reasons are presented. First, these two animal groups are situated at the nexus between single cell life and the emergence of multicellularity. Second, the unjustified thinking of immunologists would discredit these two groups with having evolved any semblance (sprout) of an immune response other than phagocytosis. Third, the information that is included, i.e. the recent discovery of Toll-like receptors (TLRs) justified their inclusion. Finally, TLRs correlate with earlier information that substantiated the immunodefense capabilities as we knew them long ago and credit them today (tables 1, 2).

Emergence of Modern Immunology May Be Indebted to Invertebrates?

These two great divisions of the immune system (innate and adaptive) that were pioneered by prescience and perhaps the coalescence of anthropocentric ideas on immunity

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Table 2. Porifera and Cnidaria early evidence of self non-self recognition in multicellular animals and evidence of signaling systems Animal group

Porifera Sponges

Assay for self non-self recognition

Results of assay(s)

Signaling system and/or adhesion protein families

Investigator

Mixing of red and yellow sponges

Disaggregated sponges do not reaggregate together

–

Wilson, 1907 Galtsoff, 1925 Spiegel, 1954 [see chapter 5 in ref. 3]

Response to bacterial lipopeptides

–

TLR, IRAK-41, effector caspase sequence (SDCA, SL) Homologies in familyspecific domains

Weins et al., 2007

Allografts and xenografts

Incompatible transplant reactions

–

Bibb and Campbell, 1973 [see chapter 5 in ref. 3]

Autografts Allografts

Compatible Incompatible

–

Theodor, 1966, 1969, 1970 [see chapter 5 in ref. 3]

Autografts Isografts Allografts

Compatible Incompatible Incompatible

–

Hildeman, 1974 [see chapter 5 in ref. 3]

Microciona prolifera Cliona celata Demosponges Suberites domuncula

Cnidaria Hydrozoa Hydra Chlorphydra pelmatohydra Anthozoa Arborescent cnidarians Staghorn corals Acropora Hydra magnipapillata Nematostella vectensis

–

–

–

–

Coral (Acropora millepora)

Canonical Toll/TLR Receptor C3, MAC/PF

Miller et al., 2007

with the genius of a zoologist, resulted from a fortuitous experiment considered as a great experiment in biology [8]. This was first recognized when Metchnikoff successfully demonstrated phagocytosis in the 19th century, a discovery worthy of the Nobel Prize in Physiology and Medicine. This discovery of invertebrate phagocytosis dramatically changed the monolithic world of immunology. His careful and detailed observations of white cell motility toward and engulfment of foreign bodies in transparent larvae of

From Darwin and Metchnikoff to Burnet and Beyond

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starfish and in the water flea Daphnia provoked a major re-evaluation of the nature of immune systems, admittedly restricted to the human good. Before his prescient observations, immune systems were believed to be wholly humoral and there was little emphasis on the role of leukocytes or white cells. Metchnikoff ’s discovery, however, added cellular immunity to the known armory of humoral immunodefense mechanisms. Serendipity surely intervened and there was probably the impulse to shout Archimedes’ eureka when the interpretation of why cells were moving toward a foreign body was easily visualized. Thus, the foundation for invoking the concept of self non-self recognition was laid [9]. Roughly a century later, this cellular component, and the animal models from which it was derived are still at the center of immunology. Moreover, there is a much greater willingness to accept that invertebrate model systems have much more to contribute than was thought, even in the early 1960s when modern immunology was beginning to develop. Metchnikoff would have relished this turn of events because immunology is infinitely richer and even biology in general has reaped substantial benefits, including the harnessing of invertebrate molecules as complementary and alternative approaches to biomedicine [10, 11]. Of course, we cannot forget the influence of Darwin that surely left its mark on the 19th century and beyond. In fact, broadly interpreted, Darwin led us into the field and Metchnikoff into the laboratory at least with respect to comparative immunology [12, 13]. Evolutionary immunology reaped the benefits of Metchnikoff and modern immunology advanced conceptually when the clonal selection theory of Burnet was advanced – in essence a Darwinian corollary [12, 14]. According to Burnet (1962), ‘The clonal-selection theory is a generalization about a wide range of biological phenomena and suffers from the inherent weakness of all biological generalizations. It can be only a partial picture of reality and its function is purely provisional one of making it easier to think effectively about a complex mass of observational and experimental data. The essence of the clonal-selection theory is that immunity and antibody production are functions of clones of mesenchymal cells. Each clone is characterized by the ability of its component cells to react immunologically with a very small number of antigenic determinants. The number may be two in all instances, but one, three, or four are also conceivable. A cell is immunologically competent because it carries on its surface a receptor – a pattern analogous to antibody and perhaps legitimately pictured as cell-bound antibody – which allows it to react with a given antigenic determinant. It is immaterial for the moment whether it has any other types of receptor. Contact with the right antigenic configuration acts as a trigger to action and it is the essence of a clonal theory that such stimulation plays a major part in determining the observed changes in type and numbers of the mesenchymal cells of the body. The trigger of immunological contact is believed to provoke actions which, depending on many associated factors, may take one or other several forms. The cells may be killed or damaged, with release of cell-damaging or stimulating products; they may be stimulated to proliferate, with or without change of morphological type; or they may be converted to the plasma-cell form, with its capacity for active synthesis and liberation of antibody. Which particular reaction

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ensures will depend essentially on the physiological state of the cell and the nature of the internal environment to which it is exposed after stimulation.’ [15]. Then came the network theory and the opening up of immunologists to the pervasive extension of the immune system throughout the body of organisms. Additional components and functions existed so that indeed the immune system was broader than plaque-forming cells in the spleen, one of the first assay systems that led us to further breakthroughs in immunology. As a natural consequence to the clonal selection theory, immunologists needed a simple model that would clarify the point that cells of the immune system were responsible for elaborating a response that is specific for a specific antigen. Organs of the vertebrate immune system such as the spleen were chosen and isolated from animals that had been immunized in early experiments with antigen (e.g. sheep erythrocytes). Very cleverly spleens were dissociated and lymphocytes were isolated and purified and then mixed with the original sheep erythrocytes in addition to complement a lytic component found in serum. Applying the technique developed by microbiologists, antigen-primed lymphocytes would then lyse surrounding erythrocytes forming clear plaques around an antibody-secreting lymphocyte. Early electron microscopy revealed antibody-secreting B cells in the center of the plaques, and thus ushered in analytical micro-methods for evaluating and pinpointing single immune cell clones.

Innate Immunity and Adaptive Immunity

We therefore have a more firm handle on the finely tuned mechanics of the immune system. This allows a more expansive view of the totality of immune responses. Thus, we envision the innate found throughout the animal kingdom that functions exclusively in invertebrates. But there is an extended capacity to collaborate with the adaptive system of vertebrates. A more expanded classification can now include for all animals: innate, natural, nonspecific (no memory) nonanticipatory, nonclonal, germline (hard-wired host defense functions); for vertebrates: adaptive, induced, specific (memory), anticipatory, clonal, somatic (flexible host defense functions) [13]. With the advent of multicellularity, clearly there are other traits of the immune response that can be examined and presented but not possible in unicellular forms or even those that straddle the divide between unicellularity and multicellularity, beginning with colonial/social protozoans, then true multicellular forms, the sponges and then the cnidarians. It seems that this classification is appropriate from all existing evidence, but it lacks flexibility that could account for exceptions to these rather rigid definitions which for now seem to be dissolving (blurring) as we examine more fully the nature of definitions and outcomes once immune events have been set in motion. Nevertheless, it is instructive to elucidating a hierarchy of animals based upon immunologic characteristics and how they parallel other traits that influence current taxonomy. In this sketch, evidence is presented that the most primitive of invertebrates

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prior to the evolution of multicellular organisms possess varying degrees of complexity at the molecular level of those hallmarks that now characterize the immune system. The level of complexity parallels that which supports classification at a particular level using other functional criteria. The choice of focusing on the basics of signaling and other molecular events provides a measure of unity and continuity. Why this assertion? Clearly at these levels, for example, there is no consideration of vast areas of cellular and humoral immunity that include such well known functions as graft rejection or the cells that provoke incompatibility or the capacity to synthesize and secrete antibodies or immunoglobulins. Still, the capacity of self non-self recognition has been reported [16] and in many respects anticipated the molecular information reported here (tables 1, 2). Before we became so molecular in approaches to the immune system’s minute components, researchers were already doing some of the classical experiments of the times that sought to reveal elements of immunity. In a sense, the molecular factors revealed today are merely confirming what had been previously found at the more gross organismic level. We are experiencing a molecular taxonomy as applied to the immune system. Let us see if the two approaches remain ever in synchrony.

Origins of Immune System Components

Unicellular Colonial Protozoans Relevant to immunity and clearly a central question in animal evolution is how multicellular animals evolved from a protozoan ancestor [17]. This question includes all known functions and of essence for this discussion, the immune system. To decipher this question, one approach to origin of animals is to determine which developmental proteins predated them and were subsequently co-opted for their development. Another strategy involves comparative genomics that can identify the minimal set of intact genes from the beginning of animal evolution that reveals those shared by all animals and their nearest relatives. Resolving the mystery of origins, these workers have sampled gene diversity expressed by choanoflagellates, unicellular and colonial protozoa that are closely related to metazoa, crucial for providing a possible clue into early animal evolution. Results revealed that choanoflagellates express representatives of a surprising number of cell-signaling and adhesion protein families not previously isolated from nonmetazoans; these include cadherins, C-type lectins, several tyrosine kinases and tyrosine kinase signaling pathway components. Choanoflagellates have a complex and dynamic tyrosine phosphoprotein profile, and tyrosine kinase inhibitors selectively affect cell proliferation. The expression in choanoflagellates of proteins involved in cell interaction in metazoa demonstrates that these proteins evolved before the origin of animals and were later co-opted for development. A similar situation exists with respect to components of the signaling system with respect to immunity and development.

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Emergence of Multicellularity: Social Amoeba To approach evidence of immune-associated characteristics, a recent exciting study has just been published. According to Chen et al. [18], social amoebae feed on bacteria in the soil but aggregate when starved to form a migrating slug. They discovered an unknown cell type in social amoeba that is apparently involved in detoxification and immune-like functions; they call it the sentinel (S) cells. S cells engulf bacteria and sequester toxins while circulating within the slug, eventually being sloughed off. A Toll/interleukin-1 receptor (TIR) domain protein, TirA, is also required for certain S cell functions and for vegetative amoebae to feed on live bacteria. This apparent innate immune function in social amoebae, and the use of TirA for bacterial feeding, suggests an ancient cellular foraging mechanism that may have been adapted to defense functions well before the diversification of the animals. Multicellularity likely increased the selective pressure on an organism’s ability to avoid exploitation by pathogens. The role of TirA in Dictyostelium’s response to bacteria provides the first glimpse of an immune-related signaling system in amoeba and suggests that the use of TIR domainbased signaling for defense represents an ancient function present in the progenitor of all crown group eukaryotes. If true, it would suggest that this system of pathogen recognition was advantageous to organisms before the evolution of multicellularity. Sponges During evolution and emergence of multicellular animals, the need arose to defend the animal’s body against foreign organisms that threaten its homeostasis. TLRs play an essential role in mediating the innate immune response among many different receptors that participate in the recognition of microbial invaders. After binding distinct microbial components, TLRs activate intracellular signaling cascades resulting in induced expression of diverse antimicrobial molecules. Sponges (phylum Porifera) are filter feeders, therefore they are extremely exposed to microorganisms that represent a potential threat. Examining sponges, therefore moving to a higher taxonomic level, Wiens et al. [19] have identified, cloned and deduced the protein sequence from 3 major elements of the poriferan innate response (to bacterial lipopeptides according to these definitions): the TLR, the interleukin-1 (IL-1) receptor-associated kinase-4-like protein (IRAK-4l), and a novel effector caspase from the demosponge Suberites domuncula [19]. Each molecule shares significant sequence similarity with its homologues in higher metazoa. There were sequence homologies especially within the family-specific domains Toll/IL-1 receptor/resistance (TLR family), Ser/Thr/Tyr kinase domain (IRAK family), and CASc (caspase family). In situ hybridization (ISH) and immunohistological analyses revealed an abundance of SDTLR (TLR) transcripts in epithelial layers of the sponge surface (exopinacoderm and endopinacoderm). ISH is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e. probe) to localize specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough in the entire tissue (whole amount ISH). This is distinct from immunohistochemistry,

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which localizes proteins in tissue sections. DNA ISH can be used to determine the structure of chromosomes. Fluorescent DNA ISH (FISH) can, for example, be used in medical diagnostics to assess chromosomal integrity. RNA ISH (hybridization histochemistry) is used to measure and localize mRNAs and other transcripts within tissue sections or whole mounts. Both SDTLR and SDIRAK-4 like (IRAK) are expressed constitutively, regardless of treatment with synthetic triacyl lipopeptide Pam(3)CysSer-(Lys)(4). By contrast, SDCASL (caspase) expression is highly Pam(3)Cys-Ser(Lys)(4) inducible. Blocking the lipopeptide with recombinant TLR prior to its application completely prevented induced expression of this sponge caspase. Their conclusion suggests that the phylogenetically oldest extant metazoan phylum already possessed signaling pathways of known metazoan antimicrobial immune systems.

Hydra and Corals According to Miller et al. [20], characterization of the innate immune repertoire of extant cnidarians is of both fundamental and applied interest – it not only provides insights into basic immunological components of the common ancestor of all animals, but is also likely to be important in understanding the current global decline of coral reefs. Recently, whole genome sequences became available for two cnidarians, Hydra magnipapillata and Nematostella vectensis, and large expressed sequence tag datasets are available for them and for the coral Acropora millepora. To better understand the basis of cnidarian innate immunity, they scanned available expressed sequence tag and genomic resources for certain key components of the vertebrate innate immune repertoire, focusing on the Toll/TLR and complement pathways. A canonical Toll/TLR pathway in representatives of cnidarians of the class Anthozoa was observed. Neither a classic Toll/TLR receptor nor a conventional nuclear factor-␬␤ was identified in Hydra – an anthozoan. The detection of complement C3 and several membrane attack complex/perforin domain (MAC/PF) proteins suggests that a prototypic complement effector pathway may exist in anthozoans, but not in hydrozoans. Together with information for several other gene families, they suggest that Hydra may have undergone substantial secondary gene loss during evolution. Such patterns of gene distribution may underscore the possible significance of gene loss during animal evolution but at the same time indicate ancient origins for components of the vertebrate innate immune system.

Toll-Like Receptors: Innate Sensing

The survival of multicellular organisms depends upon their ability to recognize invading microbial pathogens and to induce numerous immune reactions [21]. Recent evidence suggests that an evolutionally ancient family of TLRs plays a crucial

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role in detecting microbial infection and inducing immune and inflammatory responses. With respect to their work on social amoeba, Chen et al. [18] review the earliest work in relation to current views. Phagocytes that engulf bacteria form part of the innate immune system of animals in the defense against pathogens. Both plants and animals also use innate signaling pathways as a means of sensing microbial pathogens; mainly through TLRs in animals and resistance (R) proteins in plants. Both TLRs and R proteins bind to bacterial elicitors through leucine-rich repeats and signal through adaptor/effector proteins such as those containing TIR domains; this, in turn, initiates the transcriptional programs that mediate specific defense responses. According to Beutler et al. [22], in humans innate immune sensing usually proceeds through the activation of 10 TLRs, and these in turn lead to the production of cytokine mediators that create the inflammatory milieu and collaborate in developing an adaptive immune response. Each TLR senses a different molecular component of microbes that have invaded the host. TLR4 senses bacterial endotoxins (lipopolysaccharide), TLR9 unmethylated DNA, and TLR3 double-stranded RNA. Each receptor has a conserved signaling element called the TIR (Toll/IL-1 receptor/resistance) motif that transduces a signal through five cytoplasmic adapter proteins, each of which has a homologous motif. With respect to TLRs, the integration of signals that receptors emit is a crucial mechanism that requires resolution. By creating random germline mutations in mice and screening for individuals with differences in signaling potential, the complex biochemical circuitry of the innate immune response can be unraveled. Up to now, more than 35,000 germline mutants have been produced, and approximately 20,000 have been screened to predict innate immunodeficiency states [22]. Now that the yardstick of vertebrates particularly humans has been presented, it is worth taking a look beginning with simpler organisms using TLR as the central focus.

Innate and Adaptive Immunity: Is Innate Immunity the Hero?

From a more personal observation, it is not clear what caused the evolution of the adaptive system as a survival strategy because the long-lived invertebrates – no doubt, extant relatives of extinct species – with a strong innate system have successfully survived millions of years without the overly bureaucratic adaptive systems of vertebrates, especially mammals. Additionally, vulnerability to cancer, once thought to be a syndrome of vertebrates, probably has relevant precursors in certain invertebrates. Caution should be exerted when trying to adhere to these rigid characteristics since there is evidence of sharing of components such as signal and mediator molecules. A biologically unifying view might predict this a priori because of the ubiquity of DNA and the shared homologies of certain molecules such as hemoglobin (e.g. earthworms and humans). Therefore, why should the cells, tissues, organs and the molecules that they synthesize and secrete not bear striking resemblances to each other when the

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immune system is the functional characteristic being scrutinized? Thus, it appears that the adaptive response requires components of the innate system in order to be activated. However, the reverse seems not to be true, i.e. the innate system can be activated completely without any help from an adaptive system.

Was the Evolution of the Adaptive Immune System Necessary? A Final Comment

The immune system, when viewed separately, may have evolved according to the theory of immunologic surveillance, to ensure the capacity to recognize self from nonself, a concept adhered to by most immunologists. In essence, this view assumes that the immune system evolved to evict internal threats such as cancer. It is canonical – except for the controversial view, the danger hypothesis that assumes that immune responses are set into motion as a result of perceiving danger. The danger hypothesis offers an alternative to the self non-self hypothesis associated with surveillance [23]. Having just suggested for consideration the real need for an innate system for its unique role in invertebrates and its necessity to activate the vertebrate adaptive system, we arrive at a first conclusion that survival of animal species is strongly dependent upon the presence of an innate system. This then leaves us open to question the utility of the adaptive system. One idea proposes that the adaptive system evolved as a backup, a fail safe strategy in case pathogens overwhelm the innate system. A second proposal suggests that the adaptive system evolved as a surveillance system against the development of cancer. To this generality we can add certain exception since cancer has been observed in certain invertebrates (Drosophilia; certain mollusks from polluted marine waters) known to possess only innate immunity, whereas cancers are not known to exist in certain vertebrates, that possess the adaptive system. Could it be that these two generalities should be challenged? That is, certain molecular markers for cancer should be searched among invertebrates and vertebrates only to perhaps reveal clues, as has recently been the revelation of TLR [24, 25].

Conclusions and Perspectives Concerning Strides in Immunology

Immunology like other disciplines had made advances, some of which were slow whereas others might be considered as great leaps. And Herculean results and conclusions and generalities that produced hypotheses seemed to have been the result of interdisciplinary approaches and a consideration for the advantages of requiring diversity. Zoology benefited immunology with the discovery of ubiquitous phagocytosis in marine animals and applying that observation to all species. The resulting spin-off: cellular/humoral lead to the T and B concept. Thus, we benefited from an understanding of adaptive versus innate and the need for both depending on the species. Despite the lack of precise anatomical congruence with respect to T and B systems of birds and mammals, the fact remains that

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without the discovery of the avian bursa of Fabricius, the B compartment of mammals and other vertebrates may have been slow to untangle. These examples argue strongly for a vigilant approach: comparative and diverse as we ponder immunological questions.

References 1 Tauber AI, Chernyak L: Metchnikoff and the Origins of Immunology-From Metaphor to Theory. New York, Oxford University Press, 1991. 2 Cooper EL: Invertebrate Immunology; in Cooper EL (ed): Contemporary Topics in Immunology. New York, Plenum Press, 1974, vol 4, p 299. 3 Cooper EL: Comparative Immunology. Englewood Cliffs, N.J., Prentice–Hall, 1976. (Translated into Russian, 1980). 4 Cooper EL: Comparative Immunology. Englewood Cliffs, N.J., Prentice–Hall, 1976. 5 Humphreys T, Reinherz EL: Invertebrate immune recognition, natural immunity and the evolution of positive selection. Immunology Today 1994;15:316–320. 6 Cooper EL, Rinkevich B, Uhlenbruck G, Valembois P: Invertebrate immunity: another viewpoint. Scand J Immunol 1992;35:247–266. 7 Beck G, Cooper EL, Habicht GS, Marchalonis JJ (eds): Primordial Immunity, Foundations for the Vertebrate Immune System. New York, New York Academy of Sciences, 1994. 8 Silverstein AM: A History of Immunology. New York, New York Academic Press, 1989. 9 Janeway Jr CA: The immune system evolved to discriminate infectious non-self from non-infectious self. Immunology Today 1992;13:11–16. 10 Cooper EL: Bioprospecting: a CAM frontier. Evid Based Complement Alternat Med 2005;2:1–3. 11 Muller WEG, Schroder HC, Wiens M, PerovicOttstadt S, Batel R, Muller IM: Traditional and Modern Biomedical Prospecting: Part II—the Benefits: Approaches for a Sustainable Exploitation of Biodiversity (Secondary Metabolites and Biomaterials from Sponges). Evid Based Complement Alternat Med 2004;1:133–144. 12 Cooper EL: Did Darwinism help comparative immunology? Am Zool 1982;22:890. 13 Cooper EL, Kauschke E, Cossarizza A: Digging for innate immunity since Darwin and Metchnikoff. Bioessays 2002;24:319–333.

14 Burnet FM: The clonal selection theory of immunity. Nashville Vanderbilt and Cambridge University Presses, 1959. 15 Burnet FM: The integrity of the body. Cambridge Harvard University Press, 1962. 16 Marchalonis JJ, Schluter SF: On the relevance of invertebrate recognition and defense mechanisms to the emergence of the immune response of vertebrates. Scand J Immunol 1990;32:13–20. 17 King N, Hittinger CT, Carroll SB: Evolutioin of key cell signaling and adhesion protein families predates animal origins. Science 2003;301:361–363. 18 Chen G, Zhuchenko O, Kuspa A: Immune-like phagocyte activity in the social amoeba. Science 2007; 317:678–681. 19 Wiens M, Korzhev M, Perovic-Ottstadt S, Luthringer B, Brandt D, Klein S, Müller WE: Toll-like receptors are part of the innate immune defense system of sponges (demospongiae: Porifera). Mol Biol Evol 2007;24:792–804. 20 Miller DJ, Hemmrich G, Ball EE, Hayward DC, Khalturin K, Funayama N, Agata K, Bosch TC: The innate immune repertoire in cnidaria—ancestral complexity and stochastic gene loss. Genome Biol 2007;8:1–13. 21 Medzhitov R, Janeway Jr C: The toll receptor family and microbial recognition. Trends Microbiol 2000; 10:452–456. 22 Beutler B, Hoebe K, Du X, Ulevitch RJ: How we detect microbes and respond to them: the Toll-like receptors and their transducers. J Leukoc Biol 2003; 74:479–485. 23 Matzinger P: The danger model: a renewed sense of self. Science 2002;296:301–305. 24 Pancer Z, Cooper EL, Muller WEG: A tunicate (Botryllus schlosseri) cDNA reveals similarity to vertebrate antigen receptors. Immunogenetics 1996;45:69–72. 25 Pancer Z, Cooper EL, Muller WEG: A urochordate putative homolog of human EB1, the protein which binds APC. Canc Lett 1996;109:155–160.

Edwin L. Cooper, PhD, ScD Laboratory of Comparative Neuroimmunology, Department of Neurobiology David Geffen School of Medicine at UCLA, University of California, Los Angeles Los Angeles, CA 90095-1763 (USA) Tel. ⫹1 310 825 9567, Fax ⫹1 310 825 2224, E-Mail [email protected]

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Egesten A, Schmidt A, Herwald H (eds): Trends in Innate Immunity. Contrib Microbiol. Basel, Karger, 2008, vol 15, pp 12–20

General Introduction to Innate Immunity: Dr. Jekyl/Mr. Hyde Quality of the Innate Immune System Kurt S. Zänker Institute of Immunology, University Witten/Herdecke, Witten, Germany

Abstract The innate immunity plays a critical role in host protection against pathogens and transformed cells. It relies amongst others on pattern recognition receptors such as Toll-like receptors, C-type lectin receptors, and nucleotide-binding oligomerization domain proteins to alert and activate defense pathways including the activation of the complement system. Innate immunity represents a trait common to plants and animals, and besides the humoral factors different cell types e.g. subspecies of dendritic cells (plasmacytoid dendritic cells), phagocytic cells, mast cells, glia cells, Kupffer cells, neutrophils and natural killer cells are involved to orchestrate the anti-infectious and antitumor response. Studies in plants, in fruit flies and in mammals reveal that the defensive strategies of invertebrates and vertebrates are highly conserved at the molecular level, which raises the exciting prospects of an increased understanding of innate immunity in a healthy or diseased organism. However, the molecular machinery, e.g. cytokines and chemokines, which triggers, amplifies, and sustains the different phases of the innate immune response could also promote a substantial imbalance between danger and inflammatory response when an infectious challenge is either chronic Copyright © 2008 S. Karger AG, Basel or not properly declining.

The Dr. Jekyl/Mr. Hyde Syndrome of Host Defense: With Complements from Innate Immunity

The devilish duo symbolizes the dangerous nature of immunity. On one hand are the possible benefits it brings to the individuum, and on the other hand, an even darker alternative. Traditionally, and nowadays more for didactical reasons, the immune system is divided into humoral and cellular immunity. However, recent advances in immunology suggest that this division may be overly simplistic when only regarding the conventional molecular and cellular hallmarks of each system. The orchestrated and interactive biological activities of these two entities are in the main focus of

research to understand immunity from molecular biology to clinical immunology, from health to diseases. The humoral and the cell-mediated immunity can also be described under evolutionary, phylogenetic and developmental perspectives and divided into an innate and an adaptive immune competence. The innate immune response is nonanticipatory, nonclonal and less specific; the adaptive immunity is induced, highly specific, anticipatory and clonal. Already flies (Drosophila) respond to infections with both humoral, activated via the imd/Relish pathway, and cellular immune defenses, including antimicrobial peptides as well as actively proliferating and differentiating blood cells, triggered via the Toll/Dif pathway [1]. Three classes of circulating cellular immune surveillance cells can be identified in Drosophila melanogaster larvae: plasmatocytes, crystal cells, and lamellocytes. Plasmatocytes are professional phagocytes most similar to the mammalian monocyte/macrophage lineage. Crystal cells, which secrete components necessary for the melanization of invading organism are a minor part of circulating hemocytes together with lamellocytes, which are involved in the encapsulation of invading pathogens. There are no mammalian counterparts for crystal cells or lamellocytes. However, major features of innate immunity appeared to be conserved between invertebrates and vertebrates [2]. Once a plan of defense mechanism was successful, it was found again in principle in mammalians. Certain large parasites, e.g. the tubercle bacillus and the bacillus of leprosy, are encapsulated by the so called process of granulomatous inflammation, a form of chronic inflammation with a distinctive histologic appearance in mammalians. The lesion of tuberculosis, the tubercle, is characteristic of this type, being composed of epithelioid cells, lymphocytes and Langhans’ giant cells. The innate immunity gains increasingly renewed interest, particularly as it became apparent that it is an evolutionary ancient defense mechanism. Interestingly, each newly evolved defense mechanism does not replace the previous one, but supplements it, resulting in a layered structure of the immune system. Therefore, the immune system is not a static one-level system but rather a sophisticated network of various defense mechanisms operating on different levels, ranging from mechanisms common for every cell in the body to specialized immune cells [3, 4] and responses at the level of the whole organism [5].

Innate Immunity

The immune system provides protection from a wide range of pathogens. One immune entity, the phylogenetically ancient innate immune response, attacks infectious DNA/RNA carriers from the moment of first contact and is the fundamental defensive weapon of multicellular organisms. Innate immunity receptors are germline-encoded receptors that can sense molecular signatures of pathogens [6] and cancer cells [7]. Comparative studies in fruit flies and in mammalians reveal that the

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defensive strategies of invertebrates are not only at a cellular level, likewise and compared to the tubercle granulome, but also at the molecular level in vertebrates conserved. This raises the exciting prospects of an increased understanding of and thereafter therapeutically manipulating the innate immune response of mammalians [8–10]. Invading pathogens are recognized by diverse germline-encoded pattern recognition receptors (PRRs) which are distributed in three different cellular compartments: extracellular, membrane, and cytoplasma. In mammalians, the first extracellular defense line comprises the complement cascade, which opsonizes pathogens for clearance by phagocytosis which is mediated by membrane-associated phagocytic receptors, including complement receptors. The major membrane-associated PRRs, Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) recognize diverse pathogens and generate inflammation signals to coordinate innate immune response and shape adaptive immune response assisted by microvascular endothelial cells [11]. When this more forefront detection system fails, cytoplasmic PRRs may play an important role. Cytoplasmic caspase recruitment domain helicases such as melanoma differentiation-associated gene 5 mediate antiviral immunity by triggering the production of type I interferon [12]. Certain members of nucleotide-binding oligomerization domain (NOD)-like receptors such as NALP3 present in the cytosol form inflammosomes to induce inflammatory responses upon ligand recognition.

Toll-Like Receptors, C-Type Lectin Receptors and CpG Oligodeoxynucleotide Motifs

The inflammatory response to microbes is largely initiated by a single class of membrane-spanning not catalytic receptors, named for their similarity to the prototypic Toll receptor of Drosophila. The mammalian TLRs are key players in the innate immunity and are ultimately responsible for most phenomena associated with infections. TLRs are a type of PRRs and recognize molecules that are broadly shared by microbes. Molecular domains of TLRs are represented in bacteria and plants, and in the latter kingdom [13, 14]. They are well known to be required for host defenses against infection, even for the defense against cancer cells. This includes both ‘good’ responses to infections (shaping and balancing cell-mediated immunity with induction of lasting specific immunity) and ‘bad’ effects (systemic and chronic inflammation and shock). Thirteen TLRs (TLR1–13) have been identified in humans and mice and their equivalent forms have been found in other mammalian species. The TLRs transduce the toxicity of lipopolysaccharides, lipoproteins, double-stranded RNA, unmethylated CpG islands of bacterial and viral DNA [15]. The TLRs recognize molecules that are consistently associated with threats and therefore evolutionarily highly conserved. The TLRs appear to be one of the most ancient conserved compounds of the immune system. They impact on

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adaptive immune reactions and contribute to the initiation and maintenance of the inflammatory response to a multitude of potential microbial pathogens through recognition of pathogen-associated molecular patterns. TLRs also interact with a variety of endogenous human ligands and influence the activity of a wide range of tissues and cell processes. Among the common and important pathological processes in which TLRs play a role are asthma, acute respiratory distress syndrome, cardiac ischemia, coronary heart disease, inflammatory bowel disease, psoriasis, rheumatoid arthritis, diabetes, cancer and transplant rejection [16–18]. Their role as potential targets for therapeutic intervention is just beginning to be appreciated. It has been clear for many years that carbohydrate structures are not merely structural components of the molecules which bear them but are a source of information to be decoded by biological systems including the immune system [19]. A family of proteins known as the C-type lectins are implicated as the main players in carbohydrate recognition with the immune system [20]. Human CLRs characteristically bind glycosylated ligands in a calcium-dependent way via their carbohydrate recognition domain (CRD). Their carbohydrate preference is dependent on the amino acid sequence in the CRD domain and on the ability and flexibility of the CRD domain to accommodate sugar moieties that are located at different distances from each other on the glycoconjugate. Although microbial and vertebrate cells are able to produce similar polysaccharide chains, the density of carbohydrates present on both cell types can be recognized by the CLRs. This usually results in internalization of the pathogen, lysosomal degradation and subsequent loading of pathogen-derived peptides into major histocompatibility complex molecules for antigen presention. CLRs are predominantly expressed by antigen-presenting cells such as dendritic cells (DCs) [21]. DCs have a unique character of immunoregulation. DC-SIGN, a molecule designated as CD209, is one member of the C-type lectin superfamily. It is not only a pattern recognition receptor but implicated in immunoregulation of DCs [22]. DC-SIGN is in the focus of recent studies because of its important role in mediating DC adhesion, migration, inflammation, activating primary T cell, triggering immune response and participating in immune escape of pathogens and tumors [23, 24]. The C-type lectin DC-SIGN is known to be the major M. tuberculosis receptor on human DCs [25]. CD209 plays also a crucial role in dengue pathogenesis, which discriminates between severe dengue fever and dengue hemorrhagic fever [26]. In recent years, a growing number of CLRs have been identified on myeloid cells both of human and mouse origin [27]. The CLRs appear to have a far more diverse range of functions and ligands, including those of exogenous origin. Some CLRs possess activating/inhibitory signaling motifs that trigger downstream signaling events, suggesting the role for these receptors as positive/negative regulators of granulocyte and monocyte functions. DCs express CLRs for antigen capture and presentation, whereas TLRs are involved in pathogen recognition and DC activation. Recent data demonstrate that communication between TLRs and CLRs can affect the direction of

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immune response. Several pathogens specifically target CLRs to subvert this communication to escape immune surveillance either by inducing tolerance or skewing the protective immune response [24].

Cells, Signaling and Molecular Components in the Orchestration of Innate Host Defense

DCs and the complement cascade proteins are essential components of innate immunity; both participate in local inflammation. Recent studies have demonstrated the ability of DCs to synthesize C1q, C3, factor I, factor B and complement receptors 3 and 4 and are a source of soluble C4b binding protein, C7 and C8 [28]. Nuclear macromolecules can translocate into the extracellular space where they can activate the innate immunity [29]. Of nuclear molecules, DNA and the DNA-binding protein High Mobility Group Box 1 (HMGB1), display distinct patterns of immune activation. For DNA, immune activity depends on sequence, base methylation/acetylation and structural context. While bacterial DNA is an immune activator, mammalian DNA is either inert or inhibitory when free. In contrast, mammalian DNA can trigger innate immune cell activation (NK cells, NKT cells, phagocytes); HMGB1 is an abundant chromatin protein that acts as a cytokine when released in the extracellular milieu by necrotic and inflammatory cells. Extracellular HMGB1 and its receptor for advanced glycation end products (RAGE) induce both migration and proliferation of vessel-associated stem cells (mesangioblasts) and thus may play an important role in tumor angiogenesis, and in diabetic patients suffering from complications as a result of insufficiently controlled disease. Central to innate immunity is the sensing of pathogen-associated molecular patterns. DNA is in particular a potent activator of innate immune response during infection and tissue damage. In addition to the membrane-associated TLR9, it has been recently described that a Z-DNA-binding protein (Zipcode-binding protein 1) can trigger the induction of type 1 interferon (IFN) and other immune responses; the authors proposed an alternative name DAI (DNA-dependent activator of IFN-regulatory factors) [30].

The Important Role of Neutrophils in Innate Immunity

Neutrophils play a central role in host defense against infectious microbial pathogens (parasites, bacteria and virus) and tumor cells. Neutrophils patrol through the body in order to detect pathogens which release chemoattractant molecules and upon recruitment of neutrophils at the site of inflammation stimulate either phagocytosis or the segregation of neutrophil serine proteases as elastase, cathepsin G, and proteinase3 killing invading microbes directly. Migration and phagocytosis of neutrophils are important cellular features of these immune competent cells. Coronins, a family of

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conserved actin-associated proteins, are expressed (Coronins 1–4 and 7) and coronin-1 accumulated at the leading edge of migrating neutrophils as well as at the nascent phagosome [31]. Inhibition of coronin function leads to inhibition of chemotaxis and phagocytosis and to a reduction of neutrophil spreading and adhesion. Microbial pathogens can often be not detected within the body by neutrophils because of a molecular impairment of the migratory and/or chemotactic machinery. The intracellular mechanisms of cell-structuring molecules, executing migration have been reviewed in comparison to tumor cells [32]. The molecular basis for triggering signals to activate neutrophils are still incompletely understood. Recently, it was shown that myeloid-associated immunoglobulinlike receptor (MAIR)-IV is preferentially expressed on neutrophils in the peripheral blood, bone marrow, peritoneal cavity and spleen. MAIR-IV is physically associated with immunoreceptor tyrosine-based activating motif-bearing adaptor protein FcR␥ chain. Cross-linking of MAIR-IV significantly induced secretion of proinflammatory cytokines as TNF-␣ and IL-6 from neutrophils [33]. Neutrophils rest between two major bursts of transcriptional and protein synthetic activities. The first occurs in the bone marrow. This equips the neutrophils with stocks of innate defense armory that are packed into different granule subsets. The second burst occurs when the neutrophils exit circulation and migrate into tissues to find, capture and phagocytose microorganisms. This burst results in the synthesis and secretion of cytokines and chemokines that support resolution of inflammation and healing of damaged tissue [34]. On the other hand, neutrophils express a variety of innate immunity proteins, including heat shock protein HSP90 [35], which can form with proteins of the mammalian NOD-like receptor family a multiprotein proinflammatory complex, called the ‘inflammasome’. It has also become clear by gene expression profiling that some innate immune proteins previously thought to be specific to the neutrophils are expressed in epithelial cells during inflammation, too. Surprisingly, even cells, which have been known for years to have potent effector functions not related to innate immunity, have been identified recently to be involved in a complex range of innate immune functions, e.g. mast cells [36] and astrocytes [37]. Astrocytes display an array of receptors characterizing innate immunity, such as TLRs, nucleotide-binding oligomerization domains, double-stranded RNA-dependent protein kinase, scavenger receptors, mannose receptor and components of the complement system. Following activation, astrocytes are endowed with the ability to secrete soluble chemokines and cytokines like CXCL10, CCL2, IL-6 and B cell activating factors belonging to the TNF-family [37] which have an impact on both innate and adaptive immune response. Interestingly, platelets recognize antigens directly via TLRs, which suggests that there are mechanistic relations between infectious inflammation and atherosclerotic vascular diseases [38]. When hepatocellular carcinoma cells were implanted into normal and cirrhotic rat livers, they grew more progressively in cirrhotic livers than in normal liver. Kupffer cells were decreased profoundly in cirrhotic livers, resulting in markedly impaired phagocytic activity [39].

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Dr. Jekyl/Mr. Hyde Quality of the Innate Immune System

Compelling experimental data and clinical studies implicate adaptive and/or innate cells, either as single entities or within a complex and complicated immunological network, as critical regulators within the healthy or diseased human body. Inflammation is a lifelong companion, because inflammation is a key element of the immune system. However, inflammation has many facets, which is best exemplified when associated with neoplastic programming of tissues. Full and specific activation of adaptive immune cells may result in eradication of malignant cells; chronic or imbalanced activation of innate immune cells at the site of premalignant lesion or during earlier stages of tumor development may actually facilitate tumor progression and metastases formation. Cross-talk positioning between tumor cells and cells of the innate immune system may even lead to the phenomenon of cell cannibalism, which generally refers to the engulfment of cells within other cells. It is well established that, for example, metastatic human melanoma cells eat live T cells, which are instead programmed to kill them [40]. From early on in evolution, organisms have had to protect themselves from pathogens. Evolution of mechanisms of immune defense has resulted in a variety of strategies. Unicellular organisms have a rich and in part highly conserved arsenal of mechanisms for protection, such as restriction endonucleases, antimicrobial peptides, phagocytosis and RNA interference [41]. In addition to that, in multicellular organisms specialized immune cells have evolved, capable of recognition and killing of foreign cells as well as removing their own cells changed by damage, senescence, infection or cancer. Additionally, many cross-talking molecules within the body, such as the complement cascade, cytokines and chemokines, evolved to co-operate with cellular immunity. Each phylogenetically new defense mechanism does not replace an evolutionary older one, but supplements it, resulting in a layered structure – from innate responses to adaptive changes – of the immune system. Upon activation, a well-balanced immune response involving initiation and termination of the evolutionary differentially evolved immune entities takes place in fighting against infection and cancer and maintaining homeostasis [42]. In the future, research will be focused on innate immunity-associated molecules and cells which are linked to adaptive immunity in order to understand better nature’s evolutionary essential of the functional dichotomy of innate and adaptive immunity in mammalians.

Acknowledgement Kurt S. Zänker is supported by the Fritz-Bender Foundation, Munich, Germany.

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20 McGreal EP, Martinez-Pomares L, Gordon S: Divergent roles for C-type lectins expressed by cells of the innate immune system. Mol Immunol 2004;41: 1109–1121. 21 Gijzen K, Gambi A, Torensma R, Figdor CG: C-type lectins on dendritic cells and their interaction with pathogen-derived and endogenous glycoconjugates. Curr Protein Pept Sci 2006;7:283–294. 22 Zhou T, Chen Y, Hao L, Zhang Y: DC-SIGN and immunoregulation. Cell Mol Immunol 2006;3: 279–283. 23 Bhardwaj N: Harnessing the immune system to treat cancer. J Clin Invest 2007;117:1130–1136. 24 Van Kooyk Y, Engering A, Lekkerkerker AN, Ludwig IS, Geijtenbeek TB: Pathogens use carbohydrates to escape immunity induced by dendritic cells. Curr Opin Immunol 2004;16:488–493. 25 Barreiro LB, Neyrolles O, Babb Cl, Tailleux L, Quach H, McElreavey K, Helden PD, Hoal EG, Gicquel B, Quintana-Murci L: Promoter variation in the DC-SIGN-encoding gene CD209 is associated tuberculosis. PloS Med 2006;3:e20. 26 Sakuntabhai A, Turbpaiboon C, Casademont I, Chuansumrit A, Lowhnoo T, Kajaste-Rudnitski A, Kalayanarooj SM, Tangnararatchakit K, Tangthawornchaikul N, Vasanawathana S, Chaiyaratana W, Yenchitsomanus PT, Suriyaphol P, Avirutnan P, Chokephaibulkit K, Matsuda F, Yoksan S, Jacob Y, Lathrop gM, Malasit P, Despres P, Julier C: A variant in the CD209 promotor is associated with severity of dengue disease. Nat Genet 2005;37:507–513. 27 Pyz E, Marshall AS, Gordon S, Brown GD: C-type lectin-like receptors on myeloid cells. Ann Med 2006;38:242–251. 28 Reis ES, Barbuto JA, Isaac L: Complement components, regulators and receptors are produced by human monocyte-derived dendritic cells. Immunobiology 2007;212:151–157. 29 Pisetsky DS: The role of nuclear macromolecules in innate immunity. Proc Am Thorac Soc 2007;4: 258–262. 30 Takaoka A, Wang Z, Choi MK, Yanai H, Negishi H, Ban T, Lu Y, Miyagishi M, Kondama T, Honda K, Ohba Y, Taniguchi T: DAI (DLM/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature 2007;448:501–505. 31 Yan M, Di Ciano-Oliveira C, Grinstein S, Trimble WS: Coronin function is required for chemotaxis and phagocytosis in human neutrophils. J Immunol 2007;178:5769–5778. 32 Entschladen F, Zänker KS: Locomotion of tumor cells: a molecular comparison to migrating pre- and postmitotic leukocytes. J Cancer Res Clin Oncol 2000;126:671–681.

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33 Nakano T, Tahara-Hanaoka S, Nahahashi C, Can I, Totsuka N, Honda SI, Shibuya K, Shibuya A: Activation of neutrophils by a novel triggering immunoglobulin-like receptor MAIR-IV. Mol Immunol 2008;45:289–294. 34 Borregaard N, Sorensen OE, Theilgaard-Monch K: Neutrophil granules: a library of innate immunity proteins. Trends Immunol 2007;28:340–345. 35 Prohaszka Z: Chaperones as part of the immune networks. Adv Exp Med Biol 2007;594:159–166. 36 Metz M, Maurer M: Mast cells – key effector cells in immune response. Trends Immunol 2007;28: 234–241. 37 Farina C, Aloisi F, Meinl E: Astrocytes are active players in cerebral innate immunity. Trends Immunol 2007;28:138–145.

38 Shiraki R, Inoue N, Kawasaki S, Takei A, Kadotani M, Ohnishi Y, Ejiri J, Kobayashi S, Hirata K, Kawashima S, Yokoyama M: Expression of Toll-like receptors on human platelets. Thromb Res 2004; 113:379–385. 39 Tsujimoto T, Kuriyama S, Yamazaki M, Nakatani Y, Okuda H, Yoshiji H, Fukui H: Augmented hepatocellular carcinoma progression and depressed Kupffer cell activity in rat cirrhotic livers. Int J Oncol 2001;18:41–47. 40 Lugini L, Matarrese P, Tinari A, Lozupone F, Federici C, Iessi E, Gentile M, Luciani F, Parmiani G, Rivoltini L, Malorni W, Fais S: Cannibalism of liver lymphocytes by human metastatic but not primary melanom cells. Cancer Res 2006;66:3629–3638. 41 Sioud M: RNA interference and innate immunity. Adv Drug Deliv Rev 2007;59:153–163. 42 Dittmar T, Zaenker KS, Schmidt A (eds): Infection and Inflammation. Impacts on Oncogenesis. Contrib Microbiol. Basel, Karger, 2006, vol 13.

Kurt S. Zänker, MD, DVM, PhD Institute of Immunology, University Witten/Herdecke Stockumer Strasse 10 DE–58448 Witten (Germany) Tel. ⫹49 2302 926 159, Fax ⫹49 2302 926 158, E-Mail [email protected]

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Egesten A, Schmidt A, Herwald H (eds): Trends in Innate Immunity. Contrib Microbiol. Basel, Karger, 2008, vol 15, pp 21–44

The Innate Immune System of Mammals and Insects Uwe Müllera ⭈ Patric Vogelb ⭈ Gottfried Albera ⭈ Günter A. Schaubb a

Institute of Immunology, College of Veterinary Medicine, University of Leipzig, Leipzig, Group of Zoology/Parasitology, Department of Evolutionary Ecology and Biodiversity of Animals, Ruhr University Bochum, Bochum, Germany

b

Abstract Infectious agents threaten any organism. Therefore, mammals and insects have evolved a complex network of cells and humoral factors termed immune system able to control and eliminate pathogens. Immunity varies between different groups of animals but always contains an innate immune system that can act fast and often effectively against a wide range of distinct pathogens (i.e. viruses, bacteria, fungi, and eukaryotic parasites). In mammals and insects, the communication between and regulation of immune cells is carried out by cytokines which orchestrate the defense against the invaders. The major challenge to recognize and to fight pathogens is the same for any host. In insects and mammals, the pathogens are recognized as non-self by recognition of pathogen-associated molecular patterns. In addition, similar pathogen recognition receptors and signaling pathways activate the immune response in insects and mammals. The pathogens have to be opsonized and/or ingested and controlled/eliminated by antimicrobial peptides or small effector molecules (reactive oxygen and nitrogen intermediates). Interestingly, even invertebrates have evolved certain forms of adaptive immunity, i.e. specific immune priming, and in some invertebrates alternative splicing of pathogen recognition receptors allows for a more specific recognition of a wide variety of pathogens. This enhanced specificity of pattern recognition conveys a special form of memory to their invertebrate hosts. In this chapter, we also consider gut immuCopyright © 2008 S. Karger AG, Basel nity of insects and compare it with the response in mammals.

Cells of the Innate Immune System in Insects and Mammals

Innate Immune Cells of Insects and Their Hematopoietic Origin The arthropods’ innate immune system consists of both cellular and humoral components. The cellular defense mechanisms are mediated by hemocytes which resemble in development and function the cells of the mammalian myeloid lineage [1, 2]. According to the different authors and investigated groups of insects, the names, classification

criteria and thereby types of hemocytes differ. While Drosophila only shows three different types termed plasmatocytes, crystal cells and lamellocytes, other species (e.g. the mosquito Aedes) show a broader variety of cells such as prohemocytes, adipohemocytes, oenocytoids, thrombocytoids, plasmatocytes, and granulocytes. However, it is not clear for each of these cell types that they have immunological functions – some authors therefore distinguish between hemocytes and immunocytes. In the lepidopteran Manduca sexta, immune responses could be detected for plasmatocytes, granulocytes and a cell type called hyperphagocytotic hemocyte. The latter is able to phagocytose large numbers of bacteria and could be involved in nodule formation, an immune reaction which shows similarities with granuloma formation in mammals. The immune system of Manduca can distinguish between foreign invaders and self dead cells. In the uninfected insect, granulocytes are the only cells to engulf apoptotic cells via a C-type lectin pathogen recognition receptor (PRR) pathway, while in the infected animal, plasmatocytes are the major players in phagocytosis of bacteria via the same pathway. Since hematopoiesis has best been studied in Drosophila melanogaster, this species will mainly be considered in the following. Hematopoiesis in Drosophila occurs in two waves, the first wave occurs in the developing embryo and the second in the larvae [3]. The hematopoiesis of the larvae takes place in the lymph glands, in which, however, hematopoietic stem cells have not been identified yet and which possess three functionally distinct parts. The medullary zone of these paired lobes consists of prohemocytes – the precursors of all differentiated hemocytes – whereas three morphologically and functionally distinct cell types – plasmatocytes, crystall cells and lamellocytes [4] – differentiate in the cortical zone and are released upon infection [5]. The posterior signaling center (PSC) region regulates the destiny of the precursors through several transcription factors such as GATA [3] and signaling pathways such as Notch and JAK-STAT. At the end of pupal development, most prohemocytes differentiate into plasmatocytes, the lymph glands disintegrate and release their cell populations into the circulation of the hemolymph [5]. Adults possess no hematopoietic organ and are totally depending on the supply of hemocytes from the larvae or the release of compounds of crystal cells which are not present in adults [2]. The cellular immunity of adult flies is exerted only by the properties of plasmatocytes [1]. Plasmatocytes Plasmatocytes are the dominant population (90–95% in Drosophila) of hemocytes of arthropods [5]. These relatively round cells with a diameter of 8–10 ␮m [4] are morphologically characterized by a big cytoplasm with a high number of lysosomes and an extensive endoplasmatic reticulum [2]. They are adhesive and able to migrate along a chemokine gradient. Plasmatocytes have several functions in development and immunity: They are phagocytic, i.e. they engulf apoptotic particles during development and microorganisms during immune responses. In addition, they produce a wide range of extracellular matrix proteins for tissue remodeling during

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morphogenesis and secrete antimicrobial peptides (AMPs) such as cecropin A1, drosomycin and diptericin in response to infection [2]. Within the communication network of the innate immune system, they are believed to act as messengers that inform the fat body about the presence of infectious agents resulting in a strong AMP response. This signaling requires the cytokine unpaired-3 which activates AMP production through the JAK/STAT pathway [6]. Furthermore, they presumably recognize parasitization and stimulate a massive lamellocyte differentiation through PSC [1]. Crystal Cells Only about 5% of the hemocytes of Drosophila are crystal cells occurring during embryonic and larval development [5]. They are termed crystal cells due to crystalline structures within their cytoplasm and express two of the three prophenoloxidase (proPO) genes in Drosophila [7], indicating that the inclusions contain the compounds of the proPO system, which plays an important role in wound healing and melanization. Cell-free hemolymph of adult Drosophila shows phenoloxidase (PO) activity that is absent in mutants that could not develop crystal cells during maturation [8]. Lamellocytes Lamellocytes only occur in larvae, rarely in the uninfected larvae, but differentiate intensively upon parasitization. Thus, in contrast to plasmatocytes and crystal cells, lamellocytes are inducible. Expression of Col (Collier, the Drosophila orthologue of mammalian early B cell factor) in the PSC region is substantial for allowing the control region to initiate this development [9]. Lamellocytes have a flattened shape, a size up to 20 ␮m and adhesive properties [5] and participate in the cellular encapsulation of foreign bodies that are too huge to be phagocytosed. They express one of the three Drosophila proPO genes and seem to initiate melanization in the absence of crystal cells [10]. Similar to crystal cells, they disappear during metamorphosis [1]. Granulocytes In different insect species besides plasmatocytes another immune-relevant cell type exists, the granulocyte. These cells contain small granules and are circulating hemocytes that are involved in phagocytosis, but to a lower degree than plasmatocytes. The main function seems to be the encapsulation of foreign particles and the removal of own apoptotic cells.

Mammalian Innate Immune Cells In mammals, a broad variety of different cell types exists that support the innate immune response against invading pathogens. Whereas in insects immune cells are

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produced in the fat body or lymph glands, generation of immune cells in adult mammals takes place in the bone marrow. Pluripotent hematopoietic stem cells differentiate into the different myeloid or lymphoid lineages. On the one hand, we can find phagocytic cells such as neutrophils, macrophages, and dendritic cells and on the other hand cells containing granules that can damage pathogens or infected host cells. The most important of the latter are eosinophils, basophils, mast cells and natural killer (NK) cells. In addition, in the mammalian innate immune system there also are primitive lymphocytes with innate immune functions. These are T cells (e.g. a subpopulation of ␥/␦ T cells [11], the intraepithelial ␥/␦ T cells) and B cells (e.g. the B1 cells, producers of natural antibodies) [12]. Neutrophils Neutrophilic granulocytes are the most numerous and most important cellular components of the innate immune response. They circulate in the blood in large numbers and are relatively short-lived. After bacterial infection occurs, these cells leave the blood stream via diapedesis at the infected location and migrate into the tissue following a chemokine gradient. Here they can phagocytose and kill high numbers of bacteria. Macrophages Macrophages belong to the large group of cells collectively called mononuclear phagocytic system. Macrophages can be found in most tissues of the mammalian organism, whereas their progenitors, the monocytes, are circulating cells of the blood. These large cells have different functions. On the one hand they are important for the normal organ function e.g. by their involvement in bone formation and remodeling. On the other hand, they eradicate apoptotic and necrotic cells, and are able to produce important factors of the innate immune system, e.g. complement factors and chemokines. Macrophages directly recognize pathogens and thereafter induce a protective immune response. These cells are professional antigen-presenting cells that interact with T helper cells to induce an appropriate adaptive immune response. They phagocytose pathogens and kill them in phagolysosomes and produce cytokines that induce a cellular immune response by activating NK cells to produce the most important mediator of this response, IFN␥. In addition, macrophages produce factors such as TNF-␣ that is important to fully activate them. As potent effector cells, they are able to produce small effector molecules such as reactive oxygen (ROIs) and nitrogen intermediates (RNIs) which are able to kill bacteria, fungi and protozoan pathogens or control bacterial growth. ROIs and RNIs are microbicidal by inducing oxidative damage to the pathogen and inactivation of essential enzymes, respectively. Macrophages also play an important role in granuloma formation where they engulf large amounts of pathogens and partially adhere to giant cells surrounded by T cells.

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Dendritic Cells Dendritic cells are important sentinel cells and can be found inter alia as immature cells, the so called Langerhans cells, in the skin and the mucosa, the major entrance site for pathogens. Here they can recognize the pathogens via their repertoire of PRRs and opsonic receptors and engulf them. Upon antigen activation, these cells mature and migrate to the draining lymph node to present the foreign antigen to naïve T cells to induce a protective immune response. Their ability to activate naïve T lymphocytes is unique and conveys them a particular role amongst the professional antigenpresenting cells. Their exceptional role in antigen presentation is accompanied by efficient production of cytokines that regulate other immune cells. Natural Killer Cells NK cells are large granular lymphocytes that, in addition to cytotoxic T cells (cells of the adaptive immune system), are very important in controlling viral infections and tumors. They contain a potent repertoire of cytotoxic molecules in their granules, e.g. perforin and granzymes, by which they are able to induce apoptosis in virus-infected or degenerated cells that show abnormal (by degeneration) or missing (induced by virus infection) MHC expression on their surface. In addition, they are important inducers of a cellular immune response and are one of the first cells during an infection that produce IFN-␥ induced by cytokines of macrophages and dendritic cells. Subsequently, IFN-␥ derived from NK cells can activate infected macrophages. NK cells can also interact with the adaptive immune system by binding to cell-bound antibodies, e.g. antibody-opsonized pathogens with Fc␥ receptors and eliminate such cells in a unique manner, the antibody-dependent cell-mediated cytotoxicity. Eosinophils Eosinophilic granulocytes seem to play an important role during helminth infections because their number increases upon invasion of a host with helminths. Albeit controversial, activated eosinophils appear to be able to detect IgE-opsonized parasites by Fc␧ receptors. After binding to the worm via these receptors, they release cytotoxic factors of their granules to kill the parasite.

In conclusion, the mammalian cells of the innate immune system (e.g. NK cells and eosinophils) and even humoral factors of the innate immune system (e.g. the complement system) are partially dependent on the interaction with the adaptive immune system (e.g. antibodies) to exert their functions.

Communication between Immune Cells in Insects and Mammals The short- and long-distance communication between the mammalian immune cells takes place by using small molecules. An important group of such molecules are

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(glyco)proteins termed cytokines. These soluble factors are able to activate cells for antigen presentation, to induce proliferation and chemotaxis. These factors are an essential component of the mammalian immune system. The search for homologous factors of the mammalian cytokines in insects was not successful. Instead, insects appear to use their own nonhomologous set of these important communicators, such as the plasmatocyte-spreading peptide PSP1 from the moth Pseudoplusia includens which induces adhesion and spreading of plasmatocytes on foreign surfaces [13]. In the silkworm Bombyx mori, the insect cytokine paralytic peptide was found to contribute to host resistance against infectious pathogens (e.g. against bacteria such as Staphylococcus aureus). Paralytic peptide is activated by peptidoglycan or glucan derived from the cell wall of fungal and bacterial pathogens [14] and induces morphological changes in plasmatocytes. In Drosophila, different cytokines are known such as Spätzle (a cysteine knot molecule with structural similarities to mammalian neurotrophins) [15], and the unpaired-family Upd1–3, which induce the JAK/STAT pathway by binding to an interleukin (IL)-6-related cytokine receptor. Spätzle has to be proteolytically cleaved before binding to the Toll molecule. Hereby Gram-positive bacteria and fungi activate Spätzle in different ways. In the case of bacterial infections circulating recognition molecules such as peptidoglycan recognition protein-SA (PGRP-SA) and Gram-negative binding protein 1 (GNBP1) bind to peptidoglycan and activate proteolytic cascades to active Spätzle (fig. 1). Fungal infections seem to induce a protease, called Persephone, to cleave proSpätzle. In addition, further cytokines were found, such as the Drosophila helical factor induced during (presumably peptidoglycan-contaminated) lipopolysaccharide (LPS) stimulation [16]. In mammals, there exists a complex network of cytokines that on the one hand activates the cellular immune response, i.e. macrophages, T helper 1 and cytotoxic T cells to eliminate intracellular pathogens. On the other hand, during infection with extracellular pathogens, cytokines orchestrate the humoral response resulting in the production of antibodies. Cytokines such as GM-CSF induce the production of monocytes and granulocytes and activate mature macrophages. Cytokines such as IL-12 play a crucial role in the type 1 immune response of NK cells and T helper cells which will result in production of other cytokines such as IFN-␥ and TNF-␣ that are important mediators to activate effector cells of the cellular immune response (especially effective against intracellular bacteria and protozoa). For infection with extracellular pathogens, the most important regulatory cytokines are IL-4, IL-5 and IL-13 that trigger antibody production and goblet cell hyperplasia for mucus production in the mucosa of the lung or the gut (e.g. to expel intestinal helminths). These two major immune responses (i.e. cellular and humoral immunity) against intracellular versus extracellular pathogens inhibit each other by the characteristic cytokines produced to ensure that the proper immune response proceeds. The immune responses are supported by a variety of additional mediators, e.g. chemokines that direct cells to the site of infection.

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Toll pathway

TLR pathway Gram-positive bacteria

PGRP-SA GNBP1 Proteolytic cascades Cleavage Pro-Spätzle Spätzle cytokine

Toll

dMyD88 Tube

TLR

MyD88

IRAK

Pelle TRAF6

IL-1R

IL-1

IKK Cactus DIF

Drosomycin

Pro-IL-1

I␬B NF-␬B

Pro-IL-1

Insects (Drosophila)

Defensins Mammals

Fig. 1. Gram-positive bacteria activate the Toll pathway in insects and the TLR pathway in mammals by peptidoglycan. Homologous molecules are indicated by the same symbol structure. dMyD88 ⫽ Myeloid differentiation factor 88 (Drosophila); DIF ⫽ dorsal-related immunity factor; MyD88 ⫽ myeloid differentiation factor 88; IRAK ⫽ IL-1 receptor-associated kinase; TRAF ⫽ TNF-receptor-associated factor; IKK ⫽ I␬B kinase; I␬B ⫽ inhibitor of NF-␬B; NF-␬B ⫽ nuclear factor ␬B; IL-1R ⫽ IL-1 receptor.

Recognition of Pathogens by the Interaction of Pattern Recognition Receptors on Innate Immune Cells with Pathogen-Associated Molecular Patterns

Pathogen-Associated Molecular Patterns Pathogen-associated molecular patterns (PAMPs) are conserved structures or motifs of microbes such as peptidoglycans, lipoteichoic acids, mannans, unmethylated CpG dinucleotides, and LPS. Two of the most potent immunostimulants of the host defense of arthropods are bacterial peptidoglycan and fungal ␤-1,3-glucan [17]. In

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mammals, the different PAMPs as immunostimulants are on a par, and help the mammalian immune system to distinguish between pathogens. Distinct recognition mechanism exist for fungal pathogens (by e.g. the mannose receptor) and Gram-negative bacteria (by CD14 and Toll-like receptor 4 – TLR4) and Gram-positive bacteria (by TLR2/1 or TLR2/6) and protozoa. In addition, viruses carrying dsDNA, dsRNA, or ssRNA are recognized by their nucleic acids by mammals which have a number of receptors to detect different types of viruses and also the necessary weapons to deal with such infections, i.e. induction of type 1 interferon production to block protein production in virus-infected cells and activation of NK cells and cytotoxic T lymphocytes to destroy such infected cells. Little is known about viral recognition in insects but it must exist since viral infections lead to activation of the JAK/STAT pathway and to the expression of antiviral genes. In addition, the RNAi silencing machinery can also be activated to target animal viruses. Peptidoglycans are polymers composed of long linear sugar chains of alternating N-acetylglucosamine and N-acetylmuramic acid in ␤1–4 linkage, cross-linked by a stem-peptide of up to five amino acids. Peptidoglycans can be divided into two types: Gram-positive bacteria contain a L-Lys (Lys-type peptidoglycans) at the third position of the peptide bridge, Gram-negative bacteria a meso-diaminopimelic acid (DAP-type peptidoglycans) [18]. The peptidoglycan of Gram-negative bacteria is covered by an outer membrane containing LPS and phospholipids, but released into the hemolymph during cell growth and division. LPS is the dominant molecular marker of Gram-negative bacteria and was assumed to induce the antimicrobial response in insects. However, usually LPS preparations are contaminated with peptidoglycans. Therefore, unlike mammals which can recognize LPS via TLR4, LPS seems to be less important than peptidoglycans for the induction of AMPs in arthropods [18], but a role in other immunological processes is still conceivable. The recognition of PAMPs by the host via germline-encoded receptors has at least three advantages: (a) the immune system of the host is able to distinguish between self and non-self by the absence or presence of these molecules; (b) PAMPs are characteristic for the members of a given class of microbes, thereby a limited number of host receptors can recognize a wide range of pathogens; (c) these structures are essential for the survival of the microorganism and can hardly be modified [19].

Pattern Recognition Receptors In arthropods, PAMPs are recognized by different families of pattern recognition proteins, thereby activating an immunological response. In Drosophila, two protein families activate intracellular pathways related to the immune response, the GNBPs and the peptidoglycan recognition proteins (PGRPs). At least two of the three GNBPs characterized by a C-terminal ␤-glucanase-like domain and a N-terminal ␤-1,3-glucan

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binding domain are involved in the recognition of either Gram-positive bacteria or fungi, both inducing the activation of the Toll pathway [7]. The PGRPs are an evolutionary conserved family of microbial recognition proteins defined by a domain with homology to a peptidoglycans-digesting enzyme, the N-acetylmuramyl-L-alanine amidases. In Drosophila, 13 PGRP are classified – some with multiple splice isoforms – according to the size, localization and enzymatic activity. At least five of the members – PGRP-SA, -SC, -SD, -LC and -LE – participate in the detection of microbial infections and the induction of effector mechanisms of the immune system [7]. PGRP-SA, a soluble hemolymph factor, and PGRP-SD are required for sensing Gram-positive bacteria and the activation of the Toll pathway. GNBP-1 seems to hydrolyze the peptidoglycans of Gram-positive bacteria, generating muramic acid residues which are then recognized by PGRP-SA (lysozyme participates in this recognition event). At least one of the Drosophila PRRs is linked to phagocytic responses; since PGRP-SC1, a PGRP-amidase, which is essential for the phagocytosis of S. aureus by hemocytes, is a soluble factor of the hemolymph, it could act as an opsonin that simplifies the uptake of bacteria by hemocytes through binding to surface receptors [20]. For mammals, previous work has proven the involvement of short-form PGRP in the intracellular phagolysosome-mediated killing of bacteria. Although the mechanisms of PGRP-S acting seem to be different between mammals and insect, it shows the multiple functions of PGRP, activating both the humoral and cellular arm of the host defense. PGRP-LC and -LE are linked to recognition events which activate the immune deficiency (Imd) pathway against Gram-negative bacteria (fig. 2). PGRP-LC is a transmembrane receptor for Gram-negative bacteria upstream of the Imd pathway. Two of the three splicing isoforms (PGRP-LCa and -LCx) are involved in triggering the Imd pathway upon Gram-negative invasion [7]. Their intracellular domain has weak similarities to the RHIM motif which is essential for the interaction of adaptor proteins in the TLR3 signaling pathway in mammals. The formation of homo- and heterodimers of PGRP-LCa and -LCx extends the spectrum of specific PAMP signatures recognized by the innate immune system [7, 18, 19]. Combining a limited number of receptors for a more differentiated pathogen recognition has the advantage to induce a more specific and adequate response to infection. PGRP-LC isoforms have been proposed to form dimers triggering an intracellular signal cascade through Imd activation (directly or indirectly). It is assumed that the binding of bacterial peptidoglycan leads to receptor dimerization that itself activates the subsequent cascade resulting in gene expression. This is consistent with a fundamental principle of mammalian immunity where cross-linking of receptors is necessary for signal transduction. PGRP-LE also has several functions in systemic reactions of the host defense, while it acts as PRR and participates in the proPO-mediated melanization cascade upon pathogen entry. Recently it was shown that Drosophila apolipophorin III, besides its affinity to several bacterial cell wall compounds, can bind to ␤-1,3-glucan of fungi [21], indicating that there are more than the two well-established PRR families in Drosophila.

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Imd pathway

TNF receptor pathway Gram-negative bacteria

TNFR1

TNFR2

PGRP-LC

Imd

Traf

TRADD

dFADD DREDD

RIP

Traf

TLR

FADD

TAK1

TAK1 MyD88

IRAK

Caspase 8

TRAF6 IKK signalosome equivalent

IKK signalosome

IKK IκB NF-κB

Relish TNF Diptericin Insects (Drosophila)

Effector genes Mammals

Fig. 2. Gram-negative bacteria activate the Imd pathway in insects by peptidoglycan and the TNF receptor pathway in mammals by LPS. In mammals after activation of the TLR pathway, besides IL-1 (fig. 1) also TNF is induced. Homologous molecules are indicated by the same symbol structure. PGRP-LC ⫽ Peptidoglycan recognition protein LC; dFADD ⫽ Fas-associated death domain (Drosophila); DREDD ⫽ homologue to caspase 8 in mammals; TAK1 ⫽ TGF-␤-activated kinase 1; IKK ⫽ I␬B kinase; TNFR ⫽ TNF receptor; Traf ⫽ TNF-receptor-associated factor; TRADD ⫽ TNF receptor-associated death domain; FADD ⫽ Fas-associated death domain.

The binding of PRRs to PAMPs triggers three cascades of serine proteases that are regulated by serpins. The Toll pathway is essential for antimicrobial peptide gene expression in response to Gram-positive and fungal infections (fig. 1), while Gramnegative bacteria most frequently stimulate the Imd pathway (fig. 2). More recently, a third pathway, called JAK-STAT was related to immune function of insects, e.g. in Drosophila and Anopheles. The Toll and Imd pathways synergize at the level of transcription factor activation, but are not strictly separated [22]. Both cooperate to produce an appropriate response in many host defense processes, e.g. wound healing, coagulation and AMP response. The Toll pathway in insects is homologous to the TLR pathway (fig. 1) and the Imd pathway is a homologue to the TNF pathway in mammals. In mammals, these two pathways are linked by TNF-␣ (see fig. 2). In mammals, up to 13 different TLR were found which detect different PAMPs and are

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therefore able to recognize the different types of microbes (virus, bacteria, fungi, protozoa). Also in insects besides the original Toll, different Toll-related genes are expressed. Eleven different Toll-related genes were found in the silkworm B. mori and were suggested to function as cytokine receptors. The signal cascade of both Toll and the TLR induces NF-␬B activation which induces AMP responses in mammals and insects. After the induction of an immune response by PRRs in mammals, the activated innate immune cells are able to trigger the activation of adaptive immune cells, i.e. T cells which can support isotype switching and hypermutation of antibodies in B cells. These cells are able to become memory cells and therefore reinfection with a pathogen is less severe than the primary infection. Such a system does not exist in insects but recent studies have revealed that an adaptive immunity exists in invertebrates in a rearrangement-like fashion as seen in T cell receptors or antibodies. PRRs of invertebrates, as shown for Down syndrome cell adhesion molecule receptor of Anopheles gambiae [23], can have more than 30,000 different splice forms due to the presence of three variable Ig exon cassettes. During infection, the PRRs undergo a process of evolution resulting in generation of the optimal receptor for a specific pathogen. When the insect has acquired such a receptor, it is much better protected against a reinfection with this pathogen than naïve counterparts. Since the specificity in protection against secondary pathogen exposure in insects is controversial, it was named specific immune priming [24].

Control of Pathogens: Opsonization, Encapsulation or Phagocytosis, Production of Antimicrobial Peptides

Opsonization The opsonization of pathogens has two major advantages: (a) A coated microorganism is much easier engulfed by phagocytes that can not recognize the pathogen itself. The agglutination of pathogens enables the phagocytes to engulf a higher number of invaders. (b) The coat can prevent the entry of intracellular pathogens such as bacteria and viruses, blocking the receptors required for this event. Opsonizations have unique properties as these endogenous ligands can improve encapsulation and activate the proPO system in insects [25–28], indicating that opsonizing molecules could play a key role in synchronizing the humoral and cellular effector systems. The participation of opsonins in immunity of vertebrates is well known and includes antibodies and complement factors. Although the process of opsonization in arthropods is less understood, they might use this powerful tool to compensate the lack of antibody-mediated features. Various proteins with opsonizing properties have been described for arthropods, including C-type lectins, C-reactive proteins and complement-like proteins [25–30]. Although these opsonins can be bound by PRRs,

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they are discussed in this chapter due to the main difference to most PRRs described in the following, especially their ability to stimulate processes like phagocytosis, encapsulation and melanization instead of induction of an AMP response. C-type lectins are carbohydrate-binding proteins and are widespread in arthropods, e.g. as a mannan-binding lectin of the fresh water crayfish Pacifastacus leniusculus, bacterial agglutinins such as tachylectin 1–5 from the horseshoe crab Tachypleus tridentatus, which recognize bacterial LPS and peptidoglycan, or insect lectins like Periplaneta lectin, BDLs, GSL or immunolectins [26–28, 30, 31]. In detailed studies of three Drosophila C-type lectins, DL1 was upregulated in larvae upon injury [29] and recombinant DL2 and DL3 bound to some Gram-negative bacteria and agglutinated Escherichia coli [25, 29]. DL2 and DL3 are not only secreted, but also located on the surface of hemocytes. In addition, these Drosophila lectins also mediate the encapsulation of agarose beads by hemocytes of M. sexta [25]. Since lectins possess distinct binding properties, they bind a wide range of different microorganisms through variable carbohydrate-binding domains, delivering them to a limited number of germline encoded receptors in an antibody-like fashion. This proposed mechanism requires several lectins with different carbohydratebinding specificities within one species. Indeed, multiple lectins were demonstrated for various arthropods of the genera Periplaneta, Blaberus, Bombyx and Tachypleus. In addition, the characterization of various isoforms of carcinolectin 5a and 5b from the horseshoe crab Carcinoscorpius rotundicauda revealed their functional diversity. The authors suggested that the interaction of the carcinolectin isoforms with different binding affinities creates a flexible oligomeric structure that allows the discrimination between microorganism [32]. The existence of more than 30 C-type lectins encoded by Drosophila [29] could be another possibility for variable binding specificities, although their involvement in immunity, beside DL1–3, needs to be proven. Vertebrates were emphasized to have evolved the complement system exclusively, but arthropods possess at least basic components of this system such as C3-like proteins. C3-like proteins can act as opsonins, given appropriate proteolytic activation. Thus, arthropods seem to display the ancient origin of the complement cascade, which has evolved to a multifunctional system in vertebrates [33]. In addition to the complement system, mammals also can opsonize pathogens via antibodies which do not exist in insects. Therefore, cells of the mammalian innate immune system, e.g. macrophages and eosinophils, have Fc receptors on their surface to recognize antibody-opsonized particles. As outlined above, macrophages recognize the antibody-opsonized particles by Fc␥ receptors to phagocytose and eradicate them in the phagosome, whereas eosinophils recognize extracellular IgE-coated pathogens by Fc␧ receptors resulting in secretion of lytic granules. Mammals also have opsonins derived from the acute-phase response that can mark a broad range of microbes for phagocytosis, e.g. mammalian mannose-binding lectin. This system is widely distributed in insects, as exemplified by a cockroach LPS-binding lectin and a mosquito complement-like protein [33].

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Cellular Encapsulation Encapsulation is the main cellular defense mechanism against foreign objects which are too large to be phagocytosed. This reaction is widespread within arthropods, although the mechanism of capsule formation and the number of cell types involved may differ. In Drosophila, in response to parasites such as wasp eggs, a massive proliferation and differentiation of precursors into lamellocytes is observed in the lymph gland, followed by subsequent crossing into the hemolymph [5]. The lamellocytes attach to the surface of the parasite and form a multilayer capsule around the invader. This capsule is initially stabilized by the formation of septate junctions between the lamellocytes, probably through integrins [10]. The capsule often blackens due to a melanization cascade. The melanization is performed by the activity of the PO which catalyses the oxidation of phenols to quinones, that in turn, polymerize to melanin. PO activity itself is strongly regulated by a serine protease cascade, which gives rise to the active form of PO by Tollmediated cascade activation. Pro-PO of the tobacco hornworm can bind to the surface of hemocytes and the activated membrane-bound PO form initiates hemocyte melanization [34]. This can be a mechanism to restrict the PO activity to the surface of a foreign object minimizing the toxic effects for the host. The encapsulation is completed after 48 h, while the final melanization can last 72 h. The parasite is attacked by toxic by-products of the melanization cascade (H2O2, NO) and is physiologically separated from the hemolymph, which affects nutrition and respiration [5]. As lamellocytes are not present in adults, the cellular encapsulation is restricted to larvae, but an analogous cell-free mechanism, called humoral encapsulation, remains functional in adults [7]. Encapsulation can also be found in mammals. In mammals, parasites like the intracellular pathogens Trichinella and Sarcocystis can be encapsulated by the host and in some cases destroyed, e.g. in the case of the nematode Trichinella spiralis. In addition, mammals have evolved granuloma formation (e.g. against Mycobacterium tuberculosis [35]) which can last for decades. This is a process analogous to nodule formation in invertebrates. Mammals are furthermore able to control infections with different pathogens by destroying infected cells or the pathogen directly by NK cells and T cells.

Phagocytosis Due to the obvious similarities of the Drosophila hemocytes to the myeloid lineage of mammals, it was suggested that invertebrate hemocytes could serve as a model for cellular immune mechanisms such as phagocytosis by macrophages of vertebrates [36]. In fact, there are many parallels in the morphology and function of invertebrate and mammalian phagocytes.

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In general, phagocytosis leads to the killing of organisms since the uptake is followed by subsequent formation of phagolysosomes. The process of phagocytosis is initiated either by strong binding of a foreign structure to receptors of the cell surface or recognition of opsonized microorganisms. Cross-linking of these receptors triggers an intracellular cascade, which in turn leads to the rearrangement of the cytoskeleton at the binding site. The actin-dependent process enables the phagocyte to surround the object with filopodes. This event is accompanied by subsequent binding of repeating structures similar to a zipper. In Drosophila, the CD36 homologue encoded by the croquemort gene is crucial for the uptake of apoptotic remnants during embryogenesis. Croquemort also mediates the phagocytosis of S. aureus, indicating that there might be multifunctional receptors [36]. Several receptors are involved in phagocytic immune reactions: The scavenger receptor dSR-CI mediates the binding of Gram-positive and Gram-negative bacteria to Drosophila Schneider cells [37]. As mentioned above, PGRP also participate in the phagocytosis of microorganisms. While PGRP-LC is involved in the engulfment of Gram-negative bacteria in Schneider cells, PGRP-SA seems to function as an opsonizing protein that increases the phagocytosis of Gram-positive bacteria. In addition, insects possess receptors with an extracellular domain containing several EGF-like repeats that mediate direct pathogen binding and bacterial uptake or aggregation. This indicates that EGF-like repeats are crucial for the tethering of microorganisms to hemocytes of arthropods. Although these receptors are important for the binding of pathogens, it is not known if they require coreceptors for appropriate actin reorganization. In A. gambiae, silencing of the transcription of the gene of TEP1, a complementlike thiolester-containing protein, reduces phagocytosis of the Gram-negative bacterium E. coli by blood cell lines. The binding of TEP1 includes a thioester bond and therefore seems to occur in a C3-like manner. Drosophila possesses four expressed tep genes, and two further DNA sequences that correlated with complement-like proteins were found in the Drosophila genome (but these seem to be pseudogenes); tep1, 2 and 4 are upregulated upon immune challenge in larvae and tep2 and 4 in adults and are proposed to be secreted. tep3 gene expression is not modified by immune challenge [38]. Thus, in addition to PGRP-SA, TEP proteins are predicted to act as opsonizing molecules facilitating the phagocytosis of pathogens. The participation of opsonizing molecules resembles the function of complement and antibodies of vertebrates and can enhance the uptake of those organisms, which are not or hardly recognized directly. In Drosophila and mammals, the phagosome is directly trafficked to fusion with primary lysosomes after engulfment. The formation of the phagolysosome depends on several fission and fusion events with lysosomes and endosomes [36]. Within this compartment, the pathogen is killed due to the acidic environment, digestive enzymes, AMPs and ROIs similar to the well-known participation of ROIs in killing of engulfed bacteria in mammalian macrophages and neutrophils. ROIs such as H2O2

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and O2⫺ occur within larval hemocytes of different insects, e.g. Drosophila and the wax moth Galleria mellonella. In Galleria larvae, several homologous components of the NADPH oxidase complex could be identified. This indicates that an intracellular killing mechanism is conserved between arthropods and mammals and is mediated by homologous proteins to generate phagocytosis-dependent ROI production, the respiratory burst. Phagocytosis of e.g. pathogenic fungi is comparable between hemocytes in insects and neutrophils in mammals and their way of killing is identical as mentioned earlier. Furthermore, as arthropods’ ROI production is not only restricted to phagocytes but also occurs in the gut epithelia to prevent microbial growth, arthropods evolved several effector systems using the powerful respiratory burst. As mentioned above, the recognition of invading organisms is very similar between mammals and insects but mammals have a broader variety of receptors to detect pathogens. Mammals also use the complement system and antibodies for opsonization which makes phagocytic cells more effective in detecting foreign particles.

Antimicrobial Peptides Insects and mammals respond to fungal and bacterial infections with strong synthesis of AMPs. In insects, the main site of AMP production is the adult fat body, although hemocytes and different epithelia are also capable of AMP synthesis. The adult fat body is not a coherent tissue but consists of several multilayered sheets which are distributed throughout the thorax and abdomen in association with the basal membrane of epithelia [39]. Upon infection, AMP gene expression is induced within 30 min and a peak of transcription is detectable after 4 h. The systemic induction can lead to hemolymph concentrations of AMPs up to 300 ␮M. Surprisingly, these concentrations are not cytotoxic for the host [40]. Most of the invertebrates seem to attack the membrane of the invader by disturbing the molecular interactions of phospholipids, resulting in the lysis through osmotic effects. The majority of AMPs of invertebrates consist of 15–45 amino acids [39]. They differ in their activity spectrum and their mode of action. While drosomycin found in Drosophila upon induction of the Toll pathway is solely active against fungi, other AMPs such as Metchnikowin exhibit activity against fungi and bacteria and are expressed in response to the three main pathogen classes [41]. In the case of Drosophila, there exist at least eight inducible AMPs. Some are activated specifically by one pathogen class, while the induction of others (e.g. attacin, cecropin, and defensin) seems to be less specific [7]. Thus, infection by different invaders leads to a distinct but overlapping pattern of AMPs [42], perhaps due to the interaction between two signaling pathways [7, 42]. The use of AMPs with a wide activity spectrum is widespread within the arthropods. Tachyplesin and tachystatins of T. tridendatus are both active against fungi, Gram-positive and Gram-negative bacteria [31]. In addition, gambicin of A. gambiae

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prevents the growth of both classes of bacteria and increases lethality of Plasmodium ookinetes. An additional feature of most of these AMPs is the existence of various isoforms such as the three tachystatins A–C, three cecropins in Drosophila or four defensins in A. gambiae [43–45]. The tissue-specific expression of AMP isoforms enables the host to adapt to different environmental conditions and increase the power of the humoral reaction. Another family of factors with antimicrobial effects are the lysozymes which can be found in every insect and also in mammals where macrophages and granulocytes use it to kill bacteria. For a long period of time, the role of AMPs in mammals was neglected. But more recently, numerous studies focused again on AMPs due to their central role in pathogenic control. The production of some of these antimicrobial factors seems to be induced by subpopulations of T cells, e.g. Th17 cells, a recently discovered subpopulation of T helper cells. These Th17 cells can produce IL-22, a cytokine that acts on nonimmune cells, e.g. keratinocytes or hepatocytes and induces the production of AMPs. But cells of the innate immune system such as macrophages can also induce the production of AMPs (e.g. defensins) after TLR4 stimulation via LPS. These macrophages produce large amounts of IL-1, which acts on keratinocytes that produce ␤-defensin-2, a mechanism similar to drosomycin production in Drosophila after stimulation of Toll via the cytokine Spätzle [15]. In mammals, these small polycationic molecules exist in a variety of families. There are inter alia defensins, which can act against bacteria, fungi and even enveloped viruses, cathelicidins from PMN, with a variety of members such as PR-39, which act mainly against Gram-negative bacteria. Protegrins are cathelicidins with broad-spectrum microbicidal activity against Gram-positive and Gram-negative bacteria, mycobacteria, fungi and enveloped viruses. AMPs such as prophenins display activity against Gram-negative bacteria. Other AMPs of mammals are NK lysin and granulysin and even cecropin homologues, e.g. the porcine cecropin P1, the first AMP that was found in pigs [46].

Comparison of the Function of Insect and Mammalian Innate Immune Cells The immune cells of insects and mammals share some features in pathogen control (table 1). As described above, the pathogens are recognized by mammalian innate immune cells in a very similar manner as in insects via PRR and/or opsonic receptors. Mammalian innate immune cells such as macrophages are also efficient producers of AMPs as shown for different insect species. Therefore, the main features of the immune system of insects and the innate immune system of mammals are very similar: (a) recognition of pathogens by specialized receptors; (b) phagocytosis of pathogens, and (c) degranulation leading to the release of microbicidal factors.

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Table 1. Innate immune cells of insects and mammals and their functions1 Insects

Mammals

Plasmatocytes Phagocytosis Encapsulation Nodule formation Tissue remodelling Production of AMPs

Macrophages Phagocytosis Granuloma formation Antigen presentation Production of proinflammatory cytokines Production of effector molecules (ROIs/RNIs)

Lamellocytes Encapsulation

Dendritic cells Phagocytosis Prime role in antigen presentation to naïve T lymphocytes Production of proinflammatory cytokines

Crystal cells Melanization

NK cells Killing of infected cells and of tumor cells by granule secretion Production of inflammatory cytokines

Granulocytes Granulocytes Phagocytosis of apoptotic cells Encapsulation (thereby killing of encapsulated pathogen) Nodule formation

Neutrophils Phagocytosis Production of effector molecules (ROIs)

Eosinophils Binding to parasites Damage of parasites via granula secretion Primitive lymphocytes B1 lymphocytes Production of natural antibodies ␥/␦ T lymphocytes Defense function in skin and mucosa 1

Only a limited selection of cells and their functions is shown.

As shown for NK cells, these cells are able to secrete factors, e.g. granulysin or NK lysin to directly kill fungal pathogens without further activation. In insects, there is a mechanism to trap and encapsulate large foreign bodies, e.g. eggs of parasitoids. Granulocytes adhere to the object and finally a melanized capsule of several layers of cells surrounds the pathogen. In mammals, similar mechanisms can be observed. Eosinophils can surround worm eggs or larvae (but in an antibody-dependent manner) and secrete factors to damage the parasite. The first line of mammalian defense against microbes consists of the phagocytic cells, especially in the skin and the mucosa, and their function is more or less comparable to plasmatocytes and granulocytes in

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insects. Invaded bacteria are removed from the insect hemolymph by nodule formation. In a first stage, cells such as granulocytes can surround invaded pathogens to discharge their granules to entrap the microbes; thereafter, degenerated hemocytes and bacteria become melanized and build a mass of dead bacteria and necrotic cells which will then be surrounded by several layers of plasmatocytes. Granuloma formation in mammals is dependent on macrophages which engulf the pathogens and later are supported by T cells. In mammals, the phagocytic cells, especially macrophages and dendritic cells are important mediators between innate and adaptive immune responses because these cells are professional antigen-presenting cells which can activate T cells via the interaction of MHC with the T cell receptor. This system is unique for vertebrates and has no homologue in insects.

Gut Immunity: Recognition and Control of Pathogens, Survival of Symbionts

All animals have to eat and therefore ingest potentially contaminated food. Insects as well as mammals have an intestinal tract with a large surface that could be used for invasion by pathogens. In insects, only the fore- and hindgut is protected by a chitinous cuticle. Therefore, both groups have to protect this possible gateway from pathogenic microbes. However, many insects and mammals depend on symbionts in their intestinal tract and have to distinguish between the normal gut flora and pathogenic agents. The symbionts must resist the antibacterial activities or the immune system has to tolerate symbionts in the gut, because otherwise this reaction would be lethal for the host. As outlined above, according to the bacterial target, insects produce different antimicrobial compounds. The reaction is not restricted to the hemocoel, but it also occurs in the intestinal tract. This has been recognized very early by Duncan in 1929 and has been investigated in more detail only nearly 40 years later. The antimicrobial activity was attributed to lysozyme [47], but later the additional presence of defensins, attacines or other AMPs was verified. Presumably, all insects produce lysozyme in the intestinal tract and several other antimicrobial compounds. There are several occasions which allow the access of bacteria to the intestine of animals and at which the level of AMPs is increased. An insect-specific event is the uptake of air during a short period of time before moulting, because insects have to increase their volume to break the old cuticle at a perforation site of the thorax. This aspect has been rarely considered in studies related to expression of genes encoding for AMPs, but in the hematophagous insect, Triatoma infestans the expression of a lysozyme gene is upregulated directly after the moult, not in the posterior, but in the anterior midgut, in which the air is taken up [48]. Another developmental occasion at which high concentrations of antibacterial factors are evident in the intestine of insects, is the pupal development of holometabolous insects. At the beginning of the metamorphosis, all larval structures are lysed. Since

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this would allow intestinal bacteria access to the hemocoel, these insects empty their gut and increase the level of antibacterial compounds. In the lepidopteran Manduca, lysozyme is synthesized in the midgut of full-grown larvae, stored in granules within these cells, and released into the gut lumen just before metamorphosis is initiated. The gene is expressed in the intestinal cells. In Drosophila, one of the lysozyme genes, lysX, is only expressed in the midgut of late larvae, just before pupation, and it continues to be expressed for a short period in early pupae. It is the only Drosophila lysozyme that is not upregulated in infected insects [48, 49]. However, as in mammals, the majority of bacteria is ingested with the food. The fight against airborne bacteria and those present on the food has well been demonstrated in cockroaches. The antibacterial activity increases from the esophagus to the midgut diverticles and decreases then until the rectum in which symbiotic bacteria and protozoa develop well [47]. The lepidopteran Hyalophora cecropia produces attacins in the intestine which kill E. coli and other Gram-negative bacteria. The intestinal antimicrobial activity has been investigated in detail in cyclorrhaphous flies which live on and ingest large amounts of bacteria from decomposing matter, e.g. blowflies, Musca and Drosophila [48, 49]. Blowfly larvae develop in wounds of dead or alive animal at a basic pH. In meat with a bacteria-induced slightly acidic pH, development of the blowfly larvae is strongly affected [Schaub, unpubl.]. The larvae have an extraintestinal digestion regurgitating digestive enzymes and AMPs into the wound. Therefore, used by surgeons prior to the discovery of penicillin, they are nowadays used again to treat wounds infected with antibiotic-resistant bacteria. Since blowflies produce several AMPs of low molecular weight, it remains to be investigated which of these peptides kill airborne bacteria in the wounds and support the growth of specific bacteria there and which kill the ingested bacteria within the intestinal tract of the larvae. The larvae of Musca domestica are suggested to lyse the bacteria in the middle region of the midgut via the combined action of low pH, lysozyme and cathepsin D-like proteinases [48, 49]. Drosophila possesses a high number of lysozyme genes which do not appear to be expressed in the hemocytes or in the fat body where many other antibacterial compounds are synthesized. Since many lysozyme genes are expressed in the intestinal wall, the midgut epithelium is the most likely source of hemolymph lysozymes. Therefore, it is assumed that their lysozymes may be more involved in digestion than in immune response and that lysozymes are exported from the midgut epithelial cells in two directions: to the lumen and to the hemolymph. The major lysozyme genes lysB, C, D and E are expressed at high levels in the anterior midgut of larvae and/or adults, but not in late pupae, and lysS is mainly expressed in the larval gastric ceca. LysP, the basic lysozyme gene expressed in the adult salivary glands is also active at an acidic pH and is thought to be secreted with saliva and possibly to function in predigestion of food in the crop rather than in the more acidic midgut [48, 49]. This situation is similar to that in herbivorous vertebrates, e.g. rabbits as hindgut fermenters and ruminants or leaf-eating monkeys as foregut fermenters. In these mammals,

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bacterial symbionts digest the plant food in specialized regions of the gut and are then killed by lysozyme to gain access to the components of these microorganisms as a part of the food. The interesting parallels between Drosophila and the ruminants are the intensive synthesis of the enzymes in the digestive tract, the involvement in the digestion of bacteria and the high number of lysozyme genes, at least seven in the genome of Drosophila and ten in the cow. The latter finding perhaps indicates the need for a high synthesis level. These lysozymes are less basic and perhaps thereby more resistant to the acidic proteolytic conditions in the respective region of the digestive tract. However, the specific amino acid substitutions which are associated with this specific function and environment differ between Drosophila and the vertebrates [48]. Considering the bacterial load of food, only low levels of antibacterial compounds seem to be necessary in insects ingesting sterile food, e.g. the hemolymph of other insects or blood. However, many blood-sucking insects also ingest honeydew or nectar which is digested in the crop, whereas blood is digested in the midgut. In A. gambiae, the lysozyme gene is strongly expressed in sugar-fed mosquitoes and very weakly in blood-fed ones, but besides a sugar-feeding induction of the transcription of this lysozyme mRNA a blood feeding-induced repression is also possible. In the intestinal wall of the mosquito Aedes aegypti, two lysozyme genes are expressed [49]. In the midgut of the stable fly Stomoxys calcitrans, defensin and attacins are important AMPs, and at least two isoforms of defensin encoding genes are expressed in this fly [50]. In contrast to the dipteran hematophagous species, all postembryonic developmental stages of the biggest hematophagous insects, the triatomines, solely depend on blood as food. However, in addition to the uptake of air before moulting (see above) they also need a fine balanced action of antibacterial compounds, because they can only complete the larval development if they have symbionts. These bacteria (all actinomycetes) are obtained via coprophagy [48, 49], another occasion of giving bacteria access to the gut. The uptake of fresh feces of other triatomines is often done after blood ingestion. This should be the wrong period of time, since a contamination of ingested blood by air-borne bacteria rapidly kills the triatomines [Schaub, unpubl.]. However, the majority of air-borne bacteria seem to be killed and the symbionts survive. After blood ingestion, the number of symbionts increases up to 18fold in the two anterior midgut regions, the cardia and the blood-storing distensible stomach. In the following blood digestive posterior midgut, nearly all bacteria are killed, but about 0.01% of the stomach population survive there and are passaged to the rectum, ready for a coprophagic transmission [51]. The symbionts and/or the airborne bacteria are controlled by different antimicrobial compounds, e.g. lysozyme and defensin [48, 49]. In the stomach, these compounds seem to be mainly active against nonsymbiotic bacteria, since the increase in the number of symbionts after blood ingestion is accompanied by an up to 8-fold increase in antibacterial activity [Schaub, unpubl.], up to 15-fold increase in the expression of the gene encoding lysozyme [48], and up to 12-fold increase in the expression of the gene encoding defensin [49]. In contrast to the situation with A. gambiae, gut lysozyme in T. infestans

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is upregulated after feeding with the highest expression of lysozyme 15 days after feeding of fifth instar larvae. However, the posterior midgut, in which the passaged symbionts are killed, possesses a much lower antibacterial activity than the stomach. Also the expression levels of lysozyme and defensin are much lower, being about 3 and 0.3% of the expression levels in the stomach of Triatoma brasiliensis, respectively [49]. This discrepancy between the killing of symbionts and the level of antibacterial compounds may be solved by a lysozyme from T. infestans, which has been characterized most recently and has been termed lysozyme 2. It is expressed in the small intestine of the insect [52]. The deduced protein sequence shows up to 70% similarity to lysozymes from other species, but the pI is much more acidic (4.9) than that of lysozyme 1 in the stomach (pI 9.1) and the conserved amino acid residues of the active site of classical c-type lysozymes, glutamate and aspartate, are replaced by valine and tyrosine. However, such changes do not exclude a bactericidal activity. Whereas so far a maximum of two lysozymes have been found in triatomines, the triatomine Rhodnius prolixus has at least three isoforms of defensin, T. brasiliensis at least two and T. infestans at least one [49] [Schaub, unpubl.]. In insects, the production of antimicrobial compounds in the intestine is not only induced by bacteria and components of the bacterial cell wall, but also by protozoan parasites, e.g. in sand flies, defensins are produced after ingestion of the protozoan parasite Leishmania major and in R. prolixus the NO synthase is activated after infection with Trypanosoma rangeli and Trypanosoma cruzi [48, 53]. In addition, also an infection of the hemocoel with bacteria induces a reaction in the intestine, e.g. in R. prolixus a strong increase in the expression level of the gene encoding for defensin. This reaction may be due to the intimate contact of hemocytes to the intestinal wall. In contrast to mammals, the intestine of insects is not surrounded by a net of blood capillaries but by the hemocoel itself, and hemocytes are regularly attached to all organs, thereby inducing a shortly delayed reaction after the initiation of the immune reaction in the hemocoel. Considering this, an expression of antibacterial compounds by cells of the intestinal wall has to be investigated by intestines from which the hemocytes have detached, e.g. by a warming up of the insect. In mammals, a gut-associated lymphoid tissue exists with Peyer’s patches and lymph nodes that guard the mucosa and secrete e.g. antibodies (secretory IgA) into the mucus. This mucus is also used to expel helminths. Furthermore, a specialized cell type exists in the gut wall, the microfold cells (M cells). These M cells can take up pathogens from the gut lumen and transport them to the opposite side where immune cells, e.g. macrophages and T cells wait to attack these pathogens after activation via PRRs and induce an adequate immune response against the infection, e.g. secretion of AMPs into the gut lumen and uptake of the antigens by dendritic cells which transport them to the draining lymph node, as described above. In addition, infected enterocytes of the gut synthesize stress-induced proteins and express atypical class I molecules (MIC-A and MIC-B) which can be bound by intraepithelial ␥/␦ T cells carrying the NK receptor NKG2D. This binding induces apoptosis in the infected enterocyte [12].

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Mammals like other animals with symbionts in their intestinal tract have to distinguish between the normal gut flora (consisting of more than 400 species of commensal bacteria especially in the colon and ileum) and pathogenic agents. This normal gut flora protects the host by competing against pathogenic bacteria for space and nutrients (‘competitive exclusion’). However, in particular situations also the normal gut flora can cause severe diseases, e.g. after breakdown of the integrity of the gut wall and invasion of the bloodstream by normally nonpathogenic bacteria or after immunodeficiencies. To prevent invasion of the body by microorganisms, the immune system develops a response against such events but does not affect the commensal bacteria in the gut lumen. During its postpartum development, the immune system recognizes the antigens of the symbionts in the intestine and becomes tolerant to them (as seen in the blood groups). Thereafter invading microbes are recognized as foreign and therefore will be attacked by immune cells. The way by which the host distinguishes between apathogenic and pathogenic microbes is not well understood yet. But it seems to be clear that any pathogenic invasion of the gut is accompanied by an inflammatory response. Thus, infection of enterocytes leads to the induction of a prominent cytokine and chemokine production that, for example, results in the migration of neutrophils to the site of infection.

Concluding Remarks

Insects and mammals accomplish the same task to get rid of pathogens by both distinct and common mechanisms. Invertebrates and vertebrates have evolved a complex system of cellular and humoral factors, the so called immune system. As described in this chapter, the innate immune system of insects and mammals share some major features: (a) distinction between self and non-self; (b) recognition of pathogens via PRRs; (c) opsonization of pathogens; (d) uptake of pathogens by phagocytosis and secretion of reactive effector molecules to kill the microbes, and (e) surrounding and control of microbes, i.e. nodule/granuloma formation. But some features such as alternative splicing of PRRs only evolved in insects. This may in part compensate for their lack of a highly specific adaptive immune system involving gene rearrangement of B cell and T cell receptors in the adaptive mammalian immune system. Insects try to encapsulate pathogens that they can not eliminate, whereas the mammalian immune system in most cases where the innate immune system fails uses the adaptive immune system, i.e. antibodies and cytotoxic T cells to control and eliminate invaded pathogens. Insects may lack the mechanistic basis for immune memory of mammals, but may use other strategies leading to specific immune priming that allows for stronger phagocytic responses upon reinfection with the same pathogen. The homologies found between host defense of mammals and insects show that important parts of the immune system are hundreds of million years old and are capable of protecting us and also the small fly from being overwhelmed by pathogens of any kind.

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32 Zhu Y, Ng PM, Wang L, Ho B, Ding JL: Diversity in lectins enables immune recognition and differentiation of wide spectrum of pathogens. Int Immunol 2006;18:1671–1680. 33 Fujita T: Evolution of the lectin-complement pathway and its role in innate immunity. Nat Rev Immunol 2002;2:346–353. 34 Ling E, Yu XQ: Prophenoloxidase binds to the surface of hemocytes and is involved in hemocyte melanization in Manduca sexta. Insect Biochem Mol Biol 2005;35:1356–1366. 35 Seiler P, Aichele P, Bandermann S, Hauser AE, Lu B, Gerard NP, Gerard C, Ehlers S, Mollenkopf HJ, Kaufmann SH: Early granuloma formation after aerosol Mycobacterium tuberculosis infection is regulated by neutrophils via CXCR3-signaling chemokines. Eur J Immunol 2003;33:2676–2686. 36 Stuart LM, Ezekowitz RA: Phagocytosis: elegant complexity. Immunity 2005;22:539–550. 37 Ramet M, Pearson A, Manfruelli P, Li X, Koziel H, Gobel V, Chung E, Krieger M, Ezekowitz RA: Drosophila scavenger receptor CI is a pattern recognition receptor for bacteria. Immunity 2001;15: 1027–1038. 38 Lagueux M, Perrodou E, Levashina EA, Capovilla M, Hoffmann JA: Constitutive expression of a complement-like protein in toll and JAK gain-of-function mutants of Drosophila. Proc Natl Acad Sci USA 2000;97:11427–11432. 39 Dimopoulos G: Insect immunity and its implication in mosquito-malaria interactions. Cell Microbiol 2003;5:3–14. 40 Hetru C, Troxler L, Hoffmann JA: Drosophila melanogaster antimicrobial defense. J Infect Dis 2003; 187(suppl 2):S327–S334. 41 Lemaitre B, Reichhart JM, Hoffmann JA: Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. Proc Natl Acad Sci USA 1997;94: 14614–14619. 42 De Gregorio E, Spellman PT, Tzou P, Rubin GM, Lemaitre B: The Toll and Imd pathways are the major regulators of the immune response in Drosophila. EMBO J 2002;21:2568–2579. 43 Evans JD, Aronstein K, Chen YP, Hetru C, Imler JL, Jiang H, Kanost M, Thompson GJ, Zou Z, Hultmark D: Immune pathways and defence mechanisms in honey bees Apis mellifera. Insect Mol Biol 2006;15: 645–656.

44 Kylsten P, Samakovlis C, Hultmark D: The cecropin locus in Drosophila; a compact gene cluster involved in the response to infection. Embo J 1990; 9:217–224. 45 Osaki T, Omotezako M, Nagayama R, Hirata M, Iwanaga S, Kasahara J, Hattori J, Ito I, Sugiyama H, Kawabata S: Horseshoe crab hemocyte-derived antimicrobial polypeptides, tachystatins, with sequence similarity to spider neurotoxins. J Biol Chem 1999;274:26172–26178. 46 Mohrig W, Messner B: Immune responses of insects. II. Lysozome as an antimicrobial agents in gastro-intestinal tract of insects. Biol Zbl 1968;87: 705–718. 47 Mohrig W, Messner B: Immune responses of insects. II. Lysozyme as an antimicrobial agent in gastro-intestinal tract of insects. Biol Zbl 1968;87: 705–718. 48 Kollien AH, Fechner S, Waniek PJ, Schaub GA: Isolation and characterization of a cDNA encoding for a lysozyme from the gut of the reduviid bug Triatoma infestans. Arch Insect Biochem Physiol 2003;53:134–145. 49 Araujo CA, Waniek PJ, Stock P, Mayer C, Jansen AM, Schaub GA: Sequence characterization and expression patterns of defensin and lysozyme encoding genes from the gut of the reduviid bug Triatoma brasiliensis. Insect Biochem Mol Biol 2006;36:547–560. 50 Lehane MJ, Wu D, Lehane SM: Midgut-specific immune molecules are produced by the bloodsucking insect Stomoxys calcitrans. Proc Natl Acad Sci USA 1997;94:11502–11507. 51 Eichler S, Schaub GA: Development of symbionts in triatomine bugs and the effects of infections with trypanosomatids. Exp Parasitol 2002;100:17–27. 52 Balczun C, Knorr E, Topal H, Meiser CK, Kollien AH, Schaub GA: Sequence characterization of an unusual lysozyme gene expressed in the intestinal tract of the reduviid bug Triatoma infestans (Insecta). Parasitol Res 2008;102:229–232. 53 Whitten M, Sun F, Tew I, Schaub G, Soukou C, Nappi A, Ratcliffe N: Differential modulation of Rhodnius prolixus nitric oxide activities following challenge with Trypanosoma rangeli, T. cruzi and bacterial cell wall components. Insect Biochem Mol Biol 2007;37:440–452.

Uwe Müller Institute of Immunology, College of Veterinary Medicine University of Leipzig An den Tierkliniken 11, DE–04103 Leipzig (Germany) Tel. ⫹49 341 9731 224, Fax ⫹49 341 9731 229, E-Mail [email protected]

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Egesten A, Schmidt A, Herwald H (eds): Trends in Innate Immunity. Contrib Microbiol. Basel, Karger, 2008, vol 15, pp 45–60

Pattern Recognition Receptors and Their Role in Innate Immunity: Focus on Microbial Protein Ligands Thomas Areschouga ⭈ Siamon Gordonb a

Department of Laboratory Medicine, Division of Medical Microbiology, Lund University, Lund, Sweden; bSir William Dunn School of Pathology, University of Oxford, Oxford, UK

Abstract Antigen-presenting cells, such as macrophages and dendritic cells, represent a central and important part of the immune defence against invading microorganisms, as they participate in initial capture and processing of microbial antigens (innate immunity) and then activation of specific T and B cell effector mechanisms (acquired immunity). Recognition of microbial molecules by antigen-presenting cells occurs through so called pattern recognition receptors (PRRs), which recognize conserved structures, or pathogen-associated molecular patterns, in pathogenic microbes. The Toll-like receptors are the most extensively studied of these receptors, but accumulating evidence shows that other PRRs, such as scavenger receptors, C-type lectin receptors and NOD-like receptors, also play important roles in the innate immune defence. Here, we summarize current knowledge of the role of various PRRs in the defence against pathogenic microorganisms and we report recent advances in studies of different receptor-ligand interactions. In particular, we Copyright © 2008 S. Karger AG, Basel focus on the importance of microbial proteins as ligands for PRRs.

Antigen-presenting cells (APCs), i.e. macrophages and dendritic cells, are widely distributed in the body, including at sites of possible entry for pathogenic microorganisms. APCs express several surface molecules collectively referred to as pattern recognition receptors (PRRs), including the Toll-like receptors (TLRs), scavenger receptors (SRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs) and ␤2integrins, which recognize conserved structures designated pathogen-associated molecular patterns (PAMPs) on microbes. Recognition of pathogens by PRRs on macrophages leads to an inflammatory response and is followed by phagocytosis, processing of antigens and subsequent presentation on MHC class I and II molecules. Thus, PRRs on APCs play an important role in the protection against invading microorganisms by linking the innate and adaptive parts of the immune system [1, 2]. Broadly, the PRRs can be divided into two categories on the basis of their function. Some PRRs, such as the TLRs, are involved in signalling, resulting in an inflammatory

response and subsequent cellular activation. Other PRRs, such as scavenger receptorA (SR-A), act as phagocytic receptors mediating direct non-opsonic uptake of pathogenic microbes and/or their products. There are also some examples of PRRs, such as the ␤-glucan receptor dectin-1, that are both signalling and phagocytic receptors [3]. With advancing characterization of different receptor-ligand interactions it has become increasingly clear, however, that the original concept of pattern recognition needs to be somewhat refined. The ligand-binding specificity of many PRRs is rather broad, including various polysaccharides (e.g. lipopolysaccharide – LPS, and ␤-glucan), lipoteichoic acid (LTA), peptidoglycan (PG), nucleic acids, and proteins. However, many of these molecules are not only present in pathogenic microbes, but also in commensals and opportunistic pathogens. Moreover, many PRRs interact not only with exogenous microbial molecules, but also with endogenous structures, including plasma glycoproteins, modified lipoproteins and stress-induced heat shock proteins, which may affect the complex spectrum of pro- and anti-inflammatory, immunogenic and suppressive responses induced within the host [3]. In this review, we focus on the exogenous microbial ligands for PRRs. Most studies have analyzed the various non-proteinaceous microbial ligands for PRRs, but emerging evidence indicates that microbial proteins, in particular surface proteins, are common target molecules for PRRs. We summarize the current knowledge of the most important mammalian PRRs and their microbial ligands, and focus on the reported evidence for a role of these receptors in the innate immune defence against invading microorganisms.

Toll-Like Receptors

There are 11 TLRs, designated TLRs 1–11, known in mammals and each of these receptors recognizes PAMPs derived from a unique class of microbial antigens. Recognition of pathogenic microbes by TLRs on APCs triggers an intracellular signalling pathway which culminates in induction of proinflammatory cytokines, chemokines, type I interferons and subsequent activation of APCs. The TLRs have been described in detail elsewhere and several excellent reviews are available [4–6]. Here, we will briefly summarize the TLRs and their role in innate immune recognition and mainly focus on the members of this family recognizing microbial proteins. The TLRs are type I transmembrane proteins with an extracellular domain containing leucine-rich repeats and a cytoplasmic tail with homology to the interleukin-1 receptor family (IL-1R). Each TLR recruits a distinct set of Toll/IL-1R (TIR)- containing adaptor molecules such as myeloid differentiation primary response gene 88 (MyD88), TIR-containing adaptor protein, TIR-containing adaptor-inducing IFN-␤ (TRIF), TRIF-related adaptor molecule. This initiates signal transduction pathways resulting in activation of the transcription factor NF-␬B, Jun N-terminal kinase, MAP kinases and interferon regulatory factors. Recently, a fifth adaptor protein, SARM, was identified, which negatively regulates TRIF [6].

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The first TLR to be discovered was TLR4, which in the presence of an adaptor protein, MD2, recognizes LPS from many different Gram-negative bacteria [5]. Initial binding of LPS occurs via the LPS-binding protein and CD14, which transfer LPS to the TLR4/MD2 signalling complex. Interestingly, TLR4 is also triggered by a potent virulence factor of the Gram-positive pathogen Streptococcus pneumoniae, pneumolysin, which is a cholesterol-dependent cytolysin. The TLR4-dependent recognition of pneumolysin is believed to protect the host against pneumococcal infection [7]. TLR1 is able to form heterodimers with TLR2, resulting in recognition of bacterial tri-acyl lipopeptides, whereas TLR2 forms heterodimers with TLR6, leading to recognition of bacterial di-acyl lipopeptides and lipoproteins. TLR2 has further been shown to be a sensor of pure LTA and PG [5]. Interestingly, recent studies employing a lipoprotein glycerol transferase (lgt) mutant of Staphylococcus aureus, which is unable to carry out lipid modification of pro-lipoproteins, showed that it is the surface lipoproteins, rather than LTA or PG, that trigger activation of TLR2 by this Gram-positive pathogen [8]. Another microbial surface protein agonist for TLR2 is the outer membrane protein A (OmpA) of Klebsiella pneumoniae [9]. OmpA is a highly conserved surface protein in the Enterobacteriaceae family believed to be important for structural cell integrity and virulence [10]. Moreover, some outer membrane proteins, porins, of Neissera meningitidis have been suggested to activate TLR2 on B cells [11]. A recent study indicates that meningococcal porins bind directly to TLR2, but require TLR1 for signalling [12]. TLR5 has been shown to sense flagellin from both Gram-negative and Gram-positive bacteria [5]. Interestingly, the TLR5 recognition site on flagellin has been mapped to a conserved site that is required for bacterial motility [13]. A recent paper identified the region on TLR5 involved in recognition of flagellin to a conserved surface on the extracellular domain [14]. Unlike the other TLRs, TLR5 is not expressed on APCs, but rather on epithelial cells in the airways, intestine and urogenital tract [15], and has been shown to be important for protection against Escherichia coli urinary tract infection [16]. TLR11 is expressed in mice, but not in humans, and has been shown to recognize uropathogenic E. coli [17] and a profilin-like protein from Toxoplasma gondii [18]. TLR3, TLR7, TLR8 and TLR9 all reside in endosomal compartments where they recognize nucleic acids and are believed to be important in antiviral defence [5]. TLR3 recognizes viral double-stranded RNA generated during viral replication, whereas TLR7 and TLR8 recognize viral single-stranded RNA. In addition, TLR7 and TLR8 also recognize imidazoquinoline-like molecules, such as imiquimod (R-837) and resiquimod (R-848). TLR9 mediates recognition of viral and bacterial CpG DNA motifs [5].

CD14

CD14 is a glycosylphosphatidylinositol-linked glycoprotein highly expressed on macrophages shown to function as a co-receptor to TLR4 in response to LPS. Moreover, CD14 has also been suggested to enhance activation of TLR2 in response

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to PG, and to be a receptor for LTA [19, 20]. Other described ligands for CD14 include lipoarabinomannan from Mycobacteria and mannuronic acid from Gramnegative bacteria [21, 22]. Interestingly, CD14 has also been described as a receptor for bacterial tri-acylated lipopeptides, an interaction that facilitates recognition of the lipopeptides by the TLR2/TLR1 complex [23, 24]. Thus, CD14 may act as a co-receptor for the TLR2/TLR1 complex in recognition of bacterial lipoproteins.

Scavenger Receptors

The SRs were initially functionally defined by their ability to bind and endocytose modified forms of low-density lipoproteins (mLDLs), such as oxidised LDL and acetylated LDL, and most studies were devoted to their role in foam cell formation and atherosclerosis. More recently, many SRs have gained interest as PRRs important for the antimicrobial host defence [25]. The SRs are classified into classes A–G on the basis of similarities in the multidomain protein structure [26] (fig. 1).

Class A Scavenger Receptors SR-A is expressed on most macrophage populations and was first implicated as a PRR by the demonstration that it binds to the purified lipid A moiety of LPS from E. coli [27]. In a subsequent study, it was shown that soluble LTA from Streptococcus pyogenes is a ligand for SR-A [28]. Zhu et al. [29] reported that SR-A may be involved in recognition of bacterial CpG DNA. The generation of SR-A knockout mice has enabled the demonstration that SR-A is important for resistance to experimental infection with Gram-positive bacterial pathogens [30, 31], and acts as a phagocytic receptor mediating direct non-opsonic phagocytosis of several bacterial species, including the Gram-negative pathogen N. meningitidis [25, 32]. In addition, a recent study suggests that SR-A may also recognize double-stranded RNA [33], implicating a role for SR-A in antiviral defence. SR-A may also have anti-inflammatory host-protective properties, as proposed by Haworth et al. [34], who showed that SR-A–/– mice primed with BCG (bacillus of Calmette and Guérin) were more susceptible to endotoxic shock compared to their wild-type counterpart, because of an increased secretion of pro-inflammatory cytokines in response to the additional LPS challenge. Moreover, a recent paper suggests that SR-A may also dampen the pro-inflammatory response to the fungal pathogen Pneumocyctis carinii [35]. Macrophage receptor with collagenous structure (MARCO) is another distinct member of the class A SR family implicated as a PRR [36]. In contrast to SR-A, MARCO is constitutively expressed only on some subpopulations of macrophages, but expression of MARCO can be upregulated by bacterial stimulation [37, 38]. MARCO binds to soluble LPS and LTA, CpG DNA, intact Gram-positive and -negative bacteria [19,

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Fig. 1. Schematic structure of different classes of SRs involved in recognition of pathogenic microorganisms. Adapted from Pluddemann et al. [19].

25], and has been shown to contribute to the defence against experimental infection with S. pneumoniae in the mouse model [39]. SCARA-5 (SR-A 5) and SRCL-I/II (SR with C-type lectin) are two recently described class A SRs that have been shown to bind to both Gram-positive and -negative bacteria [19], but the role of these receptors in host defence is still unclear.

Other Scavenger Receptors Two members of the class B SR family, SR-BI and CD36, have been shown to bind to isolated bacterial molecules. SR-BI binds to LPS [19] and CD36 has been suggested to be a sensor of a di-acylated lipopeptide (MALP-2) and LTA, two PAMPs from Grampositive bacteria [40]. Interestingly, Hoebe et al. (2005) also show that CD36 is essential for TLR2 signalling to MALP-2 and LTA, suggesting that CD36 may act as a co-receptor for TLR2 in recognition of microbial diacylglycerides [40]. CD36-deficient mice are more susceptible to infection with S. aureus [40], and a subsequent study showed that

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CD36 acts as a phagocytic receptor for whole S. aureus bacteria, but not for E. coli [41]. Thus, CD36 might act as a PRR for Gram-positive pathogens, but not Gram-negative bacteria. Several other SRs have been shown to bind to whole inactivated bacteria or bacterial molecules, including LOX-1, SREC-I, SR-PSOX, and FEEL-1 [19].

Microbial Protein Ligands for Scavenger Receptors As outlined above, many SRs bind to purified LPS and/or LTA, but it is unclear whether these molecules act as SR ligands on the surface of intact bacteria. Indeed, SR-A-dependent binding and non-opsonic phagocytic uptake of whole N. meningitidis bacteria is independent of surface LPS, as shown employing a lipid A-deficient isogenic mutant of N. meningitidis [32]. Thus, SR-A is dependent on bacterial surface structures distinct from LPS on N. meningitidis for its ability to directly bind and phagocytose these bacteria. Interestingly, a recent study by Peiser et al. [42] identified three conserved meningococcal surface proteins as ligands for SR-A. Moreover, SR-A also binds to a surface lipoprotein from the Gram-positive pathogen Streptococcus agalactiae [Areschoug et al., unpubl. data], suggesting that bacterial surface proteins may be general target molecules for SR-A. In an interesting paper by Jeannin et al. [43], it was shown that the SR-E LOX-1 and the SR-F SREC-I both bind to recombinant outer membrane protein A (OmpA) from K. pneumoniae. Employing an OmpAnegative mutant of E. coli, it was demonstrated that OmpA also acts as a target molecule for LOX-1 and SREC-I on the bacterial surface. As mentioned above, OmpA has been shown to activate APCs via TLR2, but it does not bind to TLR2 [10, 43], suggesting that LOX-1 and SREC-I may act as sensors for OmpA in TLR2 signalling. Thus, LOX-1 and SREC-I may, as described for CD36, act as co-receptors for a TLR2 agonist. In addition to its role in recognition of Gram-positive bacteria, CD36 has also been shown to bind to the Plasmodium falciparum erythrocyte membrane protein-1 on malaria-parasitised red blood cells [44] and to be involved in clearance of infected erythrocytes in vivo [45].

C-Type Lectin Receptors

The term C-type lectin receptors denotes carbohydrate binding molecules that bind their ligands in a Ca2⫹-dependent manner. The classical CLRs contain a hydrophobic fold called the carbohydrate recognition domain responsible for the Ca2⫹-dependent binding to their ligands. However, many CLRs contain carbohydrate recognition domains that do not bind Ca2⫹ and some members have been shown to also bind to non-carbohydrate ligands [46], resulting in the designation C-type lectin domain (CTLD) to denote this common fold among the CLRs without referring to functional similarities. A range of CLRs are expressed on APCs, of which three members, the mannose

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Fibronectin type II C-type lectin domain

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Fig. 2. Schematic structure of CLRs shown to recognize pathogenic microorganisms. Adapted from McGreal et al. [46].

ITAM C

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receptor (MR), dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN), and Dectin-1 (fig. 2), have been extensively characterised with regard to pathogen interactions [46, 47] and therefore will be discussed here.

Mannose Receptor The MR belongs to a subfamily of CLRs with multiple CTLDs sharing the same overall structure, including an N-terminal cystein-rich domain, a fibronectin type II repeat domain, multiple extracellular CTLDs, a transmembrane region and a short cytoplasmic tail. It binds to terminal mannose, fucose, N-acetyl glucosamine or glucose moieties via the CTLD domain and all pathogen interactions reported so far have been shown to occur through this domain [46, 48]. The MR binds to several serum glycoproteins, and has been shown to be important for serum glycoprotein homeostasis. It has also been suggested to play a role in antigen uptake and presentation [48]. The MR was first implicated as a PRR when it was shown that COS-7 cells transfected with human MR cDNA bound and to some extent mediated ingestion of Candida albicans and P. carinii organisms [47, 48]. Subsequent studies have shown that the binding structure for MR on C. albicans is mannan and on P. carinii the main MR target molecule seems to be the surface glycoprotein A [49]. MR has also been reported to interact with two parasites, Trypanosoma cruzi and Leishmania spp. On Trypanosoma amastigotes, the main MR ligand was reported to be surface SA85–1 glycoproteins [50], but the MR-binding structure(s) on Leishmania is still unknown.

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Moreover, the MR has been reported to bind gp120 of HIV-1 [51], an interaction that may facilitate a non-infectious entry of HIV-1 into peripheral and brain macrophages [52]. The MR also binds to purified capsular polysaccharides S. pneumoniae and purified LPS from K. pneumoniae [53], but evidence is lacking that the MR bind to the whole organisms. The MR has been shown to mediate phagocytosis of Mycobacterium tuberculosis via the terminal mannosyl residues of the surface lipoglycan lipoarabinomannan, an interaction suggested to block the phagolysosomal fusion, thereby enhancing survival of these bacteria in the phagosome [54]. However, although several interactions have been described between the MR pathogenic microorganisms, its role in the innate immune defence is unclear. Although there is some evidence that the MR may act as a phagocytic receptor on APCs, studies using MR⫺/⫺ mice have shown that the MR does not seem to be essential for protection against experimental infection with C. albicans, P. carinii or Leishmania [46, 55], but its in vivo role in limiting bacterial infection has not been addressed.

Dendritic Cell-Specific Intercellular Adhesion Molecule-Grabbing Non-Integrin DC-SIGN is a tetrameric endocytic receptor highly expressed on most dendritic cell populations and by subpopulations of macrophages. It has a CTLD, a stalk region, a transmembrane domain and a cytoplasmic tail containing an internalization motif [46]. DC-SIGN was originally discovered as a receptor interacting with ICAM-3 (intracellular adhesion molecule 3), mediating DC-T-cell interactions. It specifically binds to high-mannose structures and fucose-containing glycans via its CTLDs, but also discriminates between carbohydrate ligands via secondary binding sites [46]. DC-SIGN interacts with a wide range of pathogenic microorganisms, including M. tuberculosis, S. pneumoniae, Helicobacter pylori, C. albicans, and Schistosoma mansoni [46, 47]. On M. tuberculosis, DC-SIGN binds to the surface mannose-capped lipoarabinomannan and phosphatidylinositol mannosides [56, 57]. The main target molecule for DC-SIGN on H. pylori seems to be the nonsialylated Lewisx antigen on LPS [58]. DC-SIGN was shown to bind whole S. pneumoniae of capsular serotypes 3 and 14, in contrast to strains representing other serotypes including 19F. In S. pneumoniae type 14, the main DC-SIGN ligand probably is the serotype 14 capsular polysaccharide, but the ligand on S. pneumoniae type 3 is unknown [59]. DC-SIGN also binds to several viral pathogens, including HIV-1 [47]. In the mouse model, it has been shown that SIGNR1, one of five mouse homologues of DC-SIGN, contributes to protection against experimental infection with S. pneumoniae [60], indicating that DC-SIGN may have a similar function in humans. However, other pathogens, such as M. tuberculosis and HIV-1, may exploit DC-SIGN as part of an immune evasion strategy [61]. For example, it appears that mannose-capped lipoarabinomannan can induce the secretion of the anti-inflammatory cytokine IL-10 in a DC-SIGN-dependent manner

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[61], which may inhibit the expression of co-stimulatory molecules required for adaptive immune responses.

Dectin-1 A major advance in the field of CLRs and innate immune recognition was made when dectin-1, the receptor for ␤-glucan, a major constituent of fungal cell walls, was identified [62]. It is a small type II transmembrane receptor with a single extracellular CTLD, a transmembrane region and a cytoplasmic domain with a tyrosine-based activation motif (ITAM) [63]. It specifically binds to ␤-1,3-linked and ␤-1,6-linked glucans and the binding to these carbohydrate structures is Ca2⫹ independent. Dectin-1 promotes the phagocytosis of live yeast and fungal-derived zymosan particles [63], as well as promoting proinflammatory responses to ␤-glucan and whole fungal pathogens by macrophages via co-operation with TLR2 to mediate this response [64, 65]. The ITAM motif in the cytoplasmic tail is required for interactions with the TLR2 signalling pathway. Independently of TLR2, the ITAM-motif is also required for cytoskeletal rearrangements triggered through dectin-1-mediated phagocytosis [66]. The in vivo importance of dectin-1 for the induction of protective immune responses against fungal pathogens was demonstrated in two recent studies employing dectin-1⫺/⫺ mice [67, 68]. Interestingly, a recent study suggests that dectin-1, in co-operation with TLR2, is also important for proinflammatory responses to mycobacteria by macrophages [69]. However, the Dectin-1 ligand on mycobacteria is not known.

Complement Receptor Type 3

CR3 (CD18/CD11b) is a ␤2-integrin that has been shown to be an important phagocytic receptor for particles opsonized with complement, but it has also received attention as a non-opsonic receptor binding directly to pathogens [1]. It has been reported to interact with yeast zymosan, and to mediate non-opsonic binding and phagocytosis of several bacterial species including M. tuberculosis, Pseudomonas aeruginosa, Bordetella pertussis, Coxiella burnetii and Salmonella typhimurium [19, 70]. In M. tuberculosis, two ligands for CR3 have been described. The M. tuberculosis surface protein antigen 85C has been shown to bind to the ␣-chain of CR3 [71]. The 85C antigen is part of a three protein complex anchored in the cell wall of M. tuberculosis. Also, the membrane-proximal region of the CR3 ␣-chain contains ␣-lectin-like domain that binds to oligosaccharides from M. tuberculosis [72]. The main CR3 ligand in B. pertussis may also be a surface protein, namely the filamentous haemagglutinin, a major virulence factor of this Gram-negative pathogen [73]. In P. aeruginosa, the binding is strain dependent and was reported to correlate with variations in LPS structures between strains [74]. The ligand for CR3 on the surface of Salmonella has not been identified.

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Siglecs

The Siglecs are a family of sialic acid-binding receptors belonging to the Ig superfamily. They are type I transmebrane receptors with an amino-terminal V-set immunoglobulin domain that binds sialic acid and variable numbers of C2-set immunoglobulin domains [75]. There are 13 members of the Siglec family identified in humans and most cells in the human immune system express at least one member of the Siglec family. A number of Siglecs are expressed on APCs and many of these contain signalling motifs (ITAMs, ITIMs) in their cytoplasmic tails. They are believed to play an important role in cell–cell interactions, modulating immune responses [75]. A growing body of evidence indicates that the Siglecs also interact with sialic acid on pathogens, such as N. meningitidis, Campylobacter jejuni, S. agalactiae (group B streptococcus) and T. cruzi [76]. There is little knowledge of the biological outcome of these interactions, but it is possible that the Siglecs promote phagocytosis of sialic acid-expressing pathogens by APCs [76].

NOD-Like Receptors

The NLRs represent a family of cytosolic receptors that are important in innate recognition of intracellular bacteria and their breakdown products and many studies indicate a pro-inflammatory role for the NLRs [77]. Each family member contains a C-terminal leucine-rich repeat region required for ligand sensing, a NOD domain, and an N-terminal signalling module, which varies between different molecules in the group and may be caspase-activating and recruitment domain (CARD), pyrin domain (PYD) or baculovirus inhibitor of apoptosis protein repeat (BIR). Activation of NLRs by bacterial products leads to activation of either of two major signalling pathways: the NF-␬B pathway and activation of caspase-1 by the inflammasome [77]. The NOD1 and NOD2 proteins are archetypical members of the NLR family and are sensors of PG. NOD1 is expressed in most cell types and senses PG-derived meso-diaminopimelic acid-containing muramyl peptides, a structural component in the cell wall found only in some Gram-negative bacteria. NOD2 is expressed in monocytes, macrophages, DCs and intestinal epithelial cells. NOD2 has been shown to be involved in sensing of a muramyl dipeptide found in the cell wall PG of both Gram-positive and some Gram-negative bacteria [77]. The importance of the NOD receptors in regulating inflammation is illustrated by the correlation between mutations in the human NOD genes and several autoimmune syndromes, but to date no correlations have been found between mutations in the NOD genes and increased susceptibility to infections. However, studies with nod2–/– mice have shown that NOD2 protects against experimental infection with the intracellular Gram-positive pathogen Listeria monocytogenes when administered orally, but not when administered intravenously, indicating that the main function of NOD2 in anti-bacterial

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immunity might be at the intestinal barrier, not systematically [78]. NALP1 and NALP3 are two other members of the NLR family shown to sense bacterial RNA and PG [77]. The NLR family members NAIP5 and IPAF are both sensors of flagellin [77]. Polymorphisms in the mouse naip5 locus have been shown to result in increased susceptibility to infection with the intracellular Gram-negative pathogen Legionella pneumophilia [79, 80]. NAIP5-dependent sensing of flagellin results in activation of caspase-1 and macrophage cell death. Recognition is independent of TLR5 and MyD88 and requires delivery of flagellin to the cytosol [81–83]. IPAF has also been implicated in TLR5-independent intracellular recognition of flagellin, as shown for NAIP5 [84, 85].

Conclusions

PRRs represent an important first line of defence against pathogenic microorganisms. Although the TLRs have received most attention in this field, it is clear that other types of PRRs, such as SRs, CLRs and NLRs, are also important for innate immune recognition and protection against infection. Interestingly, many studies indicate that PRRs are able to co-operate to achieve an optimal host response. For example, several different PRRs, such as CD14, CD36, Lox-1, SREC-I, have been suggested to act as co-receptors to the TLRs to enhance the cellular response to various TLR ligands. It is also clear that PRRs are able to discriminate between different classes of microorganisms, but the molecular basis for this remains poorly understood. The ligand binding specificity of PRRs is rather broad and a great deal remains to be learned about the relative contribution of various microbial ligands in immune recognition. While many of the non-proteinaceous ligands for PRRs, such as LPS, LTA, PG, ␤-glucan and nucleic acids, are important structures recognized by these receptors, a growing body of evidence shows that microbial proteins are common targets for PRRs (table 1). In particular, several of the TLRs and SRs have been shown to have microbial protein ligands, mostly microbial surface proteins, interactions suggested to be important for both sensing and cellular activation as well as direct non-posonic phagocytosis by PRRs on APCs. However, more research of different receptor-ligand interactions at the molecular level are required to obtain a better understanding of pathogen recognition in innate immunity.

Acknowledgements T.A. is supported by the Swedish Society for Medical Research, the Anna-Greta Crafoord Foundation for Rheumatological Research, the Royal Physiographic Society, and the trusts of PE Lindahl, Lars Hierta, Magnus Bergvall, Alfred Österlund, Emil and Wera Cornell, Crafoord and Goljes Minne. Work in the laboratory of S.G. is supported by the Medical Research Council, UK, and Wellcome trust.

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55

Table 1. Microbial protein/peptide ligands for PRRs Receptor

Microbial protein/peptide ligand

References

TLR1/TLR2

Bacterial triacyl lipopeptides Neisseria meningitidis porins

[5] [11]

TLR2

Bacterial surface lipoproteins Klebsiella pneumoniae OmpA

[5, 8] [9]

TLR2/TLR6

Bacterial diacyl lipopeptides

[5]

TLR4

Streptococcus pneumoniae pneumolysin

[7]

TLR5

Flagellin

[5, 13, 14]

TLR11

Toxoplasma gondii profilin-like protein

[18]

CD14

Bacterial triacyl lipopeptides

[23, 24]

SR-A

N. meningitidis outer membrane proteins

[42]

CD36

Bacterial diacyl lipopeptides Plasmodium falciparum erythrocyte membrane protein-1

[40]

LOX-1

K. pneumoniae OmpA

[43]

SREC-I

K. pneumoniae OmpA

[43]

CR3

Bordetella pertussis filamentous haemagglutinin Mycobacterium tuberculosis surface protein antigen 85C

[73] [71]

NAIP5

Flagellin

[81–83]

IPAF

Flagellin

[84, 85]

References 1

2 3

4

5 6

56

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70 van Bruggen R, Zweers D, van Diepen A, van Dissel JT, Roos D, Verhoeven AJ, Kuijpers TW: Complement receptor 3 and Toll-like receptor 4 act sequentially in uptake and intracellular killing of unopsonized Salmonella enterica serovar Typhimurium by human neutrophils. Infect Immun 2007;75: 2655–2660. 71 Hetland G, Wiker HG: Antigen 85C on Mycobacterium bovis, BCG and M. tuberculosis promotes monocyte-CR3-mediated uptake of microbeads coated with mycobacterial products. Immunology 1994; 82:445–449. 72 Thornton BP, Vetvicka V, Pitman M, Goldman RC, Ross GD: Analysis of the sugar specificity and molecular location of the d-glucan-binding lectin site of complement receptor type 3 (CD11b/CD18). J Immunol 1996;156:1235–1246. 73 Ishibashi Y, Claus S, Relman DA: Bordetella pertussis filamentous hemagglutinin interacts with a leukocyte signal transduction complex and stimulates bacterial adherence to monocyte CR3 (CD11b/ CD18). J Exp Med 1994;180:1225–1233. 74 Pollard AJ, Currie A, Rosenberger CM, Heale JP, Finlay BB, Speert DP: Differential post-transcriptional activation of human phagocytes by different Pseudomonas aeruginosa isolates. Cell Microbiol 2004; 6:639–650. 75 Crocker PR: Siglecs in innate immunity. Curr Opin Pharmacol 2005;5:431–437. 76 Crocker PR, Paulson JC, Varki A: Siglecs and their roles in the immune system. Nat Rev Immunol 2007; 7:255–266. 77 Delbridge LM, O’Riordan MX: Innate recognition of intracellular bacteria. Curr Opin Immunol 2007; 19:10–16. 78 Kobayashi KS, Chamaillard M, Ogura Y, Henegariu O, Inohara N, Nunez G, Flavell RA: Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract. Science 2005;307:731–734. 79 Diez E, Lee SH, Gauthier S, Yaraghi Z, Tremblay M, Vidal S, Gros P: Birc1e is the gene within the Lgn1 locus associated with resistance to Legionella pneumophila. Nat Genet 2003;33:55–60. 80 Wright EK, Goodart SA, Growney JD, Hadinoto V, Endrizzi MG, Long EM, Sadigh K, Abney AL, Bernstein-Hanley I, Dietrich WF: Naip5 affects host susceptibility to the intracellular pathogen Legionella pneumophila. Curr Biol 2003;13:27–36. 81 Molofsky AB, Byrne BG, Whitfield NN, Madigan CA, Fuse ET, Tateda K, Swanson MS: Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection. J Exp Med 2006; 203:1093–1104.

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Dr. Thomas Areschoug Department of Laboratory Medicine, Division of Medical Microbiology Lund University, Sölvegatan 23 SE–22362 Lund (Sweden) Tel. ⫹46 46 173 234, Fax ⫹46 46 189 117, E-Mail [email protected]

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Egesten A, Schmidt A, Herwald H (eds): Trends in Innate Immunity. Contrib Microbiol. Basel, Karger, 2008, vol 15, pp 61–77

Antimicrobial Peptides in Innate Immune Responses Ole E. Sørensena ⭈ Niels Borregaardb ⭈ Alexander M. Colec a

Division of Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden; bDepartment of Hematology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark; cDepartment of Molecular Biology and Microbiology, Burnett School of Biomedical Science, University of Central Florida College of Medicine, Orlando, Fla., USA

Abstract Antimicrobial peptides (AMPs) are ancient effector molecules in the innate immune response of eukaryotes. These peptides are important for the antimicrobial efficacy of phagocytes and for the innate immune response mounted by epithelia of humans and other mammals. AMPs are generated either by de novo synthesis or by proteolytic cleavage from antimicrobially inactive proproteins. Studies of human diseases and animal studies have given important clues to the in vivo role of AMPs. It is now evident that dysregulation of the generation of AMPs in innate immune responses plays a role in certain diseases like Crohn’s disease and atopic dermatitis. AMPs are attractive candidates for development of novel antibiotics due to their in vivo activity profile and some peptides may serve as templates for further drug Copyright © 2008 S. Karger AG, Basel development.

Antimicrobial peptides (AMPs) are important effector molecules in the innate immune system [1, 2]. These peptides have broad-spectrum antimicrobial effects on Gram-positive and Gram-negative bacteria and on some fungi and enveloped viruses. Importantly, AMPs are active against microbes, which are resistant to conventional antibiotics such as methicillin-resistant Staphylococcus aureus. AMPs were discovered as a component of the innate immune defense in insects [3] and were shortly thereafter described in rabbits [4] and humans [5]. So far, AMPs are found in all investigated species from amoebae [6] to man [5]. In mammals, AMPs contribute to the antimicrobial armamentarium of phagocytes, and play a role in microbial clearance from the epithelia and their overlying fluids. This review will focus on the biology of AMPs, the role they play in health and disease, and their use as templates for the development of anti-infective therapies.

Structure and Mode of Action of Antimicrobial Peptides

AMPs are defined generally as proteins that contain fewer than 100 amino acids and exhibit broad-spectrum antimicrobial activity. Nearly all AMPs are cationic and hydrophobic. Despite these common physical properties, AMPs constitute an extremely diverse group of peptides. However, most AMPs can be broadly categorized into the following three groups based largely on structural characteristics. 1 Amphipathic ␣-helical peptides. These are some of the most widely studied AMPs and include LL-37 [7], CRAMP [8] and BMAP-28 [9]. These peptides are found in all species investigated so far, demonstrating the importance of the molecular motif. 2 ␤-Strands stabilized with a variable number of disulfide bonds. These include the cyclic dodecapeptide (one disulfide bond) [10], protegrins (two disulfide bonds) [11], defensins (three disulfide bonds) [5], and hepcidin (four disulfide bonds) [12]. 3 Peptides enriched in one or two amino acids. These include proline- and argininerich peptides (e.g. Bac5 and Bac7) [13], tryptophan-rich peptides (indolicidin) [14], and histidine-rich peptides (histatins) [15, 16]. Most AMPs exert their antimicrobial activity by disrupting bacterial, fungal or viral membranes [17], although other mechanisms of action apart from direct membrane perturbation have been recently demonstrated [17]. It is believed that the cationic charge of the peptides electrostatically attracts them to negatively charged microbial membranes and that the hydrophobic properties enable the peptides subsequently to insert and disrupt membranes [18]. In this rather simplified scenario, the selectivity of AMPs toward bacteria is due to the far more negative charge of these compared to mammalian cell surface membranes. Consequently, it has been shown that reduced negative charge of the membrane lipids in bacteria lead to increased resistance towards AMPs [19]. Many AMPs are also toxic to mammalian cells albeit at higher concentrations than those needed for antimicrobial activity. Several studies have demonstrated that the cytotoxicity of AMPs is linked to their hydrophobic properties [9, 20], which also affords insertion even in eukaryotic cell membranes.

Antimicrobial Peptides in Humans and Other Mammals

In mammals, there are two large families of AMPs, the defensins and the cathelicidins. Defensins are 2- to 6-kDa peptides with six conserved cysteines and three disulfide bridges. According to the size and the arrangement of the disulfide bridges, the majority of defensins are further subdivided into two major subdivisions: ␣-defensins and ␤defensins. In the ␣-defensins, the cysteines are paired in the following configuration: 1–6, 2–4, and 3–5. Humans express six ␣-defensins that are present in azurophil granules of the neutrophils (termed human neutrophil peptides (HNP-1 to HNP-4) [5, 21] and in the Paneth cells of the small intestine (termed human defensin (HD)5 and HD6) [22]. The ␤-defensin structure is slightly different from that of the ␣-defensins,

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a

b

c

Fig. 1. Structure of three defensins. a Solution structure of hBD-3. Sequence: GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK. The disulfide bridges are between C1–C5, C2–C4, C3–C6. The solution structure was obtained by nuclear magnetic resonance diffusion spectrometry [139]. b Solution structure of retrocyclin 2. Sequence: RCICGRRICRCICGRGIC. The solution structure was obtained by homonuclear magnetic resonance spectrometry [155]. The structure shown is solved from noncyclidized peptide. The dots indicate the presumed structure. c Solution structure of plectasin. Sequence: GFG CNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGF VCKCY. The disulfide bridges are between: C1–C4, C2– C5, C3–C6. The solution structure was obtained by nuclear magnetic resonance spectrometry [136].

with the cysteines being paired 1–5, 2–4, and 3–6, and the N-terminus being longer and in an ␣-helical configuration. Human ␤-defensins are secreted by epithelial cells of the integument and mucosal surfaces. There are more than 30 ␤-defensin genes in the human genome [23]; however, at the protein level only hBD-1, hBD-2, hBD-3, and hBD-4 have been found to be expressed in a variety of tissues [24–27]. The ␪-defensins are the most recently discovered third subfamily of defensins, and were first isolated from leukocytes and bone marrow of the rhesus monkey, Macaca mulatta [28–30]. These circular octadecapeptides arise from two precursor peptides, each of which contributes nine residues and three cysteines to form the ␤-sheet structure of a mature ␪-defensin [28–31]. Due to intracellular head-to-tail ligation of the peptide backbone, ␪-defensins represent the first truly macrocyclic peptides of vertebrate origin. Humans do not produce ␪-defensins due to a premature termination codon in the signal peptide [32]. However, due to their remarkable activity against HIV-1, synthetic ␪-defensins serve as a potential template for drug development. The structure of a ␪-defensin and two other defensins are shown in figure 1. Cathelicidins are found mainly in peroxidase-negative granules of mammalian neutrophils [33], but the human cathelicidin is also expressed in various epithelia including the lung, skin and epididymis [34–37]. The cathelicidins share a common

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N-terminal proregion of 12 kDa, named cathelin after a putative cathepsin L inhibitor isolated from porcine neutrophils [38]. While the cathelin proregion is highly conserved amongst members of the cathelicidin family, the active AMPs vary greatly in amino acid structure ranging from proline-arginine-rich peptides to amphipathic ␣-helices and ␤-strand peptides (protegrins) even for cathelicidins within the same species (e.g. pigs and cows). While porcine and bovine neutrophils contain a variety of cathelicidins [33], humans have only one cathelicidin named hCAP-18 (human cationic antimicrobial protein of 18 kDa)/LL-37 [39–41]. Humans and other mammals contain a variety of AMPs that are not members of either the defensin or cathelicidin family; these include the histidine-rich histatins present in saliva [15, 16], calgranulins (MRP8/MRP14) in epithelial cells and cytosol of neutrophil [42], psoriasin in the skin [43], and peptides generated during activation of the complement system [44] or coagulation cascade [45].

Induced Expression of Antimicrobial Peptides

AMPs are generated at sites of inflammation/infection or wound healing by induced de novo synthesis from epithelial cells, e.g. hBD-2 and hBD-3, or monocytes/macrophages, e.g. hCAP-18/LL37. Some AMPs such as the human peptides, hBD-1 [46] and psoriasin [43], appear to be constitutively expressed by epithelial cells, while the expression of other peptides, including hBD-2, hBD-3 and hCAP-18/LL-37, are induced during inflammation and wound healing [34, 47–51]. Accordingly, the inflamed skin of psoriasis patients has been a valuable source for identification of human AMPs [52]. Indeed, some of the most widely studied human AMPs, hBD-2 and hBD-3, were first isolated from psoriatic scales [25, 26]. In epithelial cells, AMP expression can be induced after direct contact with microbes or microbe-derived molecules – at least experimentally [53–57]. The expression can be induced by signaling through Toll-like receptors [56, 58]; bacterial proteases can activate protease-activated receptors (PARs) that in turn mediate AMP expression [59], or bacteria can activate the epidermal growth factor receptor (EGFR), which leads to expression of hBD-3 [60]. However, healthy, intact noninflamed epithelia like the skin or the intestinal epithelia are continuously exposed to microbes yet express only scant or no AMPs during nonpathological conditions. Consequently, other studies have found that AMPs are not produced in significant amount by direct microbial stimuli [48]. Instead, other cells associated with the epithelia such as Langerhans cells of the skin and alveolar macrophages in the lung respond to microbial stimuli by secretion of various inflammatory mediators such as IL-1. These mediators then induce the expression of AMPs in epithelial cells [48, 49, 61]. Interestingly, different inflammatory mediators induce different AMPs [49]. Accordingly, to elicit AMP expression in epithelia, the bacteria must first cause an inflammatory response or damage to the epithelial lining to gain access to

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associated monocyte-derived cells like, for example, Langerhans cells in the skin or alveolar macrophages in the lung. In human keratinocytes, AMP expression can be induced by growth factors as well as by certain proinflammatory cytokines [62]. After wounding of the skin or sterile injury to keratinocyte cultures, membrane-bound ligands of the EGFR are released from the surface of keratinocytes. These released growth factors then bind and activate EGFR [63]. This process has recently been demonstrated to induce the expression of AMPs in the human skin at antimicrobially relevant concentrations [50]. Thus, apart from microbial stimuli, simply damaging the epithelial lining is sufficient to induce the expression of AMPs in the skin. While the intestinal epithelia can also respond to EGFR activation by producing AMPs, it remains to be seen whether this is a global response of all epithelia. In 1903, Dr. Niels Finsen received the Nobel Prize for his observation that sunlight and light therapy can cure cutaneous tuberculosis. However, the molecular basis for this observation has remained obscure until recently. It has now been found that ultraviolet light (UVB) induces AMP expression in the human skin [64]. Active vitamin D is formed from a precursor in the skin by UV radiation. Starting with Wang et al. [65], several studies have now shown that vitamin D plays a role in the expression of the human cathelicidin, hCAP-18/LL-37, in both epithelial cells, such as keratinocytes, as well as in monocytes/macrophages [66–68]. Moreover, the historic observation that vitamin D mediates suppression of mycobacteria in macrophages/ monocytes has now been directly linked to the vitamin D-mediated expression of hCAP-18/LL-37 [69–71].

Generation of Antimicrobial Peptides by Proteolytic Cleavage

AMPs can also be present in bodily fluids or stored in granules of epithelial cells/neutrophils as inactive proproteins. The AMPs then become antimicrobially active upon proteolytic cleavage from the propiece, representing a rapid way of generating AMPs on demand. In neutrophils, this is exemplified by the cathelicidins present in the peroxidase-negative granules of neutrophils [72–74]. Following degranulation either to the phagolysosome [75] or to the extracellular milieu [75–77], the cathelicidin proforms are processed into active AMPs after exposure to neutrophil elastase or proteinase 3 from azurophil granules [76–78]. Likewise, the ␣-defensins in the human Paneth cells of the small intestine are stored as inactive prodefensins, and are released from the Paneth cells upon microbial stimulation [79]. The prodefensins are then processed by matrilysin [80] in mice and by trypsin [81] in humans to generate the active defensins. It should be noted that in mice the ␣-defensins are stored as active peptides in Paneth cells, while in humans it is thought that the proteolytic cleavage occurs extracellularly. In bodily fluids, hCAP-18/LL-37 is present in abundant amounts in seminal plasma as an inactive proprotein [37]. After sexual intercourse, seminal plasma is

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acidified in the vagina, which activates the enzyme gastricsin to proteolytically process the hCAP-18/LL-37 precursor to an active AMP in the vagina [82]. Interestingly, this proteolytic generation of AMPs in the vagina is independent of the presence of infection. However, sexual intercourse represents a risk situation for subsequent infection due to the risk of barrier disruption in vagina or sexually transmitted disease. The generation of AMPs following exposure of seminal plasma to the vaginal milieu is a likely mechanism by which the host can limit infection following sexual intercourse. In human plasma, classical catalytic cascades actually lead to proteolytic generation of AMPs from circulating proproteins. The complement system is an ancient innate immune response to combat infection. The differing complement activation pathways converge in the cleavage of C3. It has now been demonstrated that the cleavage of C3 leads to production of C3-derived AMPs [44]. Furthermore, in plasma the contact system of coagulation can be activated by infection [83]. Activation of the contact system generates AMPs of importance in vivo [45]. The functional importance of proteolytic generation of AMPs during infection has been further demonstrated animal studies. In mice lacking matrilysin, the prodefensins from the Paneth cells are not processed to active defensins [80]. The release of the nonprocessed and inactive prodefensins leads to decreased antimicrobial activity of intestinal preparations in vitro and increased susceptibility to intestinal infections in vivo [80]. The porcine cathelicidins, the protegrins, are very potent AMPs [84]. Active protegrins are generated by extracellular cleavage with elastase [76], and this cleavage generates potent antimicrobial activity of porcine inflammatory fluid [85]. In vivo, the inhibition of processing of the porcine neutrophils cathelicidins inhibits clearance of bacteria from skin wounds [86].

Antimicrobial Chemokines and Chemotactic Antimicrobial Peptides

Certain AMPs exhibit other functions aside from antimicrobial activity, including chemotactic and chemokine-like effects [87–90]. AMPs can boost innate immunity by binding to TLRs [91], modulate chemokine and chemokine receptor expression in macrophages [92], and enhance acquired immunity by acting as adjuvants [93]. Interestingly, it is possible to structurally dissociate the immunostimulatory activities from the antimicrobial properties of AMPs [94]. Certain chemokines have prominent antimicrobial activity and may reach antimicrobial concentrations during inflammation [95]. Indeed, in a model of inflamed pharyngeal epithelia, the chemokine CXCL9/MIG was shown to be a major contributor to the antibacterial activity generated following infection with Streptococcus pyogenes [96]. Chemokine activity is often exhibited at nanomolar concentrations, while antimicrobial activity is generally exhibited in the micromolar range. Thus, far from all AMPs present in humans will reach physiologic levels sufficient for antimicrobial activity.

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However, even though the levels of an AMP in vivo may be insufficient for antimicrobial activity, they may very well be sufficient for chemokine activity. Conversely, certain chemokines like CXCL9 will reach levels beyond what is necessary for chemokine activity and even reach antimicrobial levels [96]. It is therefore plausible that some chemokines may have their major role as AMPs and some AMPs may have their major functions as chemokines.

Importance of Antimicrobial Peptides in Health and Disease

Starting with the observation that lactoferrin and lysozyme have synergistic antibacterial effect [97], many AMPs have been found to have synergistic antibacterial effects including defensins and cathelicidins [98]. It is important to note that, in vivo, no AMP exists alone but rather as a cocktail of multiple AMPs or antimicrobial proteins, each with differing and sometimes overlapping spectra of activity. While it is possible in some cases (e.g. human airway fluid) to pinpoint the antibacterial activity to a single or few molecules [99], in other cases the antimicrobial activity of a body fluid depends on the aggregate of all AMPs present (e.g. anti-HIV activity of human vaginal fluid [100]). Experimental data suggest that synergy may lower the amount of a single peptide needed to contribute to antimicrobial activity about tenfold compared to the activity of the peptide alone [98]. However, since a single AMP needs to be present in micromolar concentrations to exert antimicrobial activity, it reasons that a substantial amount of peptide needs to present for activity (and importance) in vivo even when synergy is taken into consideration. While animal models give important clues to the possible role of AMPs, extrapolation to the human situation must be done with caution. While defensins are found in large quantities in human neutrophils, they are absent in mouse neutrophils [101]. Thus mice can be viewed as a natural knockout for neutrophil defensins. In human neutrophils, the ␣-defensins comprise 5% of the total protein in neutrophils and around 30–50% of the total protein in the azurophil granules [5]. Additional nonprocessed prodefensins are found in the specific granules [102]. Simply due to the sheer quantity in human neutrophils, it is difficult to imagine that these molecules do not play a significant role in host defense. Mice have at least 17 ␣-defensins (cryptdins) in the Paneth cells of the small intestine [103] but humans have only two, HD5 and HD6 [104, 105]. Examining the ␤-defensins, it is not totally clear which mouse ␤-defensins are the functional correlates to the intensively studied human peptides, hBD-2 and hBD-3, which are expressed in substantial quantities in human skin [25, 26]. Due to conserved primary sequence, mBD-14 has been suggested as the murine ortholog to hBD-3. However, mBD-14 has only been described on the genomic level, no mRNA or peptide has been found in mice in vivo. This makes it difficult to ascertain the role of individual human AMPs by knockout experiments. Furthermore, since there is a functional redundancy of different AMPs and antimicrobial proteins

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in bodily fluids and sites of infection, the exclusion of just one may not have a great impact in an experimental setting in vivo. Clues to the biologic importance of AMPs have been found by examining the role of AMPs in certain human diseases, and by experiments in animal models. Mice deficient in cathelicidin have increased susceptibility to necrotic skin infections caused by S. pyogenes [106] and keratitis caused by Pseudomonas aeruginosa [107]. Knockout mice for mBD-1 have reduced bacterial clearance from the lung and bladder [108, 109]. Conversely, mice overexpressing AMPs have increased bacterial clearance or resistance to bacterial infection at epithelial sites like the lung [110], intestine [111], and skin [112]. Patients with Kostmann’s disease [113] and with specific granule deficiency [114, 115] have been reported to have deficient expression of AMPs in neutrophils. However, in both of these instances, the AMP deficiency is only a part of a greater defect in neutrophil function and differentiation, and consequently it is difficult to ascertain the exact role of the AMP deficiency. A clue for the importance of AMPs in humans has come from studies of two common skin diseases, psoriasis and atopic dermatitis. Patients with atopic dermatitis are much more prone to bacterial infections and this difference has been attributed to a reduced expression of AMPs in atopic dermatitis compared to psoriasis [116–120]. Crohn’s disease is a patchy inflammatory disease of the intestine commonly located in the terminal ileum and in the colon. The disease is presumed to be trigged by intestinal microbes in genetically disposed individuals [121] – those individuals with mutations in nucleotide-binding oligomerization domain 2 (NOD2) [122, 123]. NOD2-deficient mice have both increased susceptibility to intestinal infections and decreased expression of ␣-defensins in the Paneth cells [124]. In humans, NOD2 mutations in Crohn’s disease are associated with diminished expression of ␣defensins [125] and patients with Crohn’s disease of the ileum were found to have reduced antibacterial activity of ileal mucosal extracts that was coupled to the decreased levels of intestinal ␣-defensins [126]. Additionally, in Crohn’s disease of the colon, in vitro experiments have demonstrated decreased antibacterial activity of colonic mucosal extracts independent of the inflammatory status [127]. Though the molecular background for this reduced antimicrobial activity was not identified, it is noteworthy that inflammation upregulates AMP expression in the other major inflammatory bowel disease, ulcerative colitis, but not in Crohn’s disease [128, 129]. The interplay between AMPs and bacteria in Crohn’s disease is likely very complex since certain bacteria can reduce the expression of AMPs in the intestine [130]. Another example of the intriguing relationship between microbes and expression of AMPs is in the vagina. Bacterial vaginosis is the most prevalent polymicrobial condition of women of reproductive age, and this condition is associated with increased risk of sexually transmitted infection [131, 132]. In normal circumstances, AMPs contribute to the antimicrobial activity of vaginal fluid [133], but in bacterial vaginosis the vaginal fluid is deficient of AMPs [134]. This deficiency was found to be corrected by antibiotic treatment suggesting that the low levels of AMPs were caused by the disease [134].

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Antimicrobial Peptides as Models for Novel Antiinfectious Therapies

AMPs are attractive candidates for development of novel antibiotics due to activity towards microbes resistant against conventional antibiotics. This activity is found even after culturing bacteria for multiple passages with subinhibitory concentrations of peptide [135]. One approach to search for suitable candidates is by screening nature’s library of suitable AMPs as done with plectasin, an AMP with therapeutic potential found in a saprophytic fungus [136]. As reviewed elsewhere, it is possible to modulate existing AMPs to obtain the desired antimicrobial properties [137]. Another approach would be to take an AMP and modulate it to obtain the desired antimicrobial properties. An example of such a suitable human AMP is the ␪defensin retrocyclin active against HIV-1 [32]. Humans unlikely produce mature ␪defensin peptides due to a premature termination codon in the putative signal sequence, which should prematurely arrest translation [32]. Human bone marrow expresses two different mRNA transcripts homologous to rhesus monkey circular ␪defensins [32]. These ␪-defensin transcripts have been detected in human bone marrow, spleen, thymus, testis, and skeletal muscle [138], and in cervicovaginal epithelia [Cole, unpubl. results]. To date, researchers have not been successful in isolating ␪defensin peptides from humans. Instead, these circular 18-residue peptides were resurrected by solid-phase synthesis and termed retrocyclins. The prefix ‘retro-’ was derived from the Latin for back or backward, and the suffix ‘-cyclin’ signified the macrocyclic nature of the molecule. Retrocyclins could potently protect CD4⫹ primary T cells and lymphocytic cell lines from in vitro infection by both X4 and R5 strains of HIV-1 [32]. Moreover, retrocyclins were appreciably much more active in vitro than their human ␣- and ␤defensin brethren. Synthetic analogs of retrocyclin, including the highly active congener RC-101, could remarkably protect CD4⫹ T cells against infection by clinical isolates of HIV-1 from a number of different clades [139]. Retrocyclins prevented the entry of HIV-1 by binding the heptad repeat 2 region of gp41, which precluded viral fusion by preventing six-helix bundle formation [140]. It is notable that analog RC101 induced very little resistance in HIV-1 [141], which came at a price to HIV-1: reduced viral fusion efficiency and dependence on RC-101 for cellular entry. Recently, retrocyclins have demonstrated promise as topically applied drugs to prevent the heterosexual transmission of HIV-1. RC-101 and other retrocyclin analogs were not cytotoxic or proinflammatory to cervicovaginal tissues [32, 142], completely retained their anti-HIV-1 activity in the presence of human vaginal fluid [142], and prevented organotypic cervicovaginal tissues from HIV-1 BaL infection [142]. In addition, retrocyclins exhibited little to no hemolytic activity nor cytotoxicity against H9 cells and ME-180 cervical carcinoma cells at up to 500 ␮g/ml (the highest concentration tested), an amount that is significantly greater than the concentration required for complete protection against most strains of HIV-1 (2–10 ␮g/ml). The ability of retrocyclins to inhibit entry of herpes simplex virus types 1 and 2 infections

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[143], influenza virus, Sindbis virus, and baculovirus [144] suggests that these peptides might be broad-spectrum inhibitors of viral pathogens. While we may never realize whether human susceptibility to HIV-1 infection is a direct result of the evolutionary loss of retrocyclin peptides, retrocyclin-based microbicides that can be applied topically to augment the natural antiretroviral activity of human cervicovaginal fluid would be important in reducing HIV-1 transmission.

Concluding Remarks and Future Perspectives

A substantial amount of work has been done to identify novel AMPs. To understand the role of AMPs both in the innate immune response and in chronic disease states, we need to delineate the importance of individual AMPs and to identify the disease states where AMPs are important. This requires a concerted effort to design and implement functional studies that will elucidate the true biologic function of these peptides. Since there are – in some instances considerable – differences between AMPs of mice and men, studies will need to be performed in a setting as close to the human in vivo setting as possible. Knockout mice make it possible to focus on the role of individual molecules. This approach cannot stand alone, and for many if not most AMP, would not serve adequately to represent the human condition. In addition, significant antimicrobial activity generated in bodily fluids or tissues both in healthy tissues and in disease states must be identified. Then it is possible to work ‘backwards’ to identify the molecules responsible for antimicrobial activity as for example done in the skin [43] and airway liquid fluid [99]. Delineating the role of single AMPs in humans can give clues to gene polymorphisms of AMPs responsible for altered susceptibility to infection. Though copy number polymorphisms are not common amongst innate immunity genes [145], there exists a substantial number of copy number polymorphisms within the defensin gene clusters, which can be directly correlated to the concentration of defensin peptide produced [146]. Along the same vein, genomic variations of the gene for hBD-1 have been demonstrated to be of importance in the outcome of sepsis [147]. Other gene polymorphism of AMPs with importance for susceptibility to infection will surely be identified. AMPs have shown to be versatile molecules with many functions in bridging the innate immune response and the adaptive immune system. A recent study demonstrated elegantly that LL-37 in psoriatic skin enabled dendritic cells to sense self-DNA through TLR-9 signaling, a process that could drive an autoimmune response [148]. Apart from the obvious significance of this finding, it also speaks towards the obvious DNA-binding properties of the cationic AMPs previously described [149, 150]. This affinity for DNA may have important implications for the antimicrobial function of neutrophil AMPs. Neutrophils are capable of elaborating extracellular traps called neutrophil extracellular traps (NETs) consisting of granule proteins and DNA [151]. The long polymers of DNA trap the bacteria that are killed by the granule and histone

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proteins. The NETs are made by the dying activated neutrophil [152] and are important for the killing of bacteria both in vitro [151] and in vivo [153, 154]. Future studies will hopefully elucidate both the importance of neutrophil AMPs for the function of NETs and the importance of NETs for the antimicrobial actions of neutrophil AMPs. Many studies have elucidated both how AMPs kill bacteria in vitro and the in vivo importance of AMPs. The recent discovery of NETs demonstrates that AMPs may turn out to be important in quite other settings than previously imagined. To fully understand the role of AMPs in the innate immune response and in diseases, the more exact situations and circumstances in which AMPs play a role in vivo must be carefully delineated.

Acknowledgments This work has been supported by grants from Novo Nordisk Fonden and the Swedish Research Council to O.E.S., The Danish Medical Research Council to N.B., and the National Institutes of Health: AI052017, AI065430, and AI060753 to A.M.C.

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145 Linzmeier RM, Ganz T: Copy number polymorphisms are not a common feature of innate immune genes. Genomics 2006;88:122–126. 146 Linzmeier RM, Ganz T: Human defensin gene copy number polymorphisms: Comprehensive analysis of independent variation in alpha- and beta-defensin regions at 8p22-p23. Genomics 2005;86:423–430. 147 Chen QX, Lv C, Huang LX, Cheng BL, Xie GH, Wu SJ, Fang XM: Genomic variations within DEFB1 are associated with the susceptibility to and the fatal outcome of severe sepsis in Chinese Han population. Genes Immun 2007;8:439–443. 148 Lande R, Gregorio J, Facchinetti V, Chatterjee B, Wang YH, Homey B, Cao W, Wang YH, Su B, Nestle FO, Zal T, Mellman I, Schroder JM, Liu YJ, Gilliet M: Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature 2007;449:564–569. 149 Sandgren S, Wittrup A, Cheng F, Jonsson M, Eklund E, Busch S, Belting M: The human antimicrobial peptide LL-37 transfers extracellular DNA plasmid to the nuclear compartment of mammalian cells via lipid rafts and proteoglycan-dependent endocytosis. J Biol Chem 2004;279:17951–17956. 150 Dufourcq J, Neri W, Henry-Toulme N: Molecular assembling of DNA with amphipathic peptides. FEBS Lett 1998;421:7–11. 151 Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, Zychlinsky A: Neutrophil extracellular traps kill bacteria. Science 2004;303:1532–1535. 152 Fuchs TA, Abed U, Goosmann C, Hurwitz R, Schulze I, Wahn V, Weinrauch Y, Brinkmann V, Zychlinsky A: Novel cell death program leads to neutrophil extracellular traps. J Cell Biol 2007;176:231–241. 153 Buchanan JT, Simpson AJ, Aziz RK, Liu GY, Kristian SA, Kotb M, Feramisco J, Nizet V: DNase expression allows the pathogen group A streptococcus to escape killing in neutrophil extracellular traps. Curr Biol 2006;16:396–400. 154 Sumby P, Barbian KD, Gardner DJ, Whitney AR, Welty DM, Long RD, Bailey JR, Parnell MJ, Hoe NP, Adams GG, DeLeo FR, Musser JM: Extracellular deoxyribonuclease made by group A Streptococcus assists pathogenesis by enhancing evasion of the innate immune response. Proc Natl Acad Sci USA 2005;102:1679–1684. 155 Daly NL, Chen YK, Rosengren KJ, Marx UC, Phillips ML, Waring AJ, Wang W, Lehrer RI, Craik DJ: Retrocyclin-2:Structural analysis of a potent anti-HIV thetadefensin. Biochemistry 2007;46:9920–9928.

Ole E. Sørensen, MD, PhD Division of Infection Medicine, Department of Clinical Sciences, Lund University Biomedical Center B14, Tornavägen 10 SE-221 84 Lund (Sweden) Tel. ⫹46 46 222 4472, Fax ⫹46 46 15 7756, E-Mail [email protected]

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Complement: An Efficient Sword of Innate Immunity Günter Rambach ⭈ Reinhard Würzner ⭈ Cornelia Speth Division of Hygiene and Medical Microbiology, Department of Hygiene, Microbiology and Social Medicine, Innsbruck Medical University, Innsbruck, Austria

Abstract Complement is vital for protecting individuals against pathogens and any disturbance of homeostasis associated with appearance of foreign antigens. Four antenna molecules seek for putative danger and subsequently start three activation pathways to eliminate the hostile triggering signal. To achieve this mission the complement arsenal contains soluble plasma factors as well as membrane-bound receptor molecules. Fulfilling a broad spectrum of biological functions, complement participates to construct and orchestrate an immunological network with extensive links to other elements of innate immunity, but also to its younger brother, the adaptive immune system. The body generously supports the complement activity with a high level of complement production; not only the liver as ‘the capital of complement expression’but also decentralized synthesis sites guarantee its all-over presence. On the other hand, it is of fundamental interest for the organism to limit this powerful immunological regiment by establishing a tight surveillance composed of redundantly acting regulator molecules. To find the appropriate dimension of complement activity is critical, as shown by the spectrum of diseases associated with an excess or a lack. Numerous therapeutic approaches aim to correct such an imbalance and to re-establish the antimicrobial capacity of complement without Copyright © 2008 S. Karger AG, Basel induction of chronic inflammation and autoimmunity.

General Overview: Complement Components and Functions

Complement (C) is a phylogenetically ancient system of multicellular organisms, fulfilling a dual role by being a central part of innate immunity and by bridging innate and adaptive immune defense (fig. 1). It comprises more than 35 proteins, including soluble plasma factors, cell-associated regulator molecules and receptors (table 1). Complement activation starts within seconds when pathogen-associated molecular patterns (PAMPs) or foreign cellular structures (e.g. cell debris, non-self tissue) get in contact with the appropriate sensor molecules of the complement system, which represent surface-bound or soluble pattern recognition receptors (PRRs). The nature of the molecular patterns and the corresponding receptors differs between the three activation pathways of the complement cascade: the classical pathway is triggered

INPUT: Sensing of disturbed homeostasis

Apoptotic cells/ tumor cells

Transplanted organs

Microbial infection

Antigen/antibody complexes

OUTPUT: Processes to restore homeostasis

Complement cascade Antimicrobial attack Cytotoxicity

Clearance

(opsonization for phagocytosis, direct lysis)

(immune complexes, cell debris)

Cell activation (stimulation of signaling pathways, degranulation of mast cells)

Stimulation of immune network (humoral immunity, T cell response, cytokines, chemotaxis, inflammation)

Fig. 1. Overview. Inducers of complement activation and complement effector functions.

mainly by antigen-antibody complexes, the lectin pathway is stimulated by carbohydrate structures acting as PAMPs, and the PRRs of the alternative pathway recognize other non-self surface structures (fig. 2). All these pathways merge in the enzymatic activation of C3 and C5. In the subsequent common terminal pathway, further complement components are activated in a nonproteolytic manner and assemble into the membrane attack complex (MAC). The entire cascade is controlled redundantly by an arsenal of regulatory factors [1]. Complement is a complex machinery sensing danger and disturbed homeostasis of the body. Its sensors are the starting molecules of the three different pathways and react on the presence of microbial infection, apoptotic or transformed cells, non-self tissue after organ transplantation and antigen-antibody complexes (fig. 1). The output of the complement cascade are diverse effector mechanisms aiming to eliminate pathogens and restore body homeostasis: a Clearance of immune complexes from the circulation and of cell debris from injured tissue; b Cell activation via binding of complement factors to corresponding receptors with subsequent stimulation of intracellular signaling pathways or degranulation of mast cells;

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Table 1. Complement components Component

Main function(s)

Classical pathway C1q C1r C1s C4 C2 C3

Lectin pathway MBL M-ficolin (ficolin-1) L-ficolin (ficolin-2, ficolin/p35) H-ficolin (ficolin-3, Hakata antigen) MASP-1 MASP-2 MASP-3

Binding to Fc domain of antigen-bound IgG and IgMantibodies; binding of bacterial lipopolysaccharides, myelin, CRP, viral proteins Serine protease, associated with C1q; activation of C1s Serine protease, associated with C1q; cleavage of C4 and C2 Fragment C4b: part of the C3/C5 convertases of classical and lectin pathway; fragment C4a: anaphylatoxin Fragment C2a: serine protease, active component of the C3/C5 convertases of classical and lectin pathway; fragment C2b: kinin activity Fragment C3b: starter molecule of the alternative pathway; part of the C3/C5 convertases of alternative pathway; part of C5 convertase of classical and lectin pathway; opsonization; fragment C3a: anaphylatoxin Serum lectin; binding to foreign carbohydrates structures Nonserum lectin; binding to foreign carbohydrate structures Serum lectin; binding to foreign carbohydrates structures Serum lectin; binding to foreign carbohydrates structures Serine protease, associated with MBL or ficolins; cleavage of C2 and C3, but not C4 Serine protease, associated with MBL or ficolins; cleavage of C2 and C4 Serine protease, associated with MBL or ficolins; function unclear

Alternative pathway Factor B (B) Factor D (D) Properdin (P)

Fragment Bb: part of the C3/C5 convertases of alternative pathway Plasmatic serine protease; cleavage of C3b-bound factor B Stabilization of C3/C5 convertases of alternative pathway

Terminal pathway C5 C6 C7 C8 C9

Fragment C5b: binding of C6; fragment C5a: anaphylatoxin Binding of C7 Incorporation of C5b-C7 complex into target membrane via hydrophobic domain Binding to C5b-7 complex; induction of C9 polymerization Polymerization and formation of the MAC

Complement receptors (CRs) C1qRp (CD93) cC1qR (calreticulin) gC1qR ␣2␤1 integrin CR1 (CD35) CR2 (CD21) CR3 (CD11b/CD18) CR4 (CD11c/CD18) CRIg

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Phagocytosis Phagocytosis; induction of respiratory burst; removal of apoptotic cells Chemotaxis Costimulator for mast cell activation and cytokine secretion Binding of C3b and C4b; transport and clearance of immune complexes, phagocytosis Binding of C3d, C3dg, iC3b, CD23, IFN-␣, EBV; enhancement of humoral immunity by B cell activation Binding of iC3b, ICAM-1, LPS, fibrinogen, clotting factor X, microbial proteins; phagocytosis, cell adhesion, signal transduction, oxidative burst Binding of iC3b, fibrinogen; phagocytosis, cell adhesion Binding of C3b, iC3b, C3c; phagocytosis, inhibition of T cell activation

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Table 1. (continued) Component

Main function(s)

C3aR

Binding of C3a; proinflammatory activity with chemotaxis, release of inflammatory mediators (histamine, cytokines), cell activation, increase in vascular permeability Binding of C5a; proinflammatory activity with chemotaxis, release of inflammatory mediators (histamine, cytokines), cell activation, increase of vascular permeability Binding of C5a; stimulation of triglyceride synthesis, limitation of proinflammatory response

C5aR (CD88) C5L2

Regulatory proteins (fluid phase) C1-INH Covalent binding to and inhibition of active C1s, C1r, MASPs fI Serine protease; cleavage and inactivation of C3b and C4b C4bp Cofactor for factor I-mediated degradation of C4b; acceleration of decay of C3/C5 convertases of classical and lectin pathway fH Cofactor for factor I-mediated cleavage of C3b; decay acceleration of all C3/C5 convertases Properdin (P) Stabilization of C3/C5 convertases of the alternative pathway S protein (vitronectin) Block of C5b-C7 membrane binding; prevention of C9 polymerization Clusterin (SP-40,40) Binding to C5b-C7 complex to prevent membrane attachment; inhibition of C9 assembly Carboxypeptidase N (CPN) Inactivation of C3a, C5a by cleavage of carboxy-terminal arginine Regulatory proteins (membrane bound) Membrane cofactor Cofactor for factor I-mediated cleavage of C3b and C4b protein (MCP, CD46) Decay accelerating factor Prevention of assembly and promotion of decay of C3/C5 convertases (DAF, CD55) CR1 (CD35) Prevention of assembly and promotion of decay of C3/C5 convertases; cofactor for factor I-mediated cleavage of C3b and C4b CRIg Inhibition of C3/C5 convertases of alternative pathway by interference of substrate binding Protectin (CD59) Inhibition of formation of MAC by binding C8 and C9 MASP ⫽ MBL-associated serine protease; EBV ⫽ Epstein-Barr virus; CRs ⫽ complement receptors.

c Stimulation of the immune network and thus linkage of elements of innate and adaptive immunity; complement supports the development of antibody response and B cell memory; furthermore, it helps to induce and promote an alloreactive T cell response; complement-derived anaphylatoxins attract immune cells and thus recruit phagocytes, stimulate cytokine production and enhance inflammation and histamine release; d Direct antimicrobial attack and cytotoxicity results in opsonization (i.e. immunelabeling) of the microbial or cellular surface by complement fragments and stimulates phagocytosis via complement receptors (CRs) on phagocytic cells; subsequent presentation of phagocytosed material on the cell surface will further initiate adaptive immune responses; control of infection is also aspired by direct lysis of the microbe and of infected cells via integration of a lytic pore formed by the MAC into

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CLASSICAL

LECTIN

ALTERNATIVE

Immunoglobulins bound to antigen

Microbial carbohydrates

C3b amplification on activator surface

C1q

MBL/ficolins

C3b

Associated proteases

Associated proteases

B Ba

C4b ⫹ C4a

C4

D

P

C2 C2b

C3 convertase C3b,Bb,P

C3 convertase C4b,2a

C3

C3b⫹C3a

C5 convertase C4b,2a,3b

C5 convertase (C3b)2,Bb,P

C5

C5b⫹C5a

T E R

C6 C7 Path

MAC

oge

C8 (C9)n

n

M I N A

Microbial lysis

MAC

L

Fig. 2. Overview of the complement cascade with the three activation pathways and the terminal pathway resulting in microbial lysis (detailed explanation in the text).

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the membrane. Complement-dependent opsonization and lysis also affects allogenic transplants and transformed tumor cells. The majority of plasma complement components are synthesized by hepatocytes in the liver as the main site of complement synthesis [2]. In addition, extrahepatic complement production in many cell types and organs enables an overall presence of complement, e.g. behind an intact blood brain barrier, and allows an effective upregulation of local complement synthesis at sites of infection [3]. Cell surface-bound complement regulators are expressed in a broad variety of tissues [2].

How to Fulfill Complement Functions: Activation, Cascade Steps and Receptors

Three triggering pathways to start the complement cascade have been identified as yet, which differ in their initiating mechanisms, but all merge into the cleavage step of the central complement protein C3 (fig. 2).

The Classical Pathway The starter molecule of this pathway is C1, a complex of the tulip bundle-like C1q molecule with the proenzymes C1r and C1s (fig. 3). The interaction of the globular ‘heads’ of C1q with the Fc domains of antigen-bound immunoglobulins (IgG or IgM) acting as PAMPs is the main initiating event. Moreover, bacterial lipopolysaccharide, polyanionic compounds, myelin, C-reactive protein (CRP), viral envelopes (e.g. of human immunodeficiency virus; HIV-1) and apoptotic cells can act as antibody-independent triggers. The binding of C1q to a ligand results in a conformational change leading to the sequential activation of associated C1r and C1s. In the next step, the potent serine protease C1s cleaves the C4 protein; the exposure of a reactive thioester group located within the bigger fragment C4b enables the covalent binding to the surface of the pathogen. The following attachment of C2 to C4b permits the C1s-driven proteolysis into the smaller fragment C2b (which drifts away) and the larger C2a that acts as the proteolytic component of the newly formed dimer C4b,2a. This complex is termed the classical pathway C3 convertase and mediates the proteolytic cleavage of C3 into the anaphylactic peptide C3a and the large C3b fragment. By binding to C3aR the anaphylaxotin C3a is spasmogenic, stimulates the release of prostaglandin from macrophages, induces degranulation of eosinophils, exerts a chemotactic activity towards a variety of cell types, and exhibits proinflammatory characteristics [1]. C3b participates in the further steps of the complement cascade. It is a rather instable molecule and is quickly inactivated by factor I and its regulatory cofactors (see below). The resulting iC3b however still harbors important biological functions and

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1 Classical pathway 2 Lectin pathway

C1 C1s C1r C1q

MASP-3

MASP-1 MASP-2

MASP-3 MASP-1 MASP-2

MBL

Interaction of C1 with immune complexes

Ficolin Interaction of MBL or ficolins with carbohydrates

Pathogen

Antibody

iC3

2O

Surface protein Carbohydrate

C3 e ertas n o c v

H

C3b

C3 Amplification loop driven by activator surfaces 3 Alternative pathway

Fig. 3. Starting steps of the three complement activation pathways on non-self surfaces, e.g. of a pathogen.

acts as opsonizing molecule. By interaction with its receptors on phagocytes it stimulates internalization and thus elimination of pathogens. Further splitting of iC3b first generates the C3dg fragment remaining bound to the target, followed by cleavage into C3d. All these degradation steps eliminate the complement-activating C3b, but produce fragments with other biological functions for immunocompetent cells. Through C3d-opsonized antigens, CR2 is cross-linked with the B-cell receptor complex, a process that lowers the threshold for B cell activation by the specific antigen by two orders of magnitude (see below) [1].

The Lectin Pathway The basic principle of this pathway is the binding of complement proteins with lectin domains to carbohydrate structures on microbial surfaces acting as PAMPs (fig. 3).

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The plasma protein mannan-binding lectin (MBL) represents the main PRR constituent. This multimer possesses collagen-like stalk parts and globular lectin domains and thus closely resembles the structure of C1. The second structurally similar type of sugarbinding proteins is named ficolins (lectins with a fibrinogen- and a collagen-like domain). Three forms (L-, H- and M-ficolin) are known as yet. Similar to C1q, both MBL and ficolins are associated with proteases, the so called MBL-associated serine proteases (MASPs). The two types of lectins recognize a broad range of carbohydrates on pathogen surfaces, as well as apoptotic and necrotic cells. Binding of the respective ligand leads to the activation of the MASPs and converts them into active proteases. Cleaving and activation of C2 and C4 induces the generation of the C3 convertase C4b,2a that is identical to that of the classical pathway [1].

The Alternative Pathway The alternative pathway is a positive feedback loop to augment C3b generation (fig. 3). The starting C3b is generated either through one of the other two pathways or directly via inactive C3 (iC3), a C3b-like molecule which results from a constant reaction of plasma C3 with H2O. The iC3 can associate with factor B; cleavage of factor B by the serine protease factor D and release of the small fragment Ba generates the dimer iC3,Bb that represents the initial C3 convertase of the alternative pathway. Via the enzymatically active Bb fragment, this convertase proteolytically creates C3b which is deposited on microbial surfaces, the so-called activator surfaces. This C3b in turn forms a complex with factor B which is cleaved by factor D, resulting in the ‘final’ alternative C3 convertase C3b,Bb; associated properdin stabilizes the convertase (C3b,Bb,P) against decay [1].

Cascade Steps of the Terminal Pathway The complement cascade proceeds with the composition of C5 convertases out of the corresponding C3 convertases by covalent deposition of an additional C3b molecule: C4b,2a,3b and (C3b)2,Bb,P. The (second) C3b interacts with C5 and presents it in the correct conformation for cleavage by the C2a or Bb enzyme. Proteolysis of C5 results in release of the anaphylactic peptide C5a, whereas C5b undergoes conformational changes, exposes a binding site for the late complement component C6 and thus starts the membrane-attack complex formation. C7, C8 and C9 are sequentially activated in a nonenzymatic manner and assemble into the terminal complement complex TCC, either in a soluble form or as a lytic pore, termed as MAC. Besides its lytic properties, the MAC may induce so-called sublytic effects on cells which are not definitely identified as ‘non-self ’ and thus ‘attract’ only minor amounts of MAC on their surface [4].

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Sublytic attack stimulates transcription, protein synthesis, arachidonic acid metabolism and activates cellular signaling pathways (Ca2⫹, protein kinase C, G proteins) [1].

Complement Receptors Activated complement proteins or fragments derived thereof are recognized by specific receptors found on a variety of different cell types (table 1). Consequences of this ligand-receptor interaction are changes in cellular functions and the enhancement of innate immune responses to increase removal of foreign or damaged material (microbes, apoptotic cells, immune complexes). The CRs 1–4 as well as the recently discovered CRIg all recognize C3 fragments covalently bound to surfaces in the course of complement activation. Uptake of such opsonized particles and subsequent activation of intracellular pathways are the main functions of these C3 receptors. CR1 (CD35) is a multifunctional glycoprotein with both receptor and regulator activity. It harbors ligand-binding sites for C3b and C4b. CR1 is detected on a broad spectrum of peripheral blood cells as well as on follicular dendritic cells (FDCs). Due to the numerical predominance of erythrocytes, about 90% of CR1 are found on this cell type, where they mediate the transport of C3b/C4b-opsonized immune complexes to the liver and spleen for transfer to phagocytic cells and subsequent removal. Located on phagocytes, CR1 arranges the phagocytosis of opsonized pathogens or ingestion of foreign/damaged material. CR1 expressed on B cells is suggested to participate in B cell proliferation and differentiation. In germinal centers of the lymphoid follicles, FDC-associated CR1 may be important for the induction of immunological memory [5]. The receptor CR2 is detected on B cells, FDC and, to a lesser extent, on activated T cells and epithelial cells. This receptor interacts with C3d, C3dg, and iC3b, and acts as an attachment site for Epstein-Barr virus (EBV), mediating viral entry into B cells and epithelial cells. The probably most important role of CR2 consists in bridging of innate and adaptive immunity [6]; the interaction of CR2 on B cells with C3dopsonized antigens drastically lowers the threshold for B cell activation by the specific antigen, and supports antibody maturation and induction of B cell memory [7]. CR3 (Mac-1, CD11b/CD18) belongs to the family of ␤2-integrins and consists of the ␣-chain CD11b and the ␤-chain CD18 [8]. A remarkable feature of CR3 is its promiscuous interaction with numerous ligands, including iC3b, ICAM-1 and -2, proteins of the clotting system, or various molecules of microbial origin. CR3 is found on mononuclear phagocytes, neutrophils, mast and natural killer cells, FDC and T cell subsets. It is the main receptor for phagocytosis of iC3b-opsonized pathogens, but also contributes considerably to leukocyte adherence and migration. CR4 (CD11c/CD18) belongs to the same ␤2-integrin subfamily as CR3 and harbors similar cell distribution, ligand specificities and functions.

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The recently discovered CRIg on tissue macrophages is recognized by C3b, C3c and iC3b; mediating the internalization of C3-opsonized particles, it seems to play a pivotal role in the clearance of opsonized pathogens [9]. CRIg may also represent a link between innate and adaptive immunity. It is identical to VSIG4, a potent negative regulator of T cell response. Interestingly, ligand binding triggers the internalization of the receptor, suggesting that complement activation might remove the macrophage-induced T cell inhibition and thus promote inflammation [10]. Similar to CR1, CRIg combines the receptor function with a function as a complement regulator. The receptors C3aR, C5aR (CD88) and C5L2 bind as ligands the anaphylatoxins C3a or C5a as well as their derivatives C3adesArg or C5adesArg and mediate their various biological functions. C3aR and C5aR are expressed by cells of myeloid origin, but are also present on epithelial, endothelial and parenchymal cells as well as on B and T lymphocytes. C5L2 is found on immature DCs, neutrophils and adipocytes. After binding of C5a as the most potent anaphylatoxin, C5aR organizes its proinflammatory effects such as induction of chemotactic migration, release of inflammatory mediators (histamine, cytokines), enhancement of cell adhesion and stimulation of oxidative burst [11]. The proinflammatory characteristics of C3a via binding to C3aR overlap with those of C5a, but C3a is believed to be less potent. Since the third anaphylatoxin receptor C5L2 couples only weakly to signaling G proteins, it seems likely that C5L2 serves as a nonsignalling decoy receptor and thus limits the proinflammatory effects of C5a [12]. Several reports indicate that C1q binds to a variety of different cell types, presumably via distinct receptors, and mediates e.g. enhanced phagocytosis or oxidative burst metabolism [13]. A number of intracellular and surface receptors for C1q were identified including C1qRp, gC1qR, cC1qR and the newly identified ␣2␤1 integrin (table 1). The best-characterized C1q receptor is C1qRp, which mediates the effect of C1q on phagocytosis by monocytes and macrophages. The receptor was further localized on endothelial cells. C1q also interacts with cC1qR, which is involved in uptake and removal of C1q-opsonized cell debris [14]. The integrin ␣2␤1 was recently discovered to bind to C1q and to act as a costimulator for mast cell activation and cytokine secretion [15].

Control and Regulation

The powerful weapon of the complement system is strictly limited in its activity to prevent excessive and harmful processes. There is considerable redundancy of control proteins for many steps (table 1; fig. 4). The classical pathway of complement activation is tightly controlled by C1 inhibitor (C1-INH), which inactivates the C1q-associated serine proteases C1r and C1s by covalent binding to their substrate domains [1]. C1-INH also limits the trig-

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CLASSICAL

LECTIN

ALTERNATIVE

Immunoglobulins bound to antigen

Microbial carbohydrates

C3b amplification on activator surface

C1q

C1-INH

MBL/ficolins Proteases

Proteases

C3b

fH CR1 MCP

C4bp CR1 MCP C3 convertases C4b,2a or C3b,Bb,P

fH CR1 DAF

fH CR1 DAF CRIg

C3

CPN

C3a C3b

fH CR1 MCP

fH CR1 DAF CRIg

C5 convertases C4b,2a,3b or (C3b)2,Bb,P

C4bp fH CR1 DAF

C5

C5a CPN

C5b CD59 S-protein Clusterin

C6–C9

MAC

Fig. 4. Inhibitory regulator molecules of the complement cascade with the corresponding sites of interaction. For abbreviations see table 1.

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gering of the lectin pathway by binding to the MASPs and inactivating them in a similar manner. Both classical and lectin pathways depend on active C4b to form the C3 convertase C4b,2a. The regulatory serine protease factor I (fI) cleaves two peptide bonds of C4b, thereby inactivating the protein. The regulator C4-binding protein (C4bp) exerts further control as a cofactor for fI-mediated cleavage of C4b. Other regulatory cofactors for degradation of C4b by fI are CR1 (CD35) and MCP (CD46). The alternative pathway with the self-amplifying capacity of C3b is also controlled by fI since this protease cleaves and inactivates C3b as well; cofactors for this step are factor H (fH), CR1 and MCP. The C3/C5 convertases tend towards spontaneous disassembly, but their decay is accelerated by fH, CR1 and decay-accelerating factor (DAF); in addition, C4bp enhances the disintegration of only the classical/lectin pathway convertases. Furthermore, factor I and its cofactors fH, CR1 and MCP can prevent an effective formation of the C5 convertases by degrading C3b. The inhibitory mechanism of the alternative pathway C3 and C5 convertases by CRIg might include interference with the binding function of C3b [16]. The biologically potent anaphylatoxins C3a and C5a are also subject of control; the carboxypeptidase N can cleave the carboxyterminal arginine from both molecules and thus inactivate them. The terminal pathway is monitored by S protein and clusterin that are able to bind to nascent C5b-7 and to prevent its membrane insertion. The final step of MAC assembly is controlled by CD59 which interferes with polymerization of C9 on the membrane [1].

Complement and Disease: What Can Go Wrong?

Although designed to protect the organism against harmful invaders and processes, a variety of proven or suspected relations exist between the complement system and the pathogenesis of inherited or acquired diseases.

Insufficient Levels of Functional Complement Proteins Insufficient levels of functional complement proteins, achieved either by genetic mutations or acquired by consumption, represent such a relation of complement with clinical symptoms. Putative consequences include increased susceptibility to microbial infection, but also autoimmunity, since cell debris and circulating immune complexes fail to be cleared efficiently without functional complement cascade [17]. An inherited homozygous C3 deficiency manifests already early in childhood with severe and recurrent bacterial infections, mainly by encapsulated bacteria. Infections

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of the respiratory tract, sinusitis, tonsillitis and otitis are the most frequent consequences of absent C3, together with a high incidence of meningitis [18]. Low MBL functionality is associated with predisposition to Legionnaires’ disease [19]. MBL deficiency may also be a significant risk factor for CMV infection and septic shock as well as for lethality in infective endocarditis [20–22]. Deficiencies of late complement components are typically associated with recurrent invasive infections by Neisseria meningitidis. Autoimmune diseases, as putative outcome of complement mutations, may manifest if the genes encoding C1, C4 and, to a lesser extent, C2 are affected. These deficiencies predispose any affected individual to the development of systemic lupus erythematosus (SLE). The resulting impaired immune complex handling and inefficient clearance of apoptotic cells provoke clinical symptoms in the skin, kidney (lupus nephritis) and other organs [23].

Failures in Complement Control Failures in complement control can result in exaggerated complement activity with subsequent inflammation, lysis of bystander cells and tissue destruction. Hereditary angioedema (HAE) is due to such a loss in complement regulation. The typical appearance of multiple edemas in skin and mucosa (e.g. gastrointestinal tract, larynx) after minor traumas or infections is the consequence of mutations in the gene encoding C1-INH. A deficiency of C1-INH can also be acquired by consumption, due to autoantibodies against C1-INH or to lymphoproliferative diseases that also consume C1-INH. The resulting acquired angioedema (AAE) is phenotypically similar to HAE. The reduced amounts of C1-INH in HAE or AAE are insufficient to control local inflammation at times of physiological or psychological stress. As a consequence, complement and contact systems are unrestrictedly activated and excess bradykinin, the main factor in the development of local edema, is generated [24]. Hemolytic uremic syndrome is characterized by hemolytic anemia, thrombocytopenia and acute renal failure. The atypical form (aHUS) is not associated with diarrhea and has a poor prognosis [25]. Increasing evidence suggests that aHUS is the result of mutations in the genes encoding factor H, factor I or MCP. The reduced expression or the disturbed ligand-binding capacity of these control proteins leads to inadequate control of complement activation on endothelial cells after an initiating injury [25, 26]. The cause of paroxysmal nocturnal hemoglobinuria (PNH) is a genetic defect which affects complement regulation in an indirect manner. Mutations disturb the synthesis of functional glycosylphosphatidylinositol (GPI) anchors which attach many molecules to the cell membrane. Amongst others, the missing GPI anchor interferes with the surface expression of CD55 and CD59. The lack of these two

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control proteins is responsible for the extreme susceptibility of PNH erythrocytes to complement-dependent lysis, induced either by alternative pathway activation or by acidic activation of C6, especially at the physiologically lower blood pH during the night [27].

Persistent Presence of the Complement-Activating Stimulus A chronic complement activity with continuous inflammation and tissue damage can also be a consequence of the persistent presence of the complement-activating stimulus, e.g. in autoimmune disease, organ transplantation or in chronic infections. It is a paradoxical situation that autoimmune conditions can result both from complement deficiency and from exaggerated complement activation. One example of autoimmune disease with exceeded complement activity is membranous nephropathy. Autoantibodies directed at antigens on podocytes in the kidney give rise to subepithelial immune deposits and subsequent complement activation [28]. The podocyte response to continuously present sublytic MAC includes increased oxidant production, inflammation and fibrosis with a decrease in glomerular filtration rate. Complement itself can also be the target of autoimmunity. In approximately 1/3 of the patients with SLE, the autoantibodies are directed against C1q; particularly renal inflammatory disease (lupus nephritis), a severe complication of SLE, is strongly associated with the presence of anti-C1q autoantibodies [29]. Multiple sclerosis is another inflammatory autoimmune disease associated with a complement-driven pathogenesis. The characteristic demyelination and loss of oligodendrocytes result from autoimmunity against myelin. Furthermore, the myelin oligodendrocyte glycoprotein MOG can bind directly to C1q; subsequent initiation of the classical pathway results in lysis of oligodendrocytes and chemoattraction of phagocytes which accumulate at the site of inflammation and degrade the myelin sheath [30]. Triggering of the complement cascade by direct binding to C1q is also achieved by ␤-amyloid peptide, the main component of the senile plaques in Alzheimer’s disease. Consecutive complement-driven processes are hypothesized to be a cofactor for neuronal injury, particularly since low or absent expression of CD55 and CD59 leaves neurons extremely sensitive for complement-induced bystander lysis. Unrestricted complement activation is a critical limitation in organ transplantation and contributes to rejection processes. Allograft injury occurring during donor death and during graft reperfusion by implantation in the recipient initiates the interaction of IgM with injury-induced neoantigens. The subsequent complement activation is a reason for hyperacute rejection developing within minutes to hours after transplantation [31]. An acute or chronic rejection within days and months/years after transplantation are the consequence of T cell and/or antibody alloreactivity to the graft. Again, subsequent

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complement activation by the immune complexes in the graft is a critical mediator of rejection by induction of cell lysis, C3a/C5a-triggered chemotaxis, and C3a/C5a/TCCelicited activation of endothelial cells with production of proinflammatory molecules (cytokines, chemokines, adhesion molecules). In addition, complement promotes the humoral immune response that is elicited by graft antigens. Chronic infections are another condition with perpetual presence of a complement-activating stimulus. Thus, complement is discussed to contribute to neurodegeneration induced by HIV-1. HIV-1 is detected in more than 80% of brains from infected individuals and causes meningitis, neuropathies and a subcortical dementia with cognitive, motor and behavioral dysfunctions. The cerebral expression of different complement factors is highly upregulated in the brain of HIV-infected patients [32]. Furthermore, the HIV-1 transmembrane protein gp41 downmodulates the expression of CD59 in human neuronal and astroglial cell lines, thus enhancing the susceptibility of neurons towards complement-dependent bystander cell lysis [33]. In the septic setting, all three complement activation pathways may be brought into play by the invading bacteria. Therefore the anti-infectious response is closely linked to an overwhelming inflammatory process during sepsis with high amounts of complement-derived compounds in the plasma. Overproduction of the complement fragments C3a and C5a and excessive formation of the MAC can damage a wide variety of tissues leading to multiple organ failure [34, 35].

Microbe-Driven Counter Attacks A fourth correlation of complement to pathogenesis is a functional failure of complement reaction due to microbe-driven counterattacks. To fulfill their destructive work, pathogenic microorganisms aim to evade appropriate recognition by the complement system or to inhibit an efficient attack and destruction [36]. A range of strategies has been invented during microbial evolution; they are discussed here shortly. a Microbes can degrade complement factors by proteolysis [36]. Examples are the cleavage of C1q and C3 by Pseudomonas-derived proteases, or the degradation of C3 and C9 by a serine protease of Schistosoma mansoni [37, 38]. b Some microorganisms use poorly activating surface molecules or even shed immune complexes from the surface to protect themselves from recognition by complement ‘starter’ molecules. Examples for this strategy are the encapsulated bacteria where the capsule sialic acids are insufficient to activate complement [39]. The parasite Trypanosoma brucei removes antigen-antibody complexes from their surface [40]. c Pathogens can also disturb or inhibit the proper course of the complement cascade on the microbial surface. As a consequence, they are protected against eradication by interfering with opsonization and subsequent efficient phagocytosis as well as

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with direct complement-mediated lysis. One mechanism to achieve this aim is the occupancy of host-derived complement inhibitor proteins. HIV-1 particles budding from an infected cell acquire with the lipid bilayer also the embedded host membrane proteins, amongst others the complement inhibitors CD55 and CD59 which consequently protect HIV-1 from complement lysis [41]. Attachment of factor H to the viral surface protein gp41 confers an additional protective shield against efficient destruction [42]. Similarly, microbes such as Neisseria gonorrhoeae, Bordetella pertussis, Streptococcus pyogenes, E. coli and Candida albicans bind the regulator C4bp to their surfaces and thus exploit its complement regulatory activity [43]. Some pathogens, e.g. vaccinia virus, even advanced this approach by having acquired a complement inhibitor-encoding gene during evolution which is now part of their genome [44]. Another strategy is the shedding of MAC from the surface, as shown for bacteria like Escherichia coli and Salmonella [36].

Functional Failure of Complement in Tumor Cells A functional failure of complement can also be diagnosed for tumor cells. Some pattern recognition receptors detect changes in glycosylation or other cellular changes that are concurrent with tumor development and subsequently provide signals for complement activation [45]. In order to avoid complement-mediated lysis and elimination, many tumor types overexpress membrane-bound complement inhibitors (CD55, CD59, CD46) as well as factor H [46]. Moreover, some aggressive cancer types secrete proteases, e.g. matrix metalloproteinases, which can cleave C3b and liberate the deposited C3 fragments from the cell surface, thereby preventing the efficient recognition by phagocytes. The anticomplement activity of the metalloproteinase MT1-MMP was shown to play a significant role in vivo by promoting metastasis [47].

Exploitation of Complement by Microbes Microbes may even exploit complement for their purposes and use complement or complement-related molecules for adherence to or infection of cells and thus favor their own spreading in the host [36]. C. albicans encodes a CR3-like molecule which may bind to the corresponding counterreceptor on host cells and thus facilitates adhesion. CD46 was identified as a cellular receptor for the Neisseria pilus [48]. EBV embarks on another strategy, since its surface protein gp350/220 mimics C3, thus represents a ligand for CR2-bearing B cells and allows efficient cell infection. The exploitation of complement to infect cells is even more prominent for HIV, since it not only uses host CRs, but also the opsonization by the host for attachment. Opsonizing complement fragments on the viral surface enable the interaction of the virus with CR3-positive cells and, as a consequence, the dissemination of HIV in the host [1].

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Therapeutical Approaches

The spectrum of complement-associated diseases as well as the versatility of complement functions and molecules open up promising strategies for therapeutic approaches: (a) the limitation of excessive complement activation; (b) the stimulation of complement attack against cancer cells; or (c) the usage of complement molecules to improve vaccination or to support cancer therapy (fig. 5) [49]:

Limitation of Excessive Complement Activation Dysregulated complement activation induced either by autoimmune diseases, by mutations in regulator molecules or by chronic infections inflicts damage on cells and organs. The clinical course of e.g. myocardial infarction, sepsis or stroke might also be worsened by the activated complement cascade. Furthermore, therapeutic approaches such as (xeno)transplantation, gene therapy by viral vectors or usage of biomaterials (catheters, prostheses, etc.) induce chronic and consecutive complement activation that represents a profound limitation of success. Different concepts are therefore under intensive development or even already in clinical application to cope with the need for controlled complement activation [49]: • Administration of Complement Regulator Proteins Soluble CR1(fragments) might improve e.g. autoimmune diseases (rheumatoid arthritis, SLE) or the outcome of organ transplantation; C1-INH protein is now standard substitution therapy for hereditary or AAE, but might also be applied in transplantation or myocardial infarction. In the future this regulator might also improve the outcome of septic shock induced by Gram-negative bacteria. In vitro C1INH blocked the lipopolysaccharide (LPS)-induced increase in transendothelial permeability and endothelial cell injury. Animal models showed that C1–INH protects mice from LPSinduced increased plasma leakage, multiple organ failure and lethal septic shock [50]. Soluble CD59 is regarded as putative therapeutic drug in rheumatoid arthritis or PNH. • Transfer of Genes Encoding Complement Regulators Generation of CD55-transgenic animals represents an interesting approach for xenotransplantation; alternatively a human donor organ might be ‘enriched’ with CD55 by vector-mediated transfer of the gene before transplantation. • Monoclonal Antibodies Directed against Complement Factors Antibodies against C5 represent an interesting approach to inhibit the C5-cleaving step by the convertases in stroke, myocardial infarction or paroxysmal nocturnal hemoglobinuria; blocking antibodies directed against the anaphylatoxin C5a might inhibit its devastating action in septic shock or pulmonary edema.

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CLASSICAL

LECTIN

ALTERNATIVE

Optimized mAb binding to C1q Supply of C1-INH

C1q Proteases

MBL/ficolins Proteases

C3b Supply of soluble CR1 Inhibit ion o mAb, s f fH by iRNA

Supply of soluble CR1

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ion of mAb, s DAF by iRNA

by of fH ition RNA b i h In , si mAb

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C3a

Inhibition of C3a by receptor antagonists

Supply of soluble CR1

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ion of mAb, s DAF by iRNA

y

n of fH b Inhibitio NA mAb, siR

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C5a Inhibition of C5a by receptor antagonists

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C5 convertases

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C5b Supply of soluble CD59

C6–C9

MAC

Fig. 5. Therapeutic principles targeting different steps of the complement cascade. Inhibitory approaches to downmodulate complement activity are shown with octagons; approaches to stimulate the complement activity are represented by black arrows.

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• Peptides Blocking Complement Factors or Their Receptors Synthetic peptides can bind to C3 and thereby inhibit its cleavage by the convertases to prevent hyperacute rejection in transplantation; blocking antagonists of C3a receptor or C5a receptor might improve severe inflammation, arthritis, neurodegeneration, etc.

Stimulation of Complement Attack against Cancer Cells A variety of different malignancies escape from complement attack by overexpression of membrane-bound complement regulatory proteins. Particularly CD55 is highly synthesized in many tumors and is regarded as a requisite for cancer development and progression [51]. To undermine this evasion mechanism and thus to restore tumor sensitivity to complement attack, therapies using anti-CD55 monoclonal antibodies or including a vaccination against CD55 are currently under development. Alternatively, small interfering RNA (siRNA) to CD55 might at least reduce or even inhibit its expression in the tumor cells. A similar downmodulation of factor H by blocking antibodies or siRNA sensitizes lung cancer cells to complement attack and reduces tumor growth in vivo [46]. Complement attack is also a player in cancer therapy by specific monoclonal antibodies, e.g. by rituximab against CD20 on B lymphoma cell. Rituximab and many other anticancer antibodies strictly depend on complement activation in their therapeutic activity. Some approaches therefore aim to optimize the complement-activating capacity of these antibodies, e.g. by genetically engineering the hinge region or by converting their isotype into the highly activating IgG1 subtype. Another complement-based strategy against cancer is the application of ␤-glucans which prime the CR3 on phagocytes for enhanced binding of iC3b. As a consequence, antitumor antibodies which induce opsonization of tumor cells with iC3b can act in concert with ␤-glucans stimulating phagocytes to bind via primed CR3 to opsonized cancer cells and thus killing them [52]. A further elegant way to intensify the complement attack on cancer cells is to increase the amount of antibodies deposited on the cell surface; this might be reached by concomitant application of a specific antitumor antibody (e.g. rituximab) inducing opsonization of the tumor with iC3b and a second antibody directed against the iC3b [53].

The Usage of Complement Molecules to Improve Vaccination or to Support Cancer Therapy Molecules of the complement system as well as complement activation mechanisms can also be utilized for cancer therapy and to improve the efficacy of vaccination.

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Current new strategies aim to treat cancer with oncolytic replication-competent viruses. Since membrane-bound complement regulators are highly upregulated on tumor cells, oncolytic viruses which target these complement regulators as receptors are preferentially used to more or less selectively destroy tumors while sparing nonmalignant tissue. CD55 is the receptor for the common cold virus coxsackievirus A21 which rapidly induces lysis of melanoma cells in a mouse model [54]. CD46 can act as receptor for the attenuated measles virus vaccine strain Edmonston B which effectively lyses e.g. breast cancer cells or T cell lymphomas [55]. Furthermore, most B group adenoviruses target tumors that express CD46 at high levels, inducing oncolytic effects [56]. CD46-binding adenoviruses might also deliver therapeutic tumor-suppressing genes (e.g. p53) to cancer cells [57]. Complement also represents an interesting tool to support vaccination strategies [58]. Covalent binding of the C3 fragment C3d to the vaccination antigen helps to raise an effective immune response since C3d targets the antigen to CR2 on B cells and FDCs. This influences the magnitude of humoral immune response as only polyvalent CR2 ligands have an immunostimulatory effect. Thus, complement tagging of antigens may serve as an adjuvant mechanism in immunization and increase the immunogenicity of protective antigens as shown in preliminary experiments with measles virus, influenza virus, pneumococci or HIV [45].

Conclusions

Complement is a powerful sword of innate immunity that protects the body against infections and helps to conserve homeostasis. The complexity of immune evasion strategies developed by pathogens as well as by cancer cells underlines this central importance. However, fulfillment of correct function is dependent on a strict balance of activation and inhibition to find the best compromise between functional deficiency and exaggerated activation. Any shift in this balance, induced by genetic mutations, autoimmunity, chronic infection or organ transplantation, results in clinical presentations. Increasing knowledge on functionality of complement molecules, their association with a variety of diseases and the bridging to adaptive immunity allows the development of interesting therapeutic strategies. It is a substantiated promise that future complement-based therapies will improve the outcome of a broad spectrum of medical conditions.

Acknowledgements The authors would like to acknowledge the Austrian National Bank (OeNB 11944), the Austrian ‘Fonds zur Förderung der wissenschaftlichen Forschung’ (FWF P17043-B13), the EU Network of Excellence EuroPathoGenomics (LSHB-CT-2005-512061), and the State of Tyrol for support of their research.

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Dr. Cornelia Speth Department of Hygiene, Microbiology and Social Medicine Innsbruck Medical University, Fritz-Pregl-Strasse 3 AT–6020 Innsbruck (Austria) Tel. ⫹43 512 9003 70705, Fax ⫹43 512 9003 73700, E-Mail [email protected]

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Antibacterial Chemokines – Actors in Both Innate and Adaptive Immunity Mette Eliasson ⭈ Arne Egesten Section for Respiratory Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden

Abstract Several antibacterial proteins and peptides of the human innate immune system have additional roles in the regulation of adaptive immune responses. Among peptides with innate and adaptive immune functions are chemokines, a family of structurally related peptides with conserved amino-terminal motifs. Chemokines regulate leukocyte trafficking during both health and disease. In recent years, some chemokines have been shown to exert direct antibacterial activity. On the other hand, several granulebound antibacterial proteins of granulocytes, and epithelium-expressed antibacterial polypeptides, possess chemotactic activity and stimulate cells of the adaptive immune system. It is likely that during evolution, some antimicrobial peptides and proteins of innate immunity have diverged to coordinate the actions of the innate immune system and the evolutionary younger, adaptive immunity. This review aims to describe antibacterial chemokines and antibacterial peptides possessing chemotactic activity, Copyright © 2008 S. Karger AG, Basel biologic properties that link innate and adaptive immunity.

The first chemokines were identified more than two decades ago [1–3]. Since then, this large family of peptides has come to include 50 members, and 18 corresponding receptors have been characterized. The term ‘chemokine’ is a fusion of ‘chemotactic’ and ‘cytokine’, reflecting that these small molecules are cytokines that induce directed cellular movement (chemotaxis) in target cells expressing the appropriate receptors. Chemokines are responsible for leukocyte trafficking and activation, during both health and disease. With time and an increasing number of members, the nomenclature of chemokines became complex and in 1999, a systematic classification was introduced. The classification is based on the arrangement of NH2-terminal cysteines, i.e. XC, CC, CXC, and CX3C, respectively, where ‘X’ is a nonconserved amino acid residue [4]. In addition to chemotactic activity against different subsets of leukocytes, some chemokines participate in the regulation of angiogenesis/angiostasis, and some possess antibacterial activity [5–7].

The human immune system is complex as a result of different survival strategies during evolution. A simplified view is the division of host defense into a nonspecific innate and a specific adaptive immune system. Bacteria, plants, and evolutionary older organisms possess innate immune responses, while a combination of innate and adaptive immunity is seen in vertebrates. The evolutionary starting point, adding an adaptive capacity to the immune response, is first seen among the bony fishes. These species are derived from one of the earliest divergent lineages, having both innate and adaptive immune systems [8]. In fishes, several chemokines and corresponding receptors have been cloned including CC chemokine homologues and the receptors CXCR1/CXCR2 and CXCR4, demonstrating that chemokines are an ancient part of the immune system. This review will focus on human peptides and proteins that possess both antibacterial and chemotactic activity, thereby linking functions of innate and adaptive immunity (table 1).

Chemokines

The Chemokine Family Chemokines consist of 70–130 amino acids and their resulting molecular weights range from 8 to 13 kDa. Most chemokines are highly cationic at physiologic pH due to a relatively high content of the amino acids lysine and arginine. Structurally, even though not all chemokines have been investigated, the majority contain antiparallel ␤-sheets in the NH2-terminal region, while the COOH-terminal region consists of an ␣-helix. In CC and CXC chemokines, the antiparallel ␤-sheets of the NH2-terminal region are held together by cysteines that form two disulphide bonds (Cys1 → Cys3 and Cys2 → Cys4), resulting in the characteristic three-dimensional folding (fig. 1). The conformation of the NH2-terminal region is crucial for receptor-binding and activation [4]. Chemokines have other physiochemical properties in common. In concentrated solution and on crystallization they tend to oligomerize. Oligomerization of IP-10/ CXCL10 on the surface of endothelial cells is, for example, required for induction of chemotaxis in T cells [9]. Chemokines bind glucosaminoglycans of the extracellular matrix, a property that enable these molecules to establish stable chemotactic gradients along which leukocytes can be directed during chemotaxis [10]. In the case of antibacterial chemokines, the glucosaminoglycan-binding properties may enable them to establish an antibacterial gradient on the surface of epithelial cells. Based on their functions, chemokines can be divided into two classes, inflammatory and homing chemokines, respectively. Inflammatory chemokines are produced by many different cells in response to bacterial products (e.g. peptidoglycan, lipopolysaccharide – LPS, lipoteichoic acid) and proinflammatory cytokines (e.g. inter-

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Table 1. Examples of antibacterial chemokines and antibacterial peptides/proteins possessing chemotactic activity Antibacterial peptide

Cellular source

Receptor-mediating chemotactic response

C chemokine Lymphotactin/XCL1

T, NK

XCR1 (NK)

CC chemokines I-309/CCL1 Eotaxin/CXCL11 MCP-4/CCL13 TARC/CCL17 PARC/CCL18 MIP-3␤/CCL190 MIP-3␣/CCL2 SLC/CCL21 MDC/CCL22 TECK/CCL25 CCL28

MC Ep Ep DC Mo, Ma, DC T Ep T DC, Ma Ep, DC Ep

CCR8 (Mo, T) CCR3 (B, Eo, Baso) CCR1,2,3 (Mo, Baso, T, DC, NK) CCR4 (Eo, Baso, T) Unknown (DC, T) CCR7 (B, DC, T) CCR6 (B, DC, T) CCR7 (B, DC, T) CCR4 (Eo, Baso, T) CCR9 (T) CCR10 (B, T)

ELR-positive CXC chemokines GRO-␣/CXCL1 GRO-␤/CXCL2 GRO-␥/CXCL3 GCP-2/CXCL6 IL-8/CXCL8

Ep Ep Ep Ep Ep

CXCR2 (N, Mo, MC) CXCR2 (N, Mo, MC) CXCR2 (N, Mo, MC) CXCR1 and 2 (N, Mo, MC) CXCR1 and 2 (N, Mo, MC)

ELR-negative CXC chemokines PF4/CXCL4 Megakaryocytes MIG/CXCL9 Ep, N, Eo IP-10/CXCL10 Ep, N, Eo I-TAC/CXCL11 Ep, N, Eo SDF-1/CXCL12 Stromal cells BCA-1/CXCL13 DC, B BRAK/CXCL14 Ep, Mo

CXCR3B (En) CXCR3A/B (T, NK, DC, MC, En) CXCR3A/B (T, NK, DC, MC, En) CXCR3A/B (T, NK, DC, MC, En) CXCR4 (all leukocytes) CXCR5 (B) Unknown

Defensins HNP1–4 hBD-1, -2

GiPCR(Mo, Ma, DC, T) CCR6?

N Ep

Granule proteins of leukocytes HBP N Cathepsin G N LL-37 (hCAP-18) N, Ep Granulysin T, NK

FPRL2 (Mo) FPR (N, DC, Eo, Mo, En) FPRL1 (Mo, Ma, N, Eo, Dc) GiPCR(Mo, DC, T, NK)

Complement C3a

C3aR (leukocytes)

Liver

T ⫽ T cells; B ⫽ B cells; NK ⫽ natural killer cells; MC ⫽ mast cells; Ep ⫽ epithelial cells; N ⫽ neutrophils; Eo ⫽ eosinophils; PC ⫽ Paneth cells; Mo ⫽ monocytes; Ma ⫽ macrophages; GiPCR ⫽ G protein-coupled receptor; HNP ⫽ human neutrophil peptide. Mainly compiled from [4, 11].

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Fig. 1. Arrangement of the NH2-terminal cysteine motifs of the chemokine family. The figure shows the arrangement of disulphide bonds between the cysteines of C chemokines (a), CC chemokines (b), CXC chemokines lacking the amino acid sequence glutamic acid-leucinearginine preceding the CXC motif (ELR-negative; c), ELRpositive (having a glutamic acid-leucine-arginine motif) CXC chemokines (d), and CX3C chemokines (i.e. fractalkine; e). The disulphide bonds between the cysteines provide three antiparallel ␤-sheets, a structure that is important for binding and activation of the corresponding chemokine receptors.

C

C

C

C C

a

c

b

C

C

C

CXC

ELRCXC

C

d

Chemokine domain

Mucin-like domain

C Cytoplasmic domain

C CXXXC

e

C

leukin (IL)-1␤, TNF-␣, and interferons). One function of inflammatory chemokines is to recruit leukocytes for host defense functions at sites of inflammation. Homing chemokines are constitutively expressed in lymphoid tissues, regulating the architecture of secondary lymphoid tissue as well as controlling immunosurveillance, executed by different subsets of lymphocytes and dendritic cells. However, some chemokines, for example CXCL13, have both homing and inflammatory functions, depending on the context [11]. As mentioned, chemokines can be divided into four main classes, XC, CC, CXC, and CX3C (fig. 1). The two groups of chemokines with the highest number of members are the CC and CXC chemokines (sometimes referred to as ␣- and ␤-chemokines, respectively). In addition, there are two highly related chemokines described that belong to XC chemokines (lymphotactin/XCL1 and SCM-1␤/XCL2) and one CX3C chemokine (fractalkine/CX3CL1), respectively [11]. The group of CXC chemokines can be further divided into ELR positive, containing a glutamic acid-leucine-arginine (ELR) motif, preceding the NH2-terminal cysteine while the ELR-negative CXC chemokines lack such a motif (fig. 1). The NH2terminal ELR motif is characteristic of CXC chemokines interacting with the receptors CXCR1 and CXCR2. These receptors are expressed on a variety of cells, for

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example neutrophils, monocytes/macrophages, T and natural killer (NK) cells, mast cells, and endothelial cells [11]. Activation of pattern recognition receptors (PRRs) on antigen-presenting cells, for example Toll-like receptors (TLRs), results in release of proinflammatory cytokines that induce the expression of chemokines at sites of inflammation. Binding of an ELR-positive CXC chemokine to its receptor(s) on leukocytes causes cellular activation, chemotaxis, and sometimes, depending on the context, execution of effector functions [4]. In the case of endotehelial cells, ELR-positive CXC chemokines cause a mitogenic response, resulting in angiogenesis [12].

Characteristics of Proteins and Peptides Possessing Chemotactic Activity Peptides and proteins that exert chemotactic activity interact with members of the seven transmembrane receptor family. Receptor activation causes dissemination of heterotrimeric G proteins, resulting in an influx of extracellular calcium via activation of calcium channels, phosphatidylinositol 3-kinase, and small Rho GTPases. The intracellular signaling results in rearrangement of the cytoskeleton and eventually, cellular movement [11]. In total, there are 18 different chemokine receptors described. Six CXC chemokine receptors, and 10 CC chemokine receptors, in addition to the receptors for lymphotactin/XCL1, SCM-1␤/XCL2 (XCR1) and fractalkine (CX3CL1), have been identified to date [13]. There is a large redundancy contributing to the complex picture of chemokine biology, where several chemokines in some cases share the same receptor and several different receptors may bind the same ligand. The formyl-peptide receptor (FPR) and the FPR-like receptor 1 (FPRL1) are two receptors that bind bacterial formylated peptides and belong to the family of G proteincoupled seven-transmembrane, chemoattractant receptors. Recently, several antibacterial peptides and proteins, for example cathepsin G and LL-37, have been identified as ligands for these receptors [14].

What Properties Make Peptides and Proteins Antibacterial? The different types of antibacterial polypeptides and their functional characteristics are reviewed in detail by Sørensen et al. [this vol., pp. 61–77]. In short, antibacterial peptides can be divided into classes based on their 3-D structures [15]. These are (a) linear cationic peptides, free of cysteines, and often with an ␣-helical and amphiphatic structure in solution (e.g. LL-37), (b) peptides with disulfide bonds giving the peptides a flat dimeric ␤-sheet structure (e.g. ␣- and ␤-defensins) and (c) peptides with

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enrichment of certain amino acids, such as proline, arginine, tryptophan or histidine (e.g. PR-39 and histidine-rich glycoprotein). An amphiphatic ␣-helix has a structure where one half of the circular structure is composed of nonpolar hydrophobic amino acids while the other side contains charged amino acids, thereby being hydrophilic. These properties make it possible for the peptides to associate with, and disrupt bacterial membranes. However, there are examples of both linear and anionic peptides that possess antibacterial activity [15]. Most chemokines are cationic at physiologic pH and many have an amphiphatic COOH-terminal. In addition, they have disulfide bonds providing the peptides with a flat dimeric ␤-sheet structure in their NH2-terminal. Therefore, many chemokines fit the prototypic description of antibacterial peptides.

Antibacterial Activity of CC Chemokines Description of antibacterial activity of CC chemokines started when it was demonstrated that the antibacterial human ␤-defensin-1 (hBD-1) and ␤-defensin-2 (hBD2), bind to and activate CCR6 [16]. Soon after, it was demonstrated that the known chemokine ligand of CCR6, human macrophage inflammatory protein-3␣ (MIP-3␣)/ CCL20, had an even higher antibacterial activity than human hBD-1 and hBD-2 against Escherichia coli and Staphylococcus aureus [17]. Since, antibacterial activity has been ascribed to several CC chemokines [6]. Here, we will discuss CCL20 and CCL28, which we believe, at present, are of particular interest among antibacterial CC chemokines. MIP-3␣/CCL20 is only distantly related to other CC chemokines. While other CC chemokines are clustered at chromosome 17q11.2, the human MIP-3a/CCL20 gene has been mapped to 2q33–37 [18]. Expression of this chemokine was first detected in the liver, and therefore it was named liver and activation-regulated chemokine [19]. This chemokine is constitutively expressed by many cells (keratinocytes, lung macrophages, endometrial stromal cells), and upregulation of gene expression is seen after exposure to proinflammatory stimuli such as LPS, IFN-␥, TNF-␣, and IL1␤ in cells of different phenotypes (e.g. neutrophils, monocytes, lung macrophages, dendritic cells, epithelial cells) [18]. MIP-3␣/CCL20 is the only known chemokine that binds CCR6, a receptor expressed on most B cells, subsets of T cells, and dendritic cells. As mentioned above, early investigations suggested that MIP-3␣/CCL20 share the receptor CCR6 with hBD-1 and hBD-2, thus demonstrating the functional overlap between some chemokines and epithelium-derived antibacterial polypeptides. There is no clear sequence similarity between ␤-defensins and MIP-3␣/ CCL20, apart from a high number of cationic residues and the presence of disulphide bonds. In a recent study, cells stably expressing CCR6 proved to be unresponsive to hBD-2 and hBD-3, questioning whether CCR6 indeed is a receptor for ␤-defensins [20].

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Strong expression of MIP-3␣/CCL20 by epithelial cells during inflammation points to direct antibacterial roles for this chemokine during mucosal inflammation. In an elegant study by Starner et al. [21], high and bactericidal concentrations of MIP3␣/CCL20 were detected both on the apical and basolateral surfaces of cytokinestimulated airway epithelial cells. The bactericidal activity of MIP-3␣/CCL20 was salt sensitive, which may be important for bacterial colonization of the airways in cystic fibrosis. Mucosa-associated epithelial chemokine (MEC)/CCL28 is a CC chemokine that binds to and activates CCR3 and CCR10 [22]. MEC/CCL28 is expressed by epithelial cells in certain mucosal tissues such as exocrine glands, trachea, and colon. The CCR3 and CCR10 receptors are expressed on IgA-secreting plasma cells, and MEC/CCL28 recruit these cells to the mucosal lamina propria [23]. A high constitutive expression of MEC/CCL28 is seen in the salivary glands and human saliva contains MEC/ CCL28 at high concentrations [7]. In the context of saliva, it is interesting that the COOH-terminal of MEC/CCL28 has a sequence similarity to histatin 5, a histidinerich peptide of saliva. Similarly to histatins, MEC/CCL28 has a potent antimicrobial activity against Candida albicans, Gram-negative bacteria (e.g. Pseudomonas aeruginosa and Klebsiella pneumoniae), and Gram-positive bacteria (e.g. Streptococcus mutans and Staphylococcus aureus) [7]. Taken together, MEC/CCL28 may play dual roles in mucosal immunity, being both a chemoattractant for IgA-producing plasma cells and also as a broad-spectrum antimicrobial protein when secreted into low-salt body fluids such as saliva.

Antibacterial Activity of ELR-Positive CXC Chemokines IL-8/CXCL8 was the first chemokine to be discovered and is the prototypic ELRpositive CXC chemokine [1–3]. It is considered a key regulator of neutrophil recruitment to sites of inflammation through binding and activation of CXCR1 and CXCR2 that are expressed on neutrophils [4]. In addition to regulation of neutrophil activities, IL-8/CXCL8 controls proliferation of endothelial cells, thus promoting angiogenesis. The ELR-positive CXC chemokines seem to have a common gene regulation and their expression increases after activation of PRRs on expressing cells, such as TLRs, and after stimulation by IL-1␤ and TNF-␤. Antibacterial activity of IL-8/CXCL8 has been reported and may be localized in its COOH-terminal region [24, 25]. GRO-␣/CXCL1, GRO-␤/CXCL2, and GRO-␥/CXCL3 are highly homologous and possess antibacterial activity against E. coli and S. aureus [6]. GRO-␣/CXCL1 was initially named melanoma growth stimulatory activity since it was first isolated from the supernatants of melanoma cells, acting as a mitogen [26]. GRO-␣/CXCL1, GRO-␤/ CXCL2, and GRO-␥/CXCL3 all bind the CXCR2 receptor, resulting in recruitment of neutrophils and proliferation of endothelial cells. They are expressed

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by a variety of myeloid and epithelial cells after stimulation by proinflammatory cytokines [27]. Another ELR-positive CXC chemokine is granulocyte chemotactic protein (GCP)2/CXCL6 GCP-2/CXCL6, that resembles IL-8/CXCL8 in that it binds and activates CXCR1 and CXCR2 [4]. GCP-2/CXCL6 is expressed by epithelial cells, macrophages and mesenchymal cells. Regulation of GCP-2/CXCL6 expression involves the proinflammatory cytokine IL-1␤ while IFN-␥ has a downregulating effect [28]. Recently, we found that GCP-2/CXCL6 is a potent antibacterial chemokine with effect against several, both Gram-positive and Gram-negative bacterial pathogens relevant during mucosal infections [29]. The amino acid sequence of human GCP-2/CXCL6 displays a rather weak similarity with the related ELR-positive CXC-chemokine IL-8/CXCL8 (30%) but is more homologous with ENA-78/CXCL5 (77%). In our hands, both NAP-2/CXCL7 and ENA-78/CXCL5 display some antibacterial activity against Gram-positive and Gramnegative bacteria [29]. Taken together, all ELR-positive CXC chemokines seem to possess antibacterial activity.

Antibacterial Activity of ELR-Negative CXC Chemokines In 2001, Cole et al. [5] showed that MIG/CXCL9, IP-10/CXCL10, and I-TAC/ CXCL11 possess antibacterial activity against E. coli and Listeria monocytogenes. MIG/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11 belong to the group of CXC chemokines whose expression is dependent on IFN-␥ [30–32]. They all share the ability to signal through a G protein-coupled receptor, CXC chemokine receptor 3 (CXCR3; these chemokines bind both isoforms of the receptor, i.e. CXCR3A and CXCR3B), which is present on T cells and NK cells [33]. Ligand binding to the receptor results in the activation and recruitment of these cells to sites of inflammation. In addition, MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 possess angiostatic activity and these chemokines play roles in rejection of transplants and suppression of tumor growth [11]. High production of MIG/CXCL9 and IP-10/CXCL10 is seen in airway epithelial cells after stimulation with IFN-␥, a production that is increased by IL-1␤ and TNF-␣ [34]. Recently, we found that inflamed pharyngeal epithelial cells possess antibacterial activity that is, at least in part, dependent on the expression of MIG/CXCL9 [35]. This was further underlined by the knockdown of MIG production using a siRNA technique, which markedly reduced epithelial surface-associated killing of S. pyogenes. Taken together, the results demonstrate an important protective role for MIG/CXCL9 on inflamed epithelial surfaces. Using synthetic peptides, the antibacterial activity of MIG was mapped to the COOH-terminal region of the peptide. This region is cationic, amphiphatic, and has a predicted ␣-helical structure, properties typical of peptide sequences exhibiting antibacterial activity. MIG/CXCL9 has higher antibacterial

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MIG/CXCL9

IP-10/CXCL11

I-TAC/CXCL11

Fig. 2. A comparison of the structures of MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. The figure shows the three antiparallel ␤-sheets (blue) and ␣-helical COOH-terminal (red) of MIG/CXCL9, IP10/CXCL10, and I-TAC/CXCL11. MIG/CXCL9 has the highest antibacterial activity, an activity that is confined to the ␣-helix [35]. The structure of MIG/CXCL9 is a predicted structure based on a computer model and determined structures of other members of the CXC chemokine family [35, 61–63]. The structures of IP-10/CXCL10 and CXCL11/I-TAC in solution have been determined [62, 63].

activity compared with IP-10/CXCL10 and I-TAC/CXCL11, while the latter has a higher affinity for the CXCR3 receptor [35]. The COOH-terminal of MIG/CXCL9 is more extended than those of IP-10/CXCL10 and I-TAC/CXCL11 (fig. 2). This may explain the higher antibacterial activity. The higher production and more potent antibacterial activity of MIG/CXCL9 suggest that this chemokine is more important than IP-10/CXCL10 and I-TAC/CXCL11 as an innate antibiotic in the pharynx. MIG/CXCL9 may have evolved towards an antibacterial function while ITAC/CXCL11 has come to play a more important role in the regulation of leukocyte trafficking. Upon activation, human platelets release several antibacterial peptides among them platelet factor (PF) 4/CXCL4, which also belongs to the group of ELR-negative CXC chemokines [36]. PF4/CXCL4 is expressed in megakaryocytes of the bone marrow and stored in the ␣-granules of platelets during their circulation in the bloodstream. Upon activation of platelets, PF4/CXCL4 is released. PF4/CXCL4 is chemotactic against neutrophils and monocytes. It binds an isoform of CXCR3, CXCR3B that is expressed on microvascular endothelial cells, resulting in angiostatic activity [4]. PF4/CXCL4 displays broad antimicrobial activity against both fungi as well as bacteria such as E. coli and S. aureus [32].

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Non-Chemokine Peptides and Proteins Possessing Both Chemotactic and Antibacterial Activity

Defensins – A Family of Antibacterial Peptides Possessing Chemotactic Activity Defensins are divided into two main groups, i.e. ␣- and ␤-defensins. Human ␣defensins are expressed predominantly in neutrophils (also named human neutrophil peptides 1–4) or intestinal Paneth cells (human defensins 5 and 6) [37]. In addition to their antibacterial activity, these small peptides (3–5 kDa) are chemotactic for monocytes/macrophages, T cells, as well as dendritic cells [38, 39]. Recent findings suggest that ␣-defensins and ␤-defensins share unique receptors [40]. Previous studies have demonstrated that hBD-1, hBD-2, and hBD-3 all bind and activate CCR6 [15]. However, in a recent study, macrophages and mast cells were found to migrate to hBD-1–4, but unable to induce migration of T cells and dendritic cells. In addition, cells stably expressing CCR6 proved to be unresponsive to hBD-2 and -3, questioning whether CCR6 indeed is the receptor for ␤-defensins [20].

Granule Proteins of Leukocytes Leukocytes, in particular neutrophilic granulocytes, are equipped with granulebound proteins and peptides, ready to be released by specific stimuli at sites of inflammation [41]. These are expressed during differentiation of myeloid progenitors in the bone marrow when growth factors, sequentially activate distinct genes. Several of the granule-bound antibacterial peptides and proteins stored in neutrophil granules are produced by epithelial cells, either constitutively or during inflammation, e.g. hCAP-18 and lactoferrin [42]. In the bloodstream, leukocytes are transported in a resting state. After recruitment to sites of inflammation, specific stimuli activate surface-bound receptors resulting in the release of granule proteins to the external milieu or, if a microbe is ingested, into a phagosome; here, the microbe can be killed. In addition, a second wave of transcriptional activity is seen in leukocytes, involving expression of cytokines and chemokines, at sites of infection. Heparin-Binding Protein and Cathepsin G Heparin-binding protein (HBP; also named azurocidin and CAP37), is a 28-kDa antimicrobial protein of neutrophil azurophilic granules. It belongs to the serprocidin subgroup of the chymotrypsin-like protease superfamily that also includes neutrophil elastase, cathepsin G, and proteinase 3 [41]. Unlike the other serprocidins, HBP lacks protease activity due to mutations in the catalytic site. HBP has

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several activities during host defense. In addition to its antibacterial activity, it can induce capillary leakage, a key event in a severe condition named adult respiratory distress syndrome [43]. HBP is a specific chemoattractant for monocytes and neutrophils through activation of an as yet unknown G protein-coupled receptor [44, 45]. Similarly to HBP, cathepsin G is expressed in the bone marrow by myeloid progenitors and stored in the azurophilic granules of mature neutrophils [41]. This protein possesses both antimicrobial and proinflammatory activities [46]. Cathepsin G exerts chemotactic activity against neutrophils through activation of the high-affinity fMLF receptor FPR [47]. Human Cationic Antimicrobial Protein-18/LL-37 Human cationic antimicrobial protein with a molecular size of 18 kDa (hCAP-18) is the only member of the cathelicidin family in humans, while other vertebrates have several [42]. From the molecules of COOH-terminal, an antimicrobial peptide of 37 amino acids is cleaved off. Large amounts of hCAP-18 are present in specific granules of neutrophils and it is constitutively expressed by epithelial cells of epididymis of the male urogenital tract where it is found in seminal plasma [42, 48]. In addition, hCAP-18 expression has been demonstrated in a many epithelial cells upon inflammation [49]. In neutrophils, the antibacterial peptide LL-37 is cleaved off by proteinase 3, while activated gastricsin of seminal plasma generates the somewhat larger fragment, resulting in the ALL-38 antibacterial peptide [42]. In addition to its broad antibacterial properties, LL-37 is chemotactic for, and can induce calcium mobilization in human monocytes through binding FPRL1. LL-37 is also chemotactic for neutrophils and T cells that are known to express FPRL1 [50]. The LL-37 concentrations needed to activate the receptor are rather high, i.e. micromolar concentrations of LL-37 are required to elicit chemotactic responses and calcium mobilization, suggesting low affinity interactions with the receptor. However, in the microenvironment, high concentrations of LL-37 may be achieved. Granulysin Cytolytic T lymphocytes (CTLs) are required for protective immunity against intracellular pathogens such as L. monocytogenes and Trypanosoma cruzi, pathogens known to escape from the phagosomes into the cytoplasm of phagocytes. CTLs have also been implicated in the control of organisms that are phagocytized by macrophages and remain localized within the phagosomes (for example Salmonella typhimurium, E. coli, and Mycobacterium tuberculosis). An explanation for the functional role of CTLs in immunity against intracellular infection was provided by analysis of CTLs in tuberculosis [51]. CTLs kill infected cells through the granule-exocytosis pathway, releasing effector molecules, such as granulysin, that also kill the intracellular microbial pathogen.

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Antibacterial chemokines, ␤-defensins 3.

1.

2. PRRs Epithelium

2. PRRs

Mø/dendritic cell

IFN-␥, IL-1␤, TNF-␣

T cell

Fig. 3. Key events that may occur during mucosal infection, resulting in production of antibacterial chemokines and peptides. Bacteria break through an injured mucosal barrier (1). PRRs on antigenpresenting cells (macrophages, Mø, and dendritic cells) residing in the submucosa sense the bacteria. In addition, PRRs on epithelial cells can sense the bacteria, thus increasing the inflammatory response. This results in the production of proinflammatory cytokines such as IFN-␥, IL-1␤, and TNF-␣. The cytokines induce production of antibacterial chemokines and ␤-defensins by the epithelium, killing the bacteria (3).

Granulysin is a 9-kDa cationic protein produced by activated NK cells and cytotoxic T cells. It is composed of five ␣-helices connected by short loops. The molecule contains four cysteines that form two disulphide bonds within the molecule. Granulysin has potent antibacterial activity and cytotoxic activity occurs in the micromolar range [52]. Granulysin has been shown to exert activating and chemotactic responses in the nanomolar range against monocytes, CD4⫹, CD8⫹, memory (CD45RO) but not naïve (CD45RA) T cells, NK cells, and mature monocyte-derived dendritic cells [53]. The response was sensitive to pertussis-toxin, suggesting involvement of a G proteincoupled receptor.

Epithelial Cell Expression of Defensins and Antibacterial Chemokines – A Part of Mucosal Immunity Many antibacterial chemokines are expressed by epithelial cells at mucosal surfaces during inflammation (table 1). Macrophages and dendritic cells residing in the submucosa sense and process microbial antigens, resulting in the production of proinflammatory cytokines and chemokines. In addition, the release of cytokines contributes to an inflammatory phenotype of the adjacent epithelial layer (fig. 3). Epithelial cells themselves, may also sense microbial antigens through expression of PRRs. Integrity of the reproductive system against potential pathogens present in the environment is important both for the health of the individual but also for fertilization

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and continuance of species. Interestingly, several antibacterial peptides, for example hCAP-18 and MIG/CXCL9, are constitutively produced at relatively high concentrations in the epithelium of the male reproductive tract, in particular in the epithelium of the epididymis [49, 54]. The debate concerning the mechanisms regulating this constitutive expression has not been settled.

Complement Anaphylatoxin C3a – A Complement-Derived Antibacterial Peptide with Chemotactic Activity The complement system is an important part of innate immunity. Activation of the complement system can occur through either the classical, alternative, or the lectin pathway. These pathways all converge at the step where C3 is cleaved with release of multiple proteolytic fragments, including the anaphylatoxin C3a as described in detail by Rambach et al. [this vol., pp. 78–100]. The C3a molecule consists of 77 amino acids with a molecular weight of 9 kDa. The molecule is highly cationic (pI 11.3) and contains four ␣-helical regions. After binding its receptor, C3a possesses chemotactic activity and causes histamine release from mast cells. The carboxypeptidase N cleaves off the C-terminal arginine, resulting in the C3a-desArg peptide, which does not activate the C3a receptor. In vitro, C3a efficiently kills both Gramnegative and Gram-positive bacteria [55]. In addition, C3a-desArg and C4a, but not C5a also possess antibacterial activity [56].

Danger Signals, Alarmins, Kinocidins and the Bridging of Innate and Adaptive Immunity

Since the early days of immunology, it has been discussed how the body can discriminate between self and non-self, thus avoiding triggering of inflammation against molecules of the host (‘self ’). For instance, Charles Janeway, one of the pioneers in the field of innate immunity, claimed that PRRs made it possible for the host to sense non-self, e.g. highly conserved and redundant microbial structures [57]. This concept was proven to be true when TLRs and other PRRs were discovered. Later, Polly Matzinger launched the ‘danger hypothesis’, resulting in a paradigm shift. According to the danger hypothesis, the immune system is activated by molecules that are associated with events that constitute a threat to the body, irrespective of whether these are endogenous (e.g. formylated peptides derived from mitochondria, the high mobility group box protein chromosomal protein – HMGB1) or exogenous (e.g. LPS of Gram-negative bacteria) [58]. Oppenheim and Yang suggested the term ‘alarmins’ for some of the molecules described in this chapter, including molecules released by bacteria and damaged tissue of the host [59]. The recruitment and activation of

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antigen-presenting cells, such as macrophages and dendritic cells, are crucial in the early steps in mounting an adaptive immune response. The ‘alarmins’ include for example defensins, cathelicidin, eosinophil-derived neurotoxin, and HMGB1. Another term is ‘kinocidins’ suggested for chemokines possessing antibacterial activity [60]. Antimicrobial peptides, kinocidins, polypeptide venoms and other molecules that were once thought to be distinct in form and function now appear to be members of an ancient family of host defense effectors [60]. These molecules probably descended from archetype predecessors that emerged during the beginning of life on earth.

Unsolved Issues

Several antibacterial chemokines and peptides are presently examined for use as therapeutics. However, their mode of action at the bacterial surface is not fully understood. They perturbate the bacterial membranes, but the exact mechanism(s) is not known. Nor is much known about the influence of bacterial cell wall constituents, for example how the peptides pass the cell wall and reach their membrane target. Most, but not all, antibacterial peptides show decreased activity in the presence of salt at levels of those present in plasma. In addition, many are degraded by enzymes, both bacterial proteases and proteases released by host neutrophils. An important question is therefore, how these molecules can be efficient at sites of inflammation, where one finds both high proteolytic activity and presence of salts? Even if the use of peptide antibiotics would circumvent mechanisms of resistance to conventional antibiotics, bacteria have evolved other mechanisms during evolution to counteract antibacterial peptides.

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22 Wang W, Soto H, Oldham ER, Buchanan ME, Homey B, Catron D, Jenkins N, Copeland NG, Gilbert DJ, Nguyen N, Abrams J, Kershenovich D, Smith K, McClanahan T, Vicari AP, Zlotnik A: Identification of a novel chemokine (CCL28), which binds CCR10 (GPR2). J Biol Chem 2000;275:22313–22323. 23 Lazarus NH, Kunkel EJ, Johnston B, Wilson E, Youngman KR, Butcher EC: A common mucosal chemokine (mucosae-associated epithelial chemokine/ CCL28) selectively attracts IgA plasmablasts. J Immunol 2003;170:3799–3805. 24 Yount NY, Waring AJ, Gank KD, Welch WH, Kupferwasser D, Yeaman MR: Structural correlates of antimicrobial efficacy in IL-8 and related human kinocidins. Biochim Biophys Acta 2007;1768:598–608. 25 Björstad A, Fu H, Karlsson A, Dahlgren C, Bylund J: Interleukin-8-derived peptide has antibacterial activity. Antimicrob Agents Chemother 2005;49: 3889–3895. 26 Richmond A, Thomas HG: Purification of melanoma growth stimulatory activity. J Cell Physiol 1986;129: 375–384. 27 Becker S, Quay J, Koren HS, Haskill JS: Constitutive and stimulated MCP-1, GRO alpha, beta, and gamma expression in human airway epithelium and bronchoalveolar macrophages. Am J Physiol 1994;266: 278–286. 28 Wuyts A, Proost P, Lenaerts JP, Ben-Baruch A, Van Damme J, Wang JM: Differential usage of the CXC chemokine receptors 1 and 2 by interleukin-8, granulocyte chemotactic protein-2 and epithelial-cellderived neutrophil attractant-78. Eur J Biochem 1998;255:67–73. 29 Linge HM, Collin M, Mörgelin M, Egesten A: The human CXC chemokine GCP-2/CXCL6 possesses antibacterial activity. Submitted for publication. 30 Liao F, Rabin RL, Yannelli JR, Koniaris LG, Vanguri P, Farber JM: Human Mig chemokine: biochemical and functional characterization. J Exp Med 1995; 182:1301–1314. 31 Luster AD, Ravetch JV: Biochemical characterization of a gamma interferon-inducible cytokine (IP10). J Exp Med 1987;166:1084–1097. 32 Cole KE, Strick CA, Paradis TJ, Ogborne KT, Loetscher M, Gladue RP, Lin W, Boyd JG, Moser B, Wood DE, Sahagan BG, Neote K: Interferon-inducible T cell alpha chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3. J Exp Med 1998;187:2009–2021. 33 Loetscher M, Gerber B, Loetscher P, Jones SA, Piali L, Clark-Lewis I, Baggiolini M, Moser B: Chemokine receptor specific for IP10 and Mig: structure, function, and expression in activated T-lymphocytes. J Exp Med 1996;184:963–969.

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34 Sauty A, Dziejman M, Taha RA, Iarossi AS, Neote K, Garcia-Zepeda EA, Hamid Q, Luster AD: The T cell-specific CXC chemokines IP-10, Mig, and ITAC are expressed by activated human bronchial epithelial cells. J Immunol 1999;162:3549–3558. 35 Egesten A, Eliasson M, Johansson HM, Olin AI, Mögelin M, Mueller A, Pease JE, Frick IM, Björck L: The CXC chemokine MIG/CXCL9 is important in innate immunity against Streptococcus pyogenes. J Infect Dis 2007;195:684–693. 36 Tang YQ, Yeaman MR, Selsted ME: Antimicrobial peptides from human platelets. Infect Immun 2002; 70:6524–6533. 37 Ganz T: Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol 2003;3:710–720. 38 Chertov O, Michiel DF, Xu L, Wang JM, Tani K, Murphy WJ, Longo DL, Taub DD, Oppenheim JJ: Identification of defensin-1, defensin-2, and CAP37/ azurocidin as T-cell chemoattractant proteins released from interleukin-8-stimulated neutrophils. J Biol Chem 1996;271:2935–2940. 39 Yang D, Chen Q, Chertov O, Oppenheim JJ: Human neutrophil defensins selectively chemoattract naive T and immature dendritic cells. J Leukoc Biol 2000; 68:9–14. 40 Grigat J, Soruri A, Forssmann U, Riggert J, Zwirner J: Chemoattraction of macrophages, T lymphocytes, and mast cells is evolutionarily conserved within the human alpha-defensin family. J Immunol 2007; 179:3958–3965. 41 Borregaard N, Cowland JB: Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 1997;89:3503–3521. 42 Borregaard N, Sørensen OE, Theilgaard-Mönch K: Neutrophil granules: a library of innate immunity proteins. Trends Immunol 2007;28:340–345. 43 Gautam N, Olofsson AM, Herwald H, Iversen LF, Lundgren-Akerlund E, Hedqvist P, Arfors KE, Flodgaard H, Lindbom L: Heparin-binding protein (HBP/ CAP37): a missing link in neutrophil-evoked alteration of vascular permeability. Nat Med 2001;7: 1123–1127. 44 Pereira HA, Shafer WM, Pohl J, Martin LE, Spitznagel JK: CAP37, a human neutrophil-derived chemotactic factor with monocyte specific activity. J Clin Invest 1990;85:1468–1476. 45 Chertov O, Ueda H, Xu LL, Tani K, Murphy WJ, Wang JM, Howard OM, Sayers TJ, Oppenheim JJ: Identification of human neutrophil-derived cathepsin G and azurocidin/CAP37 as chemoattractants for mononuclear cells and neutrophils. J Exp Med 1997;186:739–747.

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46 Odeberg H, Olsson I: Microbicidal mechanisms of human granulocytes: synergistic effects of granulocyte elastase and myeloperoxidase or chymotrypsin-like cationic protein. Infect Immun 1976; 14:1276–1283. 47 Sun R, Iribarren P, Zhang N, Zhou Y, Gong W, Cho EH, Lockett S, Chertov O, Bednar F, Rogers TJ, Oppenheim JJ, Wang JM: Identification of neutrophil granule protein cathepsin G as a novel chemotactic agonist for the G protein-coupled formyl peptide receptor. J Immunol 2004;173:428–436. 48 Malm J, Sørensen O, Persson T, Frohm-Nilsson M, Johansson B, Bjartell A, Lilja H, Ståhle-Backdahl M, Borregaard N, Egesten A: The human cationic antimicrobial protein (hCAP-18) is expressed in the epithelium of human epididymis, is present in seminal plasma at high concentrations, and is attached to spermatozoa. Infect Immun 2000;68:4297–4302. 49 Frohm Nilsson M, Sandstedt B, Sorensen O, Weber G, Borregaard N, Ståhle-Bäckdahl M: The human cationic antimicrobial protein (hCAP18), a peptide antibiotic, is widely expressed in human squamous epithelia and colocalizes with interleukin-6. Infect Immun 1999;67:2561–2566. 50 Yang D, Chen Q, Schmidt AP, Anderson GM, Wang JM, Wooters J, Oppenheim JJ, Chertov O: LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. J Exp Med 2000;192:1069–1074. 51 Stenger S, Hanson DA, Teitelbaum R, Dewan P, Niazi KR, Froelich CJ, Ganz T, Thoma-Uszynski S, Melian A, Bogdan C, Porcelli SA, Bloom BR, Krensky AM, Modlin RL: An antimicrobial activity of cytolytic T cells mediated by granulysin. Science 1998; 282:121–125. 52 Clayberger C, Krensky AM: Granulysin. Curr Opin Immunol 2003;15:560–565. 53 Deng A, Chen S, Li Q, Lyu SC, Clayberger C, Krensky AM: Granulysin, a cytolytic molecule, is also a chemoattractant and proinflammatory activator. J Immunol 2005;174:5243–5248. 54 Linge HM, Collin M, Giwercman A, Malm J, Bjartell A, Egesten A: The antibacterial chemokine MIG/CXCL9 is constitutively expressed in epithelial cells of the male urogenital tract and is present in seminal plasma. J Interferon Cytokine Res 2008;28: 191–196. 55 Nordahl EA, Rydengård V, Nyberg P, Nitsche DP, Mörgelin M, Malmsten M, Björck L, Schmidtchen A: Activation of the complement system generates antibacterial peptides. Proc Natl Acad Sci USA 2004; 101:16879–16884.

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61 Qian YQ, Johanson K, McDevitt P: Nuclear magnetic resonance solution structure of truncated human GRO-beta [5–73] and its structural comparison with CXC-chemokine family members GROalpha and IL-8. J Mol Biol 1999;294:1065–1072. 62 Swaminathan GJ, Holloway DE, Colvin RA, Campanella GK, Papageorgiou AC, Luster AD, Acharya KR: Crystal structures of oligomeric forms of the IP-10/CXCL10 chemokine. Structure (Camb) 2003;11: 521–532. 63 Booth V, Clark-Lewis I, Sykes BD: NMR structure of CXCR3 binding chemokine CXCL11 (ITAC). Protein Sci 2004;13:2022–2028.

Arne Egesten, MD, PhD Section for Respiratory Medicine, Department of Clinical Sciences Biomedical Center B14, Lund University Tornavägen 10, SE–221 84 Lund (Sweden) Tel. ⫹46 46 222 4445, Fax ⫹46 46 15 7756, E-Mail [email protected]

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The Role of Neutrophils and Monocytes in Innate Immunity Chahrazade Kantaria ⭈ Magali Pederzoli-Ribeilb ⭈ Véronique Witko-Sarsata a INSERM U845, Necker Hospital, Paris, France, and bWilliam Harvey Research Institute, London, UK

Abstract Polymorphonuclear neutrophils (PMNs) and monocyte/macrophages (MMs) are professional phagocytic cells that are able to phagocytose and destroy infectious agents. Therefore, they are key anti-infectious actors in host defense but can mediate tissue damages. In addition, it is now clear that the role of these cells goes far beyond phagocytosis and pathogen killing. PMNs and MMs are essential cells for immunity, absolutely required to build and modulate the innate response. The respective roles of PMNs and MMs in the inflammatory process are discussed: their common features and their differences are reviewed, both in terms of origins and functions with special emphasis on novel concepts about neutrophil survival and resolution of inflammation. The recognition and the subsequent engulfment of apoptotic PMNs by macrophages is a key event of the resolution of inflammation, which can be associated with autoimmunity or inflammatory diseases. During the past years, significant efforts have been made to dissect the molecular mechanisms governing phagocytosis and pathogen killing. Although these effector functions are crucial, more work has to be done to understand the respective role of PMNs and MMs to regulate and inhibit the inflammatory process as well as the immune response. This might be the future challenge for the next years in phagocyte research and this will presumably open new avenues of research in the Copyright © 2008 S. Karger AG, Basel modulation of inflammation.

Polymorphonuclear neutrophils (PMNs) and monocyte/macrophages (MMs) are professional phagocytic cells that are able to phagocytose and destroy infectious agents. Therefore, they are key anti-infectious actors in host defense but are also inflammatory cells that can mediate tissue damages. In addition, it is now clear that the role of these cells goes far beyond phagocytosis and pathogen killing. PMNs and MMs are essential cells for immunity, absolutely required to build and modulate the innate response. In this chapter, we will discuss both the common features and the differences between neutrophils and monocytes, both in terms of origins and functions. Inflammation is a manifestation of the body’s response to tissue damage and infection that may be beneficial for the defense against agents deranging its homeostasis, but also

harmful for the surrounding tissues [1]. Herein, we will focus on the critical role of PMNs [2, 3] and monocytes [4] which are two professional phagocytic cells [5]. First, we will provide a survey of the mechanisms involved in adhesion, migration and phagocytosis, with novel concepts arising. Second, we will provide a description of the molecular pathways involved in the resolution of inflammation and especially the phagocytosis of apoptotic PMNs by macrophages [6]. Failure in the resolution of inflammation could explain, at least in part, many prevalent diseases, such as chronic inflammatory diseases, autoimmune but also degenerative diseases [7]. This is the paradox of the inflammatory reaction: it is an essential process for the well-being and survival, but at the same time it can trigger tissue damage and be more deleterious than the pathogenic agent itself [8].

Polymorphonuclear Neutrophils: The Most Potent Microbicidal Phagocyte

PMNs represent 70% of the population of circulating granulocytes, the others being the eosinophils and the basophils with 2 and ⬍1%, respectively. In the hours following tissue damage, PMNs are massively mobilized from the ‘marginated pool’ of PMNs, thus resulting in a rapid and dramatic neutrophilia that is a known feature of acute inflammation. Because of their powerful microbicidal equipment, they have a major role in the anti-infectious defense [9]. Their lifespan is short: after phagocytosis, PMNs undergo apoptosis and must be eliminated from the inflammatory site to avoid tissue damage: this anti-inflammatory disposal of PMNs is achieved by macrophages. It thus becomes evident that neutrophil apoptosis and phagocytosis by macrophages are key steps in the resolution of inflammation. Numerous studies have clearly pointed out the paradox of PMNs: their powerful molecules including oxidants and proteinases are indispensable for their bactericidal functions but are also potentially very toxic for the host tissues, when neutrophil activation is not controlled. This proinflammatory role of PMNs is also illustrated by their capacity to generate proinflammatory cytokines.

Monocytes/Macrophage: Mononuclear Phagocytes with Different Possible Fates

Monocytes recruited from the bloodstream to tissues differentiate either in macrophages or in dendritic cells that will next migrate into lymph nodes. Monocytes migrate to inflammatory sites after the PMNs. Like PMNs, they phagocytose foreign particles, including pathogens but also apoptotic cells, including apoptotic PMNs. Their bactericidal potential is less elaborated than that of PMNs, and thus monocytes can be considered as a second line of defense against pathogens. However, monocytes are an important source of cytokines that trigger the recruitment of other inflammatory cells, especially PMNs. In contrast to PMNs, MMs can display foreign antigens

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Monocyte PMN

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Endothelium activation PMN chemoattractants: CXC chemokine: IL-8, F-MLP, LPS, LTB4

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Monocyte recruitment via CC chemokines IL-6, TNF-␣, IL-1

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Lipid mediators Resolvin, protecin, lipoxin Annexin

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Macrophage

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Microbicidal mechanisms Oxidative burst: superoxide anion, H2O2, HOCl Proteinase, antibiotic proteins, defensins

Lymphocyte recruitment via CC chemokines (MCP1)

Antigen presentation instruction of the immune response

Phagocytosis of apoptotic PMN Anti-inflammatory process IL-10, TGF-␤, IL-13 Lipid mediators Time

Lymphocytes within lymphatic circulation

Fig. 1. Schematic view of the role of PMNs and MMs in inflammation. The time course of leukocyte traffic is represented by five different steps. (1) PMN adhesion, diapedesis and migration to the inflammatory site occur in the first hours after infection: activated endothelium by IL-8, plateletactivating factor (PAF), leukotriene B4 (LTB4) express adhesion molecules which stop and prime PMNs. (2) Phagocytosis and killing of the pathogens by PMN involving both oxygen-dependent and -independent mechanisms (24 h). (3) Monocyte mobilization from blood (48 h): they migrate to the inflammatory site and generate both proinflammatory mediators (IL-6, TNF-␣, IL-1) and cytokines to attract monocytes and lymphocytes; monocytes ‘switch’ to produce lipid mediators with antiinflammatory activities which stop PMN recruitment. (4) Recognition and engulfment of apoptotic PMNs by macrophages. (5) Recruitment of lymphocytes and antigen presentation to connect with the adaptive immune response (after 72 h).

on their surface in a form that can be recognized by antigen-specific T lymphocytes and are therefore antigen-presenting cells (APCs). Monocytes/macrophages are involved in numerous physiological processes such as the lipid metabolism, the immunoglobulin catabolism and the synthesis of transferrin, complement components, and coagulation factors. Both PMNs and MMs can be stimulated by T cell-derived cytokines, for instance INF-␥, and phagocytose opsonized particles, so that these cells serve important effector functions in specific immune response. The cooperation between PMNs and MMs in the inflammatory response is illustrated in figure 1.

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Neutrophil Granulocytes and Monocytes: A Common Origin but Separate Fate

PMNs and blood monocytes are generated within hematopoetic tissues and are released in the circulation after a series of different maturation and differentiation steps [10]. Granulocytic and monocytic cells differentiate from a common myeloid precursor because of the influence of growth factors such as the granulocyte colonystimulating factor (G-CSF) and the monocyte colony-stimulating factor (M-CSF) on PMNs and monocytes, respectively [11]. Cells orientated towards the granulocytic pathway are successively the myeloblast, promyelocyte, metamyelocyte, nonsegmented granulocyte and the mature PMN. Granule biogenesis follows the granulocyte differentiation pathway [12]. The azurophilic granules first emerge at the stage of promyelocytes and contain myeloperoxidase, serine proteases and antibiotic proteins. Azurophil granules are thus considered as the true microbicidal compartment mobilized upon phagocytosis. Later in differentiation, at the metamyelocyte stage, specific granules containing lactoferrin and collagenase emerge followed by the tertiary granule population containing gelatinase. A fourth type of granules, called the secretory vesicles, appear at the stage of mature neutrophil. Their origin might be endocytic since they contain plasma proteins such as albumin. However, recent findings have shown that this strict compartimentalization is not a dogma. For instance, proteinase 3, a serine protease described in azurophil granules is also localized in the membrane of secretory vesicles, the most mobilizable compartment of neutrophils [13] and in the plasma membrane [14]. The mechanisms underlying the secretion of the four morphologically distinct populations of granules may be under separate control. The order of exocytosis observed after ionophore-induced progressive elevation of cytosolic calcium was secretory vesicles, gelatinase granules, specific granules, and lastly azurophilic granules [15]. This order is of crucial importance to have a proper timing between degranulation of a given compartment and its associated function: for instance, secretory vesicles which contain receptors and adhesion molecules will be mobilized first to help the PMN to adhere to endothelium. In contrast, azurophilic granules which contain most of the antibiotic proteins or toxic proteinases will be the last compartment to be mobilized within the phagocytic vacuole, thus precluding surrounding tissue damages. Cells orientated towards the monocytic pathway are successively the monoblast, promonocyte and monocyte. The biogenesis of monocyte granules has not been extensively studied as compared to that of PMN. Monocytes have peroxidase-positive granules containing myeloperoxidase which decreases after differentiation. Interestingly, macrophages have a lower amount of MPO After leaving the bone marrow compartment, mature monocytes stay 2–3 days in blood before being recruited within tissues to differentiate into macrophages. Most resident macrophages in the normal adult are derived from circulating bone marrow-derived monocytes and are thus

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called monocyte/macrophages. Blood monocyte-like cells also give rise to related dendritic cells and osteoclasts. Studies of the expression of differentiation antigens and surface receptors with monoclonal antibodies have shown that tissue macrophages become markedly heterogeneous and express very different phenotypes, reflecting specialization of function within particular microenvironments [4]. As well as distinct subpopulations in lymphoids organs, MMs are found in nonlymphoid organs like the liver (Kupffer cells), lung (alveolar MMs) nervous system (microglia), epidermis (Langerhans cells), reproductive organs, and serosal cavities. MMs are also found within the lamina propria of the gut and the interstitium of organs such as the heart, pancreas, and kidney [16]. In response to inflammatory and immune stimulation, additional monocytes are recruited to local sites, where they display different phenotypes. Some data strongly support the notion of two subsets of mammalian monocytes: the ‘inflammatory’ and the ‘resident’ monocytes. In the mouse, the ‘inflammatory’ subsets that express the surface marker Gr-1 (also known as Ly6c), take up antigen in peripheral tissues, differentiate into inflammatory dendritic cells (DCs) and then migrate into lymphoid organs [17]. The second subset of monocytes has been termed resident because they were found in both resting and inflamed tissues. This subset is defined by the lack of expression of Gr-1 and high expression of the molecule LFA-1 [18].

Adhesion and Migration Allow the Critical Positioning of Polymorphonuclear Neutrophils and Monocyte/Macrophages

Analysis of the time course of leukocyte traffic in mouse model of acute inflammation, the zymosan-induced peritonitis [19], has shown that during the first hours of inflammatory reaction, the number of leukocytes increased and this infiltrate is composed predominantly by PMNs at 24 h. The number of monocyte/macrophages increased more slowly to become the major cell type present between 48–72 h after zymosan treatment. This cell profile switch from PMNs to monocytes and phagocytosing macrophages is regulated by the interaction of IL-6 with its soluble receptor [20, 21]. Binding of IL-6 to a solubilized receptor suggests a downregulation of IL-6driven inflammation. This interaction promotes chemokine shift by inhibiting CXC chemokine synthesis and promoting CC chemokines, which trigger PMN recruitment suppression in favor of mononuclear cell influx.

Adhesion and Migration through the Endothelium toward the Inflammation Site Neutrophil recruitment and monocyte trafficking are two crucial events of the innate immunity since they allow the positioning of these two immunocompetent cells in

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the body, and especially at the site of inflammation. These two events require adhesion and transmigration through blood vessel walls, by the traditional and sequential three steps of rolling, activation and firm adhesion. Both PMNs and MMs use identical adhesion mechanisms to migrate from the blood to the tissues [22]. Here, we will underline the classical steps in this process and we will also emphasize the more recent concepts that are now emerging. Traffic and Margination Mature PMNs are partioned between the ‘circulating’ and the ‘marginated’ pool which is the result of PMN sequestration in pulmonary capillaries. These latter can be quickly and efficiently mobilized upon inflammation. Contrary to PMNs, circulating monocytes are released from bone marrow as non-differentiated cells. Whereas PMNs are recruited within hours at the site of inflammation, monocytes can circulate in blood from 1 to 3 days [10]. Two subsets of circulating monocytes have been identified: the inflammatory monocytes, which are mobilized to inflamed peritoneum and the resident monocytes recruited to tissues independently of inflammatory stimuli [18]. Following recruitment to peripheral tissues, inflammatory monocytes can differentiate into macrophages or myeloid dendritic cells, as has been shown in vitro [23–25]. Adhesion to the Endothelium The dual neutrophil functions of immune surveillance and in situ elimination of microorganisms require a rapid transition between a circulating nonadherent state to an adherent state, allowing them to migrate into tissues where necessary. Rolling Selectin-Mediated Rolling. The early steps of rolling are mediated by selectins, namely L-selectin expressed in most leukocytes and P-selectin and E-selectin expressed by inflammatory endothelial cells that interact with P-selectin glycoprotein ligand 1 (PSGL1), located at the tip of leukocyte microvilli [26]. The binding of these cell surface proteins to their ligands allows PMNs and monocytes to bind weakly to endothelial cells and to initiate the adhesion cascade. Rare deficits in neutrophil selectin ligand expression, due to a metabolic defect in a synthetic pathway common to all selectin ligands, lead to faulty neutrophil trafficking in humans. P-selectin, readily mobilized in a few minutes to the endothelial cell surface following stimulation by thrombin, histamine or oxygen radicals, interacts primarily with PSGL1. Rolling subsequently involves E-selectin, which appears on endothelial cells 1–2 h after cell stimulation by interleukin-1 (IL-1), TNF-␣ or lipopolysaccharide (LPS) [27, 28]. In addition to PSGL1, E-selectin can bind to E-selectin ligand 1, a molecule highly homologous to the cysteine-rich FGF receptor located on neutrophil microvilli, and to the proteoglycan CD44 [29]. The kinetics of neutrophil recruitment in selectin-deficient mice suggests that P- and L-selectin contribute sequentially to

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leukocyte rolling and shows that L-selectin is involved in the prolonged neutrophil sequestration in inflamed microvasculature [30, 31]. The binding of PSGL1 to Lselectin nucleates leukocyte-leukocyte interaction that facilitates secondary leukocyte capture or tethering, terms that are used synonymously. Secondary tethering also enables leukocytes that do not express ligands for P-selectin and E-selectin to reach sites of inflammation. This secondary tethering would synergistically enhance accumulation on the inflamed endothelium. Integrin-Mediated Rolling. The notion of ‘slow rolling’ has recently emerged in the literature. This step, defined as a rolling mediated by selectins and integrins most commonly seen in PMNs, precedes the arrest of leukocytes on the endothelium. When mice were treated with TNF-␣, endothelial cells expressed E-selectin and increased the amount of ICAM1, and the rate of neutrophil rolling in the venules became slow. This slow rolling in vivo was shown to require not only E-selectin, but also engagement of ␤2-integrin [32], specifically lymphocyte function-associated antigen 1 (LFA1; also known as ␣L␤2-integrin) or macrophage receptor 1 (MAC1; also known as CD11b/ CD18 and ␣M␤2-integrin) [33]. It has been previously shown that monocytes and monocyte-like cell lines can roll on immobilized vascular celladhesion molecule 1 (VCAM1) by engaging their cell surface ligand very late antigen 4 (VLA-4; also known as ␣4␤1-integrin) [34, 35]. Interestingly, the ␣L␤2-integrin (LFA1) is also required for the step of crawling for the ‘resident’ monocyte subset. Indeed, intravenous injection of blocking antibodies to either CD11a or CD18 resulted in the rapid, complete and prolonged release of ‘resident’ monocytes from the endothelial wall [36]. Leukocyte Activation and Firm Adhesion to Endothelium First Step: Leukocyte Activation by Chemokines. During inflammation, endothelial cells are activated by inflammatory cytokines and they present on their luminal surface chemoattractants such as platelet-activating factor, leukotriene B4, bacterial peptides and various chemokines. Chemokines are presented to leukocytes by proteoglycans such as CD44, syndecan 1, 2 and 4 [37, 38]. Among chemokines, IL-8 specifically attracts PMNs, while having no effect on monocytes [39] and being unable to promote lymphocyte migration through endothelium. IL-8 is a major neutrophil chemoattractant, as shown by the complete inhibition of neutrophil recruitment in inflammation sites by anti-IL-8 monoclonal antibodies in animal models [40, 41]. There is evidence that microvascular endothelial cells not only synthesize IL-8 in response to IL-1 or LPS, but also store IL-8 in Weibel-Palade bodies and release it upon stimulation by histamine or thrombin [42]. Second Step. Integrin-mediated arrest leads to firm adhesion with the notion of ‘inside-out’ and ‘outside-in’ activation. In vitro and in vivo studies have established that leukocyte arrest during rolling is rapidly triggered by chemokines or other chemoattractants and is mediated by the binding of leukocyte integrins to proteins from the immunoglobulin superfamily such as ICAM1 and VCAM1, expressed by

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endothelial cells [43, 44]. Integrins most relevant to leukocyte arrest belong to the ␤1and ␤2-subfamilies [45]. The ‘Inside-Out’ Signaling ␤2-Integrins are unable to interact with their physiological ligands in unstimulated PMNs, a safety mechanism that controls acute and chronic inflammatory responses. But ligation of chemokines to a specific G protein-coupled receptor expressed at the cell surface of leukocyte activates integrins by triggering a complex intracellular signaling network. This process is referred to as ‘inside-out’ signaling. This induces integrins to undergo a dramatic transition from a bent low-affinity conformation to extended intermediate- and high-affinity conformations, which leads to opening of the ligand-binding pocket [46]. Modulation of the affinity of an integrin to its ligand is widely recognized as a crucial step in chemokine-induced arrest under flow [34, 47]. The intracellular signaling cascades from GPCRs to integrin activation are still incompletely understood. One pathway involves phospholipase C signaling (PLC), activation of small GTPases and induction of transitional integrin conformational changes through the association with actin-binding proteins such as talin-1. Another signaling pathway involves the activation of PI3 kinase, which then mediates the activation of the atypical protein kinase C-␰ that enables the clustering of activated integrins, and achieves the highest level of integrin avidity. When the integrin is in its high affinity form, the protein tyrosine kinase 2 is recruited to the integrin clusters that are formed and mediate signaling that further stimulates the formation of the polarity complex necessary for extravasation. The ‘Outside-In’ Signaling Ligand-induced integrin clustering and allosteric conformational changes probably contribute to the initiation of ‘outside-in signaling’, defined as the process by which ligation of a cell-surface receptor activates signaling pathways inside the cell. The induction of conformational changes to the cytosolic tail of the LFA-1 heterodimer upon ICAM1 binding may have a role in the rapid arrest of leukocytes under flow [47]. The role of integrins is not restricted to adhesion. It is now well established that activation of integrins generates intracellular signaling that regulates various cellular functions such as cell motility, proliferation and apoptosis [48]. Transendothelial Cell Migration Leukocytes tightly adhering to the endothelium need to cross the vascular barrier to reach the underlying inflamed tissues. This can be achieved by active, integrin-mediated cell migration. Migration requires polarization of the cell body and the formation of a lamellipodium. It also requires the formation of a uropod at the tail of the leukocyte [49, 50]. The formation of these structures is driven by integrins and the cytoskeleton. When integrins are activated and the cell migration machinery is functionally polarized, the leukocyte travels along the luminal, and then transmigrates

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passing between adjacent endothelial cells. Leukocytes have to pass through three distinct barriers: endothelial cells, the endothelial cell basement membrane and pericytes. Notion of Crawling Crawling on the endothelium is defined as an extension of leukocyte membrane protrusions into the endothelial cell body mediated by ICAM1 and MAC1 in order to seek preferred sites of transmigration. This event is a crucial step in the adhesion cascade of monocytes [51]. This observation was confirmed in vivo and extended to PMNs. When the crawling is disabled, transmigration is delayed and occurs preferentially through the transcellular pathway as opposed to the paracellular pathway [52]. The notion that integrin, and especially the ␣L␤2-integrin (LFA1), are required for ‘resident’ monocyte crawling has recently emerged. These mechanisms also implicate the interaction of the chemokine receptor CX3CR1, which binds fractalkine, highly expressed in the ‘resident’ monocytes and its ligand expressed on endothelial cells. These data demonstrate a new mechanism of leukocyte crawling on endothelial cells and a new role for LFA-1 [36]. Paracellular Migration Using these pathways, leukocytes will have to disrupt three types of endothelial junctions: tight junctions, adherent junctions and gap junctions [53, 54]. The mobilization of endothelial junctional molecules for which leukocytes express ligands to the luminal surface creates a chemotactic gradient that guides leukocytes to the junctions. Three types of molecules that are concentrated at the lateral borders of endothelial cells have been implicated in this process of transmigration: JAMs, PECAM-1 and CD99. JAMs and PECAM-1 belong to the IgG superfamily, whereas CD99 is a unique molecule. All of these molecules are capable of homophilic interactions; that is, a molecule on one endothelial cell binding to the same molecule on the apposing endothelial cell. The JAMs family consist of three members, namely JAM-A, JAM-B and JAM-C that are tight junctional molecules. JAM-A is involved in the transendothelial migration of leukocytes by interacting with the integrin LFA1 [55]. It has been shown that a JAM-A-specific monoclonal antibody (BV11) inhibits spontaneous and chemokine-induced migration of monocytes across the endothelium [56] JAM-B and JAM-C by interacting respectively with VLA-4 and MAC1 on the leukocyte participate to transendothelial migration [57]. The interendothelial adhesion molecule platelet/endothelial cell adhesion molecule PECAM-1 (also known as CD31) has been described as a crucial mediator of monocyte and neutrophil transendothelial migration [58]. It is expressed both on the leukocyte surface and at the endothelial cell, where it is largely concentrated at intercellular junctions. It mediates extravasation of PMNs via PECAM-1/PECAM-1 homophilic interactions. The absence of PECAM-1 in knockout mice only slightly affects the number of monocytes undergoing transmigration but the migration of PMNs is

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blocked at the level of the basement membrane of postcapillary venules [59]. Similar to PECAM-1, CD99 is concentrated in the regions between endothelial cells. Blocking CD99 was found to suppress monocyte and neutrophil transmigration through cytokine-stimulated human umbilical vein endothelial cells and blockade of PECAM-1 and CD99 exerts an additive effect on monocyte and neutrophil transmigration [60, 61]. These data suggest that PECAM-1-mediated leukocyte migration promotes the CD99-mediated step. Other molecules such as ICAM-1 and ICAM-2 play a role in transmigration by interacting with their ligand LFA-1. Some of these molecules mediate transmigration through a stimulus-dependent manner. PECAM1, ICAM-2 and JAM-A mediate leukocyte transmigration in response to IL-1␤ but not TNF-␣ [62]. Transcellular Migration Until recently, leukocyte transendothelial cell migration was thought to occur through the paracellular pathway only, but it is now well established that leukocyte transcellular migration occurs in various inflammatory scenarios [63] and in in vitro models [64–66]. Transcellular leukocyte migration represents the route of emigration for only a minority of emigrating cells (5–20% of transmigrating cells through cytokine-activated human umbilical vein endothelial cells [65], although the response can be very rapid – less than 1 min) [64]. Transcellular migration occurs in thin parts of the endothelium, and therefore there is less distance for a leukocyte to migrate. Migration through the Endothelium Basement Membrane Migration through the endothelial basement membrane and pericyte sheath can occur through gaps between adjacent pericytes and regions of low protein deposition within the extracellular matrix. This response can be facilitated by ␣6␤1 integrin and possibly proteases, such as matrix metalloproteinases and neutrophil serine proteinases.

Migration to Tissues in Noninflammatory Conditions: A Specific Feature of Monocytes Whereas most leukocytes use the general mechanisms of adhesion and migration that we have discussed, the positioning of monocyte-derived cells in tissues is controlled by different chemokines than those that activate the leukocyte integrin LFA1, MAC1 and VLA-4 [10]. Macrophages and dendritic cells originate from the bone marrow and therefore need to be recruited from circulating precursors in the blood. Among the chemotactic molecules that are constitutively expressed and act on circulating monocytes, the chemokine CXCL14 has been proposed to be involved in the constitutive trafficking of monocytes. The migration of monocytes to noninflammed tis-

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sues to differentiate in resident dendritic cells or resident macrophages is mediated by the interaction between ␤2-integrin and ICAM2.

Phagocytosis of Pathogens: A Common Feature of Polymorphonuclear Neutrophils and Monocyte/Macrophages

Contact of Pathogens with Phagocytes Is Mediated by Different Receptors The surface receptors of PMNs and monocyte/macrophages regulate a range of functions, including differentiation, growth and survival, adhesion, migration, phagocytosis, activation and cytotoxicity [67]. Their ability to recognize a wide range of endogenous and exogenous ligands and trigger the antimicrobial effect of both PMNs and monocytes is a crucial step in innate immunity [68]. Many different receptors recognize microbes and phagocytosis is usually mediated simultaneously by multiple receptors. Phagocytes (PMNs and MMs) express many different receptors that participate in particle recognition and internalization [5]. Some of these receptors are able to transmit intracellular signals that trigger phagocytosis, while others appear primarily to participate in binding or to increase the efficiency of internalization. We will briefly mention the most important receptors implicated specifically in phagocytosis. Fc␥ Receptors Fc␥ receptors are cell surface receptors that bind to the Fc region of IgG. By expressing these receptors, PMNs and macrophages are able to recognize IgG-opsonized particles. There are two classes of Fc␥ receptors: receptors that contain ITAM motifs in their intracellular domains that recruit kinases and activate phosphorylation cascades and receptors that contain ITIM motifs that recruit phosphatases and inhibit signaling [69]. Activating receptors with high affinity (Fc␥-RI) and low affinity (Fc␥-RIIA and Fc␥-RIIIA) bind IgG-opsonized particles and trigger internalization through actin polymerization, membrane recruitment to the site of particle contact, membrane extension outward to surround the particle, and particle engulfment [70]. Complement Receptor PMNs and monocytes express three types of complement receptors (CRs): CR1 (also known as CD35); CR3 (␣M␤2 integrin, CD11b/CD18 or MAC1) and CR4 (␣M␤2 integrin, CD11bc/CD18). Via these receptors, phagocytes bind to complementopsonized microbes. CR1 binds to complement components C1q, C4b, C3b and Mannan-binding lectin. Inflammatory cytokines (TNF-␣) and microbial products (LPS) stimulate phagocytosis through CR3. In vitro, this activation signal can be stimulated by phorbol miristate acetate and is therefore likely to involve PKC activation

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[70]. CR and Fc␥ receptor colligation can produce cooperative effects, since a study showed that macrophages do not internalize particles coated with suboptimal concentrations of IgG, but do internalize these particles when they are also coated by complement [71]. Scavenger Receptors Defined originally by their ability to bind and internalize modified lipoproteins, scavenger receptors additionally bind to such diverse ligands such as polyribonucleotides, LPS and silica particles [72]. Two members of the scavenger receptor family have been implicated in binding and internalizing microbes: Scavenger A and macrophage receptor with collagenous structures (MARCO). Macrophages from mice lacking scavenger A are less efficient at phagocytosing heat-killed Escherichia coli [73, 74]. MARCO binds to Gram-positive and Gram-negative bacteria, and blocking of MARCO significantly decreases internalization of these particles [16]. Lectins Lectins mediate detection of self- and foreign carbohydrate and cooperate in detection of microbes. They are involved in the uptake of zymosans. Toll-Like Receptors and Nod-Like Receptors They are two families of innate immune recognition receptors which are required for detection of a broad range of microbial products including LPS, peptidoglycan and bacterial lipopeptides [75, 76]. These receptors are involved in the initiation of the inflammatory response upon infection. These receptors are primary sensors of microbial products and recognize pathogen-associated molecular patterns (PAMPs) that are in turn responsible for the expression of proinflammatory genes [77]. Some of them are involved in caspase-1 activation, during the inflammasome complex formation [78], leading to pro-IL-1␤ cytokine processing [79]. They are essential to induce the innate immune response [80] but at the same time they need to be tightly regulated to avoid induction of chronic inflammatory disorders. This occurs via negative feedback that involves many negative regulators, like soluble decoy receptors or intracellular molecules [81]. However, TollLike Receptors (TLRs) or Nod-like receptors do not trigger phagocytosis by themselves.

Recognition of Microbes by Receptors Triggering Different Intracellular Signaling Pathways Particle internalization is accompanied by activation of many signaling pathways that together orchestrate rearrangement of the actin cytoskeleton, extension of the plasma

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membrane and engulfment. Numerous signaling molecules including actin-binding proteins, membrane traffic regulators, ions channels, kinases and lipases are activated during phagocytosis of complex particles and may contribute to efficient particle internalization. Phosphoinositides 3-Kinase and Phospholipase C PI3 kinase catalyzes phosphorylation of PI(3,4;5)P3, a phospholipid important in recruiting signaling molecules such as kinase AKT/PKB to specific regions of membranes [82]. Pharmacological inhibition of PI3 kinase blocks phagocytosis of IgG – and complement opsonized particles, unopsonized zymosan and bacteria [83]. Phosphoinositide PLC mediates the mobilization of intracellular Ca2⫹ stores and activates PKC family members. Phosphoinositide PLC is recruited to phagosomes containing IgG-opsonized particles, and inhibition of its activity blocks particle internalization. Like protein kinase C inhibitors, PLC inhibitors completely block the formation of actin filaments at the site of particle contact. Rho GTPases Members of the Rho family of small molecular weight GTPases (Cdc42, Rac and Rho) plays central roles in phagocytosis and due to their interactions with other signaling pathways, are likely to be points of intersection for signaling pathways that regulate phagocytic efficiency [84, 85]. Protein Kinase C Protein kinase C family members are required for phagocytosis, but also participate in many other signaling transduction pathways. General inhibitors of PKC activity inhibit internalization of IgG-opsonized and complement-opsonized particles as well as unopsonized zymosan particles [86, 87]. PKC is required at the earliest stages of particle internalization since inhibition of PKC blocks the formation of actin filaments at the site of particle binding.

Phagocytosis-Induced Activation and Destruction of the Pathogen Microbe internalization by phagocytes is usually accompanied by the production of proinflammatory signals and activation of antimicrobial mechanisms. Certain phagocytic receptors such as Fc receptors trigger inflammatory responses directly, whereas others such as CRs often do not stimulate inflammatory responses. Following phagocytosis, PMNs and MMs kill newly internalized pathogens in part by the production of noxious reactive superoxide ions produced by assembly of the NADPH oxidase on phagosomal membranes [9, 88, 89]. Both oxygen-dependent and independent mechanisms will be mobilized to achieve complete killing of the

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pathogen. Oxygen-independent mechanisms involve the degranulation of cytoplasmic storage compartments within the phagolysosome, resulting in the release of preformed proteinases or antibiotic proteins able to kill and degrade the pathogen. In addition, activation of gene transcription and cytokine production during phagocytosis is a critical feature in the development of an effective immune response. While some phagocytic receptors themselves may trigger cytokine and chemokine production, others require additional coreceptors. Beside oxygen-dependent and independent killing mechanisms, PMNs can use the neutrophil extracellular traps (NETs) that are powerful to capture and kill microbes in the extracellular space [90, 91]. Nucleases are able to degrade theses structures, demonstrating that chromatin is one of the major components.

Polymorphonuclear Neutrophils and Monocyte/Macrophages in the Immune Response

Cytokine Production by Polymorphonuclear Neutrophils and Monocyte/Macrophages MMs are the main producers of inflammatory and immunoregulatory cytokines and they are characterized by an important and sustained ability to synthesize a great variety of cytokines. Several excellent reviews have been published on this topic [92–94]. With regard to PMNs, a great body of evidence has accumulated that they are both a target and a source of various proinflammatory cytokines, chemokines and growth factors, and therefore often exert their proinflammatory functions through an autoregulatory pathway [95]. Indeed, PMNs were long considered as devoid of transcriptional activity and capable of performing no or little protein synthesis, but convincing molecular evidence that PMNs either constitutively or in an inducible manner can synthesize and release a wide range of cytokines has now been afforded. The production of cytokines by activated PMN is striking in its diversity but remains much lower in its degree than that produced by the monocytes [96]. We would like to emphasize some recent aspects of cytokine production in PMNs, thus pointing out new potential immunomodulatory roles of these cells. After incubation with G-CSF, or to a lesser extent with IFN-␥, human PMNs are able to produce high levels of the B lymphocyte stimulator, a member of the TNF ligand superfamily important in B cell maturation and survival and largely produced by monocytes and dendritic cells [97]. Moreover, PMNs and monocytes can release a soluble form of TNF-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand), a member of the TNF superfamily exerting cytotoxic activities toward tumor cells [98]. Moreover, it has been described that under exposure to proinflammatory mediators such as TNF-␣, LPS or f-MPL, IFN-␣-stimulated PMNs can readily mobilize an intracellular pool of this cytokine. This release of soluble TRAIL will

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amplify and further reinforce the notion that PMNs are important cells in tumor surveillance [99]. Chemokines that are related cytokines involved in the regulation of the immune system responses, in B and T cell development and in the development of angiogenesis, are synthesized by PMNs as well. A specific feature of chemokines is their capacity to bind to their receptors on the neutrophil surface and to subsequently induce signaling leading to activation and clustering of PMN integrins. This step will allow firm adhesion to endothelium and spreading of neutrophil. Chemokines have been classified into two major groups according to the positions of the two cysteine residues in their primary sequence: the C-X-C which are chemotactic for PMNs, including the IL-8, and the C-C families which fail to attract PMNs but are chemotactic and stimulatory for monocytes, including MCP-1, RANTES and macrophage inflammatory protein (MIP) [100, 101]. It has been shown that PMNs that have migrated at the site of inflammation can upregulate their production of chemokines, supporting the notion that by this way PMNs participate in the regulation of leukocyte accumulation. In terms of production, the principal chemokine produced by PMNs is the IL-8, which activates the PMNs in an autocrine loop. Indeed the ligation of IL-8 to its receptor on neutrophil surface triggers chemotaxis, release of granule enzyme, respiratory burst activity, upregulation of adhesion molecule expression on the surface and increased adherence to unstimulated endothelial cells [100, 101]. IL-8 release represents a stereotyped response occurring as soon as PMNs recognize any sort of ligands or foreign particles. It would be a way to attract sufficient PMNs at the site of infection. Another CXC chemokine produced by PMNs is the growth-related gene product-␣, which also exerts chemotactic and activation properties on PMNs. It has been reported that a simultaneous synthesis of IL-8 and growth-related gene product-␣ has redundant effects on PMNs. Appropriately stimulated PMNs are also able to produce CC chemokines as MIP-1␣ and MIP-1␤ [102]. According to the studies published to date, MIP-1␣ and MIP-1␤ are released by PMNs in response to various agonists like LPS, TNF-␣, eosinophil major basic protein, several bacteria, fungi, protozoa, viruses and related microorganism products [95]. It has also been described that PMNs are able to express and release MIP-3␣ (CCL20) and MIP-3␤(CCL19) when there are cultures with either LPS or TNF-␣ and that the production of these two chemokines by LPS-stimulated PMNs is negatively modulated by IL-10 [103]. As these chemokines are chemotactic for dendritic cells and specific lymphocyte subsets, it has been reported that the ability of PMNs to produce them might be significant in orchestrating the recruitment of these cell types to the inflamed sites and therefore in contributing to the regulation of the immune response [103]. The MIP-3␣ (CCL20) production in LPS-stimulated PMNs is enhanced by formylated peptide (f-MLP) and IFN-␥ [104]. The diversity of cytokines synthesized by PMNs that is now well documented, illustrates the potential immunomodulatory capacity of this cell type.

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Role of Polymorphonuclear Neutrophils and Monocyte/Macrophages in the Initiation and Modulation of the Adaptive Immune Response In contrast to PMNs, MMs are described as APCs that can uptake foreign antigen, digest it and present it to T cells, in the context of MHC class II. Their high potency in antigen presentation is due to their high levels of HLA-DR [105]. Moreover, monocytes can differentiate into dendritic cells, which are ‘professional APCs’. It has been described that when monocytes were cultured 2 days in the presence of TLR2 ligands, they developed preferentially into CD1b⫹ DC with high antigen-presenting capacity [106]. Excellent recent reviews on antigen presentation have been published recently [107], [108]. Even if PMNs are not directly implicated in antigen presentation to T cells, they can regulate the development of an effective immune response. For example, it has been shown that live or apoptotic PMNs can transfer Candida antigens to dendritic cells and PMNs can activate them to become more proficient APCs by affecting the expression of costimulatory molecules [109]. Interestingly, a new concept of involvement of PMNs in adaptive immunity is emerging, since the ␣- and ␤chains of T-cell receptor (TCR) have been detected on a subpopulation of circulating PMNs [110]. Cell activation by TCR agonists, like CD3, induces Bcl-xL expression and IL-8 secretion, demonstrating that TCR signaling protects PMNs from apoptosis. This TCR-positive PMNs could represent a first-line system of antigen-specific defense, more rapid as compared with the conventional T cells, and could boost the recruitment of additional PMNs to sites of inflammation. Thus, further studies are needed to really underscore the importance of PMNs in modulating the antigen presentation step or the adaptive immune response.

Role of Polymorphonuclear Neutrophils and Monocyte/Macrophages in the Resolution of Inflammation

General Mechanisms Over the last few years, many studies have focused their attention on the resolution of inflammation. This is an active and coordinated program involving a switch from pro- to anti-inflammatory mediators, apoptosis of inflammatory cells and their subsequent engulfment by professional (macrophages and immature dendritic cells) [111] and nonprofessional phagocytes (fibroblasts, endothelial, epithelial and mesenchymal cells) [112]. Ultimately, this should result in the release of anti-inflammatory and reparative mediators [113, 114]. Apoptotic cells and phagocytes, also known as the ‘apoptophagocytic system’ [112], are tightly correlated by a ‘loop’, whereby apoptotic cells can recruit macrophages to the site of inflammation, and phagocytes induce apoptosis of their neighbors. Therefore, it is clear that the resolution of inflammation involves both inhibition of proinflammatory signaling and stimulation of proresolving

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events [115]. These events initiate within the first hours of inflammation induction, supporting the concept of ‘the beginning programs the end’ [1]. The inflammatory reaction is controlled by anti-inflammatory cytokines, antiproteases and antioxidant systems. Different mediators produced by macrophages such as IL-10, IL-13 or TGF-␤ induce extinction of the inflammatory process. Arachidonate-derived prostaglandins, such as PGE2, and leukotriens are generated to regulate early events in the inflammatory response, like recruitment of leukocytes to the inflammatory site [116]. However, recent studies have shown that a switch in lipid mediator production occurs [117, 118]. This allows the synthesis of lipoxins, which are lipid-derived mediators, by macrophages, which have dual function, i.e. anti-inflammatory and proresolving mediators. Lipoxins promote resolution by delaying PMN arrival at the inflammatory site [119] by inhibiting their adhesion and transmigration to the endothelium. Lipoxins can stimulate monocyte chemotaxis and adherence [120] and potentiate the phagocytosis of apoptotic cells by macrophages [121]. In addition to lipoxins, other two novel families of lipid mediators, involved in the resolution of inflammation, have been recently discovered: the protectins and the resolvins [19, 122, 123]. These lipids derive from ␻–3 polyunsaturated fatty acid and possess potent anti-inflammatory and proresolving properties including reduction of PMN transendothelial migration [124] and stimulation of PMN apoptosis [125]. G-CSF, produced by stromal cells, is an essential regulator of PMN precursor proliferation and mobilization from the bone marrow [126]. Recently, it has been demonstrated that glucocorticoids increase differentiation, recruitment and phagocytosis of a subtype of monocytes to the inflamed site [127], by specific gene expression [128]. To limit leukocyte transmigration to the inflamed site, the shutdown of proinflammatory mediators, such as chemokines, is necessary: by proteolytic cleavage to neutralize their activity and by abolishing chemokine receptor signaling (CCR5) [129, 130]. Interestingly, this process has been described to stop leukocyte infiltration and clear the inflammatory site [131], since lipoxins, resolvins and protectins upregulate CCR5 expression on apoptotic cell surface, which are consequently able to scavenge chemokines. Indeed, proinflammatory cytokines are removed from the environment, promoting return to tissue homeostasis [19]. Different mediators are involved in inhibition of leukocyte trafficking: chemokines, cytokines, lipid mediators and adhesion molecules, such as P-selectin and VLA-1 [132]. Annexin-1 was first defined in leukocytes as a glucocorticoid-induced protein with anti-inflammatory effects [133], such as downregulation of PMN adhesion and transmigration [134]. In inflammatory conditions, the cytosolic annexin-1 is mobilized to the PMN cell surface and interacts with the formyl-peptide receptor and the formyl peptide receptor-like 1 [135] to induce detachment of PMNs by L-selectin shedding [136]. Annexin-1 plays a role in the negative regulation of monocyte adhesion to endothelium in vitro [137], and secretion of proinflammatory mediators [138], as well. Nitric oxide, produced by endothelial cells, has also an anti-inflammatory effect by stopping PMN diapedesis [139] and monocyte adhesion and migration [140]. The removal of phagocytosing macrophages from

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the inflammatory site is a very important event in the resolution of inflammation, which prevents local tissue damage: activated macrophages can adhere to mesothelium overlying draining lymphatics and are cleared via the lymphatic circulation [141].

Neutrophil Apoptosis PMN cell life span must be a very regulated process, since extended survival is beneficial for pathogen elimination [142] and cell death necessary to prevent cytotoxic content release from nonapoptotic activated PMNs on the inflammatory site. Indeed, PMN apoptosis is a critical point for the successful resolution of inflammation since it represents a noninflammatory process providing cell removal from the inflammatory site by macrophages, thus limiting damage of neighboring tissue [143]. A specific feature of PMNs is their ability to undergo spontaneous apoptosis, due to the upregulation of death signaling and downregulation of survival signaling. PMNs treated with protein synthesis inhibitors induce an increase in the rate of apoptosis [144]. Consequently, protein synthesis is indispensable for neutrophil survival. Indeed, PMN apoptosis is delayed by a continuous expression of short life span Bcl-2 homolog, such as myeloid cell leukemia 1 (Mcl-1) [145]. PMN apoptosis is a highly regulated process involving death factors such as FasL, TNF-␣, caspases, Bcl-2 family, inhibitor of apoptosis protein, reactive oxygen species, calpain and pathogens [146]. PMN survival factors include proinflammatory mediators [147] like NF␬B, LPS, G-CSF, GM-CSF, Foxo3a [148], kinases (PI3K, MAPK, PKA, PKC, ERK), hypoxia [149] and survivin [150]. Interestingly, cytoplasts which are nuclei- and organelle-depleted PMNs can undergo apoptosis in the same way as PMNs [151], demonstrating that PMN apoptosis does not require de novo protein synthesis and can proceed, to a certain extent, via mitochondria-independent pathways. Even if the presence of mitochondria has been controversial in PMNs [152, 153], several lines of evidence described tubular structures with particular morphology and functional features, including ATP synthesis and release of proapoptotic factors in the cytosol, like low levels of cytochrome c and apoptosis-inducing factor [154]. Since caspase-9 activity is very high upon apoptosis, it has been hypothesized that the low level of cytochrome c could be compensated by the elevated expression of Apaf-1 [155]. If Bcl-2 is not expressed in PMNs, the other antiapoptotic factors, like Mcl-1, A1 and Bcl-xL are present [156]. Both intrinsic (via mitochondria) and extrinsic (via death receptor signaling) apoptotic pathways have been described in PMNs. Indeed, PMNs express both FasL and FasR [157, 158], but it appears that the extrinsic pathway is not the principal apoptotic pathway since PMNs from FasL- or FasRdeficient mice undergo constitutive apoptosis normally [159]. The role of TNF-␣ on PMN apoptosis have been debated and depends on the cellular environment. TNF-␣ is both a proapoptotic mediator by procaspase-8 cleavage or antiapoptotic by NF-␬B activation [160]. The Role of Neutrophils and Monocytes in Innate Immunity

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Once PMNs migrate towards the inflammatory site, their survival time increases because of the presence of prosurvival cytokines and chemokines in their environment, like LPS, G-CSF, GM-CSF [161], and because of their adhesion [162]. Indeed, IL-8 delayed PMN apoptosis to allow these cells to play their role and monocytes to be recruited and differentiated into macrophages. Macrophages could participate in apoptosis induction of surrounding PMNs by secretion of Fas-L, following apoptotic cell engulfment [163]. However, when PMNs undergo apoptosis, their effector functions are downregulated. They lose their capacity to synthesize chemokines, to respond to chemotactic factors, to phagocytose, to trigger oxidative burst and to degranulate [164, 165]. The membrane of apoptotic PMNs is also altered with receptors involved in adhesion and migration, such as CD16, being downregulated [166, 167], and cytoskeletal integrity is disrupted [168]. An important feature of glucocorticoids is their ability to delay constitutive PMN apoptosis [169]. In contrast, annexin-1 is able to induce apoptosis both in monocytes [170] and in PMNs [171]. A new concept in the regulation of PMN apoptosis is the involvement of cyclin-dependent protein kinase (CDK) in the resolution of inflammation. So far, cyclin-dependent kinase inhibition was effective only in proliferating cells. Indeed, CDK induces PMN apoptosis and overrides the effect of potent survival mediators, like LPS and GM-CSF, by a mechanism involving caspase activation and reduction of Mcl-1 [172]. This effect of roscovitine, a synthetic CDK inhibitor in the nonproliferating neutrophils is very interesting and opens new possibilities of therapeutic strategies. Interestingly, PMN death [173], which is a proinflammatory process because of the cytotoxic protein release, could also be related to the destruction of invading microorganisms at the inflammatory site. Indeed, after cell membrane rupture, activated PMNs produce NET (PMN extracellular trap) [90], extracellular structures constituted of chromatin and granular proteins, as already mentioned, to trigger a beneficial death. The balance between death and survival must be tightly regulated in PMNs since alterations in these processes are linked to diseases. Indeed, both elevated survival and increased death of PMNs are involved in inflammatory and immunological diseases such as rheumatoid arthritis [174].

Macrophage Phagocytosis of Apoptotic Polymorphonuclear Neutrophils The ability of macrophages to (a) recognize ligands on apoptotic cells; (b) respond appropriately, and (c) engulf these cells is a key event in host defense, which could predispose to development of autoimmunity if not efficient [67]. Engulfment of apoptotic cells by macrophages (also called ‘efferocytosis’ [175]) is an anti-inflammatory process since it induces release of anti-inflammatory mediators, like TGF-␤1, IL-10, PGE2, leukocyte protease inhibitor [176], reparative growth factor, and inhibition of

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proinflammatory mediator secretion such as TNF-␣ [114], or IL-1␤ or GM-CSF. This process confers anti-inflammatory phenotype to macrophages conducing to resolution. On the other hand, apoptotic cells release lysophosphatidylcholine to attract monocytes and macrophages [177]. More recently, the changes in membrane composition of apoptotic cells (negative surface charges) have been hypothesized to initiate electric attractive signals for phagocytes [178]. At the inflammatory site, proinflammatory cytokines, like IL-1 and TNF-␣ [179], and glucocorticoids [180] increase the phagocytic capacity of macrophages for apoptotic PMNs. Lipoxins stimulate macrophages to phagocytose apoptotic PMNs [120]. Expression of 15-lipoxygenase on nonphlogistic macrophages, which had phagocytosed apoptotic PMNs, induce lipoxin A4 release thus resulting in a decreased PMN recruitment and in an increase in apoptotic PMN removal [181]. In order to allow macrophages to ingest apoptotic PMNs, and differentiate them from viable cells, their membrane is altered in the way that they express specific molecules and receptors [111, 182]. One of the most important events in this phenomenon of recognition and engulfment is the loss of phospholipid asymmetry and phosphatidylserine exposure (‘eat me signal’) to the outer leaflet of the apoptotic cell surface [113]. However, it does not seem to be sufficient [183] for the recognition of apoptotic cells and other proteins such as calreticulin or annexin-1 might be involved [184–186]. Hence, annexin-1 is externalized on apoptotic cell membrane, where it colocalizes with phosphatidylserine [187], to participate in the removal of these cells by macrophages. Several receptors have been reported to play a role in the ingestion of apoptotic cells: some of them involved in tethering to the phagocyte, and others in signaling pathways leading to actin cytoskeleton rearrangement and engulfment, during membrane extension and fusion [188]. These receptors on phagocytes include the controversial PSR, the vitronectin receptor, scavenger receptors, like CD36 and CD14, lectins, Mer, CRs, ␤2GPI receptor and CD91 [16]. They can interact directly with apoptotic cells or indirectly through bridging proteins, like C1q [189], ␤2-GPI, Gas6, MFG-E8. Components of the innate immune system play a role in regulating clearance of apoptotic cells since the opsonization of apoptotic cells increases their removal by macrophages [190]. This process of apoptotic cell recognition by macrophages is negatively regulated by surface molecules (‘don’t eat me signals’), like CD47 or CD31, to avoid viable cell engulfment, by inducing detachment from macrophages [191]. On the other hand, it appears that this phenomenon of discrimination between apoptotic and viable cells involves redistribution of eat-me signals into patches (to increase their avidity towards receptors) as well. After apoptotic cell ingestion, macrophage phenotypes switch (‘reprogramming’) to induce tissue cicatrisation or emigrate in the lymphatic system [1], suggesting that phagocytosis of apoptotic cells is involved in the negative regulation of macrophage activation and that PMN apoptosis could be considered as endogenous ‘active’ anti-inflammatory process. PMNs are thus able to integrate complex arrays of adhesion- chemoattractant- and cytokine-driven signals as well as survival and death information. They are well

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equipped with a myriad of proteinases of different specificities which are safely kept within cytoplasmic granules. However, proteinases can regulate pathophysiological processes, not exclusively aimed at the destruction of invading microorganisms. The diversity of the functions of neutrophil-derived serine proteinases, which include human neutrophil elastase, proteinase 3 and cathepsin G, is a relevant example to illustrate this concept. These proteinases are released in the extracellular medium upon PMN activation as soluble mediators but they can also be associated with the plasma membrane via cationic interactions for elastase and cathepsin G or via strong hydrophobic interactions in the case of proteinase 3. PMN serine proteases cleave extracellular matrix proteins and destroy connective tissues. The destructive potential of serine proteinases is well illustrated in the neutrophil-dominated chronic airway inflammation observed in cystic fibrosis [192, 193]. As a result, high levels of active neutrophil-derived serine proteinases are found in the airways of CF patients [194]. In addition, PMN serine proteinases induce chemotactic activities since they are able to cleave the N-terminal part of chemokines, which in turn increase their affinity towards their receptors. Indeed, cathepsin G cleaves CCL15 (CC chemokine ligand 15) to increase monocyte chemotaxis [195], and CXCL5 (CXC chemokine ligand 5) [196], which act on PMN recruitment. Similarly, IL-8 acts as a chemotactic factor with its activity enhanced upon clipping by proteinase 3, a neutrophil serine protease [197]. Cell migration is also regulated by PMN serine proteases since they cleave cellsurface adhesion molecule (ICAM, VCAM, E-cadherin) to induce cell-cell or cellextra cellular matrix interaction termination [198, 199]. More recently, it has been reported that annexin-1 cleavage occurred in activated PMNs and that this cleavage, triggered by membrane-associated PR3, could explain the removal of regulatory inhibitory properties of annexin-1 in chronic inflammatory environment [200]. Interestingly, cathepsin G has been shown to process procaspase-3 and thus potentiate apoptosis. In contrast, membrane-associated PR3 cleaves procaspase-3 in a 23-kDa fragment which differs from the pro-apoptotic 17-kDa fragment [201]. Surprisingly, PR3-processed caspase-3 was associated with the plasma membrane and was correlated with PMN survival. Moreover, proteinase 3 can be externalized during apoptosis concomitantly to phosphatidylserine [202] in order to impair, independent of its activity, clearance mechanisms of apoptotic PMNs by macrophages [14]. The specific substrates of proteinase 3, including the anti-inflammatory protein annexin-1, the procaspase-3 and its inhibition of macrophage phagocytosis could explain the proinflammatory role of PMN membrane PR3 in vasculitis and rheumatoid arthritis [203].

Conclusion

PMNs and MMs are key players in the inflammatory response and have complementary roles. Indeed, PMNs depicted as short-lived cells which blow up to destroy

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microorganisms, having a destructive hardware but no software is not in accordance with the reality. With regard to MMs, their role in the modulation of the immune response has long been recognized but the recent notion of MM heterogeneity and functional subsets has been clearly underestimated so far. The recognition and the subsequent engulfment of apoptotic PMNs by macrophages is a key event of the resolution of inflammation which can be associated with autoimmunity [204] or inflammatory diseases. During the past years, significant progress has been made to dissect the molecular mechanisms governing phagocytosis and pathogen killing. Although these functions are crucial, more work has to be done to understand the respective role of PMNs and MMs to regulate and inhibit the inflammatory process as well as the immune response. This might be the future challenge for the next years in phagocyte research and will presumably open new avenues of research in the modulation of inflammation.

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