The Biology and Pathology of Innate Immunity Mechanisms (ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Volume 479)

THE BIOLOGY AND PATHOLOGY OF INNATE IMMUNITY MECHANISMS

ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY EditorialBoard: NATHAN BACK, State University of New York at Buffalo

IRUN R. COHEN, The Weizmann Institute of Science DAVID KRITCHEVSKY, Wistar Institute ABEL LAJTHA, N. S. Kline Institute for Psychiatric Research RODOLFO PAOLETTI, University of Milan Recent Volumes in this Series Volume 470 COLON CANCER PREVENTION: Dietary Modulation of Cellular and Molecular Mechanisms Edited under the auspices of the American Institute for Cancer Research Volume 471 OXYGEN TRANSPORT TO TISSUE XXI Edited by Andras Eke and David T. Delpy Volume 472 ADVANCES IN NUTRITION AND CANCER 2 Edited by Vincenzo Zappia, Fulvio Della Ragione, Alfonso Barbarisi, Gian Luigi Russo, and Rossano Dello Iacovo Volume 473 MECHANISMS IN THE PATHOGENESIS OF ENTERIC DISEASES 2 Edited by Prem S. Paul and David H. Francis Volume 474 HYPOXIA: Into the Next Millennium Edited by Robert C. Roach, Peter D. Wagner, and Peter H. Hackett Volume 475 OXYGEN SENSING: Molecule to Man Edited by Sukhamay Lahiri, Nanduri R. Prabhakar, and Robert E. Forster, II Volume 476 ANGIOGENESIS: From the Molecular to Integrative Pharmacology Edited by Michael E. Maragoudakis Volume 477 CELLULAR PEPTIDASES IN IMMUNE FUNCTIONS AND DISEASES 2 Edited by Jürgen Langner and Siegfried Ansorge Volume 478 SHORT AND LONG TERM EFFECTS OF BREAST FEEDING ON CHILD HEALTH Edited by Berthold Koletzko, Olle Hernell, and Kim Fleischer Michaelsen Volume 479 THE BIOLOGY AND PATHOLOGY OF INNATE IMMUNITY MECHANISMS Edited by Yona Keisari and Itzhak Ofek

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THE BIOLOGY AND PATHOLOGY OF INNATE IMMUNITY MECHANISMS Edited by

Yona Keisari and

Itzhak Ofek Sackler Faculty of Medicine Tel Aviv University Tel Aviv, Israel

KLUWER ACADEMIC PUBLISHERS New York, Boston, Dordrecht, London, Moscow

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Preface

In recent years increased scientific attention has been given to immediate defense mechanisms based on non-clonal recognition of microbial components. These mechanisms constitute the innate immunity arm of the body's defense. Identification of pathogens by these mechanisms involves primarily receptors recognizing sugar moieties of various microorganisms. Innate immunity based mechanisms are essential for the existence of multicellular organisms. They are evolutionarily conserved and designed to provide immediate protection against microbial pathogens to eradicate infection. Activation of innate immunity is crucial for transition to specific immunity and for its orientation, and to assist the specific immune response in the recognition of pathogens and their destruction. Innate immunity is regularly involved in the arrest of bacterial, mycotic, viral and parasitic infections, giving the specific immune response time to become effective. It becomes critically essential in immunocompromised patients who fail to mount specific immune responses due to congenital or acquired immunodeficiencies as a result of chemotherapy, dialysis, immunosuppressive drugs, or HIV infection. The Innate Immunity arsenal constitutes polymorphonuclear and mononuclear phagocytes, mast cells, the complement system, Natural Killer cells, antimicrobial peptides, and presumably a subset of T lymphocytes with TCRl receptors. This book includes manuscripts of lectures presented at the "Bat Sheva Seminar on Innate Immunity" held in Israel, October 1999. The major topics presented and discussed in the seminar included (i) the role of innate immune responses as a first line defense against microbial infection, and

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tumor cells; (ii) the cellular and molecular basis of the function of cells and molecules involved in innate immunity; (iii) the role of innate immunity in the immunocompromised host; and (iv) the interactions between innate immunity components and clonal immune response. This book includes the major themes of this rapidly developing area; however, we by no means intend to cover all aspects of innate immunity. The book's first section deals with receptors, lectins and collectins with emphasis on interaction of these molecules with pathogens. The second section deals with the arsenal of host cells and cytokines playing crucial roles in innate immunity, and the third section is devoted to aspects of antimicrobial peptides. Because of its special importance, innate immunity in the compromised host is the focus of the next section. The last section deals with the interrelationship of innate immunity components and tumor cells. In order to expand the scope of the volume even further, we have also included the abstracts of some of the lectures and posters presented during the seminar. We thank the authors for their collaborative efforts. We also trust that the highlights of this book will stimulate new ideas that lead to practical designs for better understanding the complex interactions of components of the innate immunity in order to develop effective agents and measures for preventing or treating infectious diseases and malignancies. We would like to express our gratitude to all our colleagues and friends, especially to the members of the Organizing Committee (E. Ezekowitz, S. Gordon, M. Fridkin, M. Shapira, A. Mantovani, E. Yefenof, A. Etzioni and N. Sharon) who suggested, argued and altogether helped a great deal, and in many ways allowed the seminar to bloom. We believe that a follow-up seminar should be held to present and discuss the results of the new ideas that were illuminated here. Itzhak Ofek and Yonka Keisari, Chairpersons.

Contents

I.

PATTERN RECOGNITION, RECEPTORS AND COLLECTINS IN INNATE IMMUNITY

1.

Mannose receptor and scavenger receptor: two macrophage pattern recognition receptors with diverse functions in tissue homeostasis and host defense S. A. Linehan, L. Martinez-Pomares, and S. Gordon ..............................1

2.

Complement receptor 3 (CR3): a public transducer of innate immunity signals in macrophages E. Yefenof ..............................................................................................15

3.

The role of C-type lectins in the innate immunity against pulmonary pathogens I. Ofek, E. Crouch, and Y. Keisari .......................................................27

4.

Modulation of nitric oxide production by lung surfactant in alveolar macrophages M. Kalina, H. Blau, S. Riklis, and V. Hoffman .....................................37

5.

Development of chimeric collectins with enhanced activity against influenza A virus K. L. Hartshorn, M. R. White, R. A. B. Ezekowitz, K. Sastry, and E. Crouch .........................................................................................49

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Contents Initial steps in Streptococcus pneumoniae interaction with and pathogenicity to the host M. Shani-Sekler, S. Lifshitz, I. Hillel, R. Dagan, N. Grossman, G. Fleminger, and Y. Mizrachi-Brauner ................................................61

11. HOST CELLS AND CYTOKINES IN INNATE IMMUNITY 7.

Role of cytokines in the maturation and function of macrophages: effect of GM-CSF and IL-4 Y. Keisari, G. Robin, L. Nissimov, H. Wang, A. Mesika, R. Dimri, and I. Ofek .............................................................................................73

8.

Mast cell modulation of the innate immune response to enterobacterial infection S. N. Abraham and R. Malaviya ...........................................................9 1

9.

The NADPH oxidase diaphorase activity in permeabilized human neutrophils and granulocytic like PLB-985 cells I. Pessach and R. Levy ........................................................................107

10. Activation of cytosolic phospholipase A2 by opsonized zymosan in human neutrophils requires both ERK and p38 MAP-kinase I. Hazan-Halevy and R. Levy. ..............................................................115 11. Cytosolic phospholipase A2 is required for the activation of the NADPH oxidase associated H+channel in phagocyte-like cells R. Levy, A. Lowenthal, and R. Dana ...................................................125 12. The role of NK cells in innate immunity N. Lieberman and 0. Mandelboim ......................................................137 13. Similarities and dissimilarities between humans and mice looking at adhesion molecules defects A. Etzioni, C. M. Doerschuk, and J. M. Harlan ....................................147 14. The role of dendritic cells at the early stages of Leishmania infection H. Moll ................................................................................................163 15. DNA-based vaccines: the role of dendritic cells in antigen presentation L. Paul and A. Porgador ......................................................................175

Contents 16. Distinct patterns of IL- 1α and IL- 1 β organ distribution – a possible basis for organ mechanisms of innate immunity M. Hacham, S. Argov, R. M. White, S. Segal, and R. N. Apte ......185 III. ANTIMICROBIAL PEPTIDES 17. Structure and biology of cathelicidins M. Zanetti, R. Gennaro, M. Scocchi, and B. Skerlavaj ...................203 18. Structure activity relationship study of polymyxin B nonapeptide H. Tsubery, I. Ofek, S. Cohen, and M. Fridkin....................................219 IV. INNATE IMMUNITY IN THE COMPROMISED HOST 19. The clinical significance of neutrophil dysfunction B. Wolach, R. Gavrieli, and D. Ross .................................................223 20. Clinical significance of functional aberrations in macrophage and NK cells, in type- 1 cytokines and in lectin-binding molecules Z. Handzel ............................................................................................227 21. Klebsiella infections in the immunocompromised host H. Sahly, R. Podschun, and U. Ullmann .............................................237 V. INNATE IMMUNITY COMPONENTS IN CANCER 22. Macrophage – recognized molecules of apoptotic cells are expressed at higher levels in AKR lymphoma of aged as compared to young mice O. Itzhaki, E. Skutelsky, T. Kaptzan, A. Siegal, M. Michowitz, J. Sinai, M. Huszar, S. Nafar, and J. Leibovici ..................................251 23. Sensitivity to macrophages decreases with tumor progression in the AKR lymphoma T. Kaptzan, E. Skutelsky, M. Michowitz, A. Siegal, O. Itzhaki, S. Hoenig, J. Hiss, S. Kay, and J. Leibovici ....................................263

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24. Opposing effects of IL-1α and IL-1β on malignancy patterns: Tumor cell-associated IL- 1 α potentiates anti-tumor immune responses and tumor regression, whereas IL- 1β potentiates invasiveness R. N. Apte, T. Dvorkin, X. Song, E. Fima, Y. Krelin, A. Yulevitch, R. Gurfinkel, A. Werman, R. M. White, S. Argov, Y. Shendler, 0. Bjorkdahl, M. Dohlsten, M. Zoller, S. Segal, and E. Voronov ...................................................................277 25. Abstracts ..............................................................................................289 26. Index ....................................................................................................323

MANNOSE RECEPTOR AND SCAVENGER RECEPTOR: TWO MACROPHAGE PATTERN RECOGNITION RECEPTORS WITH DIVERSE FUNCTIONS IN TISSUE HOMEOSTASIS AND HOST DEFENSE

Sheena A. Linehan, Luisa Martinez-Pomares and Siamon Gordon Sir William Dunn School of Pathology, South Parks Rd., Oxford, OX1 3RE, UK

ABSTRACT In this report we have reviewed our recent data which suggest a new function for MR in antigen delivery in lymphoid organs, together with highlighting three recent discoveries from our laboratory concerning the role of SR-A in adhesion, phagocytosis of apoptotic cells and protection from endotoxic shock in mice. The diversity of functions mediated by each receptor demonstrates there is much yet to be discovered about how macrophages use their cell surface receptors to ‘see’ the external environment, and yet perform a wide range of strictly regulated functions.

1.

INTRODUCTION

The macrophage (Mø), among cell types, is distinctive in its ability to perform a wide variety of functions, which can be broadly defined as homeostatic and immunological [Gordon, 1995]. Mø play a key role in tissue remodelling, in both development and repair, are active in scavenging effete cells and molecules and may play a role in regulating The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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Mannose Receptor and Scavenger Receptor

differentiation of other cell types. The migration and adhesion properties of Mø allow them to home to specific tissues, as well as sites of infection and injury. They are professional phagocytes, and when immunologically activated, contribute to host defence through killing of phagocytosed pathogens, secretion of inflammatory mediators and antigen presentation to and activation of primed T cells. Mø sense the external environment through an array of cell surface receptors, and use these to modulate their behaviour. Intriguingly, disparate functions in homeostasis and immunity may be mediated by the same receptors. This observation does not fit neatly into current hypotheses about what determines whether or not the immune system will respond to a particular antigenic insult. Medzhitov and Janeway have proposed this discriminatory function to be mediated by ‘pattern recognition receptors’, receptors which recognise a range of ligands sharing structural features which are prevalent on microorganisms but not host molecules [Medzhitov, 1997]. Matzinger, originator of the controversial “danger” theory, has suggested that the situation is more complex, as host-derived ligands for some pattern recognition receptors have been identified [Matzinger, 1998]. Work in our laboratory focusses on Me, cell surface receptors, and here we review recent data on the mannose receptor (MR) and the class A scavenger receptor (SR-A), two receptors which have been described as pattern recognition receptors and have a variety of functions in homeostasis and immunity.

2.

MANNOSE RECEPTOR AND SCAVENGER RECEPTOR ARE PATTERN RECOGNITION RECEPTORS WITH BOTH HOST-DERIVED AND MICROBIAL LIGANDS

Both MR and SR-A recognise a range of ligands sharing key structural features and can therefore be described as pattern recognition receptors. However, unlike Medzhitov and Janeway’s hypothetical pattern recognition receptors which only recognise non-self structures, MR and SR-A bind both self and non-self ligands. MR is a 175kD type I membrane glycoprotein and was first identified in liver and then alveolar Mø by its ability to endocytose lysosomal enzymes and neoglycoproteins in a sugar-specific manner [Schlesinger, 1978; Stahl, 1978]. MR consists of a cytoplasmic tail, transmembrane domain, an array of eight C-type lectin-like carbohydrate recognition domains [Taylor, 1990], a fibronectin type II-like domain and an Nterminal cysteine-rich domain which is homologous to the B chain of the

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lectin, Ricin [Harris, 1994]. MR is the founder member of a family of receptors sharing the same general structure which appear to function in endocytosis. The phospholipase A2 receptor [Ishizaki, 1994] and an endothelial receptor [Wu, 1996] have eight C-type lectin-like domains whilst DEC-205 possesses ten [Jiang, 1995]. The affinity of MR for oligosaccharides is determined by the terminal sugar residues of the oligosaccharide, and was shown to be L-fucose > Dmannose –> D-N-acetyl-glucosamine >>> D-galactose [Stahl, 1978]. A high avidity of interaction with oligosaccharides is generated by cooperative binding of several of the carbohydrate recognition domains (CRD) of MR. Studies with recombinant deletion mutants of MR showed that CRD 4 is the only lectin domain able to mediate detectable mannose binding in isolation, and that CRDs 4 to 8 are sufficient to generate the affinity of the whole receptor for natural ligands [Taylor, 1992]. MR preferentially recognises α-linked oligo-mannoses with branched rather than linear structures [Kery, 1992], giving MR a special ability to recognise host-derived asparagine-linked high mannose-type oligosaccharides and a variety of microbial and viral polysaccharides. We have recently identified a binding activity of the cysteine-rich (CR) domain of MR for specific sites within lymphoid organs, which we discuss later. CR-Fc ligands were purified from spleen and among these, novel glycoforms of sialoadhesin and CD45 were identified. A combination of enzymatic digestion and weak anionic exchange chromatography suggested that the determinant recognised is a sulphated oligosaccharide [Martínez-Pomares, In press]. A new lectin activity of CR has recently been described for Asn-linked oligosaccharides terminating in galNAc-4-S04, following the demonstration that a rat liver receptor which binds lutropin hormone bearing galNAc-4-S04 shares structural and antigenic properties with MR. Intriguingly, MR purified from lung did not share this binding activity [Fiete, 1997a]. A protein with the same properties as the liver receptor could be generated from the same cDNA as MR, and the ability to bind galNAc-4-SO4, appeared to be determined post-translationally [Fiete, 1997b]. The galNAc-4-S04 binding site was then localised to the CR by binding studies of deletion mutants of MR [Fiete, 1998]. Tissue heterogeneity with respect to cysteine-rich domain modification and binding activity may allow Mø to perform different functions in different sites. Known MR ligands are listed in Table 1.

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Mannose Receptor and Scavenger Receptor

Table 1. MR and SR-A ligands of host. microbe, inorganic and synthetic origin

microbial

propeptide

my e I operox

synthetic

bacterium

unknown cell-surface

gramnegative

(Hughes.

(Hampton.

capsular

(Smedsrød,

199 1 ;

lysosomal hydrolases

Candida albicans

The endocytosis of modified low density lipoprotein (LDL) by Mø, was first attributed to a new receptor following the discovery that

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previously characterised LDL receptors were not involved [Brown, 1983]. SR-A was subsequently characterised at a molecular level, and shown to exist as two forms, type I and type II, generated by alternative splicing of the same gene [Freeman, 1991; Emi, 1993]. These forms share an N-terminal cytoplasmic tail, transmembrane domain, spacer, alpha-helical coiled coil and collagen-like domain, but only the type I form possesses a C-terminal cysteine-rich domain (which is not similar to the MR cysteine-rich domain). The quaternary structure is predicted to be trimeric. No differences in binding properties of the type I and type II SR-A have been detected, in contrast to the isoforms of liver and lung MR [Fiete, 1997]. Work in our laboratory has recently revealed another alternatively spliced form of SR-A, type III which acts as a dominant negative receptor when expressed with type I or type II SR-A in CHO cells [Gough, 1998]. The observation that type III SR-A is trapped in the endoplasmic reticulum may help to explain its dominant negative effect. SR-A recognises polyanionic molecules via its collagen-like domains [Acton, 1993], and recognition may be determined by the spatial characteristics of the repeating charged units, although the exact determinants are not yet known [Krieger, 1994]. Known ligands of hostderived, microbial, synthetic and inorganic origin are listed in Table 1. Like MR, SR-A is a member of a family sharing functional and, in the case of MARCO, structural characteristics. MARCO has been identified as another SR-A, a portion of which shares homology with SR-A type I collagenous and cysteine rich domains [Elomaa, 1995]. The SR-B family share some functional features with SR-A, but are structurally distinct. The founder members of this family are CD36 [Endemann, 1993] and SR-Bl[Acton, 1994].

3.

FUNCTIONS OF MR AND SR-A IN HOMEOSTASIS AND IMMUNITY

MR and SR-A internalise ligands by receptor-mediated endocytosis and phagocytosis according to the size of the ligand, contributing to homeostasis and immunity. Phagocytosis mediated by MR can induce cytocidal mechanisms and proinflammatory cytokines [Maródi, 1991 ; Yamamoto, 1997]. Since MR has both host-derived and microbial ligands, induction of anti-microbial effector mechanisms can not be determined simply by receptor ligation. The ability of a Mø to respond to a MR ligand may depend on the activation or differentiation state of the Mø and the nature of the ligand (whether it is soluble or particulate), and perhaps other unknown factors. For example, Marodi found that

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Mannose Receptor and Scavenger Receptor

recombinant human myeloperoxidase, which is a ligand of MR, induced an increase in killing of unopsonized C. albicans by GM-CSF activated human monocyte-derived Mø. but not by untreated cells [Maródi, 1998]. By contrast, if opsonized C. albicans was used, myeloperoxidase significantly increased killing capacity of both activated and nonactivated Mø. In another study, Shibata and coworkers found that small chitin particles and mannan-coated phagocytosable beads induced TNFα IFN and IL-12 from murine spleen cells, whereas mannan coated beads and chitin particles of too large a dimension to be phagocytosed did not induce these cytokines. Soluble mannan could not induce these cytokines, but was able to inhibit cytokine induction by chitin particles indicating that the physical properties of the ligand were critical in determining the response [Shibata, 1997]. For a thorough review of this subject, see [Linehan, In press]. Unlike MR, uptake of microbes or their products through SR-A may not result in activation. Our study of LPS induced endotoxic shock in bacillus Calmette Guèrin-infected mice showed that normal mice were more resistant than SR-A knock-out mice, suggesting that SR-A acts in a Work in our non-activatory clearance capacity [Haworth, 1997], laboratory has shown that SR-A is able to phagocytose apoptotic thymocytes, another function which would be expected to be nonactivatory [Platt, 1996]. The original discovery of Mø SR-A activity in modified LDL uptake suggested it may be responsible for LDLcholesterol accumulation by Mø, in atherosclerotic lesions. In support of this, SR-A has been identified at these sites [Matsumoto, 1990]. Finally, Mø from SR-A gene knock-out mice were shown to degrade acety1-LDL at less than one third of the normal rate, and oxidised-LDL at around half [Suzuki, 1997]. In vivo, SR-A on endothelial cells (as well as Mø) may protect against atherosclerosis since osteopetrotic mice which lack M-CSF dependent Mø are are still protected [de Villiers, 1998]. Whereas both MR and SR-A have functions in normal clearance of host molecules and phagocytosis of microbes, SR-A has an additional function in cell adhesion. A mAb, 2F8, was identified by its ability to block divalent cation-independent adhesion of murine Mø to tissueculture plastic and shown to immunoprecipitate SR-A [Fraser, 1993], The serum dependency of the adhesion suggested that host-derived factors may be involved in anchoring SR-A expressing Mø within tissues. A further study demonstrated that 2F8 completely blocked EDTA resistant adhesion of Mø to spleen, lymph node, lung, thymic medulla and gut lamina propria, but only partially to liver and thymic cortex [Hughes, 1995]. The degree of blocking by 2F8 correlated with the level of expression of SR-A in tissues, with high levels of expression related to

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high blocking ability, but the putative endogenous ligands have not yet been identified.

4.

PARTIALLY OVERLAPPING SITES OF EXPRESSION OF MR AND SR-A IN TISSUE

Like their functions, the expression patterns of MR and SR-A in mouse are also partly overlapping. The expression of SR-A was identified using the mAb 2F8 [Hughes, 1995], whereas we examined MR expression by immunocytochemistry using a polyclonal ab and in situ hybridization [Linehan, 1999]. We found that, like SR-A, most tissue Mø express MR. There were some discrepancies, in that marginal zone Mø of spleen expressed SR-A but not MR. These are highly phagocytic Mø. which are at sites of antigen entry into the spleen and lymph node, and may play a role in polysaccharide clearance [Humphrey, 1981]. The lack of MR expression in the marginal zone, as well as in the lymph node subcapsular sinus Mer was especially surprising as mannose-specific binding to these cells has been described [Li, 1993; Kahn, 1995]. Perivascular microglia of the brain are specialised Mø and express both SR-A [Mato, 1996] and MR [Linehan, 1999]. Likewise, cultured dendritic cells have been shown to express MR [Sallusto, 1994; Caux, 1997] and SR-A [D.A. Hughes, unpublished; R. Howarth, unpublished] but the circumstances under which dendritic cells express these receptors in vivo are not yet known. We found no expression of MR on mature or immature dendritic cells in spleen, lymph nodes or epidermis of naïve mice [Linehan, 1999]. Table 2. Expression of M R and SR-A by cell type

mature M ø monocytes selected endothelial cells cultured dendritic cells perivascular microglia mesangial cells retinal pigment epithelial cells

MR +

SR-A +

-

-

+ + + + +

+ + +

-

Endothelial expression of MR was more widespread than that of SR-A, in lymphatic endothelium in addition to sinusoidal endothelium of liver and spleen, whereas endothelial SR-A expression was found to be restricted to liver sinusoids. There were a few distinct cell types which express MR but not SR-A, namely renal mesangial cells [Linehan, 1999]

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Mannose Receptor and Scavenger Receptor

and retinal pigment epithelium [Shepherd, 1991], although the latter may express CD36, another member of the scavenger receptor family. These data are summarised in Table 2.

5.

MR MAY PLAY A NOVEL ROLE IN ANTIGEN DELIVERY TO SITES OF DEVELOPING CLONAL IMMUNE RESPONSES

Our recent work has shown that murine tissues express ligands of the cysseine-rich domain of MR, the first study to suggest a function for this domain (CR) [Martínez-Pomares, 1996]. Like SR-A ligands in tissue, MR ligands could, in theory, be used for cell adhesion of MR expressing Mø. However, the precise distribution and kinetics of expression during immune responses suggested a function in immunity. When murine tissues were probed with a chimaeric probe consisting of CR fused to the Fc region of human IgG1, CR-Fc, binding of CR-Fc to spleen marginal metallophilic Mø and undefined cells in B cell areas, and to lymph node subcapsular sinus Mø, was observed in naive animals. In immunised animals, CR-Fc binding to B cell areas of spleen white pulp was upregulated. A time-course study of a secondary immune response indicated apparent migration of CR-Fc binding cells from the subcapsular sinus of lymph nodes to sites of developing germinal centres. This suggested that MR could be directed to areas where affinity maturation of B cells occurs. However, double immunocytochemical staining of CR-Fc and MR in naive mice showed that the ligand was not expressed by the same cells as the receptor [Linehan, 1999]. We have documented the existence of a soluble form of MR (sMR) and suggest that this may act as a mobile antigen capture protein for delivery to the marginal zone of spleen and lymph node subcapsular sinus, as well as to primary and secondary B cell follicles [Martinez-Pomares, 1998]. sMR is generated by proteolysis of MR from cultured Mø and is shed into the media where it retains calcium-dependent mannosyl binding activity, and also occurs naturally in serum. sMR has been identified in cell-free bronchoalveolar lavage fluid from patients infected with HIV or coinfected with HIV and Pneumocystis carinii, although samples from healthy control volunteers had very little or no detectable sMR [I. Fraser, R. A. B. Ezekowitz, personal communication]. Infection of Mø with P. carinii results in an enhancement of sMR shedding, although the significance of this is not yet known. In a further study, the phenotype of CR-Fc binding cells localized within primary B cell follicles during the first few days of a primary immune response was examined [Berney, 1999]. They were

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found to express MHC II, sialoadhesin and CD11C. Purified CR-Fc+ cells were able to prime naive T cells when injected into naive mice as well as initiate a primary antibody response. This ability to transfer naive antigen to B cells was restricted to CR-Fc+ lymph node cells, and could, in theory, provide an effective means of initiating early protective immunity when viral or bacterial infection is at a low level. Whether soluble MR, which would preferentially recognise microbial antigens, could participate in such a role remains an exciting possibility. A model of this putative mechanism is shown in figure 1.

Figure 1. Model of soluble MR delivering antigen to CR-Fc binding cell

Cell surface ligands of SR-A have not yet been characterised at a molecular level and their detailed location is not yet known. A natural soluble form of SR-A has been identified which can bind polyanion-coated beads [W. de Villiers, unpublished] It seems that the tissue ligands of SRA mediate cell adhesion rather than antigen transfer, although this possibility cannot be ruled out.

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S04- 4- Ga lNAcβ1 4GlcNAc or Man at independent sites. Proc. Natl. Acad. Sci. USA 94, 11254-1 1261. Fiete, D. J., Beranek, M. C., and Baenziger, J. U. (1998). A cysteine-rich domain of the "mannose" receptor mediates GalNAc-4-SO4 binding. Proc. Natl. Acad. Sci. USA 95, 2089-2093. Fraser, I., Hughes, D., and Gordon, S. (1993). Divalent cation-independent macrophage adhesion inhibited by monoclonal antibody to murine scavenger receptor. Nature 364, 343-346. Freeman, M., Ekkel, Y., Rohrer, L., Penman, M., Freedman, N. J., Chisolm, G. M., and Krieger, M. (1991). Expression of type I and type II bovine scavenger receptors in Chinese hamster ovary cells: lipid droplet accumulation and nonreciprocal cross competition by acetylated and oxidized low density lipoprotein. Proc Natl Acad Sci U S A 88, 4931-5. Gordon, S. (1995). The macrophage. Bioessays 17, 977-86. Cough, P. J., Greaves, D. R., and Gordon, S. (1998). A naturally occurring isoform of the human macrophage scavenger receptor (SR-A) gene generated by alternative splicing blocks modified LDL uptake. J. Lipid Res. 39, 531-543. Hampton, R. Y., Golenbock, D. T., Penman, M., Krieger, M., and Raetz, C. R. (1991). Recognition and plasma clearance of endotoxin by scavenger receptors. Nature 352, 342-4. Harris, N., Peters, L. L., Eicher, E. M., Rits, M., Raspberry, D., Eichbaum, Q. G., Super, M., and Ezekowitz, R. A. B. (1994). The exon-intron structure and chromosomal localization of the mouse macrophage mannose receptor gene Mrcl: Identification of a ricin-like domain at the N-terminus of the receptor. Biochem. Biophys. Res. Comm. 198, 682-692. Haworth, R., Platt, N., Keshav, S., Hughes, D., Darley, E., Suzuki, H., Kurihara, Y., Kodama, T., and Gordon, S. (1997). The macrophage scavenger receptor type A is expressed by activated macrophages and protects the host against lethal endotoxic shock. J Exp Med 186, 1431-9. Hughes, D. A., Fraser, I. P., and Gordon, S. (1995). Murine macrophage scavenger receptor: in vivo expression and function as receptor for macrophage adhesion in lymphoid and non-lymphoid organs. Eur. J. Immunol. 25, 466-473. Humphrey, J., and Grennan, D. (1981). Different macrophage populations distinguished by means of fluorescent polysaccharides. Recognition and properties of marginalzone macrophages. Eur. J. Immunol. 11:221-228. Ishizaki, J., Hanasaki, K., Higashino, K.-i., Kishimo, J., Kikuchi, N., Ohara, 0., and Arita, H. (1 994). Molecular cloning of pancreatic group I phospholipase A2 receptor. J. Biol. Chem. 269, 5897-5904. Jiang, W., Swiggard, W. J., Heufler, C., Peng, M., Mirza, A., Steinman, R. M., and Nussenzweig, M. C. (1995). The receptor DEC-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing. Nature 375, 151-155. Kabha, K., Nissimov, L., Athamna, A., Keisari, Y., Parolis, H., Parolis, L. A. S., Grue, R. M., Schlepper-Schafer, J., Ezekowitz, R. A. B., Ohman, D. E., and Ofek, I. (1995). Relationships among capsular structure, phagocytosis, and mouse virulence in Klebsiella pneumoniae. Infection and Immunity 63, 847-852. Kahn, S., Wleklinski, M., Aruffo, A., Farr, A., Coder, D., and Kahn, M. (1995). Trypanosoma cruzi amastigote adhesion to macrophage is facilitated by the mannose receptor. J. Exp. Med. 182, 1243-1258. Kery, V., J. J. F. Krepinsky, C. D. Warren, P. Capek and P. D. Stahl (1992). Ligand recognition by purified human mannose receptor. Arch. Bioch. Biophys. 298, 49-55.

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Krieger, M., and Herz, J. (1994). Structures and functions of multiligand lipoprotein receptors: macrophage scavenger receptors and LDL receptor-related protein (LRP). Annu Rev Biochem 63, 601-37. Larkin, M., Childs, R. A., Matthews, T. J., Thiel, S., Mizuochi, T., Lawson, A. M., Savill, J. S., Haslett, C., Diaz, R., and Feizi, T. (1989). Oligosaccharide-mediated interactions of the envelope glycoprotein gp120 of HIV-1 that are independent of CD4 recognition. AIDS 3, 793-798. Li, R.-K., and Cutler, J. E. (1993). Chemical definition of an epitope/adhesin molecule on Candida albicans. J. Biol. Chem. 268, 18293- 18299. Linehan, S. A., Martínez-Pomares, L., Stahl, P. D., and Gordon, S. (1999). Mannose receptor and its putative ligands in normal murine lymphoid and non-lymphoid organs. In situ expression of mannose receptor by selected macrophages, endothelial cells, perivascular microglia and mesangial cells, but not dendritic cells. J. Exp. Med. 189, 1961-1972. Linehan, S. A., Martínez-Pomares, L., and Gordon, S. (In press). Macrophage lectins in host defence. Microbes and Infection In press. Maródi, L., Korchak, H. M., and Johnston, R. B. (1991). Mechanisms of host defence against Candida species. I. Phagocytosis by monocytes and monocyte-derived macrophages. J. Immunol. 146. 2783-2789. Maródi, L., Tournay, C., Káposzta, R., Johnston, R. B. J., and Moguilevsky, N. (1998). Augmentation of human macrophage candidacidal capacity by recombinant human myeloperoxidase and granulocyte-macrophage colony-stimulating factor. Infection and Immunity 66, 2750-2754. Marítnez-Pomares. L., Crocker, P. R., Da Silva, R., Holmes, N., Colominas, C., Rudd, P., Holmes, N.., and Gordon, S. (In press). Cell-specific glycoforms of sialoadhesin and CD45 are counter receptors for the cysteine-rich domain of the mannose receptor. J. Biol. Chem In Press. Martínez-Pomares, L., Kosco-Vilbois, M., Darley, E., Tree, P., Herren, S., Bonnefoy, J.Y., and Gordon, S. (1996). Fc chimeric protein containing the cysteine-rich domain of the murine mannose receptor binds to macrophages from splenic marginal zone and lymph node subcapsular sinus and to germinal centers. J. Exp. Med. 184, 1927-1937. Marínez-Pomares, L., Mahoney, J. A., Káposzta. R., Linehan, S. A., Stahl, P. D., and Gordon, S. (1998). A functional soluble form of the murine mannose receptor is produced by macrophages in vitro and is present in mouse serum. J. Biol. Chem. 273, 23376-23380. Mato, M.. Ookawara, S., Sakamoto, A., Aikawa, E.. Ogawa, T.. Mitsuhashi, U., Masuzawa, T., Suzuki. H., Honda, M., Yazaki, Y., Watnabe, E.. Luoma, J., Yla-Herttuala, S., Fraser. I., Gordon, S., and Kodama, T. (1996). Involvement of specific macrophage-lineage cells surrounding arterioles in barrier and scavenger function in brain cortex. Proc. Natl. Acad. Sci. USA 93, 3269-3274. Matsumoto, A., Naito, M., Itakura, H., Ikemoto, S., Asaoka, H., Hayakawa, I., Kanamori, H., Aburatani, H., Takaku, F., Suzuki, H., Kobari, Y., Miyai, T., Takahashi, K., Cohen, E. H., Wydor, R., Housman, D. E., and Kodama, T. ( 1990). Human macrophage scavenger recepotors: Primary structure, expression, and localization in atherolsclerotic lesions. Proc. Natl. Acad. Sci. USA 87, 9133-9137. Matzinger, P. (1998). An innate sense of danger. Sem. Immunol. 10, 399-415. Medzhitov, R., and Janeway Jr, C. A. (1997). Innate Immunity: impact of the adaptative immune response. Curr. Op. Immunol. 9, 4-9.

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O'Riordan, D. M., Standing, J. E., and Limper, A. H. (1995). Pneumocystis carinii glycoprotein A binds macrophage mannose receptors. Infection and Immunity 63, 779-784. Platt. N., Suzuki, H., Kurihara, Y., Kodama, T., and Gordon, S. (1996). Role for the classA scaveneger receptor in the phagocytosis of apoptotic thymocytes in-vitro. Proc. Natl. Acad. Sci. USA 93, 12456-12460. Resnick, D., Freedman, N. J., Xu, S., and Krieger, M. (1993). Secreted extracellular domains of macrophage scavenger receptors form elongated trimers which specifically bind crocidolite asbestos. J. Biol. Chem. 268, 3538-3545. Sallusto, F., and Lanzavecchia, A. (1994). Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colonystimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J. Exp. Med. 179, 1109-1114. Schlesinger, L. S. (1994). Binding of the terminal mannosyl units of lipoarabinomannan from a virulent strain of Mycobacterium tuberculosis to human macrophages. J. Immunol. I52, 4070-4078. Schlesinger, P. H., Doebber, T. W., Mandell, B. F., White, R., DeSchryver, C., Rodman, J. S., Miller, M. J., and Stahl, P. D. (1978). Plasma clearance of glycoproteins with terminal mannose and N-acetylglucosamine by liver non-parenchymal cells. Studies with beta-glucoronidase, N-acetyl-beta-D-glucosamine, ribonuclease B and agalactoorosomucoid. Biochem J. 176, 103-109. Shepherd, V. L., and Hoidal, J. R. (1990). Clearance of neutrophil-derived myeloperoxidase by the macrophage mannose receptor. Am. J. Respir. Cell Mol. Biol. 2, 335-340. Shepherd, V. L., Tarnowski, B. I., and McLaughlin, B. J. (1991). Isolation and charachterization of a mannose receptor from human pigment epithelium. Invest. Ophthalmol. Vis. Sci. 32, 1779-1784. Shibata. Y., Metzger, W. J., and Myrvik, Q. N. (1997). Chitin particle-induced cellmediated immunity is inhibited by soluble mannan. Mannose receptor-mediated phagocytosis initiates IL-12 production. J. Immunol. 159, 2462-2467. Smedsrød. B.. Einarsson, M., and Pertoft. H. (1988). Tissues plasminogen activator is endocytosed by mannose and galactose receptors of rat liver cells. Thromb. and Haem. 59, 480-484. Smedsrød, B., Melkko, J., Risteli, L., and Risteli, J. (I 990). Circulating C-terminal propeptide of type I procollagen is cleared mainly via the mannose receptor in liver endothelial cells. Bioch. J. 271, 345-350. Stahl, P. D., Rodman, J. S., Miller, M. J.. and Schlesinger, P. H. (1978). Evidence for receptor-mediated binding of glycoproteins, glycoconjugates, and lysosomal glycosidases by alveolar macrophages. Proc. Natl. Acad. Sci. USA 75, 1399- 1403. Suzuki, H.,Kurihara, Y., Takeya, M., Kamada, N., Kataoka, M., Jishage, K., Ueda, O., Sakaguchi, H., Higashi, T., Suzuki, T., Takashima, Y., Kawabe, Y., Cynshi, O., Wada, Y., Honda, M., Kurihara, H., Aburatani, H., Doi, T., Matsumoto, A., Azuma, S., Noda, T.. Toyada, Y., Itakura, H., Krujit, J. K., van Berkel, T.J. C., Steinbrecher, U. P., Ishibashi, S., Madea, N., Gordon, S., Kodama, T. (1997). A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection. Nature 386:292-298 Taylor, M. E., K. Bezouska, and K. Drickamer (1992). Contribution to ligand binding by multiple carbohydrate-reognition domains in the macrophage mannose receptor. J. Biol. Chem. 267, 1719-1726.

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Taylor, M. E., J. T . Conary, M. R. Lennartz, P. D. Stahl, and K. Drickamer (1990). Primary Structure of the mannose receptor contains multiple motifs resembling carbohydrate-recognition domains. J. Biol. Chem. 265, 121 56-12162. Wu, K., Yuan, J., and Lasky, L. A. (1996). Characterization of a novel member of the macrophage mannose receptor type C lectin family. J. Biol. Chem. 271, 2132321330. Yamamoto, Y., Klein, T. W.. and Friedman, H. (1997). Involvement of mannose receptor in cytokine interleukin- 1 (IL- I ), IL-6, and granulocyte-macrophage colony-stimulating factor responses. but not in chemokine macrophage inflammatory protein 1 (MIP-I), MIP-2, and KC responses, caused by attachment of Candida albicans to macrophages. Infection and Immunity 6 5 , 1077-1082.

COMPLEMENT RECEPTOR 3 (CR3): A PUBLIC TRANSDUCER OF INNATE IMMUNITY SIGNALS IN MACROPHAGES

Eitan Yefenof THE LAUTENBERG CENTER FOR GENERAL AND TUMOR IMMUNOLOGY, The Hebrew University - Hadassah Medical School, Jerusalem, Israel

1.

INTRODUCTION

The complement system has been considered for years an esoteric discipline of immunology. It emerged as an auxiliary system that complements the antibody response by the enactment of lysis and opsonization of bacteria (Ross 1986), and this is reflected in its designated name. The discovery of the alternative pathway, which enables direct activation of C3 by microorganisms and altered-self cells (Pillemer et al 1954), revised this concept thoroughly. It demonstrated that the complement system is autonomous in its activation capacity and that it plays an important function as a proinflammatory system whenever recognizing a potential pathogen. In evolutionary terms, the complement system in its alternative form was first to appear and provide non-specific innate surveillance against microbes expressing complement-activating molecules (Farrier & Atkinson 1991). Later on it was recruited by the humoral immune response of vertebrates and became a major effector system for antibodies via the components of the classical pathway. A tertiary development involved the mannose binding lectin (MBL) pathway, which links pathogens with carbohydrate rich exterior to the classical pathway in an antibody independent manner (Turner 1996). The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2 0 0 0

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Complement Receptor 3 (CR3)

Together, the three pathways of complement activation represent a major humoral effector system that operates in fish, amphibians, reptiles, birds and mammals, and cross-talks with other compartments of the immune response at several intersections.

2.

COMPLEMENT RECEPTORS

A set of complement receptors provides links between the complement system and cellular immunity (Ahearn & Fearon 1989). So far, nine complement receptors have been identified, of which six were characterized. The C3a/C4a and the C5a receptors have a 7 TMR structure and, in this regard, they are similar to the chemokine/G-protein coupled receptor family (Westrel 1995). Their ligands C3a, C4a and C5a remain soluble following complement activation and induce inflammatory responses including chemotaxis of neutrophiles, eosinophiles, basophiles and macrophages (Goldstein 1992). The other complement receptors are all specific to fragments of C3 that are bound covalently to the activating substance (Ross & Medof 1985), whether it is an antigen-antibody complex or a carbohydrate on the surface of a bacterium, virus or transformed cells that activate the complement cascade via the alternative or MBL pathways. C3 is a heterodimer of α and β chains (Muller-Eberhard 1988). Upon activation its a chain is cleaved at a specific arginine residue into C3a and C3b. This cleavage exposes a thioester residue that is reactive for a few milliseconds and can bind covelently to the activating cell or substance (Law & Dodds 1997). In this form C3b is recognized by CRl (CD35), which is expressed on a variety of hemopoietic cells, including erythrocytes (Ahearn & Fearon 1989). C3b can be further cleaved by factor H and factor I to a slightly smaller variant called inactivated, or iC3b, for which CR3 is a receptor (Ross & Veticka). iC3b is further degraded to C3dg and later on to C3d, which is a ligand for CR2 (Ahearn & Fearon 1989). CR2 demonstrates how elements of the innate, immune system, were incorporated into the specific, clonal immune response and became mandatory regulators of the immune response to an antigen (Fearon & Carter 1995). Unlike CR1 and CR3, which are expressed on a wide range of hemopoietic cells, CR2 expression is rather restricted to B lymphocytes, and to a lesser extent, epithelial cells as well. Its significance has been obscure for years except the recognition that it functions as a receptor for Epstein-Barr virus (EBV), thus, restricting the tropism of EBV to human B lymphocytes and some epithelial cells (Weis et al 1988). But this has been considered a

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secondary adaptation of the EBV envelope glycoprotein to CR2. The "true" function of CR2 in the context of the immune response became apparent when Fischer and coworkers (Fisher et al 1996) found that mice deficient in C3 are severely defective in their ability to mount an antibody response against T dependent antigens. This result indicated that complement is essential for the triggering of B lymphocytes in response to an antigenic challenge. The same phenotype was observed in mice deficient of CR2 (Ahearn et al 1996). Even though the repertoire of B lymphocytes in such mice remained intact, they failed to produce a significant level of antibodies when stimulated by an antigen. The model emerging from these findings was that optimal activation of B lymphocytes requires cross-ligation of the B cell receptor complex and CR2, which can be accomplished by an antigen that has fixed C3d via the classical or alternative pathway (Fearon 1998). A single bond between an antigen and the immunoglobulin receptor, or C3d and CR2 is not sufficient for B cell activation. Thus, CR2 is a co-stimulatory receptor of B cells and in this regard parallels CD28 of T lymphocytes responding simultaneously to a peptide and to the B7 costimulatory molecule (June et al 1994). CR2 enables B cells to distinguish between non-pathogenic antigens against which there is no need to respond, and a pathogenic antigen, which activates and fixes C3 and therefore provides the dual signal required for optimal B cell response. The complement system, which evolved as an innate immunity arm that marks microbial pathogens for destruction, underwent a secondary and tertiary evolution in birds and mammals. It became a major effector mechanism employed by antibodies to eradicate specific antigens through the components of the classical pathway. On another track, it was incorporated as an element that regulates the antibody response via the expression of CR2 as a coreceptor for B cell activation. CD19 is an essential receptor in this regard because it complexes with CR2 and transmits the signal emanating from the C3d binding site via its three intracellular thyrosine residues that are phosphorylated following a CR2 trigger (Tedder et al 1994).

3.

CR3

The complement receptor most abundant in cells of the immune system is CR3 (Stewart et al 1995). It is expressed on monocytes, macrophages, granulocytes, NK cells, and certain subsets of T and B cells. CR3 is a heterodimer of CD1 lb and CDl8 transmembrane glycoproteins belonging to the 2 integrin leukocyte receptor family (Law et al 1987, Kishimoto et al 1987). The complement ligand that binds to CR3 is iC3b

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Complement Receptor 3 (CR3)

(Wrights et al 1983). It can, however, bind additional ligands such as ICAM-1, ICAM-2 and fibrinogen (Wright et al 1988, Diamond et al 1990, Diamond et al 1991). All these attach to the I region of CR3 on the extracellular portion of CD11b. An additional site at the carboxyl region is a sugar binding site, to which β-glucan and LPS combine (Wright et al 1989, Ross et al 1985). In macrophages each of these ligands triggers a host of cellular activities which are mediated by CR3. These include adhesion to endothelial cells and extracellular matrix, phagocytosis, oxidative burst, cytokine production, and cytotoxicity (Meerschaert & Furie 1995, Fällman et al 1993, Von Asmuth et al 1991). Hence, CR3 is an important component of innate immunity by virtue of its multiple ligands and versatile activities. Its significance is demonstrated in patients with leukocyte adhesion deficiency (LAD). Such individuals are deficient of CD18 and therefore do not express CR3, LFA1 and CR4 (Wardlaw et al 1990). Consequently, they suffer from recurrent infections due to the malfunction of their macrophages and neutrofiles (Anderson & Springer 1987). We studied the host response to a leukomogenic process induced by the Radiation Leukemia Virus (RadLV). RadLV induces primary thymic lymphomas that appear several months after virus infection, which is restricted the thymus and thymic lymphocytes (Yefenof 1999). During the premalignant latency a population of abnormally large bi-noculated and granular cells appear and accumulate in the thymus (Messika et al 1991). The cells were identified as activated macrophages and the granules are T lymphocytes that underwent phagocytosis. Staining with virus specific antibodies indicated that the thymic large macrophages can selectively ingest and destroy virus-infected T lymphocytes that are subsequently destroyed. This finding posed an enigma because at any given time the proportion of virus infected cells in the thymus did not exceed 3% (Yefenof et al 1991). Yet, all of the cells inside the macrophages were virus positive. It turned out that the RadLV infected cells could activate complement via the alternative pathway, thus becoming opsonized with iC3b (Messika et al 1991). This ensures specific recognition of virally infected cells by CR3 of the thymic macrophages, followed by phagocytosis immediately thereafter. This interaction also leads to an oxidative burst response in the macrophages, which produce oxygen radicals. The response does not develop if the stimulator cells are not opsonized by iC3b or in the presence of anti C3 blocking antibodies. Thus, an interplay between the alternative complement pathway and CR3 enables the discernment of a small population of virally infected cells and enables a selective macrophage response against altered self lymphocytes without effecting other cells in

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the surroundings. Another puzzle was the fact that only macrophages from a virus infected premalignant thymus could respond to the iC3b challenge. Macrophages from bone marrow of infected mice or noninfected thymus were negative, both in oxidative burst and in phagocytosis. This indicated that not only the morphology of the thymic large macrophages was different, but they were also primed for recognition of iC3b opsonized cells. In a parallel study, we found that RadLV infected lymphoma cells produce IL-4, which is an autocrine growth factor essential for their survival (Yefenof et al 1992). The effect of IL-4 on macrophages is controversial. Some researchers like Paul, Melzer and Leder reported that IL-4 activates macrophages for increased phagocytosis and TNF production (Crawford et al 1987). Others like Abbas et al, claimed that TH2 cells inhibit macrophage function via IL-4 (Abbas et al 1991). Since IL-4 is continuously made in the prelymphoma thymus we examined the possible effect of this cytokine in the priming of the thymic large macrophages. To this end we took macrophages from bone marrow, which are not stimulated by iC3b. Treatment with IL-4, however, converted them to respond both in oxidative burst and phagocytosis of iC3b opsonized cells (Messika et al 1991). The same was observed in bone marrow macrophages responding to cross-ligation of CR3 by anti CR3 antibodies. Oxidative burst developed only if the Mø were pretreated by IL-4. We have thus identified 2 factors enabling the interaction between virus infected prelymphoma cells and thymic macrophages. One is opsonization by iC3b through the alternative pathway; the other is IL-4, which plays a double role in the lymphomagenic process. It enables survival of prelymphoma cells in the thymus, but at the same time primes thymic macrophages for recognition and response through CR3. This is a form of innate immunity that operates during the prolonged latency of the disease and is successful in removing more than one half of the prelymphoma cells at any given time.

4.

CR3 - GALECTIN-1 ASSOCIATION

We did not detect a quantitative or a qualitative change in either CD11b or CD18 following IL-4 treatment. Hence, we asked whether other signaling molecules that are associated with CR3 might be affected by IL-4. The existence of CR3 associated molecules have been long sought because its α and chains have short intracellular domains that lack intrinsic catalytic activity (Dedhar & Hannigan 1996). It was therefore

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Complement Receptor 3 (CR3)

postulated that signaling via CR3 is enabled by an associated cytoplasmic molecule or another membrane receptor such as FcγRII (Zhou & Brown 1994). In search of such a molecule we immunoprecipitated the CR3 complex by a combination of antibodies to CD11 b and CDl8. Running the immunoprecipitate on a two dimensional gel revealed a unique protein that was co-immunoprecipitated with CR3 of thymic large macrophage (Messika et al 1995). The molecular mass of this proteins was 16 kD and its isoelectric point 5.1. We therefore designated it p16/5.1. p16/5.1 was missing in macrophages of a normal thymus, peritoneum or bone marrow, but it appeared in CR3 of bone marrow macrophages treated with IL-4. Analysis of six monocyte and macrophage cell lines that express CR3 revealed four that expressed p16/5.1. These lines were CR3 positive and responded with oxidative burst when stimulated with iC3b opsonized cells (Messika et al 1995). We also identified two lines lacking p16/5.1. These cells expressed CR3 but were non-responders in the oxidative burst assay. It seems, thus, that p16-5.1 converts CR3 from a non-active molecule to an active receptor, which functions in "outside-in" signaling. We upscaled purification of p16/5.1 on the 2d gel and extracted it for microsequencing. Triptic digestion yielded 7-mer and a 9-mer peptides, which displayed exclusive homology to the animal lectin galectin- 1, whose reported molecular mass is 15 kD and its P.I. 5.3 (Hirabayashi & Kasai 1990). Next, we synthesized a 14 amino acid immunodominant peptide of galectin-1 and used it to raise polyclonal anti galectin-1 antibodies in rabbits (Avni et al 1998). Such antibodies cross-reacted with p16/5.1, which was the only protein detected on the 2d gel of immunoprecipitated CR3 by Western blotting. Likewise, immunoprecipitation with anti-galectin- 1 antibodies recovered a protein that migrates to the 16/5.1 position in the two dimensional gel. Galectin-1, like all other members of the galectin family, is a betagalectoside binding lectin that can form glycoconjugates with other proteins through its carbohydrate binding site (Wilson et al 1989). We therefore wanted to find out whether it associates with CR3 via the sugar binding site. To this end, we used lactose, which is a high affinity ligand of galectin-1 and asked whether it affects the association. Indeed, the p 16/5.1 -CR3 association was disrupted if the macrophages are incubated with lactose, but not with a control sugar such as sucrose. We could therefore conclude that galectin- 1 associates with CR3 through its carbohydrate binding site. Galectin-1, as well as other members of the galectin family, does not have a signal peptide and is therefore found mainly in the cytosol of muscle, neuron, thymic, kidney and placental cells. It can, however, be

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exported to the cell surface and to the extracellular matrix via a nonclassical secretion pathway. By immunofluorescence staining we detected expression of galectin-1 on the surface of thymic large macrophages or bone marrow macrophages treated with IL-4 (Avni et al 1998). Two color fluorescence of CD1 lb (red) and galectin-1 (green) analyzed by confocal microscopy indicated co-associatian of the two molecules as anticipated from the co-precipitation experiment. What is the functional significance of the newly identified association between a β-galectoside lectin and CR3? The ability of CR3 to bind iC3b or any other of its ligand is not constitutive but regulated by rapid onand off- switches (Diamond & Springer 1994). Such modifications in receptor activity occur following activation through other cell surface molecules including cytokine receptors (Hynes 1992). A stimulus through these receptors, IL-4 in our case, evokes and "inside-out" signal in the macrophage leading to conformational changes of CR3 that convert it to an active form. In this configuration CR3 combines specific ligands which induce a cascade of "outside-in" signaling events, leading to oxidative burst, production of TNF, IL- 1, IL-6 and phagocytosis (Rosales & Juliano 1995). CR3 can also transmit signals emanating at a glycosylphosphatidyl inositole (GP1)-linked protein such as Fc RIIIB, CD14 (receptor for LPS) and the eurokinse plasminogen activator receptor (uPAR) (Stock1 et al 1995, Zarewych et a1 1996, Gyetko et al 1995). The GPI anchored proteins, which are devoid of transmembrane domain, trap the ligand while floating in the membrane lipid bi-layer and transmit inflammatory signals by a co-associated CR3 molecule. Accordingly, CR3 has also been termed "public transducer" (Petty & Todd 1996). Galectins, on the other hand, are lectins that can form glycoconjugates with other membrane receptors through their beta-galactocide binding site (Barondes et al 1994). The unique feature of galectin-1 is its ability to switch between a monomeric structure and a divalent non-covalently associated homodimer. In this latter form it can bridge between two glycoprotein receptors either in solution, in the extracellular matrix or on the surface membrane. We therefore propose two models to interpret the functional significance of galectin- 1-CR3 interaction (Avni et al 1998). In the first model a homodimer of galectin-1 acts as an extracellular adapter molecule that interacts with CD 14 or FCγRIII, enabling crosslinkage between CR3 and other membrane receptors. The inter-receptor association facilitates transmission of signals originating at a GPI-linked receptor through and adjacent signaling receptor. An alternative model implies that galectin-1 increases the affinity of CR3 to its ligand when interacting with a β-galactoside site at the extra cellular domain of the

Complement Receptor 3 (CR3)

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receptor. This association activates the CR3, which can now bind its ligand and transmit an inward signal.

5.

CONCLUSIONS

The complement system represents an ancient tool of innate immunity whose original function was, apparently, to opsonize foreign particles for effective recognition and elimination by phagocytes or other scavenger cells. In this regard, CR3 evolved as a membrane receptor that enables recognition and uptake of complement opsonized antigens. Later on, the function of the complement system has been extended by additional components, to include killing of pathogens, chemotaxis and anaphylaxis. Likewise, the function of CR3 has been extended to include adhesion, activation of oxidative burst cytokine production and cytotoxicity. The receptor-associated galectin- 1 reflects another facet of this extension by virtue of its ability to modulate the activity of CR3, thus combining CR3 to several other signaling functions. This is yet another example how basic elements of innate immunity developed into a powerful and complex machinery of response to dangerous antigens.

REFERENCES Abbas, A.K., Williams, M.E., Burstein, H.J., Chang, T.L.. Bossu. P. and Lichtman, A.H. Activation and functions of CD4+ T-cell subsets. Immunol. Rev. 123:5-22, 1991. Aheam, J.. Fischer, M., Croix, D., Goerg, S., Ma, M., Kia, J., Zhou, X., Howard, R., Rothstein, T. and Carrol, M. Disruption of the Cr2 locus results in a reduction in B-la cells and in an impaired B cell response to T-dependent antigen. Immunity 4:251-262, 1996. Ahearn, J.M. and Fearon, D.T. Stucture and function of the complement receptor, CR1 (CD35) and CR2 (CD21). Adv. Immunol. 46:183-219, 1989. Anderson, D.C. and Springer, T.A. Leukocyte adhesion deficienty: an inherited defect in Mac-I, LFA-1 and p150,95 glycoproteins. Ann. Rev. Med. 38:175-194, 1987. Avni, O., Pur, Z., Yefenof, E. and Baniyash, M. Complement receptor 3 (CDI 1b/1CD18) of macrophages is associated with Galectin-1. J. Immunol. 160:6151-6158. 1998. Barondes, S.H., Cooper, D.N.W., Gitt, M.A. and Leffler, H. Galectins. Structure and function of a large family of animal lectins. J. Biol. Chem. 269: 20807-20810, 1994. Crawford, R.M., Finbloom, D.S., Ohara, J.. Paul, W.E. and Meltzer, M.S. B cell stimulatory factor I (interleukin-4) activates macrophages for increased tumoricidal activity and expression of la antigens. J. Immunol. 139:135-141, 1987. Dedhar, S. and Hannigan, G.E. Integrin cytoplasmic interactions and bidirectional transmembrane signaling. Curr. Opin. Cell Biol. 8:657-669, 1996. Diamond, M.S. and T.A. Springer. The dynamic regulation of integrin adhesiveness. Curr. Biol. 4:506-517, 1994.

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Diamond, M.S., Staunton, D.E., de Fougeroles, A.R., Stacker, S.A., Garcia-Aguilar, J., Hibbs, M.L. and Springer, T.A. ICAM-I (CD54): A Counter-Receptor for Mac-I (CD11b/CD18). J. Cell Biol. 111:3129-3139, 1990. Diamond, M.S., Staunton, D.E., Marlin, S.D. and Springer, T.A. Binding of the integrin Mac-I (CDI1b/CD18) to the third immunoglubulin-like domain of ICAM-I (CD54) and its regulation by glycosylation. Cell 65:961-971, 1991. Fällman, M., Andersson, R. and Andersson, T. Signaling properties of CR3 (CDI1b/CD18) and CRl (CD35) in relation to phagocytosis of complementopsonized particles. J. Immunol. 151 :330-338, 1993. Farries, J.C. and Atkinson, J.P. Evolution of the complement system. Immunol. Today 12:295-300, 1991. Fearon, D.T. The complement system and adaptive immunity. Seminars in Immunol. 10:355-361, 1998. Fearon, D.T. and Carter R.H. The CD19ICR2IJAPA-I complex of B lymphocytes: Linking natural to acquired immunity. Annu. Rev. Immunol. 13: 127-149, 1995. Fisher. M., Ma, N. Goerg, S., Zhou. X.. Xia, J., Finco, O., Han, S., Kelsoe. G., Howard, R., Rothstein J., Kremmer, E.. Rosen, F. and Carrol, M. Regulation of the B cell response to T dependent antigens by classical pathway complement. J. Immunol. 157:549-456, 1996. Goldstein, I.M. Complement: Biologically active products. In: Inflammation (I.J. Gallin, I.M. Goldstein, R. Snyderman, eds.) Raven Press, N.Y. pp. 63-80, 1992. Gyetko, M.R., Sitrin, R.G., Fuller, J.A., Todd III, R.F., Petty, H. and Standiford, T.Y. Function of the urokinase receptor (CD87) in neutrophil chemotaxis. J. Leukoc. Biol. 58: 533-538, 1995. Hirabayashi, J. and Kasai, K. The family of metazoan metal-independent P-galactosidebinding lectins: structures, function and molecular evolution. Glycobiology 3:297326, 1990. Humphries. M.J. lntegrin activation: the link between ligand binding and signal transduction. Curr. Opin. Cell Biol. 8:632-640, 1996. Hynes, R.O. Integrins: versatility. modulation and signaling in cell adhesion. Cell 69:1 1-25, 1992. June, C.H., Bluestone, J.A., Nadler, L.M. and Thompson, C.B. The B7 and CD28 receptor families. Immunol. Today 15:321-331, 1994. Kishimoto. T.K., O'Connor, K., Lee, A., Roberts, T.M. and Springer, T.A. Cloning of the β subunit of the leukocyte adhesion proteins: homology to an extracellular matrix receptor defines a novel supergene family. Cell 48:681-690, 1987. Law, S.K.A. and Dodds, A.W. The internal thioester and the covalent binding properties of the complement proteins C3 and C4. Protein Sci. 6:263-274, 1997. Law, S.K.A., Gagnon, J., Hidreth, J.E.K., Wells, C.E., Willis, A.C. and Wong, A.J. The primary structure of the β-subunit of the cell surface adhesion glucoproteins LFA-1, CR3 and its relationshipt to the fibronectin receptor. EMBO J. 6:915-919, 1987. Meerschaert, J. and Furie, M.B. The adhesion molecules used by monocytes for migration across endothelium include CDI 1a/CD18, CDI 1b/CD18 and VLA-4 on monocytes and ICAM-1, VCAM-1 and other ligands on endothelium. J. Immunol. 154:40994112, 1995. Messika, E.. Gallily, R. and Yefenof, E. Radiation Leukemia Virus (RadLV)-induced leukemogenesis is associated with an increased number and activity of thymic macrophages. Int. J. Cancer 48:924-930, 1991.

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Complement Receptor 3 (CR3)

Messika, E.J., Yefenof, E., Gallily, R., Avni, O. and Baniyash, M. Identification and characterization of a novel protein associated with macrophage complement receptor 3. J. Immunol. 154:6563-6570, 1995. Muller-Eberhard, H.J., Molecular organization and function of the complement system. Ann Rev. Biochem. 57:321-397, 1988. Petty, H.R. and Todd III R.F. Integrins as promiscuous signal transduction devices. Immunol. Today 17: 209-212, 1996. Pillemer, L., Blum, L. and Lepow, H. The properdin system and immunity. I. Demonstration and isolation of a new serum protein, properdin, and its role in immune phenomena. Science 120:279-285, 1954. Rosales, C. and Juliano, R.L. Signal transduction by cell adhesion receptors in leukocytes. J. Leukoc. Biol. 57: 189-198, 1995. Ross, G.D. Introduction and history of complement research. In: Immunobiology of the complement system (G.D. Ross. editor) Academic Press. N.Y. 1-19. 1986. Ross. G.D. and Medof, E. Membrane complement receptors specific for bound fragments of C3. Adv. Immunol. 37:217-243, 1985. Ross, G.D. and Veticka. V. CR3 (CD1 Ib, CD18(: a phagocyte and NK cell membrane receptor with multiple ligand specificities and function. Clin. Exp. Immunol. 92: 181184, 1993. Ross, G.D., Chain, J.A. and Lachmann, P.J. Membrane complement receptor type three (CR3) has lectin-like properties analgous to bovine conglutinin and receptor for iC3b. J. Immunol. 134:3307-3315, 1985. Stewart, M., Thiel. M. and Hogg, N . Leukocyte integrins. Curr. Opin. Cell Biol. 7:690696, 1995. Stockl. J., Majodic. O., Pickl, W.F., Rosenkranz, A., Prager, E., Gschwantler, E. and Knapp. W. Granulocyte activation via a binding site near the c-terminal region of complement receptor type 3 a-chain (CD 1 b) potentially involved in intramembrane complex formation with glycosylphosphatidylinositol-anchored Fcγ RIIIB (CD16) molecules. J. Immunol. 154:5452-5463. 1995. Tedder, T.F., Zhou, L.J. and Engel, P. The CD19/CD21 signal transduction complex of B lymphocytes. Immunol. Today 15:437-441, 1994. Turner, M.W. Mannole binding lectin: the pluripotent molecule of the innate. Immunol. Today 17:532-540, 1996. Von Asmuth, E.J.U., Van der Linden, C.J., Leeuwenberg, J.F.M. and Burrman, W.A. Involvement of the CD1 1 b/CD18 integrin, but not the endothelial cell adhesiuon molecules ELAM-I and ICAM-1 in tumor necrosis factor-a-induced neutrophil toxicity. J. Immunol. 147:3869-3875, 1991. Wardlaw, A.J., Hibbs, M.L., Stacker. S.A. and Springer, T.A. Distinct mutations in two patients with leukocyte adhesion deficiency and their functional correlates. J. Exp. Med. 172:335-345, 1990. Weis, J.J., Toothaker, L.E., Smith, J.A., Weis, J.J. and Fearon D.T. Structure of the human B lymphocyte receptor for C3d and the Epstein Barr virus and relatedness to other members of the family of C/C4 binding proteins. J. Exp. Med. 167:1047-1066, 1988. Westerl, R.A. Structure, function and cellular expression of complement. Curr. Opin. Immunol. 7:48-53, 1995. Wilson T.Y.G., Firth, M.N., Powell, J.T. Harrison, F.L. Sequence o ft h e I4kDa pgalactoside binding lectin evidence for its synthesis on free cytoplasmic ribosomes. Biochem. J. 261: 847-852, 1989.

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Wright, S.D., Levin, S.M., Jong, M.T.C., Chad, Z. and Kabbash, L.G. CR3 (CD11b/CD18) expresses one binding site for Arg-Gly-Asp-containing peptides and a second site for bacterial lipopolysaccharide. J. Exp. Med. 169: 175-1 83. 1989. Wright, S.D.. Weitz, J.I., Huange. A.J.. Levin. S.M., Silverstein. S.C. and Loike, J.D. Complement receptor type three (CD 11 b/CD 18) of human polymorphonuclear leukocytes recognizes fibrinogen. Proc. Natl. Acad. Sci. USA. 85:7734-7738. 1988. Wrights, S.D., Roa, P.E., Van Voorhis, W.C., Craigmyle, L.S., Iida, K.. Talle, M.A., Westberg, E.f., Goldstein, G. and Silverstein; S.C. Identification of the c3bi receptor of human monocytes and macrophages by using monoclonal antibodies. Proc. Natl. Acad. Sci. USA. 80:5699-5703, 1983. Yefenof, E. Murine models of thymic lymphomas: premalignant scenarios amenable to prophylactic therapy. Adv. Immunol. 73:5 11-538, 1999. Yefenof. E.. Ela, C. Kotler. M. and Vitetta, E.S. Induction of IL-4 by the Radiation Leukemia Virus (RadLV): Role in autocrine growth stimulation of RadLV infected preleukemic cells. Int. J. Cancer 50:48 1-485. 1992. Yefenof. E., Epsztein, S. and Kotler, M. Quantitation, in vitro propagation. and characterization of preleukemic cells induced by Radiation Leukemia virus. Cancer Res. 51:2179-2184, 1991. Zarewych, D.M., Kindzelskii. A.L., Todd III, R.F.. and Petty, H. LPS induces CD14 association with complement receptor type 3, which is reversed by neutrophil adhesion. J. Immuno. 156: 430-433. 1996. Zhou, M.J. and Brown, E.J. CR3(Mac-1,α MβCDI1b/CD18) and FCα RIII cooperate in generation of a neutrophil repiratory burst: requirement for FCγRII and tyrosine phosphorylation. J. Cell. Biol. 125:1407-1416, 1994.

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THE ROLE OF C-TYPE LECTINS IN THE INNATE IMMUNITY AGAINST PULMONARY PATHOGENS

1 1

Itzhak Ofek, ²Erika Crouch, and ¹Yona Keisari

2

Department of Human Microbiology, University of Tel Aviv, Tel Aviv, Israel, Department of Pathology, Washington University , St. Louis, MO

1.

INTRODUCTION

Most serious bacterial infections in the modern world occur in immunocompromised hosts, especially in hospitalized patients receiving immunosuppressive drugs (Doebbeling, 1993). These opportunistic infections rarely occur in otherwise healthy individuals, suggesting that one or more arms of innate immunity are compromised in these patients. Thus, a comprehensive understanding of the mechanisms through which the constituents of the innate immunity protect against the development of symptomatic infections, could lead to the development of new theraputic approaches to improve the defenses of the compromised host and increase their resistance against infectious diseases. In the following we summarise studies that examined the biological consequences of the interaction between the C type lectins of the lung and the pulmonary pathogen Klebsiella pneumoniae and its role in innate immunity.

2.

C-TYPE LECTINS OF THE LUNG AND SURFACE GLYCOCONJUGATES OF KLEBSZELLA PNEUMONIAE

The C-type lectins of the lung include the mannose receptor (MR) on alveolar macrophages and the collagenous carbohydrate binding proteins The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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28

The Role of C-Type Lectins in Pulmonary Pathogen Infections

SP-A and SP-D, all of which interact with complementary sugars in a calcium-dependent manner (Linehan et al. 2000; Crouch, 1998). A comprehensive review of the structure-function of the MR is presented in this volume (Linehan et al. 2000). The detailed structure and biological role in defense of the collagenous lectins may be found elsewhere (Crouch 1998). In the following we will briefly discuss the sugar specificity of lung C-type lectins in relation to the glycoconjugate structures of K. pneumoniae recognized by the lectins. Two types of K. pneumoniae glycoconjugate structures are recognized by the C-type lectins. One of these is in the outer-membrane lipopolysaccharides (LPS) and is recognized by SP-D. The other resides in the capsular polysaccharide and is recognized by both SP-A and MR (Table 1). This is not surprising as the sugar specificity of the carbohydrate binding domains of the MR and SP-A are similar and differ from that of SP-D (Table 1). Expression of both LPS and capsular material are under the influence of regulatory genes and environmental factors. For example, the number of the oligosaccharide repeating units in the 0-antigen of LPS is influenced by growth conditions (Weiss et al. 1986). Capsule formation on the Klebsiella surface undergo phase variation whereby unecapsulated phase variants emerge in the cell population at a defined frequency during growth of capsulated organisms and vice versa (Matatov et al, 1999). Table 1. K. pneumoniae glycoconjugates recognized by the C- type lectins of the lung. K. pneumoniae glycoconjugates recognized by C Type lectins . a Sugar specificity Location Structure C-type lectin Mannose receptor Fuc>Man>GlcNAc Capsule Manα2/3Man >>>Gal Rhaα2/3Rha SP-A

Man, Fuc>Glc,Cal > >GlcNAc

Capsuleb

Manα2/3Man Rhaα2/3Rha

SP-D

Mal>Fuc,Man,>Glc >Gal>GlcNAc

Outer-membrane LPS

Coreoligosaccharide

aRelative inhibitory activity of the saccharides: Man=mannose, GlcNAc=N-acetyl glucosamine, Mal=maltose, Glc=glucose, Fuc=fucose, Gal=galactose, Rha=rhamnose b Includes capsular serotypes K3, K7, K9, K17, K21a, K21b, K24, K26, K28, K34, K35, K36, K44, K45, K50, KS3, KS9, K62, K67, K70, K71, K74, K79, K80 and K81

Ofek et al.

3.

29

INTERACTION OF KLEBSZELLA PNEUMONIAE WITH THE MANNOSE RECEPTOR

K. pneumoniae can be subtyped into at least 77 different capsular serotypes each with a distinct composition and sequence of repeating units of saccharides (Kenne and Lindberg 1983, Karunarante 1985). A number of strains belonging to different serotypes have been tested for their ability to bind to rat alveolar macrophages (AM) in a serum free system (Athamna et al. 1991). The results showed that only some of the serotypes bound to AMs. The binding of Klebsiella to AMs was calciumdependent, occurred only with mature monocyte-derived macrophages and was inhibited by mannan, consistent with the known bindingproperties of the macrophage MR. Further studies have confirmed that the K. pneumoniae serotypes (e.g. K21a) which bound to the AMs express capsular polysaccharides that contain Manα2/3Man or LRhaα2/3-L-Rha sequences. Recognition of such sequences by the MR results in ingestion and killing of the organisms. On the other hand, serotypes that lack such sequences (e.g. K2) are not recognized by the macrophage lectin and are not internalized. Isolated and purified capsular polysaccharides containing the repeating sequence Manα2/3Man or LRhaα2/3L-Rha bound to guinea pig AMs, whereas those lacking these disaccharides did not. Interserotype switching of the capsular polysaccharide genes by reciprocal recombination allowed us to produce the capsule switched recombinant strains K2(K21a) and K2 1 a(K2), which retained their respective recipient K2 and K21 strain backgrounds, but inherited genes encoding for capsular polysaccharides of the donor strain (Ofek et al. 1993). The capsule switched recombinants K2(K21 a) inherited the macrophage binding phenotype of the K21 a donor, whereas the K21 a(K2) derivative bound poorly to macrophages because they inherited the capsule genes of the donor K2 strains, which are not recognized by the macrophages lectin.

3.1

Relationship between capsular polysaccharide structure, mouse virulence and binding to MR.

The relative contribution of lectinophagocytosis mediated by the MR to the virulence of K. pneumoniae in mice was examined (Kabha et al. 1995, using serotype K2 and K2 1 a and their respective capsule switched derivatives. The results suggest that switching of cps genes in K. pneumoniae serotypes markedly affects interaction of the bacteria with macrophages and blood clearance, and thus their virulence. Moreover, Klebsiella serotypes that express capsular polysaccharides recognized by

30

The Role of C-Type Lectins in Pulmonary Pathogen Infections

the MR, were significantly less virulent as compared to serotypes expressing capsular polysaccharides not recognized by the MR. Capsule types such as K21a are recognized by the macrophage lectin and as a result decrease the virulence of the bacteria by enhancing the host cells' lectinophagocytosis and killing. Although the K2 serotype was highly virulent, the capsule switched derivative K21 a(K2) expressing K2 capsule was more virulent than the parent K21a strains but less virulent than the cps donor Klebsiella strain. Together the data suggest that the chemical structure of the capsule partially determines the virulence of K. pneumoniae in mice

4.

INTERACTION OF KLEBSZELLA PNEUMONZAE WITH SP-A

The interaction of SP-A with Klebsiella was examined employing two serotypes, K21a and K2 and their capsule switched derivatives as described (Kabha et al. 1997). The results suggest that SP-A interacts with the capsule of K21a (containing Manα2Man sequences) as shown by SPA induced agglutination of the bacteria, and binding of SP-A coated particles onto the bacterial surface. SP-A binds also to immobilized parent K21a strain and to a recombinant strain of K2 that expresses the K21a capsule. In contrast, only marginal binding of SP-A to K2 parent strain (lacking this sequence) could be detected. Furthermore, the capsular polysaccharide of K2la bound to immobilized SP-A and the binding was inhibited by mannan but not by LPS and K2 capsular polysaccharide (Kabha et al. 1997). The data taken together suggest that SP-A recognizes the same capsular structure as those recognized by the MR of macrophages. In preliminary studies we found that SP-A did not agglutinate an unencapsulated phase variant of K21a, suggesting that like MR, structures underneath the capsule are not recognized by SP-A.

4.1.

Opsonic effect of SP-A

Because SP-A binds to Klebsiella capsule and to macrophages in a lectin-dependent and lectin-independent manner (reviewed by van Golde 1995), its ability to opsonize the K21a serotype was tested. Pretreatment of the bacteria with SP-A followed by washing off excess unbound SP-A caused a significant increase in the number of bacteria associated with AMs. Further experiments showed that the increase of Klebsiella association with macrophages was followed by ingestion and killing of

Ofek et al.

31

the bacteria, suggesting that SP-A acts as an opsonin in bridging between the capsulated K21a and the AMs (Kabha et al. 1997).

4.2.

Upregulation of MR on alveolar macrophages by SP-A

A marked increase in the association of Klebsiella with AMs was also observed when the macrophages were pretreated with SP-A. The SP-Ainduced association of K21a with AMs was inhibited by mannan and did not, or only to a minor extent, occur with K2 or the capsule switched derivative K21 a(K2) that expresses the K2 capsular polysaccharide. Further experiments revealed that SP-A treated AMs also bound increased amounts of mannan, the ligand of MR. Moreover, SP-A-induced enhancement of Klebsiella and mannan binding decrease gradually over a period of 5 hours after washing off the excess SP-A (Kabha et al. 1997). Previous studies have shown that SP-A bound to macrophages is rapidly internalized (Manz-Keinke et al 1991, Wintergerst et al. 1989). The data collectively suggest that SP-A upregulates MR resulting in increased association of Klebsiella with macrophages. This conclusion is supported by the recent findings showing that SP-A upregulated MR expression in human-monocyte derived macrophages plated on SP-A matrix, by using both mannan as ligand and anti-human MR to monitor the receptor activity (Gaynor 1995).

5.

INTERACTION OF KLEBSIELLA PNEUMONIAE WITH SP-D

In our preliminary studies we employed the slide agglutination test to screen Klebsiella strains carrying K2, K3, K7, K21a, K26, K32, K36, K50, K55, K62, K61, K67 and K70 types of capsular polysaccharides, and found that none were agglutinated by up to 10 µg/ml SP-D (Ofek et al. 1997). In contrast, unencapsulated derivatives of K21a and K50 serotypes were agglutinated by 0.5 and 4 µg/ml SP-D, respectively. The SP-D induced agglutination of the unencapsulated strains was calciumdependent and inhibited by maltose, suggesting that the carbohydrate recognition domain of the collectin is involved in the agglutination reactions. Moreover, Lipopolysaccharides purified from E. coli or K. pneumoniae inhibited the SP-D-induced agglutination of either E. coli or unencapsulated K. pneumoniae (Ofek and Crouch, 2000) and SP-D agglutinated latex beads coated with purified Klebsiella LPS. Because agglutination was not inhibited by purified capsular polysaccharides from

32

The Role of C-Type Lectins in Pulmonary Pathogen Infections

K. pneumoniae, we infer that SP-D does not efficiently bind to the capsular glycoconjugates. The data taken together strongly suggest that SP-D interacts with a common structure of enterobacterial LPS, probably the core region, which is exposed on the surfaces of unencapsulated organisms (Kuan et al. 1992; Lim et al, 1994). They further suggest that this interaction can be sterically inhibited by the presence of a capsule. In this regard, capsule has been shown to interfere with SP-D-induced agglutination of Cryptococcus neoformans (Schelenz et al. 1995).

5.1.

Opsonic effect of SP-D

The interaction of SP-D with the unencapsulated phase variant enhance binding and killing of the bacteria by macrophages in vitro (Ofek and Crouch, 2000). Thus, SP-D may play a role in pulmonary host defense by either agglutinating the unencapsulated phase variant to enhance its eradication from the air ways, or by opsonizing the Klebsiella to enhance their uptake and killing by the alveolar macrophages. The process of SP-D-dependent phagocytosis of the unencapsulated K. pneumoniae is associated with stimulation of cytokine production by the macrophages (Keisari et al. manuscript in preparation). Unlike MR, however, both the fresh blood monocytes and the monocyte-derived macrophages reacted with the SP-D-coated bacteria, suggesting that expression of the SP-D receptors involved in the phagocytic process are not dependent upon maturation of the monocytes into macrophages.

6.

ROLE OF LUNG C- TYPE LECTINS IN INNATE IMMUNITY AGAINST K. PNEUMONIAE INFECTIONS

The in vitro studies suggest that C-type lectins may protect against K. pneumoniae by either interacting with certain capsular serotypes or by interacting with the core region of the bacterial LPS. The former types of interactions are mediated by SP-A, which act as opsonin, and MR, which mediates phagocytosis. These C-type lectins seem to recognize capsular serotypes that express dimannose or dirhamnose in the repeating unit of their capsular polysaccharides. If indeed this type of interaction provides innate immunity against K. pneumoniae infections by enhancing phagocytosis as discussed above, then why does we need two C-type lectins to accomplish a protective function against the same serotypes? Because protection is actually mediated by two receptors on the alveolar macrophages, SP-A receptors and MR, a clue to this dilemma may be

Ofek et al.

33

found in a study where the expression of these receptors was determined in macrophages treated with various agents (Chroneos et al. 1995). It was found that agents that suppress either receptor in vitro or in vivo, upregulates the other receptor. Thus, it seems that the defense mechanisms provided by these two receptors are directed mainly against the dimannose and dirhamnose expressing capsular serotypes. Indeed, epidemiological data showing that Klebsiella serotypes with capsular polysaccharides that are not recognized by SP-A and mannose receptor are isolated with high frequency from patients with active pulmonary and bacteremia (Ofek et al, 1995). Clearly this is an oversimplification and other factors are undoubtedly involved, but the data seems to indicate that there is a role for C-type lectins in protecting against at least a third of the capsular serotypes of K. pneumoniae.

capsulated phenotype

noncapsulated

Expression of capsular

Man 2/3Man sequences

Interaction witha

SP-D SP-A Mannose receptor

-

+

+

I

+

I

I

I

-

Predominant phenotype Asymptomaticcarriage of upper repiratory tract

LOW b

Pneumonia with bacteriemia LOW

+ Positiveinteraction

-

LOWb

HIGH

H IGH

NONE

Nointeraction

Figure 1. Predicted chain of events during natural course of infection with K. pneumoniae. a Agglutination and opsonization bCapsule interferes with the expression of adhesin (data from Matov et al, 1999)

34

7.

The Role of C-Type Lectins in Pulmonary Pathogen Infections

CONCLUSION

Based on the available information summarized above we suggest the following roles of C-type lectins in providing innate immunity in the lung as depicted in Figure 1. Colonization of the upper respiratory tract by grain negative bacteria precedes entry of the organisms into the lung (Valenti et al, 1978; Baltimore et al, 1989). Because capsule interferes with the expression of adhesins required for colonization of epithelial cells by the organisms, it is likely that most of the bacteria colonizing the upper respiratory tract (or other mucosal surfaces) are in the unencapsulated phase (Favre-Bonte et al. 1999; Matatov et al. 1999). Klebsiella opsonization and agglutination by SP-D might, therefore, provide early protection against all strains of unencapsulated phenotypes because the LPS core region, which reacts with SP-D, is conserved (Susskind et al. 1992, Holst et al. 1995). Encapsulated bacteria that emerge during the infection as a result of the phase variation phenomenon (Mattatov et al. 1999) are expected to escape SP-D recognition. Mannose receptor-equipped macrophages in conjunction with SP-A may provide additional protection by eliminating specific encapsulated Klebsiella through recognition of the dimannose and dirhamnose sequences in the capsular polysaccharide. SP-A, which opsonizes and agglutinates the dimannose-containing Klebsiella, may also augment expression of MR, which in turn mediates phagocytosis of the organisms. Thus, Klebsiella serotypes that are not recognized by SP-A and MR (e.g. lack the dimannose or dirhamnose sequences in their capsular polysaccharides) may become the predominant infective capsular serotypes. Epidemiological data confirm this prediction as discussed above (Ofek et al. 1995). Opportunistic pathogens, such as K. pneumoniae, primarily attack immunocompromised individuals who are hospitalized and have severe underlying diseases (Podschun et al., 1998). It is still unclear what specific factor(s) predispose hospitalized individuals to develop severe pneumonia often associated with bacteremia. However, our data suggest that perturbations in the interactions of mannose receptor and lung collectins with these organisms could predispose to infection or lead to abnormal inflammatory responses to colonizing bacteria. Further studies on C-type lectin interactions with Klebsiella may provide additional clues on the identity of the predisposing factors that render hospitalized patients susceptible to bacterial pneumonia.

Ofek et al.

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ACKNOWLEDGMENTS The work from our laboratory was partially supported by grants from the National Institutes of Health (HL29594 and HL52646).

REFERENCES Athamana, A., Ofek, I., Keisari, Y., Markowitz, S., Dutton, G. S., and Sharon, N. 1991. Lectinophagocytosis of encapsulated Klebsiella pneumoniae mediated by surface lectin of guinea pig alveolar macrophages and human-monocyte-derived macrophages. Infect. Immun. 59:1673-1682 (1991) Baltimore R.S., Duncan R.L. , Shapiro E.D., and Edberg S. C. 1989. Epidemiology of pharyngeal colonization of infants with aerobic gram-negative rod bacteria. J. Clin. Microbiol 27:91-95 Doebbeling. B.N., Epidemics: identification and management, in Prevention and control of nosocomial infections, R.P. Wenzel, Editor. 1993, Williams & Wilkins: Baltimore. p. 177-206. Favre-Bonte, S., B. Joly, and C. Forestier. 1999. Consequences of reduction of Klebsiella pneumoniae capsule expression on interaction of this bacterium with epithelial cells. Infect. Immun. 67:554-56 1 Holst O., and Brade H. 1992. Chemical structure of the core region of lipopolysaccharides. In Bacterial Endotoxic Lipopolysaccharides (D.C. Morisson and L.L. Ryan, eds) Vol I, CRC Press, Boca Raton, FL, pp135-170. Karunarante, D. N.: Structural investigation of the capsular polysaccharides of K. pneumoniae. PhD Thesis. Univ. British Columbia, Vancouver: Canada 1985 Kenne, L., Lindberg, B.: Bacterial polysaccharides. In: Aspinall, G. O., and Lindberg, B.(Eds): The Polysaccharides. 2:287-363. New-York: Acad. Press, Inc.1983. Kuan, S.F., K. Rust. and E. Crouch. 1992. Interactions of surfactant protein D with bacterial lipopolysaccharides. Surfactant protein D is an Escherichia coli- binding 103 protein in bronchoalveolar lavage. J.Clin.Invest.90:97Lim, B.L., J.Y. Wang, U. Holmskov, H.J. Hoppe, and K.B. Reid. 1994. Expression of the carbohydrate recognition domain of lung surfactant protein D and demonstration of its binding to lipopolysaccharides of gram-negative bacteria. Biochem.Biophys.Res.Commun.202: 1674-1678 Matatov, R., J. Goldhar, E. Skutelsky, I. Sechter, R. Perry, R. Podschun, H. Sahly, K. Thankavel, S. N. Abraham, and I. Ofek. 1999. Encapsulated klebsiella pneumoniae to assemble functional type 1 fimbriae on their surface. FEMS Microbiol. Letters 179: 123- 130. Ofek, I., and E. Crouch. 2000. Interaction of microbial glycoconjugates with collectins: implications for pulmonary host defence. In Glycobiology (R. J. Doyle, ed)KluwerPlenum Co. London. (in press) Ofek, I., K. Kabha, Y. Keisari, J. Schlepper-Schaefer, S.N. Abraham, D. McGregor, D. Chang, and E. Crouch. 1997. Recognition of Klebsiella pneumoniae by pulmonary Ctype lectins. Nova Acta Leopold.NF 75:43-48 Ofek, I., Kabha, K., Athamna, A., Frankel, G., Wozniak, D. J., Hasty, L. D. and Ohman, E. D. 1993. Genetic exchange of determinants for capsular polysaccharide

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The Role of C-Type Lectins in Pulmonary Pathogen Infections

biosynthesis between Klebsiella pneumoniae strains expressing serotypes K2 and K21a. Infect. Immun. 61:4208-4216 Podschun, R. and U. Ullmann,l998KlebsielIa spp. as nosocomial pathogen: Epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev, 11 :589-603 Schelenz, S., R. Malhotra, R.B. Sim, U. Holmskov, and G.J. Bancroft. 1995. Binding of host collectins to the pathogenic yeast Cryptococcus neoformans : human surfactant protein D acts as an agglutinin for acapsular yeast cells. Inlfect.Immunol.63 :33603368 Susskind M., S. Muller-Loennies, W. Nimmich, H. Brade, and 0. Holst. 1995. Structural investigation on the carbohydrate backbone of the lipopolysaccharide from KIebsiella pneumoniae rough mutant R20/01-. Carbohydrate Res. 269:C1 -C7. Valenti, W.M., Trudell R.G. and Bentley D.W. 1978. Factors predisposing to ortopharyngeal colonization with gram-negative bacilli in the aged. N. Engl. J. Med. 298: 1108-1 11 1 Weiss, J., Hutzler, M., and Kao, L. 1986. Environmental modulation of lipopolysaccharide chain length alters the sensitivity of Escherichia coli to the protein. Infect.Immunol. 51:594-599 neutrophil bactericidal/permeability-increasing

MODULATION OF NITRIC OXIDE PRODUCTION BY LUNG SURFACTANT IN ALVEOLAR MACROPHAGES

¹Moshe Kalina, ²Hanna Blau, ¹Shoshana Riklis and ¹Vered Hoffman ¹ Department of Cell Biology and Histology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel. ²Pulmonary Department, Schneider Childrens Medical Center, Israel

1.

INTRODUCTION

Accumulating evidence suggests that the lung surfactant may play a modulatory role in the first line defense system of the lungs against infiltrating pathogens (Wright 1997, Crouch 1998). As such, its components may be an important part of the innate immune response as well as participate in other aspects of immune and inflammatory regulation within the lung. The surfactant components include the hydrophilic surfactant protein A (SP-A), surfactant protein D (SP-D) and surfactant lipids. A growing number of reports suggested the apparent stimulatory effect of SP-A and SP-D (Wright 1997, Crouch 1998) . In vitro they were found to stimulate phagocytosis, chemotaxis, production of reactive oxygen species as well as cytokine release by various cells. The surfactant lipids, however, were found to have a suppressive influence on a variety of immune cell functions (Thomassen et al 1992, Thomassen et al 1994, Thomassen et al 1995, Kremlev & Phelps 1994, Kremlev et al 1996). Recently, various aspects of the immune cell functions were studied, including proliferation, cytokine production, phagocytosis and expression of cell surface markers by various immune cells. Nitric oxide (NO) has been demonstrated to exert in vitro microbicidal or microbiostatic activity against a rapidly expanding list of pathogens The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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Modulation of Nitric Oxide Production by Lung Surfactani

(MacMicking & Nathan 1997). Recently we found that nitric oxide production by rat alveolar macrophages can be modulated in vitro by SPA (Blau et a1 1997). SP-A was found to upregulate nitric oxide production in a concentration-and time-dependent manner. This increase was associated with elevation in the expression of iNOS in alveolar macrophages. The stimulatory effect of SP-A was found to be lower than the known nitric oxide agonists interferon- (IFN-γ) and lipopolysaccharide (LPS). However, the cytokines interleukine 1(IL-1) and granulocyte macrophage colony-stimulating factor (GM-CSF) elevated the levels of nitric oxide production to that of LPS or IFN The non-surfactant related function of SP-A encouraged us to test a possible modulatory effect of the surfactant components, SP-A and SP-D as well as surfactant lipids on nitric oxide production by alveolar macrophages cell line NR-8383. This cell line is a well established normal rat cell line, which exhibits various characteristics of macrophage cells : phagocytosis, oxidative burst as well as cytokine secretion (Helke et al 1987). This cell line provides a high yield homogenous source of highly responsive alveolar macrophages, therefore, it represents a useful model for investigating rat alveolar macrophages. Our results indicate that both SP-A and SP-D may indeed upregulate iNOS and nitric oxide production, which was suppressed by surfactant lipids. A synergistic effect was observed between the surfactant proteins, as well as proteins and IFN-

2.

MATERIALS AND METHODS

2.1

Cells and culture conditions

The rat alveolar macrophage cell line NR 8383 (AML) was derived from normal Sprague-Dawley rats and has been shown to possess characteristics typical of rat alveolar macrophages. The cell line was obtained from the American Type Culture Collection (ATCC) and was maintained and grown as described previously (Helke et al 1987). Briefly, the cells were grown as a mixed population of adherent and suspended cells in F-12 medium supplemented with 15% fetal calf serum (FCS). Cultures were maintained by transferring both floating and adherent cells (after scraping) to additional flasks. For measurement of NO production, the cells were plated in 96 well tissue culture plates (5: l04 cells/well) in F12 medium supplemented with 5% FCS.

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39

Stimulation of the cells to product NO

Macrophages were stimulated to produce NO by addition of the various agonists to the cells after 18h in culture. In most experiments the cells were incubated with the agonists for 48h. unless otherwise stated. Both LPS (Escherichia coli, 55 : 135, Difco, Detroit MI) and recombinant rat INF- (Genzyme, Cambridge, MA) were used to stimulate the cells to generate NO as positive controls to SP-A and SP-D. SP-A was isolated from patients with alveolar proteinosis as previously described (Wright et al 1987). Rat SP-D was kindly provided by F. van Iwaarden, Vrije University, Amsterdam. The content of contaminating LPS in the surfactant proteins was tested by using the Limulus amebocyte lysate (LAL), and the kinetic methodology using the LAS-5000E automated endotoxin detection system (Atlas Bio-scan) was employed for the detection. LPS content in the SP-A and SP-D preparation was found to be 70% sequence identity) cathelicidin peptides with two disulfide bonds have been identified in pigs and named protegrins (PG). Three such peptides have been isolated from porcine leukocytes (PG-1 to PG-3) (Kokryakov et al 1993), and two have been deduced from cDNA (PG-4) and gene cloning (PG-5). They are 16-18 residue cationic peptides with an amidated C-terminus. These peptides resemble the antimicrobial tachyplesins from hoseshoe crab, and a ten residue segment of PG-3 has eight amino acids identical to those found in positions 1-10 of rabbit defensin NP-3a. The NMR solution structure of PG-1 has been determined as a monomeric ß-hairpin in which the central region is hydrophobic, while the two ends are hydrophilic (Aumelas et al 1996). Further NMR studies have indicated that PG-1 dimerizes when it binds to dodecylphosphocholine micelles and have suggested a possible association of these dimers (Roumestand et al 1998). Protegrins display a broad-range antimicrobial activity against various Gram-negative bacteria including Neisseria gonorrhoeae (Qu et al 1996) and Chlamydia trachomatis (Yasin et al 1996), Gram-positive bacteria such as MRSA and VREF (Steinberg et al 1997), Mycobacterium tuberculosis (Miyakawa et al 1996), fungi (Cho et al 1998), and enveloped viruses. Electrophysiologic studies indicate that protegrins induce membrane permeabilization by forming weakly selective ion channels (Mangoni et al 1996). The disulfide bonds are a prerequisite for membrane permeabilization, but not for the antimicrobial activity. These peptides have been selected by a Biotechnology company for their potential as antimicrobial agents. Experiments with animal models of infection have shown that i.v. administration of PG-1 protects mice from a lethal challange of MRSA or VREF. Similarly, the peptide, injected i.p. protects

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mice from lethal peritoneal infections caused by either S. aureus or P. aerugnosa (Steinberg et al 1997). The in vivo activity against clinically relevant, antibiotic-resistant bacteria and other desiderable features, such as small size, broad spectrum of activity, rapid bactericidal activity, and inability to select resistant-mutants, indicate that PG- 1 is an attractive candidate for the development of novel drugs. An analog of PG-1 is currently under clinical trials to prevent oral mucositis, a polymicrobial infection with no effective treatment, suffered by cancer patients receiving chemotherapy or radiation therapy.

5.

CONCLUDING REMARKS

Cathelicidins are a recently discovered family of effector molecules in innate immunity. In the past few years, a great deal of investigations have elucidated several aspects of their biology, such as the gene structure and activation mechanism. Despite substantial progress in the field, several issues remain to be clarified, including the biological role of the conserved proregion and the molecular mechanisms responsible for diversification of the peptide domain. The cathelicidin-derived peptides have been deeply investigated with respect to structure, spectrum of activity and mechanism of action. In general, they show a potent in vitro activity against antibiotic-resistant microorganisms. The widespread diffusion of multi-resistant strains has highlighted their potential as lead compounds for the development of novel antiinfective agents. Indeed, some of these peptides, or analogs, are already under advanced clinical trials for the treatment of topical infections. Finally, several reports suggesting that cathelicidin peptides may play additional roles in host defense, such as wound healing and chemotactic activity, have opened new fields of investigations. Further studies however are required to clearly establish the physiological relevance of the observed effects.

ACKNOWLEDGMENTS Work in the authors laboratories was supported by grants from the CNR Target Project on Biotechnology, from the Italian Ministry for University and Research (MURST), from the Istituto Superiore della Sanita', National Research Project AIDS.

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Wu, H., Zhang, G., Ross, C. R., and Blecha, F., 1999, Cathelicidin gene expression in porcine tissues: roles in ontogeny and tissue specificity. Infect Immun. 67:439-442. Yasin, B., Harwig, S. S. L., Lehrer, R. I., and Wagar, E. A., 1996, Susceptibility of Chlamydia trachomatis to protegrins and defensins. Infect. Immun. 64:709-713. Zanetti, M., Litteri, L., Gennaro, R., Horstmann, H., and Romeo, D., 1990, Bactenecins, defense polypeptides of bovine neutrophilis, are generated from precursor molecules stored in the large granules. J. Cell Biol. 111 : 1363- 137 1. Zanetti, M., Litteri, L., Griffiths, G., Gennaro, R., and Romeo, D., 1991, Stimulus induced maturation of probactenecins, precursor of neutrophil antimicrobial polypeptides. J. Immunol. 146:4295-4300. Zanetti, M., Del Sal, G., Storici, P., Schneider, C., and Romeo, D., 1993, The cDNA of the neutrophil antibiotic Bac5 predicts a pro-sequence homologous to a cysteine proteinase inhibitor that is common to other neutrophil antibiotics. J. Biol. Chem. 268:522-526. Zanetti, M., Gennaro, R., and Romeo, D., 1995, Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain. FEBS Lett. 374:1-5. Zanetti, M., Gennaro, R., and Romeo, D., 1997, The cathelicidin family of antimicrobial peptide precursors: a component of the oxygen-independent defense mechanisms of neutrophils Annuls New York Acad. Sci. 832: 147-162. Zarember, K., Elsbach, P., Shin-Kim K., and Weiss, J., 1997, p15s (15-kD antimicrobial proteins) are stored in the secondary granules of Rabbit granulocytes: implications for antibacterial synergy with the bacterial/permaebility-increasing protein in inflammatory fluids. Blood 89:672-679. Zhao, C., Ganz, T., and Lehrer, R.I., 1995a, The structure of porcine protegrin genes. FEBS Lett. 368: 197-202. Zhao, C., Ganz, T., and Lehrer, R.I., 1995b, Structures of genes of two cathelinassociated antimicrobial peptides: prophenin-2 and PR-39. FEBS Lett. 376: 130- 134.

STRUCTURE ACTIVITY RELATIONSHIP STUDY OF POLYMYXIN B NONAPEPTIDE

¹Haim Tsubery, ²Itzhak Ofek, 2Sofia Cohen and ¹Mati Fridkin

1 Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot, 76100, Israel. ² Department of Human Microbiology, Sackler Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel

1.

INTRODUCTION

Polymyxin B (PMB) is a naturally occurring cyclic decapeptide isolated from Bacillus polymyxa (Stanly et al 1947). It consists of a seven member ring containing 4 positive charges of diaminobutyric acid residues one threonine residue and a hydrophobic segment, i.e. dPheLeu, and a linear N-terminal region composed of three amino acids together with a 8 or 9 carbon fatty acid (6-methyl heptanoic and octanoic acid, respectively) forming a long hydrophobic tail. PMB is bactericidal to gram-negative bacteria and considered one of the most efficient cellpermeabilizing compounds (Danner et al 1989). Its structural features permit competitive displacement of the divalent cations from their binding sites on surface LPS (Moore et al 1986). Since PMB is a far larger molecule than a divalent cation, it disorganizes the bacterial outer membrane structure and its hydrophobic tail penetrate into the cytoplasmic membrane. This penetration causes a leak of cytoplasmic components, which leads to bacterial death (Schindler et al 1975). Although PMB binds to the lipid A moiety of LPS with relatively high affinity [0.37-0.5x 10-6M] (Vaara et al 1985), detoxify its biological effects and protects animals from endotoxemia, its use for treatment of septic shock is limited due to its high it toxicity mainly causing to renal damage and neurotoxic reactions (Carig et al 1974). The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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Structure Activity Relationship Study of Polymyxin B Nonapeptide

In order to reduce its high toxicity, a derivative of PMB was prepared by enzymatic processing which cleaved the fatty tail component (Chihara et al 1973). Indeed, polymyxin B nonapeptide (PMBN) (Figure 1) was 15 times less toxic than PMB in an acute-toxicity assay in mice and 100 times less toxic in a eukaryotic cytotoxicity assay. Although PMBN retained the ability to bind to bacterial LPS (however, with smaller affinity then PMB) it lost almost completely its bactericidal activity. However it was stile able to renders the gram-negative bacteria susceptible to several hydrophobic antibiotics and serum (Stokes et al 1989, Lynn et al 1992). The latter antimicrobial activity of PMBN is referred to as "sensitizing activity". PMBN was found remarkably active in sensitizing 53 clinical isolates of gram negative bacteria to novobiocin, a hydrophobic antibiotic, and protect mice from infection (Ofek et al 1994). Here we performed a structure activity study in attempts to locate key structural features and amino acid residues essential for sensitizing activity of the PMBN molecule.

Figure 1. Structure of Polymyxin B nonapeptide.

2.

RESULTS AND DISCUSSION

PMBN and twelve analogs (peptides 2-14) were synthesized on Wang resin using orthogonal (Fmoc, Boc and Cbz) amine protecting groups followed by solution cyclization. The study focused on the peptides’ ability to sensitize gram negative bacteria to novobiocin compared to the

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potency of PMBN (Table 1). In addition, the peptides' ability to displace Dansyl-PMBN bound to E.coli LPS was examined (Table 1). PMBN was able to inhibits the growth of Pseudomonas aeruginosa (MIC, 8 µg/mL) however all of the PMBN analogs were not active (MIC, >1000 µg/mL). Table 1. The relative potency of PMBN analogs to sensitize bacteria to novobiocin and

cyclic peptide a 1 2 3 4 5 6 7 8 9 10 11 12 13

PMBN sPMBN ["all D”]PMBN [Lys²,3,4,7,8 ]PMBN [Orn 2,3,4,7,8]PMBN [Dap2,3,4,7,8 ]PMBN [Lys3]PMBN [Lys 2,3,4,7,8‚ ]PMBN [cycloDab4,Thr9]PMBN [cycloDab2 ,Thr ]PMBN [Lys2,4]PMBN [Lys7,8 ]PMBN [Phe5 ]PMBN

Escherichia coli 100 96 11 4 2 11 17 5 4 7 9 40 5

Klebsiella pneumonia 100 75 14 3 6 5 18 3 5 5 13 26 9

IC50 (µM)

c

4.5 5.5 12 150 100 100 50 >200 >100 50 50 50 50

a Numbers indicate the positions where substitution with the indicated amino acid took place. b The relative potency is determined as a percent of PMBN potency that at 50 g/ml reduces the MIC of E.coli and K. pneumonia from 125-250 g/ml down to 1 and 4 g/ml, respectively. c The concentration required to displace 50% of Dansyl-PMBN (0.55µM) bound to E.coli LPS.

As a role, the potency of all PMBN analogs to increase the penetration of novobiocin through the bacterial outer-membrane was reduced. All the peptides exhibit similar random coil structure as detected by circular dichroism measurements. In addition, all PMBN analogs showed reduced affinity to free E.coli LPS as indicated from the displacement assay (IC50 , Table 1). The above data indicate that at least four factors are essential for the membrane disorganizing activity of PMBN. The length of the alkyl chains of the charged amino acids is an important one and the optimum seem to contain 2 methylene groups. It probably promotes the availability of the NH3 groups to the phosphates of the lipid A portion. The second factor is the ring size, which probably affects the distance between the two charged domains or their spatial arrangement, where the original 23-atom ring is preferred. The third factor is the hydrophobic

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Structure Activity Relationship Study of Polymyxin B Nonapeptide

segment dPhe-Leu. The configuration of the phenylalanine residue is essential for disorganizing activity. It seems that the interaction of the phenyl group with the hydrophobic core of the LPS was eliminated at the L-configuration. The forth factor is the overall structure orientation of the peptide according to the loss of activity of the ["all D"]PMBN analog. This indicates that it requires a specific structure orientation for effective activity. It can be concluded that the structure of the semi natural peptide, PMBN, is highly specified to disorganizing gram negative outer membrane.

REFERENCES Carig, W. A., Turner, J. H. and Kunin, C. M. (1974) Infect. Immun. 10, 287. Chihara, S., Tobita, T., Yahata, M., Ito, A. and Koyana, Y. (1973) Danner,R.L., Joiner,K.A., Rubin, M., Patterson, W.H., Johnson, N., Ayers, K.M. and Parrillo, J.E., (1989) Antimicrob. Agents Chemother. 33 1428-1434. Lynn, W.A. and Golenbock, D.T., Immunology Today 13 (1992) 271- 276. Moore, R.A., Bates, N.C. and Hancock, R.E. (1986) Antimicrob. Agents Chemother. 29(3), 496-500) Ofek, I. S. Cohen, R. Rahmani, K. Kabha, Y. Herzig and E. Rubinstein., Ant. Microb. Agents. Chemothr. 38 (1994) 374-377. Schindler, P.R.G. and Teuber, M., (1975) Antimicrob. Agents Chemother., 95-104 Stanly, P.G., Shepard, R.G. and White, H.J. (1947) Bull. Johns Hopkins Hosp. 81, 43-54 Stokes,D.C., Shenep, J.L., Fishman, M., Hildner, W.K., Bysani, G.K. and Rufus, K., J. Infec. Dis. (1989) 160, 52-57. Vaara, M., and P. Viljanen., Antimicrob. Agents Chemother. (1985) 27, 548-554.

THE CLINICAL SIGNIFICANCE OF NEUTROPHIL DYSFUNCTION

Baruch Wolach, Ronit Gavrieli and Dirk ROSS* Department of Pediatrics, Laboratory for Leukocyte Functions, Meir General Hospital, Sapir Medical Center, Kfar Sava, Israel and *The Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Holland

Neutrophils and monocytes are important effectors and regulators of the host defense system. Because of the major role they play in the phagocytic arm of the immune system, both primary and acquired defects in their function could result in recurrent as well as persistent or opportunistic infections. Evaluation for phagocytic cell disorders should be initiated among those patients who have recurrent respiratory tract bacterial infections, such as pneumonia, sinusitis and suppurative otitis media; skin infections, as cellulitis or abscesses; lymphadenitis or infections presenting at unusual sites (renal, hepatic, brain abscesses) or caused by unusual pathogens (ie, Aspergillus pneumonia, disseminated candidiasis, Serratia, marcescens, etc). The primary disorders of the phagocytic function include Chronic Granulomatous Disease (CGD), Hyperimmunoglobulin E Syndrome (HIgES), Leukocyte Adhesion Deficiencies (LAD), Chediak- Higashi Syndrome (CHS), Myeloperoxidase deficiency (MPO) and white cell G6PD deficiency (enzyme level less than 1%). During the last 10 years, 540 patients (children and adults) were referred from hospitals and clinics of Israel to our laboratory for leukocyte functions and were investigated because of recurrent or opportunistic infections. In 47% of this selected population, impairment of one or more steps of the phagocytic activity was disclosed. In 30 patients (5.6 % of all patients tested) a primary phagocytic disorder was diagnosed (Figure 1). Chronic Granulomatous Disease was diagnosed in 9 patients, in whom a significant decrease of superoxide generation was The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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The Clinical Significance of Neutrophil Dysfunction

found, with consequent diminished killing activity. In 6 of them, the subtype X-9 1" (gp9 1phox absence) was established. Three patients had, presumably, subtype X- 9 1' (gp9 1phox dysfunction). DNA analysis is currently performed; so far, in 2 male brothers a C-insertion frameshift in CYBB was found, and a de novo C688T-point mutation in CYBB was detected in another child, who was successfully bone marrow transplanted. A two-year old male developed Acute Lymphoblastic Leukemia, to the best of our knowledge, for the first time reported in CGD. Hyperimmunoglobulin E syndrome was diagnosed in 15 subjects (five are members of the same Muslim family) with variable chemotactic response. Three patients were successfully treated with Cyclosporin A with an excellent but drug-dependent clinical and immunological response. Three patients with Chediak-Higashi showed impaired chemotaxis and reduced bactericidal activity, and 2 subjects with complete white cell G6PD deficiency showed a defective bactericidal activity and oxidative burst. One child with WBC-myeloperoxidase deficiency disclosed a defective bactericidal activity. Currently, we are supervising 6 patients with suspected Hyperimmunoglobulin E syndrome and one with a probable leukocyte adhesion defect (LAD). In additional 24 patients (4%), leukocyte dysfunctions could be attributed to well defined entities, such as Congenital Myelokathexis (2), Glycogen Storage Disease (2), Systemic Lupus Erythematosus (l), Juvenile Periodontitis (I), Diabetes Mellitus (2), IgG1 subclass deficiency (1), Hemophagocytic syndrome (2), Hashimoto thyroiditis (l), SCID (1), Cyclic Neutropenia (4), Cystic Fibrosis (2), Vegetative state (3), Interstitial Nephritis (1) and Failure to Thrive (1). In an additional 200 referred patients (37%) leukocyte dysfunctions were detected, although no primary disorders could be established on them. Repeated tests were possible in 30% of these patients; abnormalities recurred in 55% of them. We speculate that the impaired function could be related to several factors, as persistent neutrophil activation and exhaustion in patients with recurrent severe infections, pharmacological deleterious effects of therapeutic agents taken by them, or other basic disorders not established yet. Specific laboratory tests and clinical follow-up should be done in specialized and experienced centers, since attempts to establish precise diagnosis often can be difficult because of the similar clinical presentation of phagocytic disorders, and the variability of the biological tests.

Wolach et al. Figure I: Leukocyte function of 540 referrals

PPID: Primary Phagocytic Immune Deficiency SPID: Secondary Phagocytic Immune Deficiency in established disorders PID-UD: Phagocytic Immune Deficiency in undiagnosed disorders

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CLINICAL SIGNIFICANCE OF FUNCTIONAL ABERRATIONS IN MACROPHAGE AND NK CELLS, IN TYPE-1 CYTOKINES AND IN LECTINBINDING MOLECULES

Zeev T. Handzel Clinical Immunology & Allergy Unit and the Pediatric Research Institute, Kaplan Medical Center, Rehovot, Israel

1.

INTRODUCTION

Primary defects in specific responses within the human adaptive immune system have been described extensively and the molecular basis of most of them has been identified (Ochs et al 1999). Also defects in the complement cascade and in neutrophil function, which are components of the innate immune system, as listed in Table 1(Ezekowitz & Hoffmann 1998, Carroll & Janeway 1999), belong to this category. We will turn our attention to other parts of innate immunity, which recently have started to arouse the interest of clinical immunologists. Table 1 . Components of the innate immune system in humans CELLS MOLECULES Neutrophils Complement cascade Monocytes/Macrophages Adhesion molecules Dendritic cells Collagens/Collectins Astrocytes/Microglia MBL & receptor(R) NK cells Toll-like Rs Mast cells Defensins Eosinophils Cathelicidin Granulosin Histatins The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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,

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2.

Clinical Aberrations in Macrophages, Cytokines and Collectins

NEWBORN INFANTS

The newborn immune system demonstrates an immaturity of most of it’s components, such as decreased levels of the complement proteins, decreased neutrophil chemotaxis and an impaired production of IgG and IgA immunoglobulins, amongst others (Kovarik & Siegfrist 1998, Arkachaisri & Ballow 1999). These defects increase in premature babies, in correlation with the degree of prematurity. Dysfunction is also demonstrable in macrophages (Ma) and natural killer (NK) cells. One study has shown that while NK cytotoxicity against HIV-infected targets is mostly normal in term newborns, as compared with adult peripheral blood mononuclear cells (PBMC), antibodydependent cytotoxicity (ADCC) is impaired (Merrill et al 1996). However, in premature babies both these functions are seriously affected (Merrill et al 1996), but may be corrected by interleukin-12 (IL-12). In another study (Lau et al 1996) a lack of production of interferon gamma (IFNγ) by cord blood was completely corrected by the addition of IL12. This demonstrates that neonatal Ma/NK cells have the capacity to respond to the appropriate stimuli, although their spontaneous responses may be blunted. It was also found that cord-blood monocytes were extremely sensitive to both Ma-tropic and T-cell tropic HIV strains (Sperduto et al 1993), while another study demonstrated a preferential sensitivity of cord-blood mononuclear cells to Ma-tropic HIV (Reinhardt et al 1995). In addition, IFNγ was found to upregulate the expression of the chemokine receptor CCR5, which is a coreceptor for HIV, on cord-blood monocytes (Harihan et al 1999). These findings may contribute to the understanding of the factors governing the vertical (mother-to-child) transmission of HIV.

3.

INNATE IMMUNITY IN THE ELDERLY

With advancing age, immunity declines (Lesourd 1999, Pawelec et al 1998, Albright & Albright 1998), the thymus-dependent adaptive one being mostly affected, with an attrition of type-I T-cell (Th-1) responses and a predominance of the Th-2 type profile. To this has to be added a commonly occurring malnutrition, which causes further T-cell deterioration. Macrophage function is better preserved, although IL-1, IL-6 and IL-18 secretion in response to various stimuli, such as LPS, becomes impaired. However, prostaglandin PGE2 secretion is increased.

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Thus, the resulting immune imbalance sets the stage for the establishment of chronic inflammatory conditions, such as osteoarthritis. These are characterized by an increased secretion of tumor-necrotising factor alpha (TNFα) and soluble TNFα receptor-] (TNFαR1) and the IL-1 receptor (IL- 1R), which are probably partly responsible for the wasting syndrome, often occurring in old age. At the same time the NK cytotoxic capacity also declines, together with reduced secretion of IFNγ and impaired responses to cytokines, such as IL-2, which results in a decreased potential for the formation of LAK (lymphocyte-activated killers). All these factors taken together may explain the marked increase in serious infections witnessed during old age.

4.

INNATE IMMUNITY IN NEURODEGENERATIVE DISEASE

The macrophage-derived microglia and astrocytes and the numerous cytokines they secrete (Table 2) are the main components of innate immunity in the central nervous system (CNS) (Gendelman & Folks 1999). In addition to their central role in the local non-specific antimicrobial defense, they participate in the pathogenesis of inflammatory degenerative diseases of the CNS (Table 2) (McGeer & McGeer 1999, Cotter et al 1999). We will discuss here two of them: Alzheimer’s Disease (AD) and HIV-associated Dementia (HAD). A few variants of AD exist, of which the common type is age-related and of late-onset. In this type neurofibrillary tangles replace the normal synaptic network and in these tangles p amyloid plaques are formed. In most, but not all cases, activated microglia are enmeshed in the tangles and components of the activated complement cascade are bound to the plaques. Thus emerged the concept that central to AD is an inflammatory process and the devastating neurodegeneration and loss of cognition are secondary (McGeer & McGeer 1999, Cotter et al 1999). It seems that the activated microglia is the main initiator of this vicious chain of events, although the basic etiology of the disorder remains elusive. The microglia produce excessive amounts of oxygen radicals and of glutamate, which are neurotoxic and secrete proinflammatory cytokines (Table 2). They express more adhesion molecules and MHC class I and class II molecules, thereby presenting more antigens to T cells, which in turn become activated by the cytokine milieu. The clinical proof of the inflammatory paradigm may lie in the observation that non-steroidal anti-inflammatory drugs (NSAID) may benefit patients with AD (Cotter et al 1999).

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Clinical Aberrations in Macrophages, Cytokines and Collectins

In HAD the pathogenic sequence is essentially similar, namely HIVinfected glia cells and astrocytes initiate the inflammatory process described hereabove. In HAD no amyloid is deposited, but a chronic encephalopathy is established, with ensuing progressive cortical atrophy. The choroid plexus may harbor activated HLA-DR positive DC, which becomes a reservoir for the virus (Hanly & Petito, 1998). In children, this process is especially insidious and destructive early in the course of the disease (Persidsky et al 1997). Numerous chemokine receptors CCR5 and CXCR4-positive macrophages have been identified in brains of children with severe HIV encephalopathy (Vallat et al 1998). Table 2. Innate immunity in Neurodegenerative disorders CNS components

CELLS

CYTOKINES

Macrophages Microglia Astrocytes DISEASES Alzheimer's disease (AD) Multiple Sclerosis (MS)

IFNa & p Interleukins (IL) 1,2,3,5,6,8,10,12,15 TNFa & p, G-CSF, GM-CSF, MCP-1, MIPa & p

5.

HIV-associated Dementia (HAD) Progressive Multiple Leukoencephalopathy (PML)

INNATE IMMUNITY IN HIV DISEASE

The numerous insults dealt by HIV to the immune system have been described extensively (Gotch et al 1997, Bofill et al 1999). The focus is usually directed to the T-helper cell, which is the main target of the virus, attaching itself to the cell membrane via the CD4 and CCR5 receptors. However, usually the immune cell originally encountered by the virus is the submucosal dendritic cell (DC), which will bind and phagocytise the virus but is unable to kill it. Therefore the DC spreads the virus to the local lymph node where it presents the Ma-tropic strains to the macrophages and the T cell-tropic ones to the CD4 cells. This will initiate the gradual attrition of cognate immunity and the decline of the CD4 cell subpopulation. Macrophages are also directly affected by the infection, becoming activated with increased secretion of cytokines. Later their capacity for antigen processing and presentation becomes impaired. A downregulation of the expression of the important mannose receptor on alveolar macrophages has been observed in HIV positive individuals (Koziel et al 1998). This reduced the capacity of these cells to bind microbes, especially P. carinii. Another study showed a decreased capacity in such cells of intracellular killing of fungi (Pietrella et al 1998). These findings may explain the increased tendency for respiratory

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infections both with common bacteria and with intracellular parasites in HIV disease.

6.

DEFECTS IN INNATE IMMUNITY LEADING TO INFECTIONS WITH INTRACELLULAR PATHOGENS

These defects are relatively rare and each of them has been described in a limited number of families or individuals. However their consequences may be clinically striking and much can be learned from them about the function of these components of innate immunity. Resistance to mycobacterial infection (Nelson & Summer 1998) depends upon the integrity of macrophages, T-cells (the link to adaptive immunity) and the timely secretion of a cluster of cytokines, namely IFNγ, TNFα, IL-2, IL-12, IL-18 and IL-10 the later dampening overactivity of the proinflammatory cytokines. Also growth factors, such as G-CSF, are of importance. The macrophages of one child, who suffered from disseminated Mycobacterium avium infection, were shown to fail to produce TNFα under E. coli toxin (LPS) stimulation (Tuerlinckx et al 1996). In another child who had recurrent respiratory infections, together with severe complications after EBV and Varicella-Zoster infections, as well as after BCG vaccination, a nucleotide substitution was found in an epitope of the FcγRIIIa receptor, which was not detectable by the CD16/CD56 monoclonal antibodies (de Vries et al 1996). A fatal outcome of a chronic disseminated M. avium infection, due to defective production of IFNγ and IL-2 by stimulated monocytes, has been described (Heurlin et al 1996). Defects in the production of IFNγ (Vilcek et al) and IL-2 (Toosi et al) in tuberculosis patients have already been described in the eighties. Recently, specific complete or partial genetic defects in the INFγreceptors 1 and 2 (R1 & R2) have been described in several families (Ottenhoff et al 1998). This resulted in fatal or near-fatal infections with Mycobacteria, including BCG, and with Salmonellae. Similar infections, albeit of lesser severity, were found in other families demonstrating null mutations in IL-12 receptor chain Rβ1 and p40 genes (Ottenhoff et al 1998, de Jong et al 1998). Thus, it was possible to describe a gradient of severity of these type-1 cytokine defects. Lack of sufficient production of IFNγ or of TNFα by monocytes/macrophages and complete IFNγR1 & R2 defects led to fatal mycobacterial, and/or salmonellar infections.

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Clinical Aberrations in Macrophages, Cytokines and Collectins

Partial defects in these receptors or in the IL-12 receptor and cytokine resulted in similar infections, but less severe.

7.

DEFECTS IN COLLECTINS

Prior to the onset of a specific humoral anti-microbial response, the phagocytic cells enter into action, complement is activated and groups of lectin-binding molecules, which are able to bind to the surface of the microbes, are secreted. At the same time receptors for these molecules are expressed on macrophages, enabling the latter to bind pathogens in a strong complex, as a prerequisite for phagocytosis and intra-cellular killing. A number of clusters of lectin-binding molecules have been described (Lu 1997 ): the Clq component of complement, collectins and the related ficolins. The collectins include five proteins: mannan-binding lectin (MBL), conglutinin and collectin-43 (CL-43) are serum proteins produced by the liver, surfactants A and D (SP-A & D) are found mainly in the lungs and recently detected also in other tissues (Eggleton & Reid 1999). MBL and its receptor seem to be of prime importance. MBL is able to bind a large number of bacteria, viruses and protozoa. It activates complement and facilitates microbial opsonization via its receptor on macrophages. SP-A & D stimulate chemotaxis of phagocytes, followed by production of oxygen radicals. These activities enhance host defenses in the lungs. Recently they have been also found in other tissues, where their function is not, as yet, clearly understood. MBL and SP-A &D are so far, the collectins for which decreased production or levels have been found to be associated with clinical significance. In 5-7% of the general population an opsonization defect of bakers’ yeast has been described, which was associated with low serum levels of MBL, previously called mannan-binding protein (Super et al 1989). The addition in vitro of purified MBL corrected the defect, which seems to cause reduced generation of C3b opsonins. Surprisingly, most of these individuals remain asymptomatic throughout most of their life. However, a series of children with this defect have been reported to suffer from bouts of fever, diarrhea and recurrent infections. Some suffered also from an increased incidence of atopy (Richardson et al 1983). Infants with low MBL levels may be at increased risk of infection around the age of physiologic hypogammaglobulinemia (Turner et al 199 1). Also, MBL gene mutations in adults correlate with susceptibility to severe infections (Summerfield et al 1995). One study found 5.4% of anonymously screened blood donors carrying homozygous or compound heterozygous MBL gene mutations (Babovic-Vuksanovic et al 1999).

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The clinical significance of this finding remains to be determined. Another study reported increased MBL serum levels in infants with the “sudden infant death syndrome (SIDS)”(Kilpatrick et al 1998). All premature newborn babies demonstrate a defect in SP-A & D maturation, which used to be responsible for a high incidence of pulmonary morbidity and mortality (Dekowski & Holtzman 1998). These infants suffer from increased pulmonary infections, an increased airway sensitivity to oxygen, leading to bronchopulmonary dysplasia (BPD) and many of the survivors became respiratory cripples. This situation has been greatly remedied by intrapulmonary administration of synthetic SP preparations and the prenatal treatment of high-risk pregnant women with steroids, which enhance SP maturation. Undoubtedly, much remains to be learned about the clinical significance of potential aberrations, genetic or others, in the collectins and other lectin-binding molecules.

8.

CONCLUSION

The aberrations and defects in the parts of innate immunity, which are of clinical significance, described above, probably represent the tip of the iceberg. The relatively high incidence of gene mutations and decreased production of MBL in the general population, may be paralleled by a similar situation for other lectin-binding molecules, remaining to be determined. We are faced in the clinics by numerous cases of recurrent infections, the cause of which remains unexplained, when submitted to the current routine immunological investigations. More intensive research efforts in defects of innate immunity may contribute to the deciphering of some of those cases.

REFERENCES Albright, J.W., and Albright, J.F., 1998, lmpaired natural killer cell function as a consequence of aging. Exp. Gerontol. 33: 13-25. Arkachaisri, T. and Ballow M., 1999, Developmental immunology of the newborn. In: (Ed) Kevin K.J., Pediatric Allergy and Immunology, Immunol. Allergy Clin. N. Amer. 19: 253-280. Babovic-Vuksanovic, D., Snow, K. and Ten, R.M., 1999, Mannose-binding lectin (MBL) deficiency. Variant alleles in a midwestern population of the United States. Ann. Allergy Asthma lmmunol. 82:134-143. Bofill, M., Borthwick, N.J., and Simmonds, H.A., 1999, Novel mechanism for the impairment of cell proliferation in HIV- 1 infection. Immunology Today 20:

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Carroll, M.C., and Janeway, C.A. Jr., 1999, Innate immunity: Editorial overview. Curr. Opin. Immunol., 1 1: 1 1-12. Cotter, R.L., Burke, W.J., Thomas, V.S., et al, 1999, Insights into the neurodegenerative process of Alzheimer’s disease: a role for mononuclear phagocyte-associated . inflammation and neurotoxicity. J. Leukoc . Biol. 65:416-427. Dekowski S.A. and Holtzman R.B., 1998, Surfactant replacement therapy. In: (Ed) Hageman J.R., Neonatology Update, Pediatr. Clin. N. Amer. 45:549-572. Eggleton P. and Reid K.B.M., 1999, Lung surfactant proteins involved in innate immunity. Curr. Opin. Immunol. 11:28-33. Ezekowitz, R.A.B., and Hoffmann, J., 1998, The blossoming of innate immunity. Immunology 10:9-11. Gendelman, H.E., and Folks, D.G., 1999, Innate and acquired immunity in neurodegenerative disorders. J. Leukoc. Biol. 65:407-408. Gotch, F.M., Koup, R.A., and Safrit, J.T., 1997, New observations on cellular immune responses to HIV and T-cell epitopes. AIDS 1 1:S99-S107. Hanly, A., and Petito, C.K., 1998, HLA-DR-positive dendritic cells of the normal human choroid plexus: a potential reservoir of HIV in the central nervous system. Hum. Pathol. 29:88-93. Hariharan, D., Douglas, S.D., Lee, B., et al, 1999, Interferon-gamma upregulates CCR5 expression in cord and adult blood mononuclear phagocytes. Blood 93:1137-1144. Heurlin, N., Dahlqvist, G., Elinder, G. et al, 1996, Fatal outcome of disseminated Mycobacterium avium infection in childhood. A case of primary incompetent monocyte/macrophage function? Acta Paediatr.85:151 1-1513. de Jong, R., Altare, F., Haagen, I.A. et al, 1998, Severe mycobacterial and Salmonella infections in interleukin-12 receptor-deficient patients. Science 280: 1435-1438. Kovarik, J., and Siegrist, C-A., 1998, Immunity in early life. Immunology Today 19: 150-154. Koziel, H., Eichbaum, Q., Kruskal, B.A. et al, 1998, Reduced binding and phagocytosis of Pneumocystis carinii by alveolar macrophages from persons infected with HIV-1 correlates with mannose receptor downregulation. J. Clin. Invest. 102: 1332- 1344. Lau, A.S., Sigaroudinia, M., Yeung, M.C., and Kohl, S., 1996, Interleukin-12 induces interferon-gamma expression and natural killer cytotoxicity in cord blood mononuclear cells. Pediatr. Res. 39: 150-1 55. Lesourd, B., 1999, Immune response during disease and recovery in the elderly. Proc. Nutr. SOC. 58:85-98. 6 Clinical Aberrations in Nk/Macrophages, Cytokines and Collectins Lu J., 1997, Collectins: collectors of microorganisms for the innate immune system. BioEssays 19:509-518. McGeer, P.L., and McGeer, E.G., 1999, Inflammation of the brain in Alzheimer’s disease: implications for therapy. J. Leukoc. Biol. 65:409-412. Merrill, J.D., Sigaroudinia, M., and Kohl, S., 1996, Characterization of natural killer and antibody-dependent cellular cytotoxicity of preterm infants against human immunodeficiency virusinfected cells. Pediatr. Res. 40:498-503. Nelson, S., and Summer, W.R., 1998, Innate immunity, cytokines, and pulmonary host defense. Infect. Dis. Clin. N. Am. 12:555-567. Ochs, H.D., Smith, C.I.E., and Puck, J.M., 1999, Primary immunodeficiency diseases: A molecular and genetic approach. Oxford University Press, New York. Ottenhoff, T.H.M., Kumararatne, D., and Casanova, J-L., 1998, Novel human immunodeficiencies reveal the essential role of type- 1 cytokines in immunity to intracellular bacteria. Immunol. Today 19:49 1-494.

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Pawelec, G., Solana, R., Remarque, E., and Mariani, E.. 1998, Impact of aging on innate immunity. J. Leukoc. Biol. 64:703-712. Persidsky, Y., Buttini, M., Limoges, J. et al, 1997, An analysis of HIV-1-associated inflammatory products in brain tissue of humans and SClD mice with HIV-1 encephalitis. J. Neurovirol. 3:401-416. Pietrella, D., Monari, C., Retini, C. et al, 1998, Human immunodeficiency virus type-1 envelope protein gp 120 impairs intracellular antifungal mechanisms in human monocytes. J. Infect. Dis. 177:347-354. Reinhardt, P.P., Reinhardt, B., Lathey, J.L., and Spector, S.A., 1995, Human cord blood mononuclear cells are preferentially infected by non-syncytium-inducing, macrophage-tropic human immunodeficiency virus type- 1 isolates. J. Clin. Microbiol. 33:292-297. Richardson, V.F., Larcher, V.F., and Price, J.F., 1983, A common congenital immunodeficiency predisposing to infection and atopy in infancy. Arch. Dis. Child. 58:799-802. Sperduto, A.R., Bryson, Y.J., and Chen, I.S., 1993, Increased susceptibility of neonatal monocyte/macrophages to HIV- 1 infection. AIDS Res. Hum. Retroviruses 9: 12771285. Summerfield, J.A., Ryder, S., Sumiya, M. et al, 1995, Mannose-binding protein gene mutations associated with unusual and severe infections in adults. Lancet 345:886889. Super, M. et al, 1989, Association of low levels of mannan-binding protein with a common defect in opsonisation. Lancet 2: 1236- 1239. Toose, Z., Kleinhenz. M.E., and Ellner. J.J., 1986, Defective interleukin 2 production and responsiveness in human pulmonary tuberculosis. J. Exp. Med. 163: 1162-1 172. Tuerlinckx, D., Vermylen, C., Brichard, B. et al, 1997, Disseminated Mycobacteriurn avium infection in a child with decreased tumor necrosis factor production Eur. J. Pediatr. 156:204-206. Turner, M.W., Super, M., Singh, S. and Levinsky, R.J., 1991, Molecular basis of a common opsonic defect. Clin. Exp. Allergy 21(Suppl 1):182-188. Vallat, A.V., De Girolami, U., He, J., Mhashilkar, A.et al, 1998, Localization of HIV-1 co-receptors CCR5 and CXCR4 in the brain of children with AIDS. Am. J. Pathol. 152:167-178. Vilcek, J., Klion, A., Hendriksen-DeStefano, D., et al., 1986, Defective gammainterferon production in peripheral blood leukocytes of patients with acute tuberculosis. J. Clin. lmmunol. 6: 146-1 5 1. de Vries, E., Koene, H.R., Vossen, J.M. et al, 1996, Identification of an unusual Fc gamma receptor IIla (CD16) on natural killer cells in a patient with recurrent infections. Blood 88:33022-3027.

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KLEBSIELLA INFECTIONS IN THE IMMUNOCOMPROMISED HOST

Hany Sahly, Rainer Podschun and Uwe Ullmann Department of Medical Microbiology and Virology, Christians-Albrechts-University Kiel, Brunswiker Str. 4, 24105 Kiel, Germany

1.

of

INTRODUCTION

The term immunocopromised host describes individuals with defects of either the nonspecific (phagocytes, complement, cytokines, skin, or mucosa) and/or of the specific (humoral or cellular) immunity to infections. Such individuals are at increased risk of infections with various pathogens, including micro-organisms with no-pathogenicity for healthy individuals. There are several major predisposing factors which render the immunocompromised host susceptible for infection. The recognition of these factors is a useful approach to infections in these individuals, because each is associated with a different spectrum of causative agents that usually do not overlap. The most important predisposing factors are: • granulocytopenia and qualitative phagocyte defects • cellular immune dysfunction • humoral immune dysfunction • splenectomy Undoubtedly, the most common and serious abnormality is granulocytopenia. The risk of infections increases significantly after the granulocyte count drops below 500 cells/µl ad rises rapidly as the count approaches zero, and the duration of the granulocytopenic phase correlate with infection (Maschmeyer, 1999). The most prevalent gramnegative bacteria causing infections in granulocytopenic patients are The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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Escherichia coli, Klebsiella pneumortiae and Pseudomonas aeruginosa (Hughes et al., 1997).

2.

CLINICAL SIGNIFICANCE OF KLEBSIELLA

Klebsiella has been known primarily as a pathogen causing severe pyogenic community-acquired pneumonia, which mainly affects chronic alcoholics and has a high fatality rate if untreated (Ishida et al., 1998; Prince et al., 1997; Torres et al., 1991). The vast majority of Klebsiella infections nowadays, however, are nosocomial (Podschun and Ullmann, 1998). As an opportunistic pathogen, Klebsiella primarily attacks immunocompromised individuals who are hospitalized and have severe underlying diseases. It is estimated that Klebsiella species cause 8% of all hospital-acquired infections(Eisenstein, 1990; Prince et al., 1997). In the USA they comprise 3% to 7% of all nosocomial bacterial infections (Horan et al., 1988), placing them among the eight most important pathogens in hospitals and second only to E. coli as the most common cause of gram-negative sepsis (Sahly and Podschun, 1997). Klebsiella infections are observed in almost any body site, although infections of the urinary and respiratory tracts predominate. Depending on the type of infection and study, its prevalence ranges from 3% to 17% of all such infections (Gikas et al., 1998). Klebsiella is ubiquitous in nature. They have two common habitats, one being the environment where they are found in surface water, sewage, soil, and on plants and the other being mucosal surfaces of mammals such as humans, horses, or swine, which they colonize. In humans the nasopharynx and the intestinal tract are the most common habitant sites. The carriage rates in stool samples rang from 5 to 38%, while in the nasopharynx it is detected between 1 to 6%. Due to the lack of good growth conditions on the human skin Klebsiella spp. are rarely found there and are regarded as transient members of the skin flora (Podschun and Ullmann, 1998). The principal pathogenic reservoirs for transmission of Klebsiella are the gastrointestinal tract of patients and the hands of personnel (Montgomerie, 1979). The ability of this genus to rapidly spread (Kuhn et al., 1993) often leads to nosocomial outbreaks. Of the 145 epidemic nosocomial infections reported in the English speaking literature between 1983 and 1991, 13 were caused by Klebsiella (Doebbeling, 1993). The Center for Disease Control (CDC) in Atlanta puts the percentage of endemic hospital infections caused by Klebsiella at 8%, of epidemic outbreaks at 3% of all pathogens (Podschun and Ullmann, 1998).

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6 - 17% 7 - 14% 4 - 15% 2-4%

5-7 2-4 3-8 6 - 11

4 - 17%

4-9

Especially feared are epidemic hospital infections caused by multidrugresistant Klebsiella strains which emerged mainly from the extensive use of antibiotics. In the 1970s these were mainly aminoglycoside-resistant Klebsiella strains (Christensen and Korner, 1972; Noriega et al., 1975). A new type of resistant Klebsiella strains producing so called extended-spectrum beta-lactamases (ESBL) was first described in Germany in 1983. Since then, various types of plasmidencoded extended-spectrum-ß-lactamases (ESBL) have been described worldwide, especially TEM and SHV enzymes (Jacoby and Medeiros, 1991). Another type of resistance to oxyimino-ß-lactams in Klebsiella arises from plasmid acquisition of normally chromosomal AmpC genes of Citrobacter and Enterobacter species (Jacoby, 1996), as has been sporadically detected in Klebsiella pneumoniae isolates worldwide (Sahly et al., 1999). In pediatric wards, particularly in premature infants and neonatal intensive care units, nosocomial Klebsiella infections became a serious problem. Klebsiella species are often the pathogens involved in neonatal sepsis (Table 1) in both early-onset and late-onset infections. They are among the top four pathogens causing infections in neonatal ICUs and represent the second most common causative agent of gram-negative neonatal bacteremia. Especially troublesome are outbreaks of ESBLproducing Klebsiella species in neonatal units. Remarkably, a highly virulent, multiresistant Klebsiella oxytoca clone expressing the capsular type K55 has been isolated with increasing frequency in several countries (Sahly and Podschun, 1997). The genus Klebsiella consist of five species : K. pneumoniae (subsp. pneumoniae, ozaenae, and rhinoscleromatis), K. oxytoca, K. terrigena, K. planticola and K. ornithinolytica (Podschun and Ullmann, 1998). K. pneumoniae is considered to be the medically most important Klebsiella species. To a much lesser degree, K. oxytoca has been isolated from clinical specimens. K. terrigena, and K. planticola were originally considered to be without clinical significance and to be restricted to aquatic, botanic, and soil environments. However, recent reports describe

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them as occurring in human clinical specimens (Podschun and Ullmann, 1992a; Podschun and Ullmann, 1994). While K. terrigena is rarely found among clinical Klebsiella strains (0.4%), K. planticola accounts for up to 20% of all clinical Klebsiella isolates. More than half of these isolates were recovered from respiratory tract secretions; wound infections and urine isolates were next most common. However, the clinical significance of these species remains unclear, since most of the isolates were cultured from polymicrobial specimens. All members of the species produce polysaccharide capsule which structurally form the basis for classification into 77 capsular serotypes. The lipopolysaccharide of this species also form the basis for classification into 8 0-serotypes.

3.

PATHOGENICITY FACTORS OF KLEBSIELLA

In susceptible host, symptomatic K. pneumoniae infections are characterized by severe inflammatory process. In addition to phagocytosis by polymorphonuclear granulocytes, the first line of defense, e.g. innate immunity, by the host against the invading microorganisms includes the bactericidal effect of serum, which is mediated primarily by complement proteins. Two pathways of complement activation has been described; in the classical pathway the so called natural Klebsiella-specific antibodies participate in a process where by the complement system is activated by antibody-antigen complexes formed on bacterial surfaces. In the alternative pathway, activation is achieved by antigens on bacterial surface via the properdin system. Both complement pathways lead, via the activation of C3, to the formation of C3b on bacterial surface, which either mediates phagocytosis by bridging the bacteria to C3 receptors on PMNL or by forming the terminal C5bC9 complex attack which (Joiner, 1988). The alternative pathway of activation is considered the major innate immunity against K. pneumoniae infections. The host also deprive the bacteria from iron in this process by secreting iron binding proteins. A number of virulent factors that enable the bacteria to overcome the innate immunity the host were described (Figure 1).

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Figure 1. Schematic representation of Klebsiella pathogenicity factors.

3.1.

Adhesins

Adherence of the microorganisms to the host cells is considered a critical step in the infectious process (Ofek and Doyle, 1994) . As a member of the enterobacteriacea, K. pneumoniae produces multiple adhesins some of which are fimbrial (pili) and others are nonfimbrial each with distinct receptor specificity (Figure 1). In K. pneumoniae two types of pili predominate. The Type 1 pili agglutinates guinea pig erythrocytes. The agglutination is inhibited by mannose and therefore are mannose specific (Duguid and Old, 1980). The mannose sensitive or specific (MS) type 1 fimbriae is common in many members of enterobacteria. Their role in the pathogenesis of UTI was clarified mostly in studies of E. coli but has also been described for K. pneumoniae in animal models (Fader and Davis, 1980; Fader and Davis, 1982; Maayan et al., 1985). They may also play a role in mediating adhesion to the upper respiratory tract (Ayars et al., 1982). The type 3 fimbriae is characterized by its ability to agglutinate tannin-treated erythrocytes and designated mannose-resistant, Klebsiellalike (MR/K-HA)fimbriae (Duguid and Old, 1980). Although this name implies that these pili are synthesized only by K. pneumoniae, Clegg et al demonstrated their expression by many enteric genera (Clegg and Gerlach, 1987). It was shown that this type of pili is capable of binding to various human cells, such as endothelial cells, epithelial cells of the respiratory and urinary tract (Hornick et al., 1992; Tarkkanen et al.,

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1997; Wurker et al., 1990). However, the role of this fimbrial adhesin in the pathogenic process is largely unknown. The only correlation between MR/K-HA and disease is the observation of expression of type 3 pili in Provedencia stewartii in catheter-associated bacteriuria, which mainly appears in patients with long-term indwelling catheters (Mobley et al., 1988). Three new types of K. pneumonia eadhesins have been recently reported. The R-plasmid-encoded non-fimbrial CF29K adhesin which was shown to mediate adherence to human intestinal cell lines (DarfeuilleMichaud et al., 1992). It is suggested that the CF29K adhesin is the product of the transfer of genetic determinants coding for the CS31-A adhesin from E. coli to K. pneumonaie. A new capsule-like extracellular has been described and seems to confer aggregative pattern of adhesion to intestinal cell lines (Favre-Bonte et al., 1995). Another fimbria-like adhesin designated KPF-28, was found in the majority of K. pneumoniae strains producing CAZ-5/SHV-4 type Extenden-Spectrum-ß-Lactamase, and suggested to mediate adhesion to and colonization of the human gut (Di Martino et al., 1996).

3.2.

Lipopolysaccharides

In K. pneumoniae 8 different Lipopolysaccharides serotypes (LPS, Oantigen) have been described from which the 01 serotype is the most common O-antigen found among clinical isolates (Mizuta et al., 1983). LPS has been implicated as a major factor in the ability of the bacterium to resist the host serum bactericidal activity (Ciurana and Tomás, 1987; Porat et al., 1987; Tomás, et al., 1986). Since LPS is generally able to activate complement, C3b is subsequently deposited onto the LPS molecules. However, since it is fixed preferentially to the longest Opolysaccharide side, C3b is far away from the bacterial cell membrane. Thus, the formation of the lytic membrane attack complex (C5b-C9) is prevented, and subsequent membrane damage and cell death do not take place (Merino et al., 1992).

3.3.

Sidrophores

The availability of iron increases the susceptibility of the host to K. pneumoniae infections as was shown in animal model (Khimji and Miles, 1978). Since the major amount of the host iron is bound by intracellular (e.g. hemoglobin, ferritin, hemosidirin, myoglobin) and extracellular proteins (lactoferrin and transferrin), many bacteria secure their supply for iron in the host by secreting high-affinity, low-molecular-weight iron

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chelators, called siderophores, that are capable of competing effectively for iron bound to host proteins (Griffiths, 1987). Two types of siderophores have been described, the phenolate-type and the hydroxamate-type, from which the former is the most common type. The best known of the phenolate-type is enterobactin, and aerobactin is the most important siderophore within the hydroxamates. In K. pneumoniae aerobactin-positive strains have been described rarely (Podschun et al., 1993). While the contribution of enterobactin to the virulence of the bacteria is uncertain (Miles and Khimji, 1975), the virulence enhancing effect of aerobactin has been documented in animal model (Nassif and Sansonetti, 1986).

3.4.

Capsular Polysaccharides

K. pneumoniae is capable to produce a prominent capsule composed of complex acidic polysaccharides. The capsular repeating subunits consist of four to six sugars and usually uronic acids as negatively charged constituents. Based on the structural variability of the capsular polysaccharides subunits, K. pneumoniae has been classified into 77 serotypes (Ørskov and Ørskov, 1984). Among the virulent factors described above, the capsular polysaccharides appear to ultimately determine the pathogenicity of this species (Cryz et al., 1984; Domenico et al., 1982; Ehrenwort and Baer, 1956; Highsmith and Jarvis, 1985; Podschun and Ullmann, 1998). A number of biological activities are described to the presence of capsular polysaccharide (CPS) on bacterial surface. The capsular massive layer presumably protect the bacterium from phagocytosis by polymorphonuclear granulocytes(Podschun et al., 1992; Podschun and Ullmann, 1992b; Simoons-Smit et al., 1985; Simoons-Smit et al., 1986). Furthermore, they prevent killing of the bacteria by bactericidal serum factors mainly by inhibiting the activation of or uptake of complement components, especially C3b (Tomás et al., 1986; Williams et al., 1983; Williams and Tomas, 1990). Capsule may also act as anti virulent factor. This notion was first noticed by the observations showing predominance of specific serotypes in severe infections (Casewell and Talsania, 1979; Cryz et al., 1986; Maschmeyer, 1999; Podschun et al., 1986; Rennie and Duncan, 1974; Riser and Noone, 1981; Simoons-Smit et al., 1985; Ullmann, 1986). Strains expressing the capsular antigens K1 and K2 were found to be especially virulent to mice (Mizuta et al., 1983). K2 serotype is among the most common capsule type isolated from patients with pneumonia and bacteremia as well as from UTI.

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Attempts to clarify differences in the virulence of the various capsular serotypes focused on nonopsonic pahgocytosis of K. pneumoniae by macrophages. It has been shown that capsule polysaccharides of certain Klebsiella serotypes with the sequences Mana2/3Man and/or Rhaa2/3Rha bind specifically to the mannose receptor of macrophages, leading to phygocytosis and destruction of the bacterium (Ofek et al., 1995). This feature may explain why certain capsule types can be isolated more frequently from clinical material than others. Capsule may also modulate the transcription of the recently described CF29K adhesin of K. pneumoniae (Favre-Bonte et al., 1999). Regulation of capsule formation is under complex genetic control. It involves the products of rmpA and RcsA genes which also regulate the synthesis of polysaccharides in enterobacteria (Arakava et al., 199 1 ; McCallum and Witfield, 1991). It is also regulated by two-component system and thus influenced by environmental conditions (Favre-Bonte et al., 1999). In vitro, noncapsulated spontaneous variants arise from capsulated clones at a frequency higher than mutation rate suggesting that capsule formation is under phase variation control (Matatov et al., 1995). Because capsule is considered a major virulence factor and because the emergence of antibiotic resistant strains, new therapeutic approaches are targeted against the capsule. A polysaccharide-based vaccines has been tested (Cryz et al., 1985). Although Klebsiella pneumoniae is considered an extracellular pathogen, recent studies demonstrated its ability to internalize into epithelial cells (Fumagalli et al., 1997; Oelschlaeger and Tall, 1997). Internalization of nonphagocytic cells such as epithelial cells by bacterial pathogens probably enable the bacteria to escape deleterious agents such as antibodies and antibiotics. Recently, it was suggested that in the case of Streptococcus pyogenes it may also lead to asymptomatic carriage (Neeman et al., 1998). It has been suggested that although Klebsiella colonize asymptomatically a number of body sites, the main reservoir for severe symptomatic infections is the bowel (De Champ et al., 1989; Podschun and Ullmann, 1998). Although capsule interfere with adhesion, it is not clear whether and how they might also interfere with internalization of the bacteria by intestinal epithelial cells.

4.

CONCLUDING REMARKS

Klebsiellae are oportunistic pathogens which can give rise to severe infections such as septicemia, pneumonia, UTI, and soft tissue infections.

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Hospitalized, immunocompromised hosts with severe underlying diseases are the main target of Klebsiella. They are cosidered to be the causative agent for 5 -7% of all hospital-aquired infections and are among the most important nosocomial pathogens. Of particular importance are new trend which have been observed in context with nosocomial Klebsiella infections such as the emergence of extended spectrum beta-lactamase-producing strains, neonatal septicemia caused by K. oxytoca capsule type K55, and new Klebsiella species as causative agents of human infections (K. planticola and K. terrigens). A number of pathogenicity factors have been identified in Klebsiella. The capsule is considered to determine the pathogenicity of the bacterium. Based on the structural variability of the capsular polysaccharides Klebsiella sp. has been classified into 77 serotypes which differ in their pathogenicity and epidemiological relevance. The serotypes K1 and K2 are considered especially likely to be virulent. The lipopolysaccharides have been implicated as a major factor which render the bacterium resistant against the bactericidal activity of the host serum.8 different LPS have been described from which 01 is the most common one among clinical isolates. Other pathogenicity factors are five types of adhesins from which the type 1 and type 3 pili play a role in mediating adhesion to various epithelial cells. Moreover, two types of iron binding siderophores have been described in Klebsierlla from which aerobactin is considered to have a virulence enhancing effect.

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Nassif, X., and Sansonetti, P. J. (1986). Correlation of the virulence of Klebsiella pneumoniae K1 and K2 with the presence of a plasmid encoding aerobactin. Infect Immun 54, 603-608. Neeman, R., Keller, N., Brazilai, A., Korenman, Z., and Sela, S. (1998). Prevention of internalisation-associated gene, prtF1, among persisting group-A streptococcus strains isolated from asymptomatic carriers. Lancet 352, 1974-77. Noriega, E. R., Leibowitz, R. E., Richmond, A. S., Rubinstein, E., Schaefler, S., Simberkoff, M. S., and Rahal, J. J. (1975). Nosocomial infection caused by gentamicin-resistant, streptomycin-sensitive Klebsiella. J Infect Dis 131 (Suppl), S45-S50. Oelschlaeger, T., and Tall, B. (1997). Invasion of cultured human epithelial cells by Klebsiella pneumoniae isolated from the urinary tract. Infect. Immun. 65, 2950-2958. Ofek, I., and Doyle, R. (1994). Bacterial adhesion to cell and tissues. Chapman and Hall, London. Ofek, I., Goldhar, J., Keisari, Y., and Sharon, N. (1995). Nonopsonic phagocytosis of microorganisms. Ann Rev Microbiol 49, 239-276. Ørskov, I., and Ørskov, F. ( 1984). Serotyping of Klebsiella. In “Methods in Microbiology” (T. Bergan, ed.), pp. 143- 164. Academic Press, London. Podschun, R., Heineken, P., Ullmann, U., and Sonntag, H. G. (1986). Comparative investigations of Klebsiella species of clinical origin: plasmid patterns, biochemical reactions, antibiotic resistances and serotypes. Zbl Bakt Hyg A 262, 335-345. Podschun, R., Penner, I., and Ullmann, U. (1992). Interaction of Klebsiella capsule type 7 with human polymorphonuclear leucocytes. Microb Pathog 13, 37 1-379. Podschun, R., Sievers, D., Fischer, A., and Ullmann, U. (1993). Serotypes, hemagglutinins, siderophore synthesis, and serum resistance of Klebsiella isolates causing human urinary tract infections. J Infect Dis 168, 1415-1421. Podschun, R., and Ullmann, U. (1992a). Isolation of Klebsiella terrigena from clinical specimens. Eur J Clin Microbiol Infect Dis 1 I, 349-352. Podschun. R., and Ullmann, U. (1992b). Klebsiella capsular type K7 in relation to toxicity, susceptibility to phagocytosis and resistance to serum. J Med Microbiol 36, 250-254. Podschun, R., and Ullmann, U. (1994). Incidence of Klebsiella planticola among clinical Klebsiella isolates. Med Microbiol Lett 3, 90-95. Podschun, R., and Ullmann, U. (I 998). Klebsiella spp. as nosocomial pathogen: Epidemiology. taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 11, 589-603. Porat, R., Johns, M. A., and McCabe, W. R. (1987). Selective pressures and lipopolysaccharide subunits as determinants of resistance of clinical isolates of gramnegative bacilli to human serum. Infect Immun 55, 320-328. Prince, S., Dominger, K., Cunha, B., and Klein, N. (1997). Klebsiella pneumoniae pneumonia. Heart Lung 26, 413-7. Rennie, R. P., and Duncan, I. B. R. (1974). Combined biochemical and serological typing of clinical isolates of Klebsiella. Appl Microbiol 28, 534-539. Riser, E., and Noone, P. (1981). Klebsiella capsular type versus site of isolation. J Clin Pathol 34, 552-555. Sahly, H., Boehme, V., Podschun, R., A. Bauernfeind, Fölsch, U. R., and Ullmann, U. (I 999). Infection and colonization of an intensive care unit patient’s respiratory tract by a Klebsiella pneumoniae strain producing an novel AmpC-type B-lactamase different from that known. Clin Inf Dis 28, 1338-9.

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MACROPHAGE-RECOGNIZED MOLECULES APOPTOTIC CELLS ARE EXPRESSED AT HIGHER LEVELS IN AKR LYMPHOMA OF AGED AS COMPARED TO YOUNG MICE

OF

O. Itzhaki, E. Skutelsky, T. Kaptzan, A. Siegal, M. Michowitz, J. Sinai, M. Huszar, S. Nafar and J. Leibovici Department of Pathology, Sackler Faculty of Medicine, Tel-Aviv University, 69978 TelAviv, Israel

1.

ABSTRACT

While a direct relation between aging and tumorigenesis is well established, a slower tumor progression rate was reported in old as compared to young cancer patients. The mechanisms responsible for the less aggressive behavior of tumors in the aged, are largely unknown. We have recently shown an increase in apoptotic cell death in tumors derived from aged as compared to young animals in the AKR lymphoma. This was shown by DNA flow cytometry and by the ladder type DNA fragmentation in agarose gel electrophoresis. Analysis of the expression of genes involved in apoptosis in tumors derived from young and old animals showed a lower bcl-2 expression in those from the aged. The Fas antigen, on the contrary, displayed higher expression levels on lymphoma cells derived from old than on those from young mice. Apoptotic cells are recognized and phagocytosed mainly by macrophages. One molecular property of apoptotic cells which is recognized by macrophages is a loss in cell surface sialic acid concomitantly uncovering galactose residues. While comparing the "eat The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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me status” phenotype of the tumor cells derived from young and aged animals, by the use of lectins recognizing sialic acid and galactose residues, FACS analysis showed a decrease in cell surface sialic acid and a gain in galactose residues in aged as compared to young mice. Moreover, Western blot analysis showed that a 130 Kda sialylated membrane glycoprotein was expressed at a lower level in tumors from the old as compared to young mice. Our results, at both the cellular and molecular levels, particularly with regard to molecules recognized by macrophages, indicate that increased apoptotic cell death in tumors from old as compared to those from young animals constitutes, as we have previously suggested, one of the mechanisms of the age-related decrease in tumor progression rate.

2.

INTRODUCTION

The relation between aging and tumorigenesis is well established. Cancer incidence is known to rise with increasing age of the host, mainly due to prolonged exposure to carcinogens (Peto et al 1975) and immunosenescence (Kaesberg & Ershler 1989). Paradoxically however, it has been found that tumor growth rate and metastatic dissemination proceed at a slower rate in aged patients (Herbsman et al 1981, Ershler et al 1983). This phenomenon has also been described in experimental tumors by other groups (Ershler et al 1984) and by ourselves (Donin et al 1997, 1995). Only few studies have dealt with the elucidation of the mechanism(s) underlying the reduced tumor progression rate in old as compared to young organisms. Several mechanisms, such as decreased cell proliferative rate (Cameron 1972) or modifications in immune response (Kalsberg & Ershler 1989, Herbsman et al 1981), have been suggested. We examined the possibility of another mechanism for the relatively more benign behavior of neoplasms in the aged, namely an increased tendency for apoptotic cell death (Donin et al 1996). Apoptotic cells are recognized and phagocytosed mainly by macrophages by a nonphlogistic mechanism of endamaged cell disposal (Lin et al 1999). Several molecules on the apoptotic cell surface have been reported to serve as recognition sites for the macrophage (Savill et al 1993). Exposure of phosphatidylserine on the outer leaflet of the cell membrane, thrombospondin receptor and decrease in cell surface sialic acid content concomitantly with a rise in galactose residues have been described as molecular markers of the “eat me status” phenotype of apoptotic cells.

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In the present study we compared AKR lymphoma cells which grew in young or aged mice with regard to one of these molecular cell surface properties of apoptotic cells recognized by macrophages, the content in sialic acid and galactose residues. These saccharidic residues were detected by suitable lectins by FACS and Western blot analysis.

3.

MATERIALS AND METHODS

3.1

Mice and tumors

AKR/J mice were purchased from the Tel-Aviv University Breeding Center. Two age groups were used: 1) 4-6 weeks; 2) 6-8 month. The variants of the AKR lymphoma differing in degree of malignancy were obtained in our laboratory from spontaneous tumors (Leibovici 1984). Tumor cell suspensions were prepared as previously described (Leibovici et al 1980). Tumor cells (2x10 5 in 0.2 ml RPMI medium) were inoculated S.C. in the back of mice. Tumor growth was evaluated by recording the incidence and by measuring 2-3 times a week the diameter of the tumors formed at the S.C. site of inoculation (primary tumors), by measuring the size of inguinal lymph nodes as a criterion for metastatic growth and by recording daily the mortality of mice.

3.2

DNA flow cytometry

Cells derived from primary tumor growths of the AKR lymohoma variants, grown in young or old mice were incubated with propidium iodide ( 50 µg/ml) following the procedure of Vindelov (Vindelov et al 1983). The data were analyzed on a Cell Software BP MultiCycle Phoenix Flow Systems Phenix Arz.

3.3

DNA fragmentation analysis by gel electrophoresis

DNA from primary tumors derived from AKR lymphoma grown in young versus old mice, was analyzed by horizontal electrophoresis during 4 h on 1.5% agarose gel and visualized by UV fluorescence after staining with ethidium bromide (0.5 µg/ml).

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Analysis of Bcl-2 protein expression by flow cy tome t ry

Single cell suspensions were washed with PBS and red blood cells were removed by RBC buffer. Cells were suspended at a concentration of 7 2x10 cells/ml in SB (1%BSA (Sigma) in PBS, PH=7.4) and saponine 0.03% (Sigma) in order to permeabilize the cells (15). The cells were incubated with hamster anti - mouse - Bcl-2 mAb (Pharmingen, USA, clone 3F11) for 30 minutes at 4°C. After washing with SB+saponine, cells were incubated with FITC-conjugated goat anti - Armenian hamster IgG (Pharmingen, Jackson ImmunoResearch Laboratories) for 30 min. at 4°C. After two addional washings, samples were analyzed on a FACSort Becton Dickinson, San Jose CA, with WINMDI Joseph Trotter Scripps data processing.

3.5

FACS analysis with fluorescent lectins

Fluorescent lectins (labeled by FITC) were purchased from Vector, Burlingame Calif. The tumor cell suspensions were washed in PBS and treated with fluorescent lectins for 30 min at 4"C with the appropriate concentrations of lectins: 1 µg/106 cells/ml for the Maakia amurensis (MAL-I), 0.1 µg/10 6 cells/ml for the Sambucus nigra (SNA) and 10 µg/106cells/ml for the Soybean agglutinin (SBA). Comparison of the lectins binding to the tumor cells was done by FACS analysis (Becton Dickinson Sunnyvale CA USA FACSort) and data processing was done by the Becton PC-lysis program.

3.6

Western blot analysis

Preparation of membrane proteins was done as follows. First, the tumor cell suspensions were washed in lysis buffer with antiproteases without Triton and 2-3 freezings and thawing were done followed by centrifugation at 13.000 rpm for 30 min at 4oC. Second, lysis buffer containing Triton (1%) was added to the precipitate of the tumor cells for 30 min at 4oC with mixing by Vortex every 10 minutes. The membrane proteins were prepared by centrifuging at 13.000 rpm for 30 min at 4°C. The concentration of the proteins in the supernatant was determined by Lowry’s method using the Bio-Rad kit (16). The proteins were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membranes were blocked for 1 h with 3% bovine serum albumin and then incubated for 2 h with biotinylated

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Itzhaki et al. lectins (1:100 from a stock solution of 500µg/ml) in Following five washes in TTBS (10mM Tris HCL, 0.15M Tween, pH 7.4), the proteins were revealed by incubating at complex avidin-biotin conjugated to horseradish peroxidase The proteins were detected by enhanced chemiluminescence the substrate of Pierce.

4.

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0.1% BSA. NaCL, 0.1% 37 oC with a for 30 min. (ECL) using

RESULTS

The biological behavior of the AKR lymphoma in young and old mice is presented in Fig 1. A reduced growth of both primary and metastatic tumors was observed in aged as compared to young animals. The difference was significant statistically only in the case of the metastatic tumor growth ( p< 0.0025).

Figure I: Comparison of the biological behavior between primary and metastatic tumors of the AKR lymphoma in young and old mice 5 Groups of 5 mice were inoculated S.C. with 2x10 tumor cells. The data presented were observed on day 13 after tumor inoculation.

Figure 2 presents a representative experiment of a comparison of a DNA flow cytometry analysis of the AKR lymphoma derived from young or old animals. Two differences can be noted between the tumors growing in mice of different ages: 1) Tumor cells from old mice possess a lower proliferative capacity (S+G2M phases) than those originating from young animals ; 2) The apoptotic cell population ( the sub G1

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fraction ) is markedly higher in the old than in the young animals. Figure 3 summarizes quantitatively the average values of several experiments (45) with regard to both cell proliferative activity and to spontaneous apoptosis. With relation to both characteristics, the differences between tumors of young and old mice was statistically significant ( p< 0.025).

FLUORESCENCE INTENSITY

Figure 2: Comparison of DNA flow cytometry analysis in primary tumor cells of AKR lymphoma derived from young (Y) and old (O) mice

A comparison between DNA degradation, as seen by agarose gel electrophoresis, between tumors derived from young and aged animals is shown in Figure 4. The degradation of DNA is much more intense in the tumor taken from old than in those from young mice. Examination of the expression of genes related to apoptosis supported the results at the cellular level. Tumors from aged mice expressed a lower level of Bcl-2 protein as compared to those derived from young animals (Fig 5). The difference was statistically significant (p< 0.025). The expression of the fas gene, was, on the contrary, higher in lymphoma cells originating from the old than in those grown in young mice (not shown). Since macrophages recognize and phagocytose apoptotic cells, we tried to assess whether cell surface molecules known to determine this recognition differ in their expression on tumor cells derived from animals of different ages.

Itzhaki et al. a

257 b

Figure 3: Comparison of content in S plus G2M cell cycle phase cells (a) and apoptotic cell content (b) according to DNA flow cytometry in primary tumor cells of AKR lymphoma derived from young and old mice The data of apoptotic cell content represent the percentage of cells with DNA content below G0/G1. These data constitute average values of 5 experiments

Figure 4: Comparison of DNA fragmentation in tumor cells from AKR lymphoma grown in young versus old mice Horizontal electrophoresis was done during 4h on 1.5% agarose gel. Lane 1 represents the DNA marker of different sizes ( pUC18 DNA marker Hea III digest, 102-587 b.p.) , lane 2 - normal spleen DNA, lane 3 - AKR lymphoma DNA of young mice and lane 4 - AKR lymphoma DNA from old mice. The data represent the average of 5 experiments.

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Figure 5: Comparison of Bcl-2 protein expression, according to percentage of Bcl-2 positive cells, in metastatic tumor cells of AKR lymphoma derived from young and old mice - FACS analysis

Examination of binding of lectins recognizing sialic acid, such as SNA, showed a lower attachment to old mice – derived tumor cells than to lymphoma cells taken from young animals. By contrast, cell surface galactose residues as detected by SBA, were seen at higher levels in tumors from old than in those from young animals ( Figure 6 ).

FLUORESCENCE INTENSITY

Figure 6: Comparison of binding of Sambucus nigra agglutinin (SNA) and Soybean agglutinin (SBA) to the metastatic tumor cells of AKR lymphoma derived from young (Y) and old ( O) mice- FACS analysis

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Figure 7 presents a Western blot analysis of the cell membrane glycoproteins of AKR lymphomas from young and aged mice as detected by the MAL-I and PNA lectins. The sialylation of a glycoprotein reactive with MAL-I of 130 kDa is at a lower level on tumor cells from old than on those from young mice ( Figure 7a ). By contrast, a membrane glycoprotein of 88 kDa, detected by PNA, is found at a higher level of galactosylation on tumor cells of old than on cells from young animals (Figure 7b ).

Figure 7: Western blot analysis of cell membrane proteins of AKR lymphoma derived from young (Y) and old (O) mice, identified by Maackia Amurensis lectin I (MAL-I) (a) and Peanut Agglutinin (PNA) (b).

5.

DISCUSSION

While tumorigenesis rises with increasing age, tumor progression is inversely related to advanced age. The mechanism(s) of this age-related reduced tumor progression rate has not yet been elucidated. While decreased cell proliferation and changes in immune response with host’s age have been suggested (Kaesberg & Ershler 1989, Herbsman et al 1981, Donin et al 1995, Cameron 1972), we have proposed that increased apoptotic cell death may occur in tumors of old as compared to those of young animals. We have indeed reported that increased apoptotic cell death in old mice constitutes a mechanism of the reduced tumor progression rate with age (Donin et al 1996). Normal nontumoral cell populations were shown to undergo more elevated apoptotic cell death rates in aged organisms. This has recently been shown for polymorphonuclear granulocytes (Fulop et al 1997).

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Singhal et al. have shown an increase in apoptotic cell death of macrophages, splenocytes and thymocytes (Singhal et al 1997). We have found in the AKR lymphoma ( by DNA flow cytometry and by DNA fragmentation in agarose gel electrophoresis ) a higher apoptotic cell fraction in tumors derived from old mice as compared to those derived from young ones. These data at the cellular level were supported by results obtained by examining the expression of genes involved in apoptosis: Tumor cells taken from old animals expressed lower levels of Bcl-2 protein but displayed higher Fas receptor cell surface content than lymphoma cells from young mice. Macrophages can recognize, phagocytose and neatly nonphlogistically degrade apoptotic cells (Liu et al 1999). One molecular property of apoptotic cells which is recognized by macrophages is a loss in cell surface sialic acid concomitantly uncovering galactose residues (Savill et al 1993). We compared, according to this criterion, the "eat me status" phenotype of the tumor cells derived from young and old animals, by using lectins recognizing sialic acid and galactose residues. FACS analysis showed a reduction in cell surface sialic acid and a gain in galactose residues in aged as compared to young mice. Moreover, Western blot analysis demonstrated that a 130 Kda membrane glycoprotein was sialylated at a lower level in tumors from old as compared to those from young mice. By contrast, a 88kDa membrane glycoprotein had higher galactose levels in tumor cells from old as compared to growths from young animals. Our results, at both the cellular and molecular levels, particularly with regard to molecules recognized by macrophages, indicate that increased apoptotic cell death in tumors from old as compared to those from young animals constitutes, as we have previously suggested (Donin et al 1996), one of the mechanisms of the age-related decrease in tumor progression rate. Our results may have implications for the design of antimetastatic therapy particularly appropriate for the aged patient.

ACKNOWLEDGMENTS This study was performed in partial fulfillment of the requirements for a Ph.D. degree of Mrs. Orit Itzhaki, Sackler Faculty of Medicine, TelAviv University, Israel. The study was partially supported by the Shauder Grant for Medical Research and by the Hylda Portnoy Grant for Cancer Research.

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REFERENCES Cameron, I.L. Cell proliferation and renewal in aging mice. J. Gerontol., 27: 162-172, 1972. Donin, N., Sinai, J., Staroselsky, A., Mahlin, T., Nordenberg, J. and Leibovici, J. Comparison of growth rate of two B16 melanomas differing in metastatic potential in young versus middle- aged mice. Cancer Invest. 15: 416-421, 1997. Donin, N., Itzhaki, O., Sinai, J. and. Leibovici, J. Involvement of apoptosis in the agerelated reduction in the tumor progression rate. The XXIV Meeting of the International Society for Oncodevelopmental Biology and Medicine, Coronado California, USA, p.1 12, 1996. Donin, N., Sinai, J., Michowitz, M., Hiss, J., Nordenberg, J. and Leibovici. J. Role of immune response as determinant of tumor progression in function of age in the B16 melanoma. Mech. Ageing Dev., 80, 121-137. 1995. Ershler, W.B., Socinski. M.A., Greene,C.J. Bronchogenic cancer. Metastasis and aging. J. Am. Geriat. SOC., 31: 673-676, 1983. Ershler, W.B., Stewart, J.A., Hacker, M.P., Moore, A.L., and Tindle, B.H. B16 melanoma and aging -slower growth, longer survival in older mice. J. Natl. Cancer Inst., 72: 161-164, 1984. Fulop, T.Jr, Fouquet, C., Allaire, P., Perrin, N., Lacombe, G., Stankova, J., RolaPleszczynski, M., Gagne, D., Wegnar, J.R., Khalil, A., Dupuis, G. Change in apoptosis of human polymorphonuclear granulocytes with aging. Mech. Ageing. Dev. 96: 15-34, 1997. Herbsman, H., Feldman, J., Seldera,J., Gardner, B., and Alfonso, A. Survival following breast cancer surgery in the elderly. Cancer,47: 2358-2363, 1981. Kaesberg, P.R. and Ershler. W.B. The importance of immunosenescence in the incidence and malignant properties of cancer in hosts of advanced age. J. Gerontol. Biol. Sci., 44: 63-66.1 989. Leibovici, J. Serial passage of tumors in mice in the study of tumor progression and testing of antineoplastic drugs. Cancer Res. 44: 1981-1984, 1984. Leibovici, J., Susskind-Brudner, G. and Wolman, M. Direct antitumor effect of highmolecular weight levan on Lewis lung carcinoma cells in mice. J. Natl. Cancer Inst. 65:391-396, 1980. Liu, Y., Cousin, J.M., Hughes, J., Van Damme J., Seckl J.R., Haslett C., Dransfield I., Savill J. and Rossi A.G. Glucocorticoids promote nonphlogistic phagocytosis of apoptotic leukocytes. J. Immunol., 162: 3639-46, 1999. Lowry, O.H., Rosebrough N.J.. Farr, A.L., Randall, R.J. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265-75, 1951. Peto, R., Roe, F.J.C., Lee, P.N., Levy, L., and Clack, J. Cancer and aging in mice and men. Br.J. Cancer, 32: 41 1-426; 1975. Savill, J., Fadok, V., Henson, P. and Haslett, C. Phagocyte recognition of cells undergoing apoptosis. Imrnunol. Today, 14: 131-136, 1993. Singhal, P.C., Reddy, K., Franki, N., Sanwal, V., Kapasi, A., Gibbons, N., Mattana, J., and Valderrama, E. Age and sex modulate renal expression of SGP-2 and transglutaminase and apoptosis of splenocytes, thymocytes and macrophages. J. Investig. Med. 45: 567-575, 1997. Veis, D.J, Sentman, C.L, Bach, E.A and Korsmeyer, S.J : Expression of the bcl-2 protein in murine and human thymocytes and in peripheral T lymphocytes. J. Immunol 151: 2546-2554,1993.

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Vindelov, L.L, Christensen, L.J, Nissen, N.l: Standartisation of high-resolution flow cytornetric DNA analysis by the simultaneous use of chicken and trout red blood cells as internal reference standard. Cytornetry 3 :328-331, 1983.

SENSITIVITY TO MACROPHAGES DECREASES WITH TUMOR PROGRESSION IN THE AKR LYMPHOMA

T. Kaptzan, E. Skutelsky, M. Michowitz, A. Siegal, 0. Itzhaki, S. Hoenig, J. Hiss, S. Kay and J. Leibovici Department of Pathology, Sackler Faculty of Medicine, Tel-Aviv University, 69978 Tel Aviv, Israel

1.

ABSTRACT

Resistance to immune reactions, innate or acquired, may be one of the mechanisms responsible for the progression of tumors. We have, indeed shown higher numbers of macrophages surrounding low- as compared to high-malignancy cells. In the present study we examined the level of cell surface molecules known to determine sensitivity to macrophages, namely galactose (GAL) and sialic acid (SA) residues. A histochemical assay for identification of SA by electron microscopy showed a higher cell surface content on metastatic (MT) than on primary (PT) tumor cells. The FACS data seen with fluorescent lectins showed a higher binding of Sambucus nigra agglutinin, which identifies SA attached to terminal GAL in -2.6 or -2.3 linkage, in MT than in PT cells. Binding of Maakia amurensis lectin (MAL-I), which identifies SA at position 3 of GAL, showed that the MT cells contain two subpopulations, one binding more MAL-1 and another less. Cell sorting showed a more aggressive behavior of the first population. The comparison of Peanut agglutinin (PNA) binding, which identifies GAL, demonstrated a decreased amount of PNA receptors in MT as compared to PT cells. Western blot analysis of the membranal proteins with different lectins, identified The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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3 sialylated glycoproteins. The 88 kDa glycoprotein had no significance for metastatic potential. The 130 kDa glycoprotein was higher in MT than on PT cells. The 220 kDa glycoprotein was practically present only on MT cells. The tendency observed was of a higher level of membranal glycoconjugates terminally sialylated with subterminal galactose residues, in MT cells as compared to PT cells. This may explain the recently found decrease in apoptotic cell death with increasing aggressiveness of the AKR lymphoma and suggests a lower sensitivity to macrophages with tumor progression. Treatment based on the reduction in sialic acid content might render the tumor cells more vulnerable to macrophages. We found, indeed, that Wheat germ agglutinin (WGA) injected in vivo, exerted an inhibitory effect on growth of the lymphoma. We found morever that WGAtreated tumor cells were more sensitive than nontreated cells to macrophages in vitro.

2.

INTRODUCTION

Macrophages are known to be able to exert an inhibitory effect on tumors. Several lines of evidence prove the antitumoral effect of macrophages. Leukocyte infiltrates, including macrophages, where for instance found in the vicinity of numerous neoplasms. As for the role of macrophages in tumor progression, opposite effects have been suggested. The process of tumor progression may be related to an evolution of resistance of the neoplastic cells to various components of the immune system. Decreased sensitivity to T lymphocytes (Bosslet & Schirrmacher 1981, Gregory et al 1988), natural killer cells (Gorlik et al 1979, Hanne & Fidler 1980) and macrophages (Urban & Schreiber 1983, North & Nicolson 1985, Yamashina et al 1985) have been reported to accompany increasing malignancy of tumors. However, the mechanism of this resistance at the tumor cell level has not often been explored. Although a good prognosis has often been attributed to tumors containing host cell infiltrates (Yan Nagell et al 1978, Ran & Witz 1972, Ohtani 1998), opposite ideas were expressed as well (Joachim 1976, Husby et al 1976, Wei et al 1986, Dony et al 1999, Bardos et al 1998, Hildenbrand et al 1998). Certain tumors were found to secrete factors which inhibit macrophage function, enhancing thereby tumor growth (Crawford et al 1998). Macrophage-based tumor immunotherapy was used or suggested to be used by various groups (Killion & Fidler 1998,

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Nakashima et al 1998) and by ourselves (Leibovici et al 1986) as antitumoral treatment modalities. The most threatening aspect of neoplastic growths is their capacity to metastasize. The process of tumor dissemination most often involves therapeutic failure. Information regarding the metastatic phenotype could contribute both to a better understanding of the metastatic process and to the identification of cellular molecules possibly suitable as targets for antimetastatic therapy. Various aspects of the metastatic phenotype of a series of AKR lymphoma malignancy variants (Leibovici 1984, Leibovici et al 1992, Klein et al 1998) as well as of cells derived from primary as compared to metastatic growth (Leibovici et al 1985) were studied in our laboratory. In the AKR lymphoma system it has been found that cellular functions implied in metastatic potential, such as homotypic aggregation of tumor cells, their attachment to endothelial cell monolayer and to extracellular matrix, their migration as well as their ability to adhere to potential target organs for metastasis, varied in the different malignancy variants (Klein et al 1992, 1996). Resistance to immune reactions, innate or acquired, may be one of the mechanisms responsible for the progression of tumors. We have indeed shown higher numbers of macrophages surrounding low- as compared to high-malignancy cells. A higher sensitivity of low- malignancy as compared to highly malignant AKR lymphoma variants to a macrophage stimulator, levan, has been shown by us (Leibovici et al 1986). In the present study we examined the level of cell surface molecules known to determine –positively or negatively- sensitivity to macrophages, namely galactose (GAL) and sialic acid (SA) residues. In addition, since treatment based on the reduction in sialic acid content might render the tumor cells more vulnerable to macrophages, we tested the effect of Wheat germ agglutinin (WGA) on the growth of the Iymphoma.

3.

MATERIALS AND METHODS

3.1

Mice and tumors

AKR/J mice, 6-10 weeks old, were purchased from the Tel-Aviv University Breeding Center. The variants of the AKR lymphoma differing in degree of malignancy were obtained in our laboratory from spontaneous tumors (Leibovici 1984). Tumor cell suspensions were

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prepared as previously described (Leibovici et al 1980). Tumor cells (2x105 in 0.2 ml RPMI medium) were inoculated S.C. in the back of mice. Tumor growth was evaluated by recording the incidence and by measuring 2-3 times a week the diameter of the tumors formed at the S.C. site of inoculation (primary tumors), by measuring the size of inguinal lymph nodes as a criterion for metastatic growth and by regarding daily the mortality of mice.

3.2

Electron Microscopy

Tissue preparation Tumor cell suspensions were prepared as previously described. This tumor cell suspensions were washed once in phosphate-buffered saline (PBS), pH 7.4 and fixed with Karnovsky’s fixative (Dvorik et al 1970) for 2 h at 4 0 C. Then they were washed twice with PBS and labeled with colloidal ferric hydroxide, pH 1.8, and postfixed with 1% OsO4 in the same buffer. Dehydration was carried out with graded ethanols and propylene oxide, and tissues were embedded in araldite. Stainingprocedures Ultrathin sections (0.075±0.015µm) were prepared by a LKB III Ultratome, using diamond knife, and the sections were mounted on Formvar-coated, 200 mesh nickel grids. The sections were rinsed with doubly-distilled water and post-stained for 40 min with saturated uranyl acetate in 50% methanol. Examination of the sections was carried out using a JEOL- 100CX electron microscope, at 80 kV.

3.3

FACS analysis with fluorescent lectins

Fluorescent lectins (labeled by FITC) were purchased from Vector, Burlingame Calif. The tumor cell suspensions were washed in PBS and 0 treated with fluorescent lectins for 30 min at 4 C with the appropriate concentrations of Iectins: 1 µg/l06ceIIs/mI for the MAL-1, 0.1 µg/l06ceIIs/mI for the SNA and 10 µg/l06ceIIs/mI for the PNA. Comparison of the lectins binding to the tumor cells was done by FACS analysis (Becton Dickinson Sunnyvale CA USA FACSort) and data processing was done by the Becton PC-lysis program.

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3.4

267

Western blot analysis

Preparation of membrane proteins was done as follows. First, the tumor cell suspensions were washed in lysis buffer with antiproteases without Triton and 2-3 freezings and thawing were done followed by centrifugation at 13.000 rpm for 30 min at 4O C. Second, lysis buffer containing Triton (1%) was added to the precipitate of the tumor cells for 30 min at 4OC with mixing by Vortex every 10 minutes. The membrane proteins were prepared by centrifuging at 13.000 rpm for 30 min at 4OC. The concentration of the proteins in the supernatant was determined by the Lowry’s method using the Bio-Rad kit (Lowry et al 1951). The proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membranes were blocked for 1 h with 3% bovine serum albumin and then for 2 h with biotinylated lectins (1:100 from a stock solution of 500µg/ml) in 0.1% BSA. Following five washes in TTBS (1 0mM Tris HCL,0. 15M NaCL, 0.1 % Tween, pH 7.4), the proteins were revealed by incubating at 37OC with a complex avidin-biotin conjugated to horseradish peroxidase for 30 min. The proteins were visualized with Diethylaminobenzidine (DAB) as substrate in PBS containing H2O2. The reaction was stopped by TTBS.

3.5

Treatment with WGA

Fluorescent Wheat Germ Agglutinin was purchased from Vector, Burlingame Calif. Hundred micrograms of this lectin were added to l06 tumor cells in 1 ml PBS and the mixture was incubated for 30 min at 4OC. The treated tumor cells (2x105 cells in 0.2 ml PBS containing 20 µg lectin) were inoculated to groups of 5 mice. Tumor growth was compared to development of tumors in mice inoculated with non-treated cells. Incidence and size of S.C. primary and metastatic tumors (tumorally enlarged inguinal lymph node) were evaluated 2-3 times per week. The data represent an average diameter of the tumors. Statistical evaluation was done by Student's t-test.

4.

RESULTS

An electron microscopy histochemical determination of sialic acid residues on the surface of primary and metastatic tumor cells is presented in Figure 1. We used colloidal iron oxide at pH=1.8 to localize cell surface

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SA. A higher cell surface sialic acid content was identified on MT than on PT cells.

Figure.1. Electron microscopy of cell surface sialic acid residue (collodial iron oxide) in primary (PT) and metastatic (MT) tumor cells (x40.000)

Figure 2 presents a comparison of the binding capacity of two lectins which recognize sialic acid, SNA and MAL-1 to primary and metastatic tumor cells of the TAU-44 AKR lymphoma variant. Both lectins bind at higher levels to the MT than to the PT cells. This is particularly evident for the Sambucus nigra agglutinin. By contrast, the Peanut agglutinin (PNA), which recognizes galactose residues, attaches less avidly to metastatic than to primary tumor cells (Figure 3).

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Figure 2. Comparison of the binding of lectins specific for sialic acid, to the primary (PT) and metastatic (MT) cells of the TAU-44 variant of AKR lymphoma The concentration of the SNA lectin was 0.1 µg/l06ceIIs/mI and of the MAL I lectin 1 µ g/10 6cell/ml.

PNA

Figure 3. Comparison of the binding of the PNA lectin to the primary (F'T) and metastatic (MT) cells of the TAU-44 variant of AKR lymphoma The concentration of the lectin was 10 µg/l06ceIIs/mI

The binding of MAL-1 showed that the metastatic tumor cells were heterogenous, presenting two subpopulations of cells differing in affinity to this lectin. In order to verify whether the content in cell surface

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molecules recognized by MAL- 1 has implications for the biological behavior of the cells, we performed sorting of the two cell subpopulations, injected them to different group of mice and followed tumor development. Figure 4 shows the results of the cell sorting. A significantly more rapid evolution of tumors was seen in the mice inoculated with the cell subpopulation binding higher amounts of MAL- 1 than in those injected with the cells having a lower affinity for this lectin (p60% (p0.045) and confocal microscopy. No difference was observed in CXCR2 expression. These results emphasize the role of innate immune mechanisms for the resistance to UTI, and diminish a role of lymphocytes and specific immune mechanisms. The results suggest that deficient IL-8 receptor expression may account for the increased susceptibility to pyelonephritis observed in some children.

312

Abstracts

FAILURE TO ERADICATE GROUP A STREPTOCOCCI- A ROLE FOR BACTERIAL INTERNALIZATION ? Revital Neeman¹ , Nattan Keller², Asher Barzilai3, Ethan Rubenstain4 and Shlomo SeIa¹

1 Department t of Human Microbiology, Sackler school of Medicine, Tel-Aviv University, Tel-Aviv, 2Depts. Clinical Microbiology, ³Pediatric infection, and 4Unit of Infectious Diseases, Chaim Sheba Medical center, Tel-Hashomer Hospital, Israel

Asymptomatic carriage following antibiotic treatment occurs in up to 30% patients with pharyngotonsillitis caused by group A streptococcus (GAS). Numerous theories have been proposed to explain this phenomenon, thought none gained wide acceptance. Recently, GAS was shown to internalize cultures epithelial cell. We hypothesize that persistence of GAS might be associated with streptococcal internalization. To examine this hypothesis, we have compared the adherence, internalization and survival capabilities of 42 GAS isolates derived from patient with acute pharyngotonsillitis. Twenty-none isolates were derived from patient with bacterial eradication following beta-lactame therapy, and 13 were derived from patients who became carrier following treatment. It was found that isolates derived from carriers were able to adhere, internalize and survive in Hep-2 cells, significantly better than those of the eradication group were. The results implicate that the development of the carriage state is correlated with adhesion, internalization and survival capabilities of GAS strains.

Abstracts

313

HOW DO ANTIMICROBIAL PEPTIDES SELECTIVELY LYSE BACTERIA: FROM NATIVE TO DE-NOVO DESIGNED PEPTIDES Yechiel Shai Dept. of Biological Chemistry. Weimann Inst., Rehovot, Israel

Antimicrobial peptides are natural antibiotics that constitute a major part of the innate immunity of a wide range of organisms including humans. During the last two decades numerous studies have demonstrated the essential role of antimicrobial peptides in the first line of defense against invading pathogens and their proliferation. An important property of most antimicrobial peptides is their ability to selectively kill bacteria. Despite numerous studies on the structure and activity of antimicrobial peptides, our knowledge on their mode of action and their cell specific activity is incomplete. The most studied group includes the linear, mostly alpha-helical peptides. Although developed by distant and diverse species such as plants, insects, amphibians and human, linear antimicrobial peptides share two properties, namely, a net positive charge and a high propensity to adopt amphipatic alpha-helical conformation in hydrophobic environments. Although the exact mechanism by which antibacterial peptides kill bacteria is not clearly understood, it has been shown that peptide-lipid interactions, rather than receptor-mediated recognition processes, play a major role in their function. Their net positive charge facilitates their binding to bacteria and their hydrophobic character is responsible for their ability to disrupt and permeate bacterial membranes.Membrane permeation by amphipatic alpha-helical peptides has been proposed to occur via one of two general mechanisms; (i) transmembrane pore formation via a "barrel-stave" mechanism; and (ii) membrane destruction/solubilization via a "carpet" mechanism. Recent studies on linear alpha-helical antimicrobial peptides will be presented in light of these two proposed mechanisms. In addition, the different stages of membrane disintegration by antimicrobial peptides will be evaluated based studies with a novel group of diasteriomeric antimicrobial peptides. This group includes a-helical non-cell selective lytic peptides in which D-amino acids were incorporated in specific sites along the peptide chain. The resulting diasteriomers lost their cytotoxic effects on mammalian cells but retained high antibacterial activity, thus providing a basis to design novel peptide antibiotics composed of D and L amino acids which are selective to microorganisms.

314

Abstracts

THE ROLE OF LINEARITY IN SELECTIVE BACTERIA LYSIS BY AMPHIPATHIC BETAHELICAL ANTIMICROBIAL PEPTIDES Ziv Oren & Yechiel Shai Dept. of Biological Chemistry, Weizmann Institute of Science, Rehovot 761 00, Israel

The major and the most studied group of antimicrobial peptides is the linear, amphipathic beta-helical antimicrobial peptides. However, despite numerous studies on the contribution of structure, amphipathicity, and positive charges to their activity, the importance of linearity has not been examined. In the present study, we functionally and structurally characterized de-novo designed amphiphatic linear and cyclic peptides composed of either all L-amino acids or their diastereomers. We found that both linear peptides lyse bacteria and have significant hemolytic activity. Cyclization substantially decreased the hemolytic activity of both wild type peptide and its diastereomer but had a minor effect on their activities towards Gram-positive and Gram-negative bacteria. In order to gain information on the cause for selective lytic ability of the peptides, their affinity to phospholipid membranes was examined. The results reveal that only the wild type peptide could bind both negatively charged and zwitterionic peptides. ATR-FTIR spectroscopy revealed lower --helical content of the cyclic peptides and the linear diastereomer compared to the linear wild type peptide when bound to PE/PG membranes. Overall our results indicate that peptide linearity is not crucial for antibacterial activity, but linearity seems to effect selectivity between mammalian cells and bacteria.

Abstracts

315

THE ROLE OF HYDROPHOBICITY IN THE STRUCTURE, FUNCTION AND MODE OF ACTION OF DE NOVO DESIGNED ALL L AND DIASTERIOMERS ANTIMICROBIAL PEPTIDES Dorit Avrahami & Yechiel Shai Dept. of Biological Chemistry, Weizmann inst. of Science, Rehovot 76100, Israel

During the last two decades 400 different antimicrobial peptiaes were. discovered in the host defense system of eukaryotes and prokaryotes. The aim of my M.Sc. study was to examine the role of hydrophobicity on secondary structure, biological activity and cell selectivity of designed L-peptides and their diasteriomers. Each peptide was composed of three types of amino acids, namely, four Lys, seven identical hydrophobic amino acids (Gly, Ala, Val, Leu or Ile) and one Trp. In each case, four hydrophobic L-amino acids were substituted for their corresponding Damino acids. A correlation between hydrophobicity and biological activity was found. The higher the hydrophobicity, the higher the biological activity. Furthermore, in all the cases where the L-peptides were hemolytic their diasteriomers were not, although their antibacterial activity was preserved. FTIR spectroscopy revealed that the peptides K4L7W, and K4A7W adopt more than 80% a-helical structure. However, this may not be sufficient for biological activity since K4A7W is neither hemolytic nor antibacterial. In light of the data, we can conclude that in the attempt to achieve a selective activity, three features are necessary: (i) a certain level of hydrophobicity, (ii) a minimal percentage of a-helical structure and (iii) a very low tendency for aggregation. This study supports the “carpet-like” mechanism as the mode of action of the diasteriomers rather than the pore formation mechanism. Currently, we expanded our research into the development of antifungal peptides and the study of their mechanism.

316

Abstracts

FAS EXPRESSION IN MONOCYTIC CELLS Enrico Conte, Livia Manzella, Ann Zeuner, Benedetta Sciacca, Giuseppe Cocchiero, Etta Conticello, Luca Zammataro, Ruggero De Maria and Angelo Messina Inst. General Pathology, University of Catania, Catania, Italy

Fas (CD95 or APO-1), a component of the TNF/NGF receptor superfamily, and its ligand are required for immune homeostasis. Fas-Fas ligand interaction represents a major pathway for the induction of apoptosis in cells and tissues. The mechanisms regulating the expression of Fas in monocyte/macrophage function are still poorly understood. In this study we utilized the promyelocytic leukemia cell line U937 induced to differentiate by phorbol 12-myristate 13-acetate (PMA) and stimulated by Interferon-gamma. The differentiation state of cells was evaluated, up to five days, by growth curves, morphological analysis and FACS analysis of surface antigens, and markers of differentiation such as CD11c and CD14. Fas expression was evaluated in terms of mRNA accumulation by RT-PCR, promoter activity by reporter gene assay and protein production by FACS analysis. Apoptosis induced by anti-Fas antibodies was also evaluated.

Abstracts

317

NATURALLY OCCURRING ANTIBODIES: A HUMORAL COMPONENT OF INNATE IMMUNITY Isaac P. Witz Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv, Israel. Present address: John Wayne Cancer Institute, Santa Monica, CA, USA

Naturally occurring antibodies (NOA) are immunoglobulins (mainly .IgM) produced spontaneously by healthy individuals without deliberate immunization. Many NOA are polyreactive and react with foreign ,as well as with autoantigens. NOA are produced in many cases, by CD5 B cells and are generally encoded in germ-line configuration. The present overview will focus on two subjects. The first will deal with the general characteristics of CD5 B cells and with developmental and functional aspects of these cells. Some open and controversial questions related to CD5 cells will also be discussed. These will include functions of the CD5 protein; induced expression of CD5 on B cells and the "lineage switch" from CD5 B cells to macrophages. The second topic will address general characteristics of NOA and provide data generated at the authors' laboratory and in those of others on varied functions of NOA with respect to tumorigenesis and tumor progression. NOA reacting with trimethylammonium; phospholipids; interferons and the carbohydrate GAL epitope will also be discussed.

318

Abstracts

THE IMMUNE RESPONSE TO APOPTOTIC CELLS Dror Mevorach, MD The Laboratory for Cellular and Molecular Immunology, Division of Medicine, TelAviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel-Aviv, Israel

Programmed cell death (PCD) can be divided into two distinct but linked sequential processes, killing of the cells and removal of the dead cells, which may be a neighboring cell or a professional phagocyte. Following internalization of the apoptotic cell, the phagocyte typically triggers neither the development of a pro-inflammatory response nor the production of autoantibodies directed against apoptotic self antigens. Since apoptotic cells are characterized by translocation of autoantigens such as nucleosomes to the surface of the cell, we tested the hypothesis that excess or abnormally processed apoptotic cells can generate autoantibodies. We have found that syngeneic apoptotic load can induce transient hypergammaglobulinemia, anti-DNA, anticardiolipin, and glomerular depositions in normal mice. Furthermore, we also found that one of the important mechanisms of uptake of apoptotic cells involves opsonization by the complement system, suggesting that deficient states could lead to aberrant handling of apoptotic cells. Therefore, conditions in which apoptotic cells become immunogenic may explain antigen selection in inflammatory and autoimmune conditions, such as in systemic lupus erythematosus (SLE).

Abstracts

319

AS101 RESTORES IMMUNE FUNCTIONS OF MURINE CYTOMEGALOVIRUS (MCMV) INFECTED MICE B. Sredni (1), Rosenthal-Galili, Z. (1), Blagerman, S. (2), Kalechman, Y. (1) and Rager-Zisman, B. (2) ®1© C.A.I.R. Institute, The Marilyn Finkler Cancer Research Center, Faculty of Life Sciences, Bar Ilan University, Ramat Gan, (2) Dept. of Microbiology and Immunology, Ben-Gurion University, Beer Sheva, Israel

Murine cytomegalovirus (MCMV) infection is a widely used animal model for human cytomegalovirus (HCMV) infection. HCMV is known for its immunosuppressive activities and can act as a co-factor in enhancing susceptibility of the host to other opportunistic infections. AS 10 1 , ammonium trichloro(dioxyethylene-0-0')tellurate, a synthetic organotellurium compound developed in our laboratory, has previously been shown to possess immunoregulatory properties with minimal toxicity. We investigated whether in vivo treatment of mice with AS101 will restore immune functions affected by MCMV. The effects of sublethal MCMV infection on production of interleukin-2 (IL-2) by spleen cells, IFNg and natural killer (NK) activity were studied. Our findings show that the virus infection led to a significant decrease in IL-2 production which was restored after treatment with AS101. MCMV increased the levels of IFNg and NK for 3-5 days after infection. AS101 treatment prolonged and sustained these levels for at least 14 days. Moreover, MCMV infection led to a significant decrease in the number of bone marrow (BM) cells and in the production levels of colony forming units (CSF) and IL-6. There was also a decrease in the number of stromal cells, as reflected by the number of colony forming unit fibroblasts (CFU-F), and in the relative number of CFU-GM progenitors. Treatment of MCMV infected mice with AS101 restored CSF and IL-6 production by BM cells to levels of uninfected control mice as well as the number of CFU-F and stromal cell elements which consequently led to the restoration of the total number of BM cells. Results presented here indicate that AS101 may have immunomodulatory effects on MCMV mediated myelosuppression. These results may be explained by the ability of AS101 to inhibit IL-10 at the mRNA level. Administration of AS101 to patients with CMV associated BM damage may improve the restoration of their BM function.

320

Abstracts

CD6 ANTIGEN, A SCAVENGER RECEPTOR CYSTEINE-RICH SUPERFAMILY MEMBER, AS A POTENTIAL TARGET FOR IMMUNOTHERAPY IN AUTOIMMUNE DISEASES 5 2 Enrique Monterol¹, Leopoldina Falcon , Gil R eyes , Olga To rres¹, M. 6 i Guibert Nelson Rodriguez , Yadira Morera2 , Jorge Estrada5 , Juana 2 5, Delgado Maria Diaz3 Jorge Navarro4 , Jorge Delgado Margarita 1 Perez4 , LeoneI Torres7, Ana Matecon 6, Ada Ruiz2 Mercedes Cedeno , 1 1 1 Blanca Tormo¹, Patricia Sierra , Juan F. Amador , Rolando Perez , Alfredo Hermandez 5, Agustin Lage1 2 3 4 1

Center of Molecular Immunology, C.J. Finlay Hosp; ,Hnos Ameijeiras Hosp; 6 5 7 Enriquez Hosp; CIMEQ; Institute of Rheumatologv, CIC; Havana, Cuba

M.

CD6 antigen is a type I cell membrane glycoprotein belonging to the scavenger receptor cysteine-rich (SRCR) superfamily group B, predominantly expressed by T cells and a B cells subset. CD6 binds activated leukocyte cell adhesion molecule (ALCAM), a member of the immunoglobulin superfamily (IgSF). ALCAM is expressed on activated T cells, B cells, monocytes, skin fibroblasts, keratinocytes and rheumatoid arthritis synovium, and mediates homophilic and heterophilic adhesion. CD6-ligand interaction has been implicated in cell adhesion, T cell maturation and regulation of activation, constituting an uncommon type of protein—protein superfamilies interaction. The ior tl is a murine IgG2a mAb recognizing a different epitope compared to other anti-CD6 mAbs. It is in a Phase II Clinical Trial (PIICT) for Cutaneous T-cell Lymphomas treatment. Recently, we reported its intravenous therapeutic effect in a Psoriasis Vulgaris patient. Skin lesions remission of psoriatic patients after topical treatment with ior tl mAb observed in two PIICT (versus Calcipotriol and versus placebo) are shown. The topical use of this mAb induces a prolonged clinical and histological improvement without local side effects. Preliminary data about a PIICT in rheumatoid arthritis patients is also shown, including therapeutic effects, technetium99m-labeled ior-tl mAb joint uptake and body distribution.

Abstracts

321

IMMUNOMODULATION INDUCED BY IgG POLYSPECIFIC ANTI-IDIOTYPIC ANTIGANGLIOSIDE GM2 MONOCLONAL ANTIBODIES Enrique Montero¹’², Francisco Quintma², Hila Amir-Kroll², Amparo 1 Maciasl, Constantin Fesel², Rolando Perezl, Agustin Lage , Irun R. ¹Cohen²

2 Center of Molecular Immunology, Havana, Cuba The Weizmann Institute of Science, Rehovot, Israel

Natural autoantibodies (NAb) are characteristically polyspecific and highly connected. They are naturally found in all normal individuals and constitute a subfraction of normal serum. NAb are directed against several self antigens and also against microbial antigens. Therapeutic infusions of pooled normal IgG (ivIg) enriched in NAb are effective in autoimmune diseases and infections. Recently, we obtained two highly connected anti-idiotypic IgG monoclonal antibodies (mAbs) by immunizing syngeneic Balb/c mice with an anti-ganglioside GM2 specific IgM antibody. The anti-idiotypic mAbs named B7 and 34B7 belong to the IgG2a and IgG1 subclasses. Here we present some striking properties of these mAbs, such as their recognition of a wide panel of evolutionarily conserved self antigens including self antigens that may be targets of autoantibodies in autoimmune diseases, similar to ivIg. In addition, a dose-dependent effect of these mAb in nonobese diabetic (NOD) mice was found, in association to a modulation in the immune response to heat-shock proteins 60 and 70. Moreover, these mAbs protected against Streptococcus pneumoniae type 4 infection in Balb/c mice. Finally we discuss implications of the immunomodulation through ganglioside polyspecific antibodies associated to other structures of the innate immunity and their interplay with the adaptive immune system.

322

Abstracts

PATTERN RECOGNITION MOLECULES IN HOST DEFENSE R. Alan B. Ezekowitz Laboratory of Developmental Immunology, Department of Pediatrics, Massachusetts General Hospital, Harvard Medical School. Boston, MA

The role of innate immunity is to restrict and limit infection. Many molecules and cellular processes conspire and act in concert to defend the host in the first minutes or hours after exposure to an infectious challenge. We have been interested in two mammalian molecules that may be considered as pattern recognition molecules in that they appear to distinguish the patterns of carbohydrates that adorn certain microorganisms selectively. The serum mannose-binding protein may be considered as an ante-antibody and acts like a broad spectrum polyvalent antibody. The macrophage mannose receptor by contrast is a membrane protein that mediates endocytosis and phagocytosis and appears to play a role in first line host defense. Furthermore, we have used Drosophila as a model system to identify putative primitive pattern recognition molecules. I will discuss our progress in these areas of investigation.

INDEX

Acquired immunity 165 Activating peptide, neutrophils 191 Adhesin 24 1-242, 244-245 Adhesion molecules 147- 148, 165, 188, 227, 229 Aerobactin 243, 245 Agglutination 30-34, 50, 55, 64, 241, 292 Alveolar macrophages 27, 29, 31, 37, 38, 40-46, 76, 232, 295, 297 Alzheimer's disease 229-230 Amyloid 4, 229-230 Antibiotic 62, 64, 153, 208213,220, 239,244, 312, 313 Antibodies, monoclonal 40, 65, 80, 93, 141, 231, 308, 321 Antibodies, natural 240, 3 16, 32 1 Antibody 9, 15-17, 40, 43, 62, 65, 94, 100, 104, 116-117, 138, 140, 142, 144, 150, 154, 157, 188, 228, 240, 321, 322

Antigen presenting cells (APC) 93, 164-165, 176, 179, 191 Antimicrobial peptides203, 205208, 212, 220, 313-315 Antibody dependent cytotoxicity (ADCC) 138, 188, 228 Apoptosis 151, 191-198, 251, 256, 260, 316 Apoptotic cells 1, 180, 251, 259-260, 264, 291, 318 Arthritis 91, 186, 228, 320 Astrocytes 227, 229-230 Autocrine 19, 191, 287 Autoimmune 29 1 B lymphocytes 16-17, 100, 185 Bacteremia 33-34, 63, 157, 239, 243, 294, 296, 311 Bacteria4, 9, 15, 27, 29-32, 34, 45, 49-50,53-54, 57, 61-65, 69, 73, 82, 91-99,104, 150152,157, 165,167, 208-2 13, 219-221, 223, 231232,237, 245,292-294, 297, 303, 309

323

324 Bactericidal 74, 93, 210-213, 2 19-220, 224, 240,242-245 Bone marrow 19-21, 73, 9596, 99, 150, 155, 204, 206207, 224, 319 Bordetella bronchiseptica 167 Borrelia burdgoferi 167, 169 Bronchopulmonary dysplasia 233 C-reactive protein 18 8 C-type lectin 2-3, 28-29, 3233, 79, 82, 292 Candida albicans 4, 82 Capsular polysaccharide 28 34, 62, 65-66, 293, 243 Capsular serotypes 28-29, 3234 Capsule 28-34, 240, 242245, 293 Carbohydrate recognition domain (CRD) 2-3, 31, 50, 55, 57, 292-293 Cardiomyocytes 196 Cthelicidins 2 03 -2 13 Cell wall 61, 63-64, 69, 95, 165, 292, 296 Chediak-Higashi syndrome 223-224 Chemokines 16, 185, 228, 230, 300-304, 308 Chlamydia spp. 167, 169, 212 63 Cholin-binding protein Chronic granulomatous disease (CGD) 223-224 Collagen (Collagenous) 4-5, 27-28, 49, 51-54, 56-57, 186, 227, 292, Collectins 31, 34, 41, 4446, 49, 52, 56, 227, 232, 233, 292, 293 Colony stimulating factor (CSF) 73-84, 187

Index Complement (receptor ,CR) 15-18, 22, 49-50, 95-98, 100, 104, 115-116, 119120,166,185, 191, 227-228, 232,237,240, 242-243, 291, 297, 304, 310 Concanavalin A (Con A) 98 Congenital myelokathexis 224 Conglutinin 50 -57, 232 Crohn's disease 91 Cryptococcus neoformans 209 Curosurf 39-46 Cutaneous 50, 157, 163, 166, 168-169, 270, 320 Cyclic Neutropenia 224 Cysteine proteases 27 8 Cysteine rich domain 5 Cystic Fibrosis 209, 224 Cytokines 5-6, 14, 18-22, 32, 37-38, 46, 73-83, 93, 157, 163-166,168- 169, 185-1 86, 188-189, 191, 193-195, 197-198,227,229-232, 237, 277-280, 286-287, 300-301 Cytomegalovirus 3 18 Cytotoxic (cytolytic) T lymphocytes (CTL) 137, 176, 211, 280 Defensins 191,195,204,212, 227 Dementia (HAD) 229 Dendritic cells 7, 74-75, 80, 82-83, 163-170, 175-176, 191, 227, 230, 301 Dextran sulfate 4 Diabetes 186, 224 Diaphorase 107-113 Dimannose 32-34 Dipalmitoylphosphatidyl (DPPC) 39, 41, 46 Dithiothreitol (DTT) 127-129 175 DNA-based vaccine

Index Effector cells 91, 140, 168, 278, 281, 286, 304 Eicosanoids 82 Elderly 62, 228 ELISA 66, 79, 182, 189-190, 192-195, 279, 282, 297 Endocytosis 3-5, 63, 8283, 166, 192, 303, 322 Endoplasmatic 5, 278, 285 Endothelial cells 6-7, 18, 63, 82, 89, 148-149, 154, 188, 191,195, 241, 265,285, 304 Endothelins 19 1 Enterobacter cloaca 97 Enterobacteria 91-96, 101, 104, 239, 241, 244 Enzyme inhibitors 42 Epithelial cells 7, 16, 34, 6164, 67, 69, 190, 241, 244245, 292, 308,310, 312 Epstein Bar Virus (EBV) 16, 100, 231 Escherichia coli®E. coli0 31, 39, 92, 95-101, 209211, 220-221, 231, 237, 241-242, 297, 308, 311 Exosurf 39, 41, 44, 46 Extracellular matrix 18, 20-21, 265, 286, 307 Failure Thrive syndrome 224 Fc receptor (FcR) 19, 91 Fetuin 61, 64-65 Fibronectin 2, 95, 305, 307 Fibrosarcoma cells 279-283, 285 Fimbriae (Fim A) 96-98 Flavocytochrome 107-108, 126, 298, 306 Fucose 3, 28, 154, 158 G protein 101, 301, 305

325

Galactose 3, 28, 51, 251252, 258, 260, 263-265, 268, 271-272, Galectin 19-2 1 Ganglioside 32 1 Glycogen 224, Glycosy 1-Phosphatidyl-inositol (GPI) 21, 99-101 Gram-negative sepsis 23 8 Granulocyte 17, 38, 73, 107, 127, 132, 237, 240-242, 258, 272 Granulocytopenia 23 7 Green fluorescent protein (GFP) 141, 177-179 GTP binding protein 71 Hemagglutinin 50, 55 Hashimoto thyroiditis 224 Heat shock protein (HSP) 297, 321 Helicobacter pylori 9 6 Hemophagocytic syndrome 224 Hepatocytes 190- 19 1 Histoplasma 169 HLA 77, 137-141, 230 Homeostasis 1-2, 5 Hospital infections 238-239 Hydrogen peroxide 53 Hydrophobicity (hydrphobic) 39, 208-209, 219-221, 313, 315 Hypersensitivity, delayed type (DTH) 151-153, 156-157, 185, 191 Hypogammaglobulinemia 148, 232 Hypotension 278 Hypoxia 197 IgE 91, 223, 304 IgG 8, 142, 224, 294, 321 Immunocompromised 237, 27, 34, 92, 98, 104

326

Immunoregulation 168 Immunotherapy 264, 278-279, 320 Infection 2, 8-9, 18, 27, 29, 3334,46, 49-51, 57,62-63, 69, 91-95, 139, 149-158, 163169,210-213, 223,23 1-233, 237-245,278, 292,294, 296, 301, 308-309, 311-312, 319, 321-322 Inflammation (Inflammatory)2, 15-16, 23,34, 37, 62,73-74, 76, 78-79, 82-83, 91-93, 104,116, 150-153,158, 165, 185-187,191-197, 206-207, 228-23 1,240,278, 287, 292, 300-301, 307, 307,309, 318 Influenza A virus (IAV) 49-50, 176- 180 Innate immunity, components 15, 17, 22, 27, 32, 34, 50, 137, 185, 203, 213, 227233, 240, 272, 292, 300, 313, 317, 321-322 Integrins 17, 148-149, 152, 157, 307 Interferon (IFN) 38, 44, 73, 163,168, 206, 228, 316-317 Interleukines (IL) 46, 38, 75-95, 80, 163-165, 169, 185-198, 228, 231-232, 277-287,308-3 11, 3 19, Interstitial Nephritis 224 Intestine 187, 190 Intracellular 17, 171, 149, 163, 166, 169, 186, 191195, 230, 242, 278, 297 Iodonitrotetrazolium 109- 1 10 Iron binding protein 240 Juvenile Periodontitis 224 Kidney 20, 188, 192, 197, 308, 311

Index Kinases 92, 101, 115 Klebsiella spp. 4, 27, 46, 80, 92, 96 Langerhans cells 75 Lectin 2, 15, 20, 27, 49, 61, 79, 97, 227, 252, 259, 266 Leishmaniasis 163 Leukocyte adhesion deficiency (LAD) 18, 147, 223 Leukocyte 17, 147, 196, 204, 223, 265, 277, 286, 293, 300, 320 Leukotriene 93 Leukocyte function Ag. (LFA) 18, 149 Lipopolysaccharide 4, 28, 38, 95, 164, 187, 240, 242 Lipoprotein 5, 207 Lipoteichoic acid 4, 63, 309 Listeria monocytogenes 167, 309 Liver 2, 186, 197, 232, 310 Lung 3, 27, 37, 50, 61, 150, 232, 292, 293, 297 Lymphatic organ 7, 186 Lymphoma 251, 263, 321 Lymphoreticular organ 1 8 9 Lymphoid cells 137, 281 127, 149 Mac-1 antigen Macrophages 1, 15, 29, 37, 73, 96, 126, 163,185, 227, 243, 251, 263, 280, 291, 293, 294, 297, 298, 301, 309, 310, 316, 317, 322 Malignancy, Malignant 253, 263, 277 Mannose-binding protein (MBL) 3, 15, 49, 97, 101, 322 Mannose receptor 1, 27, 79, 166, 185, 230, 244, 293, 297, 322

Index Mast cells 91, 191, 227, 3 04 Metastasis, metastatic 252, 263, 279 MHC 9, 80, 137, 164, 175, 304 Microglia 7, 227 Monocyte-derived macrophages (MoDM) 6, 29, 74, 293 Monocytes 6, 17, 29, 32, 46, 73, 127, 156, 186, 223, 227, 294, 301, 307, 309, 310, 316, 320 Muscle 20, 175, 192 Myeloperoxidase 6, 94, 223 NADPH oxidase 107, 121, 125, 211, 298, 306 Neonates 228, 239 Neoplastic 189, 264, 301 Neuron 20, 192, 212 Neutrophils 4, 16, 53, 76, 92, 107, 115, 126, 149, 185, 206, 223, 227, 279, 305, 308, 311 Nitric oxide (NO)37, 166, 185 NKcells 17, 137, 167, 185, 195, 227, 278, 319 Opportunistic infection 27, 34, 92, 211, 223, 238, 319, Opsonisation 6, 15, 30, 54,79, 96, 103, 115, 188, 232, 244, 297, 305, 318 Oxidase 107, 116, 125, 211, 255, 298, 306 Oxidative burst 18, 38,74, 224 Pattern recognition molecules 1, 96, 322 Phagocytes 2,22, 53,74, 107, 116, 125, 196, 204, 229, 232, 237, 291, 294, 298, 301, 30 Phagocytosis 1, 5, 18, 29, 37, 52, 79, 116, 126, 156, 166,

327 185, 230, 240, 251, 297, 303, 322 Phospholipase (PLC, PLA) 3, 99, 115, 125 Platelet activating factor (PAF) 63, 188, 196 Pneumonia 34, 62, 152, 223, 238, 243, 293 polymyxin B 39, 42, 219 Polymorphonuclear leukocytes (PMN) 240, 259, 295 Polysaccharides 3, 28, 62 240, 293 Psoriasis 91, 321 Pulmonary fibrosis 46, 209, 224 Respiratory tract 34, 62, 223, 23 8 Rhamnose 32 Rheumatoid arthritis 91, 186, 320 Salmonella 50, 167, 231, 303 Sarcoidosis 47 Scavenger receptor 1, 320 Selectin 93, 148 Septicemia 23 9 Serratia 97, 223 Side rop ho re 243 Spleen 3, 137, 186, 208, 281, 307, 319 Staphylococcus spp. 96, 212 Streptococcus spp. 61, 96,157, 244, 296, 312, 321 Stroma cells 185, 277, 319 Sudden Infant Death Syndrome (SIDS) 233 Superoxide 76, 107, 116, 121, 125, 223, 298, 306 Surfactant proteins (SP-A,SP-D) 30, 37, 50, 232, 292, 293 Systemic Lupus Erythematosus 224, 319

328 T helper cells 156, 163, 230 T Lymphocytes 17, 137, 176, 185, 264, 280, 293 Thymus 18, 208, 228, 307 Toxoplasma gondii 167 Transcription factors (ERK, NF) 115, 165 Transposon . 64 Tumor 168, 186, 210, 251, 263, 277, 317

Index Tumor necrosis factor (TNF) 166, 188, 229 Tyrosine Kinase 92, 101, 148 Urinary tract infections (UTI) 238, 308, 311 Vaccination (Vaccine) 62, 168, 175, 231, 244, 277, 285, 296 Wound 211, 239 Zymosan 115, 305