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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2.2

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 <25µg/l µg protein. Two preparations of surfactant lipids were used in the present study. 1. Exosurf (Wellcome, England), a synthetic surfactant containing dipalmitoylphosphatidyl choline (DPPC), cetyl alcohol and tyloxapol. When reconstructed with distilled water the surfactant preparation contains 13.5mg/ml DPPC. 2. Curosurf (Chiesi Farmaceutici S.p.A., Parma, Italy), isolated from porcine lung which contains exclusively polar lipids mainly 70% Phosphatidylcholine, (56mg/ml) and some (1 %) of the hydrophobic surfactant proteins B and C). Various phospholipids (sigma) were also tested to their effect on NO production. Liposomes were prepared from the phospholipids according to the method described by McCormack et. Al (McCormack et al 1993). In some experiments, polymixin B (Sigma), which is known to inhibit LPS (Raetz et al 1991); was added at a concentration of 1-10µg/ml. In another set of experiments, an inhibitor of iNOS, NG-monomethy1-Larginine (L-NMMA; calbiochem, La Jolla, CA), was added at a concentration range of 10-500µM.

2.3

Measurement of nitrite in cell culture supernatant

NO, quantified by the accumulation of nitrite in the culture medium, was measured using the Greiss reaction; sodium nitrite was used as the standard (Nicolas & Nason 1957). Briefly, 50µ1 of culture medium aliquots were mixed with 50µl of Greiss reagents and were incubated at room temperature for 10 min. The absorbency at 550 nm was measured in a microplate reader. Preliminary experiments indicated that surfactant lipids SP-A, or SP-D did not interfere with the Greiss reaction. Data are

40

Modulation of Nitric Oxide Production by Lung Surfactant

presented as µM nitrite. The quantitative date are presented as means ± SE; statistical comparisons were made using a two-tailed student's t-test.

2.4

Western blotting

Control and stimulated cells were washed with ice cold PBS, and then solubilized. The cells were further frozen and thawed 5 times. Aliquots (15µg protein) were subject to SDS-PAGE on 7.5% polyacrylamide slab gels and then blotted onto nitrocellulose and processed accroding to Paul et.al (Paul et al 1997).

2.5

Immunocytochemistry

The cells were grown on coverslips and were fixed in cold methanol (10 min.) followed by acetone (10 min). The cells were then immunostained for iNOS as previously described (Blau et al 1997). Briefly, the cells were incubated with rabbit polyclonal antibody (diluted 1 : 200) raised against iNOS of mouse origin (Santa Cruz, Biotechnology, Santa Cruz, CA). The cells were incubated overnight at 4°C, washed and further processed by using the ABC staining method (Ref). For quantitation, the stained cells were counted (10 fields, – 300 cells) under the microscope and were expressed as percentage of stained cells from total cell number. The purity of the macrophage preparations was assessed by immunostaining the cells with the specific anti rat macrophage monoclonal antibody ED1 (Dijkstra et al 1985).

Figure 1. Stimulation of NO production in NR-8383 alveolar macrophages cell line by SP-A, SP-D, INF-γ and LPS, and the inhibitory effect of various concentrations of curosurf

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41

RESULTS

Alveolar macrophages rat cell line NR-8383 were stimulated in vitro to produce NO by the surfactant proteins SP-A and SP-D. Both collectins showed a significant increase in NO production compared with unstimulated cells (Figure 1 a,b). This increase was concentration dependent and was comparable to that of the known iNOS synthase agonists LPS and INF- (Figure 1C, d). The effect of surfactant lipids upon the stimulating effect of these agonists was tested by using the commercial surfactant preparations Curosurf and Exosurf. Both surfactant preparations were found to suppress the stimulatory effect of SP-A and SP-D as well as LPS in a similar manner. Therefore, the data presented in figs. 1a-d is that obtained only with Curosurf. This inhibition of NO production was dose-dependent (Exosurf 25-500 µg/ml; Curosurf 100- 1000 µg/ml), reaching approximately 80% inhibition with the high lipid dose. Inhibition of SP-A, SP-D and LPS on NO production was obtained at all concentrations of the agonists. The suppression of INF- induced NO production was obtained only at high concentrations of the agonist (25U/ ml) and was low (10%-20%) as compared to SP-A, SP-D or LPS used as agonists (Figure 1d). These results which were described for NR-8383 cell line, were similar to those obtained with freshly isolated rat alveolar macrophages (data not shown). Various phospholipids were tested for their ability to inhibit NO production by the NR-8383 cell line. An effective inhibition was obtained with DPPC or a mixture of phospholipids containing DPPC with SP-A as the agonist (Figure 2).

Figure 2. The inhibitory effect of various phospholipids on SP-A (1µg/ml) inducedNO production in NR-8383 alveolar macrophages cell line. PL- a mixture o phosphatidylcholine and dipalmitoylphosphatidylcholine (7:3); DPPC- diapalmitoylphosphatidylcholine; PC-phosphatidylcholine; DPEA- diapalmitoylphosphatidylethanolamine.

42

Modulation of Nitric Oxide Production by Lung Surfactani

To confirm that the NR-8383 cell lines was producing NO via iNOS, we used L-NMMA, which is a specific enzyme inhibitor. L-NMMA was found to inhibit NO production by the cells at a concentration range of 10-500µM with all tested agonists Figure 3)

N-monomethyl-L-arginine (ug/ml

Figure 3. The inhibitory effect of N-monomethyl-L-arginine on NO production by NR8383 alveolar macrophages cell line.

A number of experiments were conducted to eliminate the possibility that contaminating LPS in the SP-A or SP-D preparations are responsible for the NO stimulated secretion by the cells. In these experiments, polymyxin B, a known inhibitor of LPS was added together with the various agonists. It was found that polymixin B (1-10µg/ml) inhibited >90% of LPS induced stimulation. However, only a relatively small inhibitory effect was observed with SP-A (<15%) or SP-D (25%) and none with INF- (Figure 4).

Figure 4. The effect of Polymixin B (1µg/ml) on NO production induced by various agonists in NR-8383 alveolar macrophages cell line.

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The increase in NO production by the various agonists was accompanied by an increase in the expression of cellular iNOS, as was determined by immunostaining of the cells with an antibody to iNOS as well as Western blotting. Control unstimulated cells were unstained as compared to the immunostained cells incubated with the various agonists (Figure 5). Only -50% of the cells were found to express iNOS after 48h with LPS, and a lower percentage of immunostained cells with the other agonists (Figure 5a). There was an increase in the number of immunostained cells in the time range of 8-48hr. after the addition of the various agonists to the cells (Figure 5a). The percentage of immunostained cells was correlated to NO production by the cells (Figure 5b). Generation of NO with time (0-48hrs.), reflected the increase in stained cells, however, there was a considerable lag in time between the expression of iNOS in the cells and the secreted NO measured in the medium. Addition of surfactant lipids decreased the number of cells expressing iNOS with all the agonists tested except for INF- that was unaffected by the added surfactant (data not shown).

Figure 5. Stimulation by various agonist iNOS expression (a) and NO production (b) in NR-8383 alveolar macrophages cell line; SP-A 1µg/ml, SP-D 1µg/ml LPS 0.1µg/ml INF 25 units/ml. The cells in Figure 5a were immunostained for iNOS and counted at various times following stimulation with the various agonists.

44

Modulation of Nitric Oxide Production by Lung Surfactani

The increase in the number of cells immunostained with iNOS, with time is also reflected in the expression of the 130 kDa isoform of the enzyme. Increase with time (0-30 hrs.) in the expression of iNOS in the NR-8383 alveolar macrophages is presented in Figure 6. The rate of increase in iNOS expression is varied according to agonist. The expression of iNOS stimulated by SP-A and SP-D appears to be slower than that of LPS and Interferon- (Figure 6, 8 hr.). Addition of Curosurf or Exosurf suppressed the expression of iNOS with all the agonists tested in the cells.

a Hours

0

8

19

SP-A

+

+

SP-D

curosurf

–

+

30

SP-A SP-D LPS

b +

+

-

+

LPS

+

+

- +

Figure 6. Expression of iNOS (130kDa) in NR-8383 cell line following stimulation with SPA (1µ g/ml), SP-D (1µ g/ml), LPS 0.1µ g/ml); a) time response, b) inhibition with Curosurf (500µ g/ml).

In another set of experiments we tested the possibility of synergism between SP-A and SP-D, as well as between SP-A, SP-D, LPS and IFN-γ Results presented in Table 1 indicate a synergism in NO production with SP-A and SP-D added together at various concentrations. An increase of 167% in NO production was observed with SP-A (1 µg/ml) and SP-D (0.3µg/ml) added together (this increase is above the level of NO production obtained with the collectins added separately). A synergism was observed between SP-A, SP-D and IFN-γ This increase was much

Kalina et al.

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higher (125%) than that observed with I F N shown).

added t o L P S (data n o t

% increase of NO production* SP-A 0.01 µgml

SP- A 0.1 µgml

SP-D0.0034µgml

4.6%

26.4%

48.9%

SP-D0.034µgml

32.5%

29 %

52.3 %

SP- A I µgml

SP-DO.34µgml 14.2% 47% 167.% * The increase in NO production by SP-A + SP-D is after the deduction of the contribution in NO production by each of the collectins added separately

4.

DISCUSSION

A major threat at the air-liquid interface is the invasion of airborneParticles and microorganisms inhaled into the lung and deposited within two cell thickness’ of the blood stream. The lung, therefore, must have an effective mechanism of recognition and clearance of potentially harmful pathogens. Our recent understanding is that some of the surfactant components, proteins and lipids could be considered as first line defense molecules which function in the alveoli as part of the innate lung defense. The data presented extend our knowledge regarding the non surfactant-related functions of lung surfactant. Both SP-A and SP-D were found to upregulate NO production by alveolar macrophage cell line NR8383 in a concentration–and time –dependent manner. The increase in NO production was associated with elevation in the expression of iNOS in the cells. Only 50% of the alveolar macrophages responded by expressing iNOS. The percentage of the responding cells were similar in all the agonists tested (surfactant proteins, LPS and IFNThese results are similar to previously published data with primary cells either in vivo or invitro (Blau et al., 1997, Warner et al., 1995). At the present time, the reason for this phenomenon is unclear. Special attention was given to the possibility that contaminating LPS in the SP-A and SP-D preparations is responsible for the NO release. The LPS inhibitor polymixin B was added in some experiments and inhibited alveolar macrophages NO production by -20% when cells were stimulated with SP-A or SP-D. Over 80% inhibition was observed when the cells were stimulated with LPS. These results are comparable to those observed previously with rat alveolar macrophages (Blau et al., 1997).

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Modulation of Nitric Oxide Production by Lung Sufactani

Similar results were also observed when various recombinant SP-A species were used to stimulate NR-8383 cell line (Damodarasamy et al., 1999). The lipid constituents of surfactant used in the present study included a) synthetic surfactant – Exosurf b) a natural derived surfactant preparation - Curosurf and c) various purified phospholipids added to the cells as liposomes. The two surfactant preparations inhibited NO production stimulated by both SP-A and SP-D as well as LPS. Inhibition of INF- stimulated NO production was much less effective. Similar downregulation by surfactant lipids was perviously observed in various experimental systems (Thomassen et al., 1992, 1994, 1995, Kremlev et al., 1994, 1996, 1997). Many of the surfactant replacement therapies have been shown to inhibit cytokine release from various immune cells (reviewed in ref.1). These cytokines included TNF-α IL-lβ and IL-6 (Kremlev et al., 1996). It has been shown that SP-A increases inflammatory cytokine production (IL- 1, IL-6, TNF-α in alveolar macrophages and human monocytes (Kremlev et al., 1994, 1996), which was down regulated by artificial surfactant. Modulation of cell surface markers by SP-A and surfactant lipids were found in THP-1 monocytic cell line (8). The use of purified phospholipids indicated that DPPC is one of the active ingredients in the suppression of SP-A induced NO production in NR-8383 cells. DPPC is an ingredient in both Exosurf and Curosurf used in the present study. NO has been demonstrated to exert invitro microbicidal or microbiostatic activity against a rapidly expanding list of pathogens (MacMicking et al., 1997). Recently NO has been associated with protection against bacterial infections. (Tsai et al., 1997) demonstrated that NO is required and a critical component for an effective innate immunity against Klebsiella pneumoniae. It has also been suggested that NO is associated with the killing mechanism of Mycobacterium bovis BCG in human alveolar macrophages (Nozaki et al., 1997). The authors found an increased production of iNOS in BCG-innoculated alveolar macrophages from patients with pulmonary fibrosis.

5.

CONCLUSION

The data presented in this paper adds to the notion that surfactant Lipids and proteins are multifunctional, acting both to reduce surface tension and to modulate immune cell functions. The modulation of NO production by the surfactant constituents is similar to that described for various inflammatory cytokines; stimulation by the collectins and downregulation by the lipids. Many pulmonary diseases are associated

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with altered levels of surfactant lipids and proteins. Changes in surfactant composition have been reported in sarcoidosis, ARDS, pulmonary fibrosis and more diseases (Hamm et al., 1992, Fhelps and Rose, 1991). This data suggests that surfactant constituents may play an important role in vivo in the local immune regulation of host defense in the alveolar spaces of the lung and that altered regulatory mechanisms could be involved in the pathogenesis of some lung diseases.

ACKNOWLEDGMENTS The work from our laboratory was supported by a grant from the Israeli Academy of Science.

REFERENCES Blau H., Riklis, S., Van Iwaarden J.F., McCormack, and F.X., Kalina, M.. 1997. Nitric oxide production by rat alveolar macrophages can be modulated in vitro by surfactant protein A. Am. J. Physiol. 272: (Lung cell Mol. Phyiol. 16). L1 198L 1204. Crouch, E.C., 1998, Collectins and pulmonary host defense. Am. J. Respir. Cell Mol. Biol. 19: 177-201. 6 Damodarasamy, Zhang, M., Kalina, M., and McCromack F.X. 1999. The Cys interchain disulfide bond and the collagen-like domain of surfactant protein A are required for stimulation of NO release from alveolar macrophages . Resp. and Crit. Care. Med. 159:No. 3, A.506. Dijkstra, C.D., Dopp, E.A. Joling, P. Kraal G., 1985. The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulation in the rat recognized by the monoclonal antibodies ED1, ED2 and ED3 . Immunology 54:589601. Hamm, H., Fabel, H., and Bartch W. 1992. The surfactant system of the adult lung. Physiology and clinic1 prospectives. Clin. Investig. 70: 637-650. Helmke, R.J., Boyd, R.L., German, V.F., and Mangos J.A., 1987. From growth factor dependence to growth factor responsiveness. The genesis of alveolar macrophages cell line. Invitro cell. Develop. Biol. 23: 867-574 Kremlev, S.G. Umstead, T.M., and Phelps, D. 1996. Surfactant protein A regulate cytokine production in the monocytic cell line THP-I. Am. J. Physiol. 272: (Lung cell. Mol. Physiol. 16) L916-L929. Kremlev, S.G., and Phelps, D. 1997. Effects of SP-A and surfactant lipids on expression of cell surface markers in the THP-1 monocytic cell line. Am. J. Physiol. 272: (Lung cell Mol. Physiol. 16) L153-L161. Kremlev, S.V., and Phelps, D. 1994. SP-A Stimulation of inflammatory cytokines and immunoglobulin production. Am. J. Physol. 267: (Clin. Cell. Mol. Physiol. 11) L 7 12-L7 19.

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MacMicking, J.D. Xie, Q.W., and Nathan, C. 1997. Nitric oxide and macrophage function. Ann. Rev. Microbiol. 15: 323-350. McCormack, F.X., Calvert, H. M., Watson P.A. Smith D.L., Mason R.J., and Voelker D. 1993. The structure and Function of Surfactant Protein A. Biol. Chem. 269: 58335841. Nicolas, D.J.D., and Nason.A., 1957. Nitrate determination by diazotization and coupling reactions . In: Methods in Enzymology (edited by Colowick, S.P. and Kaplan N.O. New York Academic. 3: 983-984 Nozaki, Y., Hasegwa, Y., and Shimokata, K. 1997. Nitric oxide dependent killing mechanism of micobacterium bovis BCG in human alveolar macrophages. Respiratory and Crit. Care. Med. 155 No. 7: A-336. Paul, A., Doherty K., Pelvin R. 1997. Differential regulation by protein kinase C isoforms of nitric oxide synthase induction in RAW 264.7 macrophages and rat aortic muscle cells. Brit. J. pharmacol. 120: 940-946. Phelps, D.S., and Rose, R.M. 1991. Increased recovery of SP-A in AIDS related pneumonia. Am. Rev. Respir. Dis. 143: 1072-1080. Raetz, C.R.H, Ulevitch, R. J. Wright, S.D., Sibley, C.H., Ding. A., and Nathan, C.F. 199 1. Gram-negative endotoxin: an extraordinary lipid with profound effects on eukaryotic signal transduction. FASEB J. 5: 2652-2660. Thomassen, M.J., Antal, J.M., Connors, M.J., Meeker D.P., and Wiedemann, H.P., 1994. Characterization of exosurf (surfactant) mediated suppression of stimulated human alveolar macrophage cytokine responses. Am. J. Resp. Cell Mol. Biol. 10:399-404. Thomassen, M.J., Antal, J.M., Divis, L.T. , and Wiedemann, H.P., 1995. Regulation of human alveolar macrophages inflammatory cytokines tyloxapol: a component of the synthetic surfactant Exosurf. Clin. Immunol. Immunopathol. 77: 201-205. Thomassen, M.J., Meeker, D.P., Antal, J.M., Connors, M.J., and Wiedemann, H.P., 1992. Synthetic surfactant (Exosurf) inhibits endotoxin-stimulated cytokine secretion by human alveolar macrophages. Am. J. Resp. Cell Mol. Biol. 7: 257260. Tsai .W. C. Strieter R. M., Zisman, D.A., Wilkowski J.M., Buckness K.A., Chen, G-H., and Standiford T. J. 1997. Nitric oxide is required for effective innate immunity against Klebsiella pneumoniae. Infection anf Immunity.65, No. 5: 1870- 1815. Warner, R.L., Paine III R., Christensen P.J., Marietta M.A., Richards M.K., Wilcoxen S.E., and Ward P.A. 1995. Lung sources and cytokine requirements for in vivo expression of inducible nitric oxide synthase. Am. J. Respir. Cell. Mol. Biol. 12: 649-661. Wright, J.R. 1997, Immunomodulatory functions of surfactant. Physiol. Rev. 77: 93 1962. Wright, J.R., Wager, R.E., Hawgood, S., Dobbs, L., and Clements J.A. 1987. Surfactant apoprotein M2-26000-36000 enhances uptake of liposomes by type II cells. J. Biol. Chem. 262: 288-294.

DEVELOPMENT OF CHIMERIC COLLECTINS WITH ENHANCED ACTIVITY AGAINST INFLUENZA A VIRUS ¹Kevan L. Hartshorn, ¹Mitchell R. White, 2R. Alan. B. Ezekowitz, 1 Kedamath Sastry, and 3Erika Crouch ¹Boston University School of Medicine, Boston, MA, , and 2Harvard Medical School, 3 Boston, MA, Washington University School of Medicine, St. Louis, MO USA

1. 1.1

INTRODUCTION The role of collectins in host defense: general considerations and role in Influenza A virus (IAV) infection

The collectins are a group of collagenous lectin proteins present in mammalian serum, pulmonary and gastrointestinal secretions which participate in first line host defense against a variety of pathogens including bacteria, viruses and yeast (Sastry and Ezekowitz 1996). The most compelling human evidence thus far for the host defense role of collectins comes from the observation that subjects who have deficiency of serum mannose-binding lectin (MBL) are at increased risk for various infections (Garred et al 1997, Turner 1996)). MBL structurally resembles Clq and can fix complement through interaction with the MBL-associated serine protease (MASP). MBL has been shown to bind to a wide variety of bacteria, yeast, and viruses through recognition of distinctive microbial surface carbohydrate patterns. MBL can participate The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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Development of Chimeric Collectins

in host defense by direct inhibition of infectivity, deposition of complement, and promotion of phagocytosis. Two additional collectins have been identified in bovine serum conglutinin and CL43. In vitro evidence suggests that these collectins may play a role in host defense. Although conglutinin cannot fix complement it can bind to iC3b which has been deposited on red blood cells or bacteria. Conglutinin levels in ox serum decline during infection and at parturition possibly as a result of deposition on complement coated cells. Conglutinin has shown opsonic activity against bacteria but only in the presence of complement (Friis-Christiansen et al 1990). Subcutaneous injections of conglutinin increased survival of mice inoculated with Salmonella typhimurium. Recent murine data strongly suggests a host defense role for the surfactant collectin, surfactant protein A (SP-A). Mice which have targeted disruption of the SP-A gene have normal lung functions but clearly increased susceptibility to bacterial infections (LeVine et a l1 997). Surfactant protein D (SP-D) knockout mice have marked abnormalities in surfactant phospholipid metabolism (Korfhagen et al 1998). Results of tests of the susceptibility of SP-D knockout mice to infection are awaited with interest. Initial evidence that calcium-dependent lectins play a role in innate immunity to influenza A virus (IAV) came with the discovery by E.M. Anders et al that bovine and mouse serum binhibitors of IAV infectivity are mannose-binding lectins (Anders et al 1990). These elegant studies demonstrated that, 1. these mammalian serum inhibitors were calciumdependent lectins, and 2. viral strains develop resistance to B-inhibitors through point mutations leading to loss of a high mannose oligosaccharide attachment on the globular domain of the viral hemagglutinin (HA) protein. Subsequent studies confirmed that both human MBL and bovine serum conglutinin inhibit IAV infectivity and hemagglutination activity (HA activity) (Hartley et al 1992, Hartshorn et al 1993a b, Wakamiya et a1 1992) and that neutralization of IAV infectivity by MBL present in serum involves activation of the classical complement pathway (Anders et al 1994). Purified SP-D and SP-A were then shown to inhibit infectivity and HA activity of IAV as well (Benne et al 1995, Hartshorn et al 1994). Human bronchoalveolar lavage fluids were shown to contain sufficient concentrations of SP-D to inhibit IAV HA activity, and depletion of SP-D from BAL reduced its HA inhibitory effects (Hartshorn et al 1994). Although all evidence thus far indicates that the serum collectins and SP-D inhibit IAV infectivity through attachment of their carbohydrate recognition domain (CRD) to carbohydrate components of viral envelope

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glycoproteins, SP-A activity is mediated by non-calcium-dependent attachment of IAV to carbohydrate attachments on SP-A (Benne et al 1995, Hartshorn et al 1994, Reading et al 1998b). SP-A has N-linked carbohydrate attachments to the N-terminus and the CRD. SP-D, in contrast, has only one N-linked oligosaccharide located in the collagen domain of the molecule. Deletion of this carbohydrate attachment through site-directed mutagenesis did not reduce the ability of SP-D to inhibit viral HA activity (Hartshorn et al 1997). Although no data is available as yet regarding IAV infection in SP-A or SP-D knockout mice, other murine studies do suggest a role for surfactant collectins in defense against IAV. Strong negative correlations have been demonstrated between the ability of SP-D or MBL to inhibit infectivity of various IAV strains in vitro and replication of these strains in mice after intranasal inoculation (Hartley et al 1997, Reading et al 1997). Co-administration of mannose-containing saccharides along with virus resulted in markedly increased replication of IAV in the lung. In additional studies, Reading et al (1 998a) demonstrated that SP-D sensitive strains of IAV replicated to higher titers in lungs of diabetic mice as compared to nondiabetic controls, that replication was positively correlated with blood glucose levels, and could be reduced with insulin treatment. These findings could account for the enhanced susceptibility of diabetic patients to IAV infection. Overall these results indicate that surfactant collectins - and in particular SP-D - contribute significantly to containment of IAV infection in vivo.

2.

VARIATIONS IN CARBOHYDRATE BINDING SPECIFICITY AND QUATERNARY STRUCTURE OF COLLECTINS: FUNCTIONAL SIGNIFICANCE WITH RESPECT TO IAV INFECTION

2.1.

Inhibition of Viral Infectivity and HA Activity: Relationship to CRD

The collectins differ in terms of the affinity for binding specific monosaccharides. SP-D has a relatively stronger affinity for binding glucose or maltose and lower affinity for binding GlcNAc, while the reverse pattern is observed for MBL and conglutinin. All of the collectins bind mannose with high affinity and have relatively low affinity for galactose. The significance of differences among collectins in

52

Development of Chimeric Collectins

monosaccharide affinity in terms of host defense functions has not been studied. As noted SP-D, MBL, and conglutinin inhibit HA activity and infectivity of IAV through attachment to virus-associated carbohydrates (Hartshorn et al 1994, 1993a, b). Among the collectins, conglutinin is most potent at inhibiting infectivity of IAV (Hartshorn et al 1996a, Reading et al 1998b). Although MBL had markedly reduced capacity to aggregate IAV particles compared to SP-D, we found that its ability to neutralize infectivity of IAV was comparable to that of recombinant rat SP-D ((Hartshorn et al 1999) and unpublished data). These results suggested to us that the carbohydrate binding profiles of conglutinin and MBL might be more favorable for inhibiting infectivity of IAV than that of SP-D. However, differences in N-terminal and collagen domains of these collectins could have substantial impact on the binding properties of their CRDs (i.e. by affecting cooperative interactions among CRD domains in a given molecule). Studies using isolated recombinant CRD preparations have demonstrated greater inhibition of HA activity of IAV for the CRD of conglutinin than that of SP-D (Eda et al 1996, 1997, Hartshorn et al 1997). However, such preparations have markedly reduced antiviral activity overall compared to fully multimerized collectins. We therefore decided to produce recombinant full length collectin chimeras which shared the same N-terminal and collagen domains and differed only in their CRDs in order to more definitively compare functional contributions of the CRDs (see below).

2.2.

Viral aggregation and enhancement of phagocytosis: relationship to N-terminal and collagen domains

The collectins can be divided into two families based on their quaternary structure. The quaternary structure of the collectins is determined by the N-terminal and collagenous domains, while carbohydrate binding is mediated by the CRD. All of the collectins initially form trimers so that the CRD contains three carbohydrate binding sites. In the case of MBL, SP-D, SP-A and bovine conglutinin the basic trimers are associated together in higher order multimeric structures. MBL and SP-A commonly are composed of 6 trimers in association (octadecamers) which form structures closely resembling that of Clq. In contrast, conglutinin and SP-D have much larger collagen domains than SP-A or MBL. Conglutinin and SP-D most commonly form cruciate dodecameric structures (i.e. with 4 trimers attached at their N-terminus) in which the distance between CRDs is much greater than for

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MBL or SP-A. SP-D also exists in vivo (or in recombinant preparations) as trimers and very high order multimeric structures (i.e. containing up to 32 globular CRD heads in one molecule) (Crouch et al 1994, Hartshorn et al 1996a). The bovine serum collectin CL43 forms trimers of similar size to those of conglutinin, but does not assemble into higher order multimeric structures (Rothmann et al 1997). Our studies have suggested that properties of N-terminal and collagen domain of collectins have important impact on the ability of collectins to aggregate viral particles and alter viral interactions with phagocytic cells. SP-D and conglutinin were found to induce massive viral aggregation, whereas SP-A and MBL - even al considerably higher concentrations than were required for SP-D or conglutinin - induced formation of much smaller viral aggregates (Hartshorn et al 1997). Similar findings have been obtained comparing the ability of MBL and SP-D to agglutinate bacteria (Hartshorn et al 1998 and unpublished data) or yeast (Schelenz et al 1995). Collectins can act as opsonins for IAV, promoting binding and uptake of the virus by neutrophils. Furthermore, pre-incubation of IAV with the collectins caused marked enhancement of the ability of IAV to act as a stimulus of neutrophil hydrogen peroxide production. The ability of collectins to enhance neutrophil binding of, or respiratory burst responses to, IAV was found to be associated with the ability of various collectins to induce aggregation of viral particles (Brown-Augsburger et al 1996, Hartshorn et al 1996a, b). The degree of multimerization of SP-D preparations correlated both with their ability to induce viral aggregation and to enhance neutrophil binding of IAV. Similar results were obtained with respect to the ability of various SP-D preparations to aggregate and promote neutrophil uptake of bacteria (Hartshorn et al 1998). MBL had a markedly reduced ability to promote neutrophil binding of IAV as compared to SP-D or conglutinin, consistent with the finding that it caused minimal viral aggregation (Hartshorn et al 1997). We hypothesised that the marked differences in the ability of MBL and SP-D to induce viral aggregation or promote viral uptake by neutrophils was due to the obvious differences in size and structure of their N-terminal and collagen domains. A more subtle but statistically significant difference was also noted between the potency of conglutinin and similarly multimerized (i.e. dodecameric) preparations of RhSP-D and RrSP-D at enhancing binding of IAV to neutrophils (Hartshorn et al 1996b). We hypothesised that differences between N-terminal and collagen domain regions of conglutinin and SP-D might account for the relatively greater viral osponizing activity of SP-D. The collagen domain of SP-D is longer than that of conglutinin by 6 amino acids and does not

54

Development of Chimeric Collectins

have an interruption in the GlyXaaYaa sequence found in the collagen domains of the other collectins. We constructed chimeric collectins to compare the specific functional contributions of the collagen domains of SP-D, MBL and conglutinin.

3.

CONSTRUCTION OF COLLECTIN CHIMERAS TO DETERMINE CONTRIBUTION OF SPECIFIC DOMAINS TO ANTIVIRAL AND OPSONIC ACTIVITIES

Two chimerae were constructed: an SP-D/Conglutinin chimera containing the N-terminal and collagen domains of recombinant rat SP-D (RrSP-D) and the neck region and CRD of bovine conglutinin, and an SP-DMBL chimera containing the N-terminal and collagen domains of recombinant human SP-D (RhSP-D) and the neck region and CRD of human MBL (Hartshorn et al 1999, White et al 1999). Our goals were to test the hypotheses that, 1. differences in ability to neutralise viral infectivity result from differences in carbohydrate binding properties, 2. differences in viral and bacterial aggregation among collectins result from differences in their N-terminal and collagen domains, and 3. that differences in opsonizing activity among collectins result from differences in N-terminal and collagen domains. The chimeric collectins were constructed by a PCR-based strategy using synthetic primers derived from sequences of SP-D and bovine conglutinin or human MBL. The collectin constructs were expressed in CHO-K 1 cells and purified using Carbohydrate affinity chromatography. Comparisons of functional activity were made using dodecameric fractions (purified by gel filtration chromatography) of the climerae and wild type recombinant conglutinin or SP-D to insure that functional differences were not the result of differences in multimerization. Extensive studies with the SP-D/Conglutinin chimera have been completed and preliminary data are available regarding the SP-D/MBL chimera

3.1.

RrSP-D/conglutinin and RhSP-D/MBL chimeras have altered carbohydratebinding specificity and enhanced IAVneutralizingactivity compared with wild type SP-D

The SP-D/Conglutinin chimera had significantly greater HA inhibitory activity than RrSP-D (See Table I). The SP-DMBL chimera had similar

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HA inhibitory activity to SP-D/Conglutinin (data not shown). To compare monosaccharide specificities of the chimeric collectins with those of wild type SP-D, we tested the ability of various monosaccharide preparations to interfere with the ability of collectins to inhibit IAV HA activity. Whereas glucose strongly interfered with HA inhibitory activity of SP-D, GlcNAc did not. In contrast, GlcNAc strongly interfered with HA inhibitory activity of the SP-D/MBL or SP-D/Conglutinin chimeras, while glucose did not (data not shown). Table 1: Concentration of Wild Type and Chimeric Collectins Required to Inhibit

RrSP-D RbConglutinin

29 ±4.3 7.3±1.8

14±5 3±0.3

* Significantly lower than all other collectins tested vs. Bangkok79 IAV strain (p<0.05)

Table 2 shows that the chimeric collectins had significantly greater potency at inhibiting infectivity of IAV than the wild type collectins from which they derived. Note in particular that RrSP-D/Conglutinin chimera was markedly more inhibitory of infectivity than RrSP-D. This result supports the concept that the relatively greater viral neutralizing activity of conglutinin as compared to SP-D results from properties of its carbohydrate recognition domain.

D/Conglut.

Chimera Infectivity was measured using a fluorescent focus assay. Results represent percent of control infectious foci in collectin treated samples as compared to controls (mean SEM of 3 or more experiments are shown). Samples of Phil82 H3N2 IAV strain were incubated in PBS alone or PBS containing the indicated concentrations of collectins for 30 minutes prior to inoculation of MDCK monolayers. Infectious foci were identified after 7 hours through fixation of cells and labeling with mAb directed against the IAV nucleoprotein. This method was adopted from Reading et al (1997).

56

Development of Chimeric Collectins

The RhSP-D/MBL chimera had significantly greater IAV neutralizing activity than RhSP-D, although this difference was less marked than that between RrSP-D/Conglutinin and RrSP-D. This may be in part attributable to the fact that RhSP-D had significantly greater neutralizing activity than RrSP-D. Of note, a marked difference was noted between neutralizing activities of RhMBL and RhSP-D/MBL. These results suggest that carbohydrate recognition domain properties alone cannot account for neutralizing activity (i.e. since both RhMBL and RhSP-D/MBL have the same CRD). RhSP-D/MBL bound with greater affinity to IAV (data not shown) and induced significantly more viral aggregation (see below) than RhMBL. Hence, placement of the MBL CRD on the scaffold of the SP-D N-terminus and collagen domain significantly altered the ability of the CRD to bind to or crosslink IAV particles. These findings could account in part for differences in viral neutralization between MBL and RhSP-D/MBL.

3.2,

The SP-D/MBL chimera has markedly greater aggregating and opsonizing activity than wild type MBL

As shown in Table 3, the chimeric collectins caused a significantly greater degree of viral aggregation than the wild type collectins from which they were derived. Table 3: Aggregation of IAV Particles and Enhancement of Neutrophil Uptake of IAV by Collectins

a. Viral aggregation was assessed by measuring decrease in light transmission through a viral suspension as described (Hartshorn el al 1993a). Results shown are mean±SEM % of control light transmission 8 minutes after addition of 0.4µg/ml (RhSP-D, RhMBL or RhSP-D/MBL Chimera) or 0.8µg/ml (RrSP-D, RbConglutinin or RrSPD/Conglutinin chimera) of collectins. b. Neutrophil uptake of virus was measured by flow cytometry after incubation of FITClabeled IAV with 1.4µg/ml (RhSP-D, RhMBL or RhSP-D/MBL Chimera) or 2µg/ml (RrSP-D, RbConglutinin or RrSP-D/Conglutinin chimera) of collectins (as described Hartshorn et al 1997). Significantly greater aggregation than with RhSP-D or RhMBL Significantly greater aggregation than with RrSP-D or RbConglutinin * Significantly greater uptake than with RhMBL * * Significantly greater uptake than with RbConglutinin

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The most striking differences were between RhMBL and RhSPD/MBL. Replacement of the N-terminal and collagen domains of MBL with those of SP-D converted RhMBL from low to high aggregating activity. This change was also associated with a marked increase in ability to promote neutrophil uptake of IAV (Table 3). RhSP-D/Conglutinin also caused a significantly greater degree of viral aggregation, and increased uptake of IAV to a greater extent, than RbConglutinin. Of interest, RhSPD/MBL and RrSP-D/Conglutinin also caused significantly greater viral aggregation than RhSP-D or RrSP-D, respectively.

4.

CONCLUSION

These studies demonstrate that it is possible to alter the carbohydrate recognition and viral neutralizing activities of SP-D through replacing the SP-D CRD with that of either conglutinin or MBL. These proteins have been useful tools in confirming that, 1. the enhanced ability of conglutinin to inhibit IAV infectivity as compared to SP-D results from differences in the CRDs of these proteins, and 2. the substantial greater viral aggregating and opsonizing activities of SP-D as compared to MBL result from differences in the N-terminal and collagen domains of these molecules. The chimeric collectins had significantly greater viral aggregating and neutralizing activity than either parent collectin from which they were derived. Hence, such constructs could be of therapeutic value in treatment of IAV infection. Although the two chimerae had similar activity against IAV, substantial differences were noted between their ability to bind to or aggregate bacteria (Hartshorn et al, unpublished data). Hence, there are probably significant differences between the functional properties of the CRD of conglutinin and that of MBL which need to be examined in further studies.

REFERENCES Anders, E.M., Hartley, C.A., and Jackson, D.C., 1990, Bovine and mouse serum B inhibitors of influenza A virus are mannose-binding lectins. Microbiology 87:44854489. Anders, E.M., Hartley, C.A., Reading, P.C., and Ezekowitz, R.A.B., 1994, Complementdependent neutralization of influenza virus by a serum mannose-binding lectin. J. Gener. Virol. 75:615-22. Benne, C.A., Kraaijeveld, C.A., van Strijp, J.A.G., Brouwer, E., Harmsen, M., Verhoef, J., van Golde, L.M.G., and van Iwaarden, J.F.,1995, Interactions of surfactant protein A with influenza A viruses: Binding and neutralization. J. Infect. Dis. 171:335-41.

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Brown-Augsburger, P., Hartshorn, K.L., Chang, D., Rust, K., Fliszar, C., Welgus, H., and Crouch, E.C., 1996, Site directed mutagenesis of Cys15 and Cys20 of pulmonary surfactant protein D: expression of a trimeric protein with altered anti-viral properties. J Biol Chem 271:13724-13730. Crouch, E, et al 1994, Recombinant pulmonary surfactant protein D. J. Biol. Chem. 269: 15808- 15813. Eda, S., Suzuki, Y., Kase, T., Ohtani, K., Sakamoto, T., Kurimura, T., and Wakamiya, N., 1996, Recombinant bovine conglutinin, lacking N-terminal and collagenous domains, has less conglutination activity but is able to inhibit haemagglutination activity of influenza A virus. Biochem J 1996:43-48. Eda, S, Suzuki, Y., Kawai, T., Kase, T., Ohtani, K., Fujinaga, Y., Sakamoto, T., Kurimura, T., and Wakamiya, N., 1997, Structure of a truncated human surfactant protein D is less effective in agglutinating bacteria than the native structure and fails to inhibit haemagglutination by influenza A virus. Biochem J 323:393-399. Friis-Christiansen, P., Theil, S., Svehag, S.E., Dessau. R., Svendsen, P., Andersen, O., Laursen, J.B., and Jensenius, J.C., 1990, In vivo and in vitro antibacterial activity of conglutinin, a mammalian plasma lectin. Scand. J. Immunol. 3 1:453-460. Garred, P. Madsen, H.O., Balslev, U., Hofmann, B., Pederson, C., Gerstoft, J., and Svejgaard, A., 1997, Susceptibility to HIV infection and progression of AIDS in relation to variant alleles of mannose-binding lectin. Lancet 349:236-240. Hartley, C.A., Reading. P.C., Ward, A.C., and Anders, E.M., 1997, Changes in hemagglutinin molecule of influenza type A (H3N2) virus associated with increased virulence in mice. Arch of Virol 142:75-88. Hartley, C.A., Jackson, D.C., and Anders, E.M. 1992, Two distinct serum mannosebinding lectins functions as B inhibitors of influenza virus: identification of bovine serum B inhibitor as conglutinin. J. Virol. 66:4358-4363. Hartshorn, K.L., Chang, D., Rust, K., White, M.R., Heuser, J, and Crouch, E.C., 1996a, Interactions of recombinant human pulmonary surfactant protein D and SPD multimers with influenza A. Amer J Physiol 271:L753-762. Hartshorn, K.L., Crouch, E.C., White, M.R., Colamussi, M.L., Kakkanatt, A., Tauber, B., Shepherd, V., and Sastry, K., 1998, Pulmonary surfactant proteins A and D enhance neutrophil uptake of bacteria. Amer J Physiol 274:L958-69. Hartshorn, K.L., Crouch, E.C., White, M.R., Eggleton, P., Tauber, A.I., Chang, D., and Sastry, K.,. 1994, Evidence for a protective role of pulmonary surfactant protein D (SP-D) against influenza A viruses. J. Clin. Invest. 94:311-319. Hartshorn, K.L., Reid, K., White, M.R., Jensenius, J.C., Moriis, S.M., Tauber, A.I., and Crouch, E.C., 1996b, Neutrophil deactivation by influenza A viruses: mechanisms of protection after viral opsonization with collectins and hemagglutination-inhibiting antibodies. Blood 87:3450-3461. Hartshorn, K.L., Sastry, K.N., Chang, D., White, M.R., Crouch, E.C., 1999, Enhanced anti-influenza activity of a recombinant pulmonary surfactant protein D and serum conglutinin fusion protein. Amer. J. Physiol. In press. Hartshorn, K.L., White, M.R., Shepherd, V., Reid, K., Jensenius, J.C., and Crouch, E.C., 1997, Mechanisms of anti-influenza activity of pulmonary surfactant proteins A and D: comparison with other collectins. Amer J Physiol 273:L1156-1166. Hartshorn, K. L., Sastry, K., Brown, D., White, M.R., Okarma, T.B., Lee, Y., and Tauber, A.I., 1993a, Conglutinin acts as an opsonin for influenza A viruses. J. Immunol. 151 :I-9.

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Hartshorn, K.L., Sastry, K., White, M.R., Anders, E.M., Super, M., Ezekowitz, R.A.B., and Tauber, A.I., 1993b, Human mannose-binding protein functions as an opsonin for influenza A viruses. J. Clin. Invest. 91:1414-1420. Korfhagen, T.R., . 1998, Surfactant Protein D regulates surfactant phospholipid homeostasis in vivo. J Biol Chem 273:28438-28443, LeVine, AM, Bruno M., Huelsman K.M., Ross G.F., Whitsett J.A., and Korfhagen T.R., 1997, Surfactant Protein A deficient mice are susceptible to group B streptococcal infection. J. Immunol. 158:4336-4340. Reading, PC, Allison, J., Crouch E.C., Anders E.M., 1998a,Increased susceptibility of diabetic mice to influenza virus infection: compromise of collectin-mediated host defense of the lung by glucose? J Virol 72:6884-6887. Reading, PC, U Holmskov, and EM Anders, 1998b, Antiviral activity of bovine collectins against rotaviruses. J Gen Virol 79:22SS-2263. Reading, P.C. Morley L.S., Crouch, E.C., and Anders E.M., 1997, Collectin-mediated antiviral host defense of the lung: evidence from influenza virus infection of mice. J Virol 71 :8204-8212. Rothmann, A.B., Mortensen, H.D., Holmskov, U., and Hojrup, P., 1997, Structural characterization of bovine collectin-43. Eur J Biochem 243:630-635. Sastry, K.N., and Ezekowitz R.A.B., 1996, Collectins. In Collectins and innate immunity. R. Ezekowitz, K. Sastry, and K. Reid, eds. Pp. 1-7. Austin, Texas: RG Landes Co. Schelenz, S., Malhotra, R., Sim, R.B., Holmskov, U., and Bancroft, G.J., 1995, Binding of host collectins to the pathogenic yeast Cryptococcus Neoformans: human surfactant protein D acts as an agglutinin for acapsular yeast cells. Infect and Immun 63: 3 360-33 66. Turner, M.W., 1996, Functional aspects of mannose binding protein. In Collectins and innate immunity. K. Sastry and R. Ezekowitz. eds. Pp. 73-97. Austin, Texas: RG Landes. Wakamiya, N., Okuno, Y.,Sasao, F., Ueda, S., Yoshimatsu, K., Naiki, M., Kurimura, T.,1992, Isolation and characterization of Conglutinin as a influenza A virus inhibitor. Biochem. and Biophys. Res. Comm. 187: 1270- 1278. White, M.R., Crouch, E.C., Sastry, K., Guo, N., Enpelich, G., Ezekowitz, R.A.B., Hartshorn, K.L., 1999, Replacement of the collagenous domain of mannose binding lectin with that of surfactant protein D markedly alters aggregating, antiviral, and opsonic properties. In Review.

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INITIAL STEPS IN Streptococcus pneumoniae INTERACTION WITH AND PATHOGENICITY TO THE HOST

Michal Shani-Sekler¹, Sarit Lifshitz¹, Iris Hillel¹, Ron Dagan¹. Nili Grossman², Gideon Fleminger³, Yaffa Mizrachi-Brauner¹.² ¹Peclintric Infect. Dis. Unit. Soroka Med. Center; ²Dept. Microbiol & lmmunol., Fac. Health Sciences; Ben Gurion Univ., Beer Sheva. ³Dept. Microbiol & Biotechnol. Tel Aviv Univ. Tel Aviv.

1.

ABSTRACT

Streptococcus pneumoniae (Pnc) is one of the leading pathogens in the world. Attachment to respiratory mucosal and lung surfaces is presumed to be involved in carriage, in disease and in the interaction with macrophages initiating innate immune responses. We hypothesized that bacterial adhesins mediate Pnc adhesion and host cell invasiveness. Initial studies have focused on the purification of cell wall and membrane proteins using fetuin affinity chromatography, SDS PAGE and western blot analysis probed with pooled healthy human sera. Using a Pnc clinical isolate, and a gpt mutant we have detected I0-lectin proteins isolated from the cell wall and adherent to the affinity column and 15 lectins isolated from membrane extracts. The fetuin-captured lectins agglutinated rabbit erythrocytes. 15 proteins in the cell wall and 18 proteins in the membrane that failed to bind to the fetuin column did not agglutinate rabbit erythrocytes. Further purification of the cell wall and membrane fetuinseparated fractions was achieved via anion exchange FPLC, was verified by SDS PAGE. These proteins maintained their agglutinating activity, and were subsequently tested for their ability to interfere with Pnc adhesion and invasion of epithelial cells in culture. Additional The Biology and Pathology of lnnate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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Initial Steps in Streptococcus Pneumoniae Infection

biochemical, immunological and molecular techniques are being used in attempt to identify relevant proteins.

2.

INTRODUCTION

Streptococcus pneumoniae (Pnc) a gram-positive bacteria, is a major cause of morbidity and mortality worldwide. Despite the availability of penicillin therapy, Pnc pneumonia causes more deaths annually than almost any other infectious disease (CDC 1995). Mortality from Pnc infection is frequently a result of antibiotics resistance (Austeriank & Gold 1964). It has been suggested that the best way to protect these individuals was to develop a preventive vaccine. A 23-valent vaccine comprised of capsular polysaccharides most commonly associated with human diseases was developed (Robins et al 1983).i. However, this vaccine is only 60% effective in preventing Pnc invasive infection in the elderly (Shapiro et al 1991).ii, and its efficacy against pneumonia is debatable. Furthermore, this vaccine is unable to elicit adequate antibody response in children younger than 2 years old (Cowan et al 1978). A potential solution has been developed, based on studies performed in animal models, in the form of polysaccharide-protein conjugate vaccines (Avery & Goebel 1929). Indeed, the Pnc conjugate vaccines have been found to be more immunogenic in children than soluble polysaccharides (Kaythy & Eskola 1996). Although efficacious, several limitations to this form of vaccine exist: a) the number of different conjugates that can be used are limited; and b) variations of the common vaccine serotypes in different parts of the world necessitate more than one vaccine preparation to be produced. Further studies are needed for the development of the next generation of “universal” Pnc vaccines. The molecular mechanisms that underlay the life cycle of Pnc and hence the molecules involved in the pathogenicity of Pnc is poorly understood. Pnc like many other bacterial respiratory pathogens may colonize the nasopharynx without causing disease. However, either the emergence of a pathogenic strain, or the activation of the nasopharyngeal tract by an additional pathogen, may in turn enable virulent strains of the Pnc to cause a clinical infection (Austrian 1986, Alonso et al 1995, Thuomanen et al 1995). Pnc bind avidly to cells of the upper and lower respiratory tract (Thuomanen 1997). The mechanisms that underlie this attachment are presumed to involve adherence, which enables carriage without an overt inflammatory response. In the case of a clinical disease, adherent Pnc are internalized apically within vacuoles in polarized epithelial cells through receptor-mediated endocytosis (Thuomanen et al

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1995). Pnc containing vacuoles traversing the cytoplasm and depositing the Pnc basally ultimately lead to clinical bacteremia. The nature of the molecules involved in this process and the mechanisms controlling such complex procedures are unknown. Our working hypothesis is that, like most bacteria, adherence of Pnc to mammalian cells is mediated through proteins residing on the bacterial surface. Immune responses to these proteins are essential for providing protective immunity to the host. Recently it has been shown that Pnc surface proteins bind to at least five eukaryotic carbohydrates in a lectin like fashion (Cundel and Thuomanen 1994). The nature of one mammalian carbohydrate (N-acetyl Glucose Amine) carrying protein has been identified as the platelet activating factor receptor (PAF-R). This receptor is expressed following inflammation of activated lung and endothelial cells, probably involved in pathogenic stages of the bacterial infection (Cundel et al). The nature of other bacterial binding proteins on non activated cells is unknown. Several recent studies have hinted at the possibility that some proteins located in the Pnc cell wall (CW) are directly or indirectly involved in Pnc and mammalian cell interactions. Among these are bacterial permeases such as the PsaA (Sampson et al 1994). Other candidates are the group of choline binding proteins (Cbp), bound to the choline component of the CW teichoic acid or lipoteichoic acids of Pnc (Ronda et al 1987). PspA, a Cbp, was shown to illicit protective antibodies that can prevent or delay lethal Pnc infection in mice (McDaniels et al 1992). Additional choline binding proteins were identified of which CbpA has been more thoroughly investigated (Rosenow et al 1997). This protein has been shown to be involved in adhesion to activated cells, colonization and immunogenicity. Interestingly, Cbp expression was shown to vary between opaque and transparent variants of the bacteria correlating with their pathogenicity (Weiser et al 1994). Thus although some information regarding Pnc surface proteins has been described, the full picture of the Pnc life cycle has not yet been revealed. Our working hypothesis is that among the Pnc surface proteins we will find adhesins that are involved in pathogenesis of Pnc colonization, pathogenicity and virulence. In the current study we have identified Pnc immunogenic CW and membrane (M) proteins. We have characterized their functional role in the Pnc life cycle and have initiated identification of the described proteins.

64

3.

Initial Steps in Streptococcus Pneumoniae Infection

METHODS

The bacterial strains used in this study are: 1. An untypable clinical isolate. 2. A parental type 3 strain. 3. An unencapsulated subtype 3 strain produced by a Tn916 transposon mutation in the gpt gene parental strain (Kindly provided by Dr. D Watson, USA). The Pnc were plated into tryptic soy agar with 5% sheep blood and incubated for 17-18 hours at 35°C with 5-10% CO2. The Pnc were transferred to Todd-Hewitt broth + 0.5% Yeast extract, and were grown up, to their mid-late log phase. Bacteria were harvested at 4000rpm for 15 at 4°C. Bacterial pellets were stored at -70°C. Separation of cell wall (CW) and membrane (M) proteins was performed as follows: Bacterial pellets were sonicated and centrifuged. The pellet was treated with RNase and DNase and centrifuged again. The pellet was treated with lysozyme to release CW proteins. This preparation was again centrifuged and the membrane pellet was solubilized using 0.5% Triton X-100. CW and diluted M protein (x10) supernatant were adhered to a fetuin agarose column for 2 hours in 50 mM phosphate buffer pH 7.4 (PB). The flow through (nonlectins; NL) was collected and the column was then washed with an additional 15 ml PB. The adhered CW proteins (lectins; L) were eluted with 50 mM ammonium acetate at pH 3.5. 0.5 ml samples were collected. Samples were dried in a Speed Vac and resuspended in 10mM Phosphate buffer. Four fractions were obtained CW L; CW-NL; M-L and M-NL. The agglutination assay was performed as follows: Rabbit whole blood was washed x3 in PBS. A suspension of 3% erythrocytes was prepared in PBS. 50µl of this suspension was mixed in U bottom, 96 well plates with protein samples at various dilutions isolated from the fetuin or FPLC columns. The adhesion assay was performed as follows: epithelial cells were grown to confluence on 96 well plates in DMEM medium supplemented with 10% fetal calf serum and 100µg/ml penicillin and 100µg/ml streptomycin. Sixteen hours prior to the adhesion experiment the culture medium was changed to culture medium without antibiotics. The cultures were blocked with DMEM medium supplemented with 2.5% bovine serum albumin (DMEM-BSA) for 4 hours. Bacteria were grown to mid logarithmic phase and incubated with human sera at the denoted concentrations for 30 minutes and were then added to the cells. Following 30 minutes incubation the cells were extensively washed with PBS-BSA (x6). 100µ1 of 1mM EDTA in PBS was added for 5 minutes at 37°C. 30µ1 of this suspension was cultured on sheep blood agar petri dishes for 18 hours. Colonies were counted and the CFU/ml were calculated. Separation of proteins was achieved by FPLC: The various fractions were separated on a Mono Q column. The samples were

Shani-Sekler et al. injected and the column washed with 50mM PB pH 8. performed with a 1M NaCl gradient.

4.

65

Elution was

RESULTS

An unencapsulated mutant (a gpt mutant from serotype 3) was used to rule out interference by the capsular polysaccharide with the fetuin column. Similar results were obtained with an untypable clinical isolate. Using fetuin affinity chromatography we separated the CW protein into fetuin adherent (lectin; CW-L) and fetuin non-adherent (non-lectin, CWNL) protein fractions and membrane fetuin adherent (lectins; M-L) and fetuin non-adherent (non-lectin; M-NL) protein fractions. The fetuincaptured lectins agglutinated rabbit erythrocytes, the non-captured proteins did not. These fractions were subjected to SDS PAGE separation and western blot analysis with pooled adult human sera (Figure 1).

Figure 1, Western blots of proteins from gpt Pnc mutant. Cell wall (A) and membrane (B) proteins seperated by fetuin affinity chromatography were reacted with pooled healthy adults sera. Lane1. NL, 1st ml. Lane 2. NL, last ml. Lanes 3-7. Fetuin column ammonium acetate pH3.5 eluted proteins fractions 1-5 respectively.

Negative controls included samples without primary human sera, or probing bacterial protein blots with an anti phosphoryl choline monoclonal antibody, both of which failed to highlight any bands on the nitrocellulose blots. Normal, adult sera were used throughout this study as a positive control. In the adult group (N=8), previous exposure to Pnc

66

Initial Steps in Streptococcus Pneumoniae Infection

was assumed by the presence of anti polysaccharides (PS) IgG (had high tested by ELISA to capsular polysaccharides). All adult sera contained anti Pnc PS antibodies. Five adults had high anti PS antibodies concentrations (>10µg/ml IgG to at least 1 of the PS antigens tested). 3 adults had low anti PS antibodies (<10µg/ml IgG to all 7 antigens tested). All the sera obtained from the healthy adult subjects reacted with the CW and membrane proteins (Table 1), while the intensity of the response was variable. There seem to be a correlation between low anti-PS antibodies and low intensity of anti Pnc surface protein antibodies as determined by densitometry. Adult # 1 2 3 4 5 6 7

anti Capsular PS (1; 5; 6B; 9V; 14; 19F; 23F ) <10µg/ml* 10<µg/ml* < 10µg/mlˆ >10µg/ml* >10µg/ml* >10µg/ml* 10>µg/mlˆ

CW-NL 14 19 15 17 16 11 18

CW-L

M-NL

M-L

14 7 0 2 6 10 8

11 11 14 13 11 11 12

15 15 16 16 11 14 15

*>10µg/ml IgG to at least 1 of the PS antigens tested IgG to all 7 antigens tested

ˆ<10µg/ml

To determine the extent of humoral immune responses in a more susceptible patient population, children attending Israeli day care centers (DCC) were tested, and belonged to 2 age groups (N=10). These children were carriers of various Pnc serotypes (Table 2). The anti Pnc protein antibodies found in these children cross-reacted with the serotype 3 proteins in our Pnc surface protein preparations. Antibody responses in these children varied considerably, as measured by Western blot densitometry. In order to longitudinally compare antibody responses to Pnc proteins sera was collected from DCC children starting at 19 months of age and followed over a period of 2 years. In the 3 children tested we observed a quantitative and qualitative enhancement in antibody responses over time to Pnc surface proteins. Over the 2-year period the intensity of antibody responses increased as measured by densitometry. In terms of qualitative responses, the children developed antibodies that recognized proteins previously undetected (Figure 2). DCC children developed antibodies against the following proteins: In the CW-L against 48, 60, 70, and 83 kDa._ In CW-NL: 50, 66, 73, 116kDa. In the M-L: 50, 55, 62 and 83kDa, and in the M-NL: 45, 60, 64, 70, 83, and 100kDa.

Shani-Sekler et al.

67

Figure 2. Analysis of sera from DCC children over time. Sera from 2 DCC children were analyzed over a period of 2 years. Panel A. CW-NL proteins from child 1. Panel B. CW-L proteins from child 2. Lane 1. initial sample. Lane 2. 1 year follow up. Lane 3. 2 year follow up. The first sample was withdraw at the age of 1.5-2 years.

To analyze whether these antibodies had neutralizing activity, we tested their ability to interfere with Pnc adhesion to epithelial cells using the HaCat epithelial cell line. We have found that sera taken from adults interfered with Pnc-epithelial cell adhesion, in a concentration dependent manner (Figure 3).

.........

,

Control

10

50

100

500

Sera reciprocal dilutions Figure 3. Inhibition of Pnc adhesion to epthelial cells (Hacat). Pnc Were treated with several dilutions of pooled adult human sera.

Sera collected from DCC children revealed inhibition of Pnc epithelial cell adhesion and this inhibition increased over time, in correlation with the increase in antibody responsiveness. Enrichment of some of the CW and M lectins and non-lectin fractions was accomplished via FPLC. Peaks

68

Initial Steps in Streptococcus Pneumoniae Infection

separated by the monoQ anion exchange column were collected and then further separated on a cation exchange mono S column and different proteins identified. A representative FPLC MonoQ column separation is presented in Figure 4.

FRACTIONS Figure 4. Fetuin bound membrane proteins from Pnc gpt mutant separated by FPLC Mono Q column. Insert A. FPLC fractions that demonstrated the presence of proteins were subjected to western blot analysis with pooled human sera. Lane 1. Pooled fractions 2-3. Lane 2. Pooled fractions 15-19. Lane 3. Pooled fractions 21-23. Lane 4. Fraction 24. Insert B. Hemagglutination of the above fractions.

Shani-Sekler et al. Child #

69

Pnc serotype

Pnc disease

0002

Blood withdrawal Date 19 months

19A

0023

19 months

23 F

1001

12.12.96

23 F

10 0 4 10 0 5

2 7. 11.96 1 9.1 1.96

6B 23F

1020 I029 3024

1 .12.96 5 .12.96 2 years after immunization

23 F 19 F 3

3045 3052

2 years after immunization 2 years after immunization

3 3

2x Otitis media respiratory distress URI 2x eye infection Otitis media URI Pneumonia Respiratory distress Otitis media Respiratory distress, eye infection None Pneumonia, URI Respiratory distress Otitis media None Otitismedia 5xURI respiratory distress 2xURI Hospitalization 3 x otitis media

All the children in the 1000 series were 1.5-2 years of age at time of immunization.

5.

SUMMARY

In accordance with our working hypothesis adhesins/lectins were found to reside in Pnc cell wall and membrane fractions. Using healthy adult sera, we have demonstrated that Pnc cell wall and membrane proteins are highly immunogenic and are able to significantly interfere with Pnc adhesion to epithelial cells. We have also demonstrated that antibodies against the higher molecular weight cell wall lectins increased qualitatively and quantitatively over a period of 2 years in DCC children. The increase in the immunogenicity of the DCC children sera correlated with their ability to interfere with bacterial adherence to epithelial cells, albeit with less inhibition than that found in adults. We thus have evidence of a developing immune response to CW and M Pnc proteins which may play a major role in bacterial adhesion and subsequent pathogenesis. Future studies include the identification and

70

Initial Steps in Streptococcus Pneumoniae Infection

characterization of several proteins for their ability to function as vaccine candidates.

ACKNOWLEDGMENT We would like to thank Dr. David Watson (Dallas, TX) for providing us with the Pnc unencapsulated mutants. We would like to thank Dr. Rachel Teitelbaum for her editorial assistance. YM is a recipient of the Goistella award. The first two authors contributed equally to this study.

REFERENCES Alonso De Velasco E. A., Verheul F.M., Verhoef J., Scippe H. 199.5. Streptococcus pneumoniae: virulence factors, pathogenesis, and vaccine. Microbiol. Rev. .59:591603. Austerian R and Gold J. 1964. Pneumococcal bacteremia with special reference to bacteremic pneumococcal pneumonia. Ann. Intern. Med. 60:759-776. Austrian R.. 1986 Some aspects of the pneumococcal carrier state. J. Antimicrob. Chemother. 18(suppl A):35-45. Avery O.T., Goebel W.F. 1929. Chemo-immunologic studies on conjugate carbohydrateproteins. II Immunological specificity synthetic sugar-protein antigens. J. Exp. Med. 50:533-550. Center for disease control and prevention. 199.5 Pneumonia and Influenza death rateUnited States 1979-1 994. Morbid. Mortal. Weekly Rep. 44:535-537. Cowan M.J., Ammann A.J., Wara D.W, Howie V.M., Schultz L., Diyle N., Kaplan M. 1978. Pneumococcal polysaccharide immunization in infants and children. Pediatrics. 62:721-727. Cundel D; Gerard N; Gerard C; Idapaan-Hiekkila I; Tuomanen E. Streptococcus pneumoniae anchors to activated eukaryotic cells by the receptor to platelets activating factor. Nature. 377:435-438. Cundel D; Tuomanen E.I. 1994. Receptor specificity of adherence of Strptococcus pneunzoniae to human type II pneumocytes and vascular endothelial cells in vitro. Microbiol. Pathog. 17:361-374. Kaythy H., Eskola J. 1996. New vaccines for the prevention of pneumococcal infections. Emerg. Infect, Dis. 2:289-298. McDaniels LL.S; Sheffield J.S; Siatlo E ; Yother J; Crain M.J; Brils D.E. 1992. Molecular localization of variable and conserve regions of PspA and identification of additional PspA homologous sequences in Streptococcus pneumoniae. Microb. Pathog. 13:261-269. Robins J. B., Austrian R, Lee C.J., Rastogi S.C., Schiffman G., Henrichsen J., Makela P.H., Broome C.V., Facklam R.R., Tiesjema R.H., Parke, Jr. J.C. 1983. Considerations for formulating the second generation of pneumococal capsular polysaccharide vaccine with emphasis on the cross-reactive type within the groups. J. Infect. Dis. 148:1136-1159.

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Ronda C; Garcia J; Garcia E; Sanchez-Puelles J; LopezR. 1987. Biological role of the pneumococcal amidase: cloning of the LytA gene. Eur. J. Biochem. 164:621-624. Rosenow C; Ryan P; Weiser J; Johnson S Fountan P; Ortqvist A; Masure H. 1997. Contribution of a novel choline binding protein to adherence, colonization and immunogenicity. Infect. Immun. 25:819-829, Sampson J; O’Connor R; Stinson A, Tharpe J, Russel H. 1994. Cloning and nucleotide sequence analysis of PsaA, the Sreptococcus pneumoniae gene encoding a 37kilodalton protein homologous to previously reported Streptococcus sp.adhesins. Infect. Immun. 62:319-324. Shapiro E. S., Berg A. T., Austrian R., Scroeder D., Parcells V., Margolis A., Adair R.K., Clemmens J.D. 199 1. Protective efficacy of polyvalent pneumococcal polysaccharide vaccine. N. Engl. J. Med. 325:1453-1460. Thuomanen E.I., Austrian R., Masure H. R. 1995. Pathogenenesis of pneumococcal infection. N. Engl. J. Med. 332: 1280-1284. Thuomanen E. I. 1997. The biology of pneumococcal infection. Ped. Res. 42:253-258. Weiser J; Austrian R; Streenivasan P; Masure H. 1994. Phase variation in pneumococcal opacity: relationship between colonial morphology and the nasopharyngeal colonization. Infec. Immun. 62:2582-2486.

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ROLE OF CYTOKINES IN THE MATURATION AND FUNCTION OF MACROPHAGES Effect of GM-CSF and IL-4 Yona Keisari, Guy Robin, Liat Nissimov, Hongbin Wang, Adi Mesika, Rachel Dimri, and Itzhak Ofek Department of Human Microbiology, Sackler Faculty of Medicine Tel Aviv University, Tel A viv, Israel

1.

INTRODUCTION

Macrophages and monocytes that infiltrate into chronic inflammatory sites might be influenced by cytokines such as GM-CSF, IL-3, IL-4 and IFN- that are abundant in these sites. Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), is a glycoprotein which is characterized by its capacity to stimulate cell precursors in the bone marrow to undergo proliferation and differentiation into granulocytes and macrophages (Metcalf 1985, Golde and Gasson. 1988). Granulocyte-macrophage CSF is secreted by various cell types, and its production might be enhanced by bacterial products (Chodakewitz et al 1988), and by cytokines such as Interleukin 1 (Zucali et al 1986, Sieff et al 1987, Fibbe et al 1988, Herrmann et al 1988), Interferon (Piacibello et al 1985), and TNF- (Lu et al 1988, Munker et al 1986). GM-CSF which is present in chronic inflammatory sites (Williamson et al 1988, Xu et al 1989) was implicated as a key cytokine in inflammatory joint disease (Campbell 1998). The role of GM-CSF as a proinflammatory cytokine was further substantiated by the findings that GM-CSF deficient mice exhibited an attenuated production of inflammatory cytokines i n response to lipopolysacchauide (LPS) (Basu 1997). The Biology and Pathology o f Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

73

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Role of Cytokines in the Maturation and Function of Macrophages

During the course of an inflammatory/immunological response other cytokines such as Interleukin-4 (IL-4), are also produced and may affect the reactivity of mononuclear phagocytes. IL-4 was described as a prototypic immunoregulatory cytokine (Paul 1991, Brown & Hural 1997). It is also considered to be an activating factor for the accessory activity of monocytes (Riancho et al 1993), and as a modulator of cytokine production by monocytes (for review see Brown & Hural 1997). In our studies we examined the effect of GM-CSF, IL-3, and IL-4 on the maturation of human peripheral blood monocytes into macrophages in long term cultures. Peripheral blood monocytes (PBM) were allowed to mature into monocyte derived macrophages (MoDM) in the presence of each cytokine alone, or a combination of cytokines. We examined the number of adherent MoDM, and assessed the bactericidal and inflammatory potential of the cytokine treated cells as expressed by their oxidative burst activity, expression of membranal receptors, and cytokine product ion.

2.

EFFECT OF CYTOKINES ON THE SURVIVAL AND DIFFERENTIATION OF MoDM IN LONG TERM CULTURES

Peripheral blood human monocytes (PBM) were obtained from normal blood bank donors (Boyum 1968, Keisari 1996), and incubated, without changing the medium, for up to 2 weeks in the presence of GMCSF, IL-3 (Behringwerke, Marburg, Germany) and/or IL-4 (PeproTech Inc. Rocky Hill, NJ, USA) to obtain human monocyte derived macrophages (MoDM). Cell counts showed that non treated MoDM cultures contained only 30% of the initially adherent cultures, while in the presence of CSF, about 80% of the cells remained in the cultures (Robin et al 1991). The effect of GM-CSF or IL-3 on the amount of adherent MoDM was dose dependent, and maximal when added to freshly harvested cells (Robin et al 1991, Dimri et al 1994). The loss of response of aged cells to the cytokines, may result from either a decrease in the number of receptors with cell maturation, or from progressive death of the cultured monocytes. GM-CSF has been reported to promote the differentiation and survival of monocytes in atopic dermatitis (Bratton et al 1995), and of adherent monocyte derived dendritic cells (Markowicz & Engelman 1990). The increased longevity of GM-CSF treated adherent MoDM in culture may be attributed to its effect in preventing monocyte programmed cell death in tissue culture (Mangan & Wahl. 1991).

Keisari et ai.

75

Hemacolor (O.D 630nm)

The capacity of colony stimulating factors to augment the viability and adherence of monocytes might improve the response and activity of the monocyte population involved in either acute or chronic inflammatory reactions, and points at a close cooperation between tissue cells and professional phagocytes in defense mechanisms. We also studied the effect of IL-4 and GM-CSF on adherent human MoDM in long term cultures (2 weeks). IL-4 alone as well as GM-CSF augmented cell survival. Nevertheless, the combination of IL-4 and GMCSF was found to be more effective than each cytokine by itself. Yet, IL4 was reported rather to enhance programmed cell death of activated monocytes (Mangan et al 1992). Studies indicated that while GM-CSF is important for monocyte differentiation into macrophages, IL-4 provoked cell fusion and formation of giant cells (Dugast et al 1997). Incubation of peripheral monocytes with GM-CSF and IL-4 for extended time periods may lead to the differentiation of dendritic cells which express augmented antigen presentation capabilities (Sallusto & Lanzavecchia 1994). Adult peripheral blood monocytes can also differentiate into Langerhans cells in the presence of GM-CSF, IL-4 and TGF-beta1 (Geissmann et al 1998). It is important to mention in this regard, that in order to obtain dendritic cells from monocytes, 10-100 fold higher concentrations of the cytokines are required, compared to those used in our studies to obtain differentiated macrophages.

0

0.1

1

5

10

IL-4 (ng/ml) Figure 1. The effect of GM-CSF and IL-4 on MoDM survival in tissue culture. Adherent monocytes cultured in the presence of GM-CSF (200 U/ml) and/or IL-4 (0-10ng/ml) for 2 weeks were assayed by the Hemacolor assay (Keisari 1992). The results represent mean±SE from 3-4 different experiments each done in triplicate.

Role of Cytokines in the Maturation and Function of Macrophages

76

3.

EFFECT OF CYTOKINES ON BIOCHEMICAL AND FUNCTIONAL ACTIVITIES OF MoDM

Cytokines that affect monocyte maturation, may also affect the activities of MoDM. In our studies we examined the effect of GM-CSF, IL-3, and IL-4 on the oxidative burst activity, expression of membranal molecules, and cytokine production by MoDM.

3.1

Effect on Oxidative Burst (OB) Activity

The oxidative burst activity is an important component of the inflammatory potential of monocytes and neutrophils. We studied the capacity of MoDM, that differentiated for 2 weeks with GM-CSF, IL-4, or both, to generate OB products in response to a TPA stimulus. The results presented in Table No. 1 show that the long term exposure to GM-CSF had no effect on 0 2- production by the cells. Table 1: The Effect of GM-CSF on the oxidative burst activity of Monocyte Derived

(100 U/ml) -5 b

Cell no. (x10 ) C

Superoxide 6 (nmol /10 cells)

none

2.87±0.4

1 .52±0.27

2.4±0.2

6.5±5.8

13.2±1

4.8±1.2 30.8±2.2

in 1.6cm wells. b Adherent cells in each well were counted using an hemocytometer. c The cultured monolayers were covered with 1ml Earles-BSS without phenol red and containing 80 µM ferricytochrome C. The cells were either triggered or not with TPA for 60 min. at 37 o C and cytochrome C reduction was assessed at 550nm as described (Babior et al 1973, Pick & Mizel 1981). The results represent mean ±SE of 3 different experiments each done in triplicates.

Short term exposure of monocytes (Nathan et al 1984, Kharazami et al 1988) or alveolar macrophages (Thomassen et al 1989) to GM-CSF also failed to affect the response of the cells to OB stimulants. In contrast, GM-CSF enhanced the generation of H2O2 and O2 - by neutrophils (Kharazami et al 1988). IL-4 have been reported to abrogate significantly the oxidative burst activity of human monocytes, and human alveolar macrophages (Abramson & Gallin 1990). Thus, we tested the effect of IL-4, with or

Keisari et al.

77

without GM-CSF, on the oxidative burst activity of MoDM. We found that the effect of IL-4 on the O2 - production of monocytes is dose dependent. Incubation of MoDM for 2 weeks with less than 1 ng/ml of IL-4, elevated their OB activity, whereas, increasing the concentration of IL-4 (5 ng/ml) was found to reduced 02 production. Reduced 02 - production was also obtained when the macrophages were cultured in the presence of both IL4 and GM-CSF (Table 2). Table 2: Effect of IL-4 and GM-CSF on

Superoxide (Control) (nmo1/mg protein)' Superoxide (TPA)

the oxidative burst of monocyte derived

19.3±2.02 7.4±3.3

74.3±24.2 3.85±2.1

47.4±15.9 15.74±7.4

85.4±18.4 6.6±2.4

35.9±5.35

8.5±3.6

45.2±1 1

16.3±4.0

CSF and/or IL-4 in 1.6cm wells. Protein concentration in each well was determined by the Bradford method. c The cultured monolayers were covered with 1ml Earles-BSS without phenol red and containing 80 µM ferricytochrome C. The cells were either triggered or not with TPA (100 nM) for 60 min. at 37 OC and cytochrome C reduction was assessed at 550nm. The results represent mean ±SE of 3 different experiments each done in triplicates. b

3.2

Effect on the Expression of Membranal Molecules

Membranal molecules and receptors play an essential role in monocyte/macrophage activities. We found that GM-CSF or IL-3 treated cells exhibited a higher expression of CD-14, HLA-DR, and IL-1 compared to control MoDM (Dimri et al 1994). Eischen et al (1991) previously reported that long term exposure (7 days and longer) of monocytes to GM-CSF elevated HLA-DR expression. We also found that IL-4 and GM-CSF, individually and combined, increased the expression of membranal CD-1a, but not the percentage of CD-1a positive cells. A similar observation was made by Kasinrerk et al (1993), who found that GM-CSF upregulated the expression of accessory cell associated CD- 1 molecules. We failed to observe any change in HLA-DR expression by IL-4 differentiated MoDM. Nevertheless, IL-4 was reported to elevate HLA-DR expression in peripheral blood monocytes (Littman et al 1989, Ruppert et al 1991), and reduce expression of CD-14, and CD-16 (Ruppert et al 1991). Monocytes cultured for 7 days with GM-CSF exhibited increased expression of IL-4Ra (Bonder 1998), and an 8-fold increase in IFN-

78

Role of Cytokines in the Maturation and Function of Macrophages

receptor expression (Finbloom et al 1993). Ligation of CD44 by hyaluronan is also a proinflammatory event. GM-CSF and IL-3 treatment of human monocytes induced hyaluronan binding by the cells, while IL4 was a potent inhibitor of this event (Lavesque & Haynes 1997). GM-CSF and IL-3 seem to promote the expression of macrophage membranal molecules involved in the amplification of both immunological and inflammatory reactions. In comparison, IL-4 promotes the expression of accessory molecules, but down regulates proinflammatory receptors.

3.3

Effect on Cytokine Production

An important parameter tested in our studies was the long term effect of GM-CSF and IL-4 on cytokine production by the cultured phagocytes. MoDM cultured with GM-CSF and triggered with LPS showed increased TNF-α production after 1 to 3 weeks in culture compared to non treated counterparts (Robin et al 1991, Dimri et al 1994). Several investigations showed that short term treatment of monocytes with GM-CSF (1 8hr) enhanced TNF- (Chantry et al 1990, Sisson & Dinarello 1988, Hart et al 1988, Cannistra et al 1987), IL-lα (Sisson & Dinarello 1988), and IL-1ß (Smith et al 1990, Sisson & Dinarello 1988) production in response to LPS or PMA. Hart et al (1 988) reported that GM-CSF alone did not stimulate IL-1 or TNFproduction by monocytes, whereas Thomassen and colleagues (1 989) found that constitutive TNF- secretion was elevated in GM-CSF treated monocytes from several donors. Monocyte derived macrophages exposed to IL-4 with or without GMCSF were also tested for the production of pro- (IL-6) and antiinflammatory (IL-10) cytokines. The results presented in Table 3. show that the production of both IL-6 and IL-10 was elevated by GM-CSF treatment. Yet, whereas a combination of GM-CSF and IL-4 resulted in reduced IL-6 production, that combination augmented IL- 10 production compared to GM-CSF treated cells. It can be concluded that IL-4 down regulates pro-inflammatory cytokines secretion and up regulates antiinflammatory cytokines. .The capacity of IL-4 to abrogate the pro-inflammatory potential of differentiated human macrophages, revealed in the present study, is in line with previous reports. IL-4 reduced TNF-α production by alveolar and peritoneal human macrophages (Hart et al 1991, Sone et al 1992), and reduced the capacity of monocytes to produce TNFa-α IL-6, and IL1 (Hart et al 1989, Donnelly et al 1990, Te Velde et al 1990).

Keisari et al.

79

Table 3: Effect of IL-4 and GM-CSF on cytokine production by monocyte derived No treatment Cytokine IL-6 5 (ng/2x 1 o cells)b

3.8±1 .8 1

IL-4 (1ng/ml) 0

GM-CSF (100 U/ml) 172.6±10.4

GM-CSF +IL-4 54.3±19.6

1.33±0.2 1

6.3S±1.11

and/or IL-4 in 96 well plates. The cultured monolayers were triggered with LPS and the supernatants were measured for cytokines by ELISA. The results represent mean ±SE of 3 different experiments each done in triplicates. c Protein concentration in each well was determined by the Bradford method.

b

The anti inflammatory activity of IL-4 was also manifested by it’s effect on other macrophage activities. IL-4 increased the expression of aminopeptidase-N (CD 13) by mononuclear phagocytes, which may result in increased capacity to inactivate inflammatory mediators (Van Hal 1994). IL-4 also augmented mRNA and surface expression of the IL-1 decoy receptor (RII) in human mononuclear phagocytes which down regulates IL- 1 driven inflammatory reactions (Colotta et al 1996). Hart et al (1993) reported that IL-4 may act differently on different cells of the monocytic lineage such as monocytes and synovial fluid macrophages.

4.

GM-CSF AND IL-4: EFFECT ON MANNOSE RECEPTOR EXPRESSION AND FUNCTION

The mannose receptor (MR) is a macrophage C-type lectin (Drickamer & Taylor 1993) expressed by all tissue macrophages examined, including alveolar, peritoneal and hepatic ones (Ezekowitz & Stahl 1988), and the differentiation of human monocytes into macrophages is also characterized by the appearance of membranal mannose receptors (Shepherd et al 1982). The MR has been shown to be involved in a number of macrophage functions, including recognition of carbohydrates frequently present on the surfaces of different microorganisms (for review see Tauber and Chernyak 1991, Ofek et al 1995, Stahl & Ezekowitz 1998, see also Linehan et al in this proceeding). In previous studies we found that the mannose receptor is expressed on the cell membrane of macrophages to accommodate non opsonic phagocytosis of microorganisms bearing Man 2/3Man sequences on

Role of Cytokines in the Maturation and Function of Macrophages

80

their surfaces, such as mannan containing yeast cells and encapsulated Klebsiella pneumoniae (Athamna et al 1991, Kabha et al 1995). The receptor is particularly well suited to direct particles to phagolysosomes and trigger a respiratory burst (Marodi et al 1993, Mosser 1994). The MR is also involved in the capture of antigens by dendritic cells that use the MR to concentrate macromolecules in the MHC class II compartment (Sallusto et al 1995). Macrophage fusion and the formation of giant cells may also depend on mannose receptor activity (McNally et al 1996). A substantial lack of data exists on the effect of cytokines on macrophage receptors to microbial cells. In our studies we examined the effect of IL-4, GM-CSF and their combination on MR expression by monocyte derived macrophage. Monocytes were cultured for up to 7 days, and MR expression was determined by FACS analysis using anti MR monoclonal antibodies. The results presented in Figure 2 show a sharp increase in the percent of MR positive cells after 4 days in culture (19.3k0.05% and 44.8±3.5 after 2 and 4 days respectively). Maximal percentage of MR positive cells was reached after 7 days (51±3.6%). Next we examined the binding of the yeast Saccharomyces cerevisiae to MoDM cultured for 2 weeks in the presence of the cytokines. GM-CSF treatment increased yeast cell binding, that was blocked by the MR ligand, mannan (Figure 3).

%Fluoresence cells

60 50 40 30 20 10 0.

0

1

2

4

5

7

Days Figure 2: The expression of mannose receptor at different days following cultivation of monocytes. Adherent monocytes cultured for 7 days, were assayed for the expression of mannose receptor by binding of anti MR antibodies (PAM-1, Biondi et al 1984). The results represent mean ± SE from 4 different experiments.

Keisari et al. Yeast binding (O.D 415nm)

81 0.50.40.30.2,

.

0.10.00

100

200

GM-CSF(U/ml) Figure 3: The combined effect of GM-CSF on yeast binding by monocytes derived macrophages. Adherent monocytes cultured in the presence of GM-CSF (0-100 U/ml) 7 days, were assayed for the expression of mannose receptor by yeast binding. The results represent mean ± SE from 4 different experiments, each done in quadruplicate.

The addition of IL-4 (5 ng/ml and higher) but not IFN-gamma (results not shown) to GM-CSF, significantly decreased yeast binding (Figure 4).

Yeast binding (O.D 414nm)

0.3

0.2

i

0.1

0

2

4

6

8

10

12

IL-4 (ng/mI) Figure 4: The combined effect of GM-CSF and IL-4 on yeast binding by monocytes derived macrophages. Adherent monocytes cultured in the presence of GM-CSF (100 U/ml) and IL-4 (0.1, 1, 5, & 10 ng/ml) for 7 days, were assayed for mannose receptor mediated yeast binding. The results represent mean ± SE from 4 different experiments, each done with quadruplicates.

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Role of Cytokines in the Maturation and Function of Macrophages

The macrophage mannose receptor can be regulated by various cytokines and inflammatory agents, that may, therefore, affect the outcome of macrophage-bacteria interactions mediated by the MR. Our results indicate that GM-CSF can increase the capacity of monocyte derived macrophages to bind microorganisms expressing the ligand for the MR, by both augmenting the viability of the adherent cell population, and by elevating the membrane expression of the MR. In contrast, IL-4 reduced the functional expression of the MR. Macrophage colony-stimulating factor (M-CSF), that promotes macrophage differentiation, also augmented MR expression in murine macrophages, and mannose inhibitable killing of Candida albicans (Karbassi et al 1987). In our studies IL-4 reduced MR expression in human macrophages. Yet, other reports indicated that in mouse macrophages MR expression increased following treatment with IL-4 (Stein et al 1992, Yamamoto et al 1997). Raveh and his coworkers (1998) demonstrated that IL-4 and IFNy, individually or combined, enhanced mannose receptor-mediated phagocytosis, although having adverse effects on MR expression and endocytosis. It should be also mentioned that dendritic cells obtained from monocytes in the presence of high concentrations of GM-CSF and IL-4, express another C-type lectin termed dendritic cell immunoreceptor (DCIR) (Bates et al 1999). Most of the studies in this field indicated that pro-inflammatory agents reduced macrophage MR expression while anti-inflammatory agents augmented it (for review see also Ofek et al 1995). For example, the macrophage activating cytokine, IFN-γ reduced the expression of the MR in human monocyte-derived macrophages, but increased the receptor mediated killing of C. albicans (Marodi et al 1993). The down regulation of the MR by IFN-γ was attributed to inhibition of gene transcription (Harris et al 1992). Inflammatory reagents such as endotoxin were found to down-regulate MR expression in macrophages (Shepherd 1990), and the expression of MR in dendritic cells declined by 50% following exposure to TNF-α and LPS (Sallusto et al 1995). In contrast to the findings that pro-inflammatory reagents reduced macrophage MR expression, in other cell types they promoted MR expression. MR mRNA was induced in a dose- and time-dependent manner in mesengial cells by IL-1 α and TNF-α , but not by plateletderived growth factor-B or IL-6 (Liu 1996). IL-1 and LPS also elevated MR expression in hepatic sinusoidal endothelial cells (Vidal-Vanaclocha 1996). The eicosanoid, PGE, which is generated during inflammation, and anti-inflammatory agents such as glucocorticoids, were found to augment

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MR expression and synthesis, and reverse the 1FN-γ induced diminution in MR expression (Schreiber et al 1993, Shepherd et al 1994). Cytokines with anti inflammatory or immunosuppressive activities, in general, upregulated MR expression. IL- 10 upregulated MR expression in human dendritic cells (Morel et al 1997), and increased MR mediated endocytosis in monocyte derived dendritic cells (Longoni et al 1998). Stein et al (1992) reported that IL-10 had only a modest or no effect on mouse macrophage MR activity. IL-13 was another cytokine reported to upregulate MR expression in differentiated human monocytes (DeFife et al 1997), and peritoneal mouse macrophages (Doyle et al 1994).

5.

CONCLUDING REMARKS

The studies summarized hereto showed that colony stimulating factors such as IL-3 and GM-CSF facilitated the long term maturation of monocytes into macrophages, augmented their capacity to capture bacterial and fungal cells, and elevated the release of cytokines involved in inflammatory and granulomatous reactions. The TH-2 cytokine ,IL4, that accelerated monocyte to macrophage differentiation, reduced the inflammatory activities of the cells, and counteracted the effects of GMCSF. It may be conceived that GM-CSF acts as a maintenance cytokine in inflammatory and granulomatotic sites, while IL-4, which although contributes to cell survival, acts to diminish their inflammatory activity. The net result of the cumulative action of the cytokines is an increase in the survival of the cell populations and a decrease in the inflammatory potency in order to minimize the damage to the surrounding tissue.

ACKNOWLEDGMENT This study was supported, in part, by a grant from the Michel Smitka Memorial Fund, Israel Ministry of Health. The authors wish to thank Dr. F.R. Seiler and Dr. D. Krumwieh from the Behringwerke, Marburg, Federal Republic of Germany, for the human recombinant GM-CSF and IL-3 used in this study. We thank Dr. Alberto Mantovani (Mario Negri Institute, Milan, Italy), for The anti mannose receptor antibody PAM-1.

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MAST CELL MODULATION OF THE INNATE IMMUNE RESPONSE TO ENTEROBACTERIAL INFECTION

Soman N. Abraham¹ And Ravi Malaviya² 1

Department of Pathology and Microbiology, Duke University Medical Center, Durham NC27710, and ²Department of Allergy and Inflammatory Diseases, Hughes Institute, 2665 Long Lake Road. St. Paul, MN 55113

1.

INTRODUCTION

Although mast cells have been associated with many biological responses (e.g. inflammation, tissue repair and angiogenesis) and clinical conditions (e.g. rheumatoid arthritis, interstitial cystitis, scleroderma, psoriasis, neurofibromatosis and Crohn’s disease), they remain one of the least understood cells of the immune system. By virtue of their high affinity IgE/FcεRI-receptors and their intrinsic capacity to release large amounts of inflammatory mediators, mast cells are primary known as major effector cells of allergy or type 1 hypersensitivity reactions (Marone et al 1997). In spite of these deleterious properties, mast cells have been preserved through evolution even among the lowest orders of animals indicating that these cells must be serving a valuable function in the body. A number of factors suggest a role for mast cells in modulating the innate immune response against bacteria. These include the fact that mast cells are present in large numbers under the epithelia of various mucosal surfaces and skin. They also have the intrinsic capacity to mobilize a rapid and vigorous inflammatory response in the host upon activation and mast cells have already been shown to contribute to certain aspects of the IgE-mediated adaptive immune response to parasites (Levy & The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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Frondoza 1983, Miller 1996). Recently, several laboratories including ours have generated data directly implicating mast cells in the host’s innate immune response to bacteria. Here we summarize some of these studies and describe our efforts at elucidating the molecular basis for mast cell bacteria interactions. For these studies, we used Escherichia coli and Klebsiella pneumoniae which are opportunistic pathogens accounting for a large proportion of infections in immunocompromized and hospitalized patients.

2.

PROTECTIVE ROLE OF MAST CELLS IN BACTERIAL INFECTION

The availability of mast cell deficient makes it possible to examine the specific role of mast cells in various host responses (Galli et al 1991, Galli & Wershil 1996, Zhang et al 1992). These mice have resulted in a mutation at the W/c-kit locus (Galli et al 1991). The mutation at the W/ckit locus results in the absence of c-kit tyrosine kinase, the receptor for stem cell factor (SCF) on the mast cell surface (Galli & Kitamura 1988). The interaction of c-kit and SCF is important for normal mast cell development. By comparing differences in biological responses between V V mast cell deficient mice WBB6F1-W/W (W/W ) and their congenic mast cell sufficient WBB6F1 -+/+ (+/+) controls and then by analyzing the v v responses in mast cell resonstituted W/W mice (W/W + MC), it is possible to define the specific contributions of mast cells to a variety of inflammatory conditions. We utilized this model to examine the role of mast cells in host defense against bacterial infections by comparing the susceptibility of WW mice and their +/+ littermate controls following intraperitoneal challenge by mouse-virulent enterobacteria. We found v that mast cell deficient W/W mice experienced as much as 80% mortality compared to no mortality in the wild type +/+ mice (Malaviya et al 1996). v Furthermore, W/W +MC mice exhibited the same resistance to infection as that exhibited by wild type mice (Malaviya et al 1996). This confirmed that the observed difference in susceptibility to bacterial infection was solely due to mast cells and not to other abnormalities that may exist in these mice. When we compared the extent of bacterial clearance in the three groups of mice, we found that W/Wv mice were 20 fold less efficient in clearing infection than the +/+ mice or mast cell WWv + MC mice (Table I). The limited bacterial clearance in WWv mice directly correlated with impaired neutrophil influx (Malaviya et al 1996). Based on these findings, we concluded that the impaired neutrophil response to

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bacteria in the WWv mice could be due to their inability to effectively clear bacteria (Malaviya et al 1996). Mast cells are capable of releasing several neutrophil chemoattractants such as Leukotriene (LT) B 4 , interleukin (IL)-8 and TNFα , (Zhang et al 1992, Lukacs et a1 1996). The role of TNFa was, however, of particular interest because the mast cells have the unique capacity to store presynthesized TNFα and thus, are able to release this cytokine immediately from preformed stores following activation (Galli et al 1991, Galli & Wershil 1996, Gordon & Galli 1987). This suggest that TNFα derived from mast cells may participate in early phases of inflammation. TNFa is also known to enhance the bactericidal properties of neutrophils and the adhesion of neutrophils to endothelial cells (Galli & Wershil 1996). This is due, in part, to expression of the adhesion proteins Eselectin (ELAM-l) and ICAM-1 by endothelial cells. We observed that following intraperitoneal bacterial inoculation, a burst in extracellular TNFα was released immediately preceding the influx of neutrophil in the mast cell sufficient mice, a phenomenon that was not detected in mast cell deficient mice (Malaviya et al 1996). Injection of mast cell sufficient mice with a monoclonal antibody directed at TNFα but not another monoclonal antibody directed at IL-1β, another mast cell proinflammatory cytokine, blocked up to 70% of the neutrophil response, confirming that mast cell-derived TNFα played a significant role in recruiting neutrophils to sites of bacterial infection (Table I; Malaviya et al 1996). In an independent report, a similar conclusion was reached utilizing a different approach by Echtenacher et al (Echtenacher et al 1996). These investigators showed that mast cells prevent death in a mouse model of septic peritonitis induced by surgical caecul ligation and puncture and this effect can be abolished by the injection of the anti TNFα neutralizing antibodies. Interestingly, the direct injection of TNFα into mice W/W V mice was also protective against enterobacterial infection, al beit only within a limited range of cytokine concentrations (Echtenacher et al 1996). These studies provide definitive evidence that mast cells are critical for the host defense in bacterial infection. Recently, we have determined that TNFα was not the only neutrophil chemoattractant produced by mast cells upon bacterial challenge. We found that the LTB4, was also a potent neutrophil chemoattractant produced by mast cells during bacterial infection. Leukotrienes are a family of biologically active compounds that are produced from arachidonic acid in a multistep process via activation of the 5lipoxygenase pathway. Compound A63162, a hydroxamic acid derivative, is an orally active compound that selectively inhibits LT formation in vitro as well as in vivo in rodents (Zhang et al 1992). We

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v

treated three groups of mice, +/+, W/W and W/W + MC mice with A63 162 and challenged them intraperitoneally with enterobacteria. Two hrs later, we determined that the neutrophil influx in A63162-treated +/+ v mice or W/W +MC mice was significantly decreased compared to untreated controls (Table I). Furthermore, the neutrophil response in v A63162-treated +/+ mice appeared close to that of control W/W mice (Table I). A63162 did not have a significant effect on the neutrophil V response of WW mice (Table I) suggesting that mast cells were the v source of the chemoattractant. The A63162-treated +/+ or W/W + MC mice also revealed a reduced level of bacterial clearance compared to untreated controls indicating, once again, a correlation between neutrophil influx and bacterial clearance in the peritoneum (Table I). Taken together, our data suggested that mast cell derived TNFα and LT are important for the influx of neutrophils and the resulting clearance of bacteria in mice peritoneum. Table I: Effect of TNF -specific antibody and compound A-63162 on Mast Cell Mediated Bacterial Clearance and Neutrophil Influx in Mast Cell Deficient and Their Bacterial Viability

Myeloperoxidase

Percent Inhibition Following Pret reatment with A-63 162 Vehicle Anti(xI0 CFU) TNFa Ab 0(4.5±2.5) ND I

Percent Inhibition Following Pretreatment with

Group

v

W/W +/+ v

w/w +MC

Vehicle (mU/Peritoneum)

Anti-TNF Ab

A-63162

6.7±2.I

9

2

0(0.3±0.S)

ND

140*

32±2.4

71

40*

0 (0.3±0.2)

ND

100*

35±8

83

43*

ND, not done; * p<0.05; values are expressed as mean +SEM.

3.

IN VITRO MAST CELL RELEASE OF TNFα AND LTS IN RESPONSE TO BACTERIA

To obtain direct evidence that bacteria-activated mast cells can release TNFα and LT, we examined cultured bone-marrow derived mast cells in vitro for TNFα and LT release after 1 h exposure to enterobacteria. Upon bacterial challenge, mast cells released TNFα in a dose dependent fashion (Figure 1A). Limited amounts of LTB4 were also detected in the extracellular medium (Figure 1B) confirming that enterobacteria can induce the release of this potent chemoattractant from mast cells.

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Because LTB4 is readily metabolized, its level in this assay represented only recently generated LTB4 . Therefore, we assayed the medium for LTC4 which is secreted concomitantly with LTB4, but which is markedly more stable. As shown in Figure lB, appreciable release of LTC4 from bacteria-activated mast cells is indicated. It is noteworthy that LTC4 is a vasoactive factor which can increase microvascular permeability. Taken together, these findings indicate that mast cell release TNFα and LT in response to bacteria and that these mediators plays an important role in modulating neutrophil influx and bacterial clearance at sites of infection.

Bacteria (1,000,000) /well

Figure I. E.coli ORN103(pSH2) induced TNFα and LT release by mast cells. Monolayers of bone marrow cultured mast cells were exposed to E.coli ORN103(pSH2) and 60 min later the extracellular media was assayed for TNFα (Figure 1A) and LTC 4 and LTB4 release (Figure IB). N=3-6

4.

MOLECULAR BASIS FOR THE INTERACTIONS BETWEEN MAST CELLS AND ENTEROBACTERIA

Like traditional immune cells, mast cells have the ability to discern and bind a variety of infectious agents even in the absence of specific antibodies to the pathogen. Mast cells may also be activated without physical contact with invading through the release of toxins. This has been demonstrated with cholera toxin (Leal-Berumen et al 1996) and by cell wall components such as lipopolysaccharides (LPS) (Leal-Berumen et al 1994). Host derived proteins generated during bacterial infection which could potentially activate mast cells include the by-products of

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complement activation, C3a and C5a, and the subfragments of fibrinogen and fibronectin that are generated following cleavage by plasmin (Galli 1993, Wojtecka-Lukasik & Maslinski 1992). Other mast cell activators such as granule-associated cationic polypeptides may originate from infla m matory cells such as macrophages, neutrophils, and eosinophils following their activation by agonists including the bacterial peptide FMLP (Zheutlin et al 1984, Fantozzi et al 1986). Mast cells exhibit two basic mechanisms of microbial recognition in naïve hosts: opsonin-dependent and opsonin-independent. The former requires soluble host components such as complement to first opsonize the microorganism before their recognition by the mast cell. For example, microbes coated with the iC3b fragment of complement will be readily recognized by the CR3 receptor on the mast cell membrane (Sher et al 1979, Sher & McIntyre 1977). The critical role of complement system in mast cell recognition of bacteria comes from the recent finding that inflammatory responses to enterobacteria in complement deficient mice was significantly reduced compared to wild type mice (Prodeus et al 1997). In opsonin-independent interactions, specific receptors on mast cells and complementary ligands on the bacterial cell surface are thought to be involved. In vitro experiments involving monolayers of cultured mouse bone marrow-derived mast cells and various Gram negative and Gram positive bacteria, we have found that mast cells possess a striking capacity to bind many bacteria even in the absence of host-derived opsonins (Malaviya et al 1994). Among Gram negative bacteria tested, mast cells bound bound effectively to E. coli, Klebsiella pneumoniae, S. typhimurium and Helicobacter pylori. Among gram positive bacteria, mast cells bound extremely well to Staphylococcus aureus and Streptococcus feacalis but exhibited considerably less affinity for Streptococcus pyogenes (unpublished findings). The molecular basis for many of these interactions is not known but it is conceivable that "pattern recognition receptors", molecules that display binding specificity for structural patterns displayed by cell-surface molecules common to many microorganisms [e.g. LPS], on the mast cell membrane are somehow involved (Medzhitov & Janeway 1997).

5.

FIMH, THE MAST CELL BINDING MOIETY ON ENTEROBACTERIA

Electron microscopy has revealed that mast cell binding to enteric bacteria correlated with expression by these organisms of hair-like appendages of attachment called fimbriae on the cell surface. Since the

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most commonly expressed fimbriae on enteric bacteria are the type 1 fimbriae, which are characterized by their capability to mediate mannoseinhibitable binding reactions, we examined the ability of 100mM of Dmannose or its analogue, α methyl D-mannopyranoside, to block the binding interaction between bacteria and mast cells. We found ‘that the binding of mast cells to E.coli, K. pneumoniae, Enterobacter cloacae and Serratia marcescens strains were mannose-sensitive (Malaviya et al 1994) indicating the involvement of type 1 fimbriae in these binding interactions. At this time, it is unclear whether bacteria or mast cells play a more active role in the binding interaction since the intensity and mannose-sensitivity of binding between dead bacteria and dead mast cells are comparable with that seen with their viable counterparts (unpublished). Type 1 fimbriae are 1-2 µm long and 7 nm thick heteropolymeric appendages. They are composed of a major subunit, FimA, and at least three minor subunits, including FimH a mannosebinding lectin. We have determined that FimH is typically 29 kDa and is presented preferentially at the tips of the fimbriae (Abraham et al 1987, Abraham et al 1988). Because of the peritrichous and radial arrangement of the fimbriae, FimH is likely to be the first component on intact bacteria to make contact with host cells. To further confirm the potency of the interactions mediated by bacterial type 1 fimbriae, we transformed E.coli ORN103, a nonadhesive and nonfimbriated laboratory K12 strain, with pSH2, a plasmid encoding all the genes necessary for the expression of fully functional type 1 fimbriae (Abraham et al 1987). We found that in contrast to E.coli ORN103, the transformed type 1 fimbriated E.coli strain ORN103(pSH2) mediated a high level of binding to mast cell monolayers (Table 11). To demonstrate the specific role of FimH in the binding interaction, we examined the binding mediated by E. coli ORNl03(pUT2002) a mutant created by specifically knocking-out the fimH gene at the distal end of the fim gene cluster in E.coli ORN103(pSH2) (Abraham et al 1987). The binding mediated by the fimbriated FimH- mutant was comparable to that mediated by the nonfimbriated ORN103 strain (Table 11). To directly demonstrate the adhesive role of FimH, we coated inert beads with recombinant E.coli FimH and examined their ability to bind mast cell monolayers. We found that compared to beads coated with bovine serum albumin, FimH-coated beads mediated a remarkably high level of binding (Malaviya et al 1994). Moreover, this binding was mannose inhibitable (Table 11). Finally, to show that FimH was capable of binding and activating mast cells, we instilled E.coli ORN103(pSH2) or E.coli ORN103(pUT2002) into the peritoneal cavities of mice. Compared to E.coli ORN103(pSH2),

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ORN 103(pUT2002) evoked significantly reduced level of histamine release from the peritoneal mast cells (Malaviya et al 1994). Table 2: Binding of FimH-expressing Bacteria and FimH-coated Beads to Mast Cells.

Particles

Properties

Bacteria E.coli ORN 103 E.coli ORN103(pSH2) E. coli ORN 103 (pUT2002)

Nonfimbriated/FimHFimbriated/FimH* Fi mbriated/FimH-

Beads FimH-coated FimH -coated FimA-coated FimA-coated The results are expressed as mean±SEM, N=3.

No of Adherent Particles /50 mast cells

Percent Inhibition by D-mannose

96±12 5 70±9

2 75 4

970±23 195±18

65 12

Thus, the component on enterobacteria largely responsible for binding and activating mast cells is the minor fimbrial component, FimH. Since, the binding between FimH-expressing bacteria or FimH-coated beads and mast cells is mannose-sensitive, the complementary “receptor” on mast cell membrane is predictably a mannose-containing moiety. It is noteworthy that expression of type 1 fimbriae by enterobacteria plays a critical role in their ability to successfully colonize various mucosal epithelia and to resist the flushing actions of mucosal secretions (Thankavel et al 1997). While the binding interactions between FimHexpressing bacteria and mast cells appear to largely benefit the host, it is not inconceivable that in immunocompromized individuals, this interaction may be co-opted by these typically opportunistic pathogens for their own benefit.

6.

CD48, THE FIMH BINDING MOIETY ON MAST CELL MEMBRANES

To begin to elucidate the signaling events triggered by bacterial FimH in mast cells, we sought to identify the mast cell receptor for bacterial FimH. We chose to isolate the FimH receptor from the rat mucosal mast cell . line, RBL-2H3. We reasoned that because the FimH receptor contained mannose, we could use the mannose binding Con A lectin to enrich for mannose containing molecules from the mast cell membrane

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preparations. A 100 x g fractions of Triton X-100 soluble membrane fraction from approximately 1011 cells was passed through a SepharoseCon A affinity column to isolate candidate mannosylated compounds. α methyl D-mannopyranoside (100 mM) was employed to elute all material bound via their mannose residues. The resulting eluate was dialyzed against PBS to remove α methyl D-mannopyranoside concentrated. The The concentrated material was then subjected to SDS-PAGE. electrophoresed material was transferred onto PVDF membranes. The immobilized material was then exposed to I125 labeled recombinant E.coli FimH. The FimH probe specifically bound to a 45 kDa band only in the absence of α methy1 D-mannopyranoside (Malaviya et al 1999). Furthermore, when the blot was exposed to FimH expressing E. coli ORN103(pSH2) and mutant FimH-deficient E.coli ORN 103(pUT2002), only the former bound to the 45 kDa band (Malaviya et al 1999). The binding reaction of E. coli ORN103(pSH2) could be inhibited by 100mM α methyl D-mannopyranoside. These data indicate that E.coli FimH binds specifically to a 45kDa rat mast cell membrane component via mannose residues. To determine the identity of the 45 kDa band, the protein from the ConA eluted fraction was purified to homogeneity and then subjected to microsequencing. A sequence of 12 amino acid residues in the amino terminus were identified as 100% homologous to rat CD48. CD48 is a glycosylphosphatidylinositol (GP1)-linked molecule that has been reported to be present primarily on cells of hematopoietic lineage (van der Merwe et al 1994). Since GPI-linked moieties are cleaved off the surface of cells with phospholipase C (PLC), we incubated bone marrow derived mouse mast cells with increasing amounts of PLC prior to exposure to FimH expressing E.coli. PLC pretreatment of mast cells was found to inhibit bacterial binding in a dose dependent fashion (Malaviya et al 1999). More direct evidence implicating CD48 as the putative E.coli FimH receptor on rodent mast cells comes from the observation that pretreatment of these mast cells with antibodies to CD48 inhibited the adherence of FimH expressing E.coli in a dose-dependent fashion while antibodies to CD117 (c-kit), a well known mast cell membrane marker did not (Malaviya et al 1999). We were also interested to know whether expression of CD48 on cells that do not normally express this molecule could promote binding of FimH-expressing E.coli. We stably transfected Chinese hamster ovary (CHO) cells with the full length cDNA encoding CD48. We examined the transfectants for their capacity to bind FimHexpressing E.coli. The association of bacteria with these CD48 expressing cells was at least 4 fold higher than the number associated with CHO cells transfected with control cDNA (Malaviya et al 1999). Thus, CD48

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molecules on transfected CHO cells are functional as FimH receptors. Taken together, the data show that the putative FimH receptor on mast cells is a GPI-linked moiety, CD48. Although human mast cells express CD48, we have, as yet, not determined if this molecule serves as the FimH receptor on these cells. We have already shown that CD48-specific antibody inhibits FimHmediated bacterial binding to mast cells (Malaviya et al 1999). To show the physiological significance of CD48-FimH interaction receptor, we examined the role of CD48 in eliciting the mast cell TNFα response to FimH expressing bacteria after (i) exposing mast cell surface CD48 to CD48-specific antibody and (ii) removing CD48 from mast cell surface with phospholipase C. Antibody to CD48, but not antibody to CD117, blocked mast cell TNFα release in a dose dependent manner (Malaviya et al 1999). Further confirmation of the critical role of CD48 in the mast cell TNFα response comes from the finding that pretreatment of BMMC with increasing concentrations of PLC significantly reduced the mast cell’s capacity to release TNFα following exposure to FimH expressing E.coli (Malaviya et al 1999). Taken together, these observations provide definitive evidence that the mast cell TNFα response to FimH expressing bacteria is mediated by CD48 molecules present on the mast cell surface. Although there are likely to be other mannosylated moieties on the mast cell membrane capable of binding FimH expressing E.coli, these studies show that CD48 is the biologically relevant receptor. CD48 is the first physiologically relevant receptor identified on mast cells for an infectious agent. CD48 has been referred to as BCM1 in mice, OX 45 in rats and Blast-1 in humans (Malaviya et al 1999). CD48 was discovered as a cell surface molecule expressed by human B lymphocytes in response to EBV infections but its physiologic role in the body is still unclear. The involvement of CD48 in bacterial recognition and in triggering TNFα release in inflammatory cells represents a novel function for this molecule. CD48 joins a growing class of GPI anchored cell surface molecules that serve as receptors for microbes and their toxins. A variety of cell surface proteins including CD14, Thy-1 and CD55 are anchored in the cell membrane through GPI (Clarkson et al 1995, Fenton & Golenbock 1998). This mechanism involves a covalent linkage from the protein to an oligosaccharide which is in turn linked to phosphatidylinositol (Low & Saltiel 1988). Although some of GPI anchored molecules are involved in cell adhesion (Malek et al 1994) or regulation of the complement system (van der Merwe et al 1995), but physiological functions of most of them are not known. The demonstration of signaling occurring through GPI anchored proteins presents an interesting dilemma. Although these

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proteins are tenuously tethered to the outer lamella of the membrane and have no direct contact with internal milieu. Engagement of these receptors by microbes or their products have been shown to trigger cellular responses (Clarkson et al 1995, Baorto et al 1997). Many GPI * anchored moieties, including CD48, are typically found in special “glycolipid enriched microdomains” in the plasma membranes of cells (Simons & Ikonen 1997). These microdomains are rich in signaling molecules such as the heterotrimeric GTP binding proteins which can potentially mediate signal transduction from GPI anchored proteins. G proteins are involved in many signal transduction pathways, including stimulation of adenylate cyclase, regulation of Ca++ channels, stimulation of phospholipase A2 and stimulation of phosphoinositol 3-kinase or phospholipase C (Tankavel et al 1997). A recent immunochemical study has shown that CD48 in lymphocytes is physically associated with GTP binding proteins (Solomon et al 1996). Thus, engagement of CD48 could potentially trigger a mast cell response via a signaling pathway involving GTP binding proteins. Engagement of CD48 has also been shown to activate Src family member tyrosine kinases which are also important effectors of signal transduction found associated with glycolipid enriched microdomains of the plasma membrane (Cebecauer & Horejsi 1998).

7.

CONCLUSION

In this review, we have provided substantial evidence that mast cells mount a protective inflammatory response against enterobacterial infection. In response to bacterial recognition mast cells release a number of proinflammatory mediators including neutrophil chemoattractants TNFα and LTB4 . Our results also show that the mast cell TNFα and LTB 4 responses were crucial for bacterial clearance and for, the survival of mice when challenged by potentially lethal doses of enterobacteria. A diagrammatic depiction of the mast cell’s role is presented in Figure 2. Although there are potentially many ways that mast cells may be activated during bacterial infection, we have identified an opsonin independent mechanism involving the direct binding of enterobacteria to mast cell plasma membrane. The specific molecules that are involved are FimH, a mannose binding lectin presented on the distal tips of bacterial fimbriae, and CD48 (at least in rodent mast cells) a molecule that is anchored to the mast cell plasma membrane via a GPI moiety. Because many potentially pathogenic enterobacteria express FimH, mast cells have the intrinsic capacity to recognize and bind a wide range of enterobacteria.

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The coupling of FimH with CD48 result in mast cell activation leading to the release of several proinflammatory mediators which can potentially play a key role in determining the nature and intensity of the early inflammatory response to the infecting bacteria. It should be emphasized, however, that while this mode of recognition between mast cells and FimH-expressing enterobacteria exists, its utility depends to a large extent on the immune status of the host. Because of the preponderence of enterobacteria amongst our endogenous microflora, most healthy individuals have relatively high levels of enterobacteriaspecific antibodies in circulation and in mucosal secretions. Thus, invading enterobacteria are liable to be opsonized first with specific antibody or by complement fragments prior to encountering any mast cells. In which case, the interactions between the opsonized bacteria and mast cells may involve IgG or complement receptors on the mast cell rather than CD48. At any given time, the interactions between FimHexpressing bacteria and mast cells in vivo are likely to involve both opsonin-dependent and independent interactions. In healthy hosts, the opsonin-dependent interactions are likely to predominate whereas in naïve or immunocompromized patients, the opsonin-independent interactions such as that mediated between FimH and CD48 are likely to predominate. Although both types of interactions can result in mast cell activation, the range and amounts of mediator release are likely to be different since distinct mast cell receptors are involved. Nevertheless, the capacity of mast cells to recognize and respond to enterobacteria even in the absence of opsonins is consistent with its proposed role in contributing to the host’s innate immune system.

ACKNOWLEDGMENTS This work was supported in part from research grants from the NIH (AI 35678 and DK 50814).

REFERENCES Abraham, S. N., Goguen, J. D., Sun, D., Klemm, P . , and Beachey, E. H . (1987). Identification of two ancillary subunits of Escherichia coli type 1 fimbriae by using antibodies against synthetic oligopeptides of fim gene products. J Bacteriol 169:5530-6. Abraham, S. N., Sun, D., Dale, J. B., and Beachey, E . H. (1988). Conservation of the Dmannose-adhesion protein among type 1 fimbriated members of the family Enterobacteriaceae. Nature 336:682-4.

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Immunity

Baorto, D. M., Gao, Z., Malaviya, R., Dustin, M. L., van der Merwe, A., Lublin, D. M., and Abraham, S. N. (1997). Survival of FimH-expressing enterobacteria in macrophages relies on glycolipid traffic. Nature 389, 636-9. Cebecauer, M., Cerny, J., and Horejsi, V. (1998). Incorporation of leucocyte GPI-anchored proteins and protein tyrosine kinases into lipid-rich membrane domains of COS-7 cells. Biochem Biophys Res Commun 243, 706-10. Clarkson, N. A., Kaufman, R., Lublin, D. M., Ward. T., Pipkin, P. A., Minor, P. D., Evans, D. J., and Almond, J. W. (1995). Characterization of the echovirus 7 receptor: domains of CD55 critical for virus binding. J Virol 69, 5497-501. Echtenacher, B., Mannel, D. N., and Hultner, L. (1996). Critical protective role of mast cells in a model of acute septic peritonitis [see comments]. Nature 381, 75-7. Fantozzi, R., Brunelleschi, S., Rubino, A., Tarli, S., Masini, E., and Mannaioni, P. F. (1986). FMLP-activated neutrophils evoke histamine release from mast cells. Agents Actions 18, 155-8. Fenton, M. J., and Golenbock, D. T. (1998). LPS-binding proteins and receptors. J Leukoc Biol 64, 25-32. Galli, S. J. (1993). New concepts about the mast cell. N Engl J Med 328, 257-65. Galli, S. J. and Kitamura, Y. (1988). Genetically mast-cell-deficient W/Wv and S1/S1d mice. Their value for the analysis of the roles of mast cells in biologic responses in vivo. Am. J. Pathol. 174, 103-7. Galli, S. J.. and Wershil, B. K. (1996). The two faces of the mast cell [news; comment]. Nature 381, 21-2. Galli, S. J., Gordon, J. R.. and Wershil, B. K. (1991). Cytokine production by mast cells and basophils. Curr Opin Immunol 3, 865-72. Gordon, J. R., and Galli, S. J. (1987). Release of both preformed and newly synthesized tumor necrosis factor alpha (TNF-alpha)/cachectin by mouse mast cells stimulated via the Fc epsilon RI. A mechanism for the sustained action of mast cell-derived TNF-alpha during IgE-dependent biological responses. Am J Pathol 127, 191-8 Leal-Berumen, I., Conlon, P., and Marshall, J. S. (1994). IL-6 production by rat peritoneal mast cells is not necessarily preceded by histamine release and can be induced by bacterial lipopolysaccharide. J Immunol 152, 5468-76. Leal-Berumen, 1., Snider, D. P., Barajas-Lopez, C.. and Marshall, J. S. (1996). Cholera toxin increases IL-6 synthesis and decreases TNF-alpha production by rat peritoneal mast cells. J Immunol 156, 3 16-2 I. Levy, D. A., and Frondoza, C. (1983). Immunity to intestinal parasites: role of mast cells and goblet cells. Fed Proc 42, 1750-5. Low, M. G., and Saltiel, A. R. (1988) Structural and functional roles of glycosylphosphatidylinositol in membranes. Science 239, 268-75 Lukacs, N. W., Strieter, R. M., Chensue, S. W., and Kunkel, S. L. (1996). Activation and regulation of chemokines in allergic airway inflammation. J Leukoc Biol 59, 13-7. Malaviya, R., Gao, Z., Thankavel, K., Merwe, P. A., and Abraham, S. N. (1999). The mast cell tumor necrosis factor alpha response to FimH-expressing Excherichia coli is mediated by the glycosylphosphatidylinositol-anchored molecule CD48. PNAS 96, 81 10-5. Malaviya, R., Ikeda, T., Ross, E., and Abraham, S. N. (1996). Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-alpha. Nature 381, 77-80. Malaviya, R., Ross, E. A., MacGregor, J. I., Ikeda, T.: Little, J. R., Jakschik, B. A., and Abraham, S. N. (1994). Mast cell phagocytosis of FimH-expressing enterobacteria. J lmmunol 152, 1907-14.

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Malaviya, R.. Ross, E., Jakschik, B. A., and Abraham. S. N. (1994). Mast cell degranulation induced by type 1 fimbriated Escherichia coli in mice. J Clin Invest 93, 1645-53. Malek, T. R., Fleming, T. J., and Codias, E. K. (1994) Regulation of T lymphocyte function by glycosylphosphatidylinositol (GPI)-anchored proteins. Semin lmmunol 6, 10513. Marone, G., Casolaro, V., Patella, V.. Florio, G., and Triggiani, M. (1997). Molecular and cellular biology of mast cells and basophils. Int Arch Allergy Immunol 114, 20717. Medzhitov, R., and Janeway, C. A.; Jr. (1997). Innate immunity: impact on the adaptive immune response. Curr Opin Immunol 9, 4-9. Miller, H. R. (1996). Mucosal mast cells and the allergic response against nematode parasites. Vet Immunol Immunopathol 54, 33 1-6. Prodeus. A. P., Zhou, X., Maurer, M., Galli. S. J., and Carroll, M. C. (1997). Impaired mast cell-dependent natural immunity in complement C3- deficient mice. Nature 390, 172-5. Sher, A., and Mclntyre, S. L. (1977). Receptors for C3 on rat peritoneal mast cells. J Immunol 119, 722-5. Sher. A., Hein, A., Moser, G.. and Caulfield, J. P. (1 979). Complement receptors promote the phagocytosis of bacteria by rat peritoneal mast cells. Lab Invest 41, 490-9. Simons. K., and Ikonen, E. (1997). Functional rafts in cell membranes. Nature 387, 56972. Solomon, K. R.. Rudd, C. E.. and Finberg, R. W. (1996). The association between glycosylphosphatidylinositol-anchored proteins and heterotrimeric G protein alpha subunits in lymphocytes. Proc Natl Acad Sci U S A 93, 6053-8. Thankavel. K., Madison. B., Ikeda. T., Malaviya. R., Shah, A. H., Arumugam. P. M., and Abraham, S. N. (1997). Localization of a domain in the FimH adhesin of Escherichia coli type 1 fimbriae capable of receptor recognition and use of a domain-specific antibody to confer protection against experimental urinary tract infection. J Clin Invest 100, 1123-36. van der Merwe. P. A., Barclay, A. N., Mason, D. W., Davies, E. A., Morgan, B. P., Tone, M., Krishnam, A. K., Ianelli, C., and Davis. S. J. (1994). Human cell-adhesion molecule CD2 binds CD58 (LFA-3) with a very low affinity and an extremely fast dissociation rate but does not bind CD48 or CD59. Biochemistry 33, 10149-60. van der Merwe. P. A., McNamee, P. N., Davies, E. A., Barclay, A. N., and Davis, S. J. (1 995) Topology of the CD2-CD48 cell-adhesion molecule complex: implications for antigen recognition by T cells. Curr. Biol, 5, 74-84. Wojtecka-Lukasik, E., and Maslinski. S. (1992). Fibronectin and fibrinogen degradation products stimulate PMN-leukocyte and mast cell degranulation. J Physiol Pharmacol 43, 173-8 1. Zhang, Y., Ramos, B. F., and Jakschik, B. A. (1992). Neutrophil recruitment by tumor necrosis factor from mast cells in immune complex peritonitis. Science 258, 1957-9. Zheutlin, L. M.. Ackerman. S. J.. Gleich, G. J., and Thomas. L. L. (1984). Stimulation of basophil and rat mast cell histamine release by eosinophil granule-derived cationic proteins. J Immunol 133, 2180-5.

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THE NADPH OXIDASE DIAPHORASE ACTIVITY IN PERMEABILIZED HUMAN NEUTROPHILS AND GRANULOCYTIC LIKE PLB-985 CELLS

Itai Pessach and Rachel Levy Infectious Diseases Laboratory, Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of The Negev and Soroka Medical Center, Beer Sheva 84105, Israel

1.

SUMMARY

The phagocyte NADPH oxidase is a multicomponent transport chain that generates superoxide, a precursor of microbicidal oxidants, important for host defense. This transport chain is contained mainly in the large membrane subunit of the oxidase (gp91 phox), and transfers electrons from cytosolic NADPH, through FAD binding and heme centers, to molecular oxygen (Babior, 1999; Fujii and Kakinuma, 1991; Rotrosen et al., 1992; Segal and Abo, 1993). Cross et al. have recently described a novel NADPH oxidase diaphorase activity present in the membrane fraction of activated neutrophils, using a cell free model (Cross et al., 1994). This diaphorase activity is measured by the artificial electron acceptor 4 -iodonitrotetrazolium violet (INT) and is attributed to the reduction of the flavin center of the flavocytochrome (Cross et al., 1994; Li and Guillory, 1997). In the present study we establish a system for detecting diaphorase activity in intact cells. Neutrophils and PLB-985 cells, that were differentiated using 1.25% dimethyl sulfoxide (DMSO) to granulocyte phenotype, were permeabilized by electroporation, and diaphorase activity was determined using INT. Neutrophils and differentiated PLB-985 cells stimulated by PMA or GTP S showed a diaphorase activity that was not present in unstimulated The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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differentiated cells. The diaphorase activity could not be detected in undifferentiated cells and was developed during differentiation. The pattern of diaphorase activity in stimulated parent differentiated PLB cells was similar to that observed in stimulated human neutrophils. The permeabilized – INT cell system offers a unique tool for the evaluation of NADPH oxidase diaphorase activity, in whole cells.

2.

INTRODUCTION

The phagocyte NADPH oxidase is a multicomponent transport chain that transfers electrons from NADPH to molecular oxygen and generates superoxide, a precursor of microbicidal oxidants important for host defense. NADPH oxidase subunits include three cytoplasmic components, p47 phox, p67 phox, and rac2, and a membrane flavocytochrome b558 composed of gp91phox and p22phox (Babior, 1999; Knaus et al., 1991; Leto et al., 1990; Lukacs et al., 1993; Nunoi et al., 1988; Rotrosen et al., 1992; Volpp et at., 1988; Volpp et at., 1989). The flavocytochrome b558 contains two redox centers (FAD and heme) and the NADPH binding site (Segal and Abo, 1993). In addition, a H + channel was identified in the 230 N-terminal amino acids of gp91phox (Babior, 1999; Henderson et al., 1997; Lukacs et al., 1993). In phagocytes, stimulation results in translocation of the cytosolic NADPH oxidase components to the membrane where they interact with the flavocytochrome to form the activated oxidase. This activated form of the enzyme is able to transfer electrons from cytosolic NADPH to extracellular or phagosomal molecular oxygen through the electron transport chain. The flavin center transfers electrons from the physiological electron donor, NADPH, which binds to a specific binding site contained in the cytochrome, to the heme of the cytochrome. The latter serves as the terminal electron donor to oxygen (Babior, 1999; Cross et al., 1994). Several studies have demonstrated a diaphorase activity associated with NADPH oxidase in a cell free system using the artificial electron acceptor iodonitrotetrazolium violet (INT) (Cross et al., 1994). This diaphorase activity is measured by the artificial electron acceptor INT and is attributed to the reduction of the flavin center of the flavocytochrome (Cross and Curnutte, 1995; Cross et al., 1994; Li and Guillory, 1997). The aim of the present study was to develop a novel system for measuring oxidase diaphorase activity in whole cells, permeabilized neutrophils and differentiated PLB-985 cells.

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INT REDUCTION IN PERMEABILIZED HUMAN NEUTROPHILS

NADPH oxidase diaphorase activity was detected in cell free systems and membrane fractions of activated human neutrophils using INT as a substrate. Cross et al have reported that INT is not reduced appreciably by intact cells suggesting its interaction with an inner site in the cytoplasmic membrane. Our experiments were designed in order to develop a system for detecting diaphorase activity in whole cells, using a permeabilized cell technique. This system was developed first in permeabilized human neutrophils and than applied to permeabilized PLB-985 cells differentiated to a granulocytic phenotype. Cells were permeabilized according to Lu et al. with slight modification (Lu and Grinstein, 1990). Stimulation of permeabilized human neutrophils with the optimal concentrations of GTPγS (330 µM) or PMA (50 ng/ml) resulted in linear INT reduction activity. These activities in stimulated cells were significantly higher than in non-activated cells (P < 0.01) (Table 1). INT reduction could not be detected in the absence of NADPH in the reaction mixture (Table 1). The requirement of NADPH as a substrate and of stimuli (PMA or GTPγS) for INT reduction indicates a close association with the mechanisms that initiate NADPH oxidase activation. Table 1. INT reductase activity of activated permeabilized Human neutrophils.

- NADPH

Unstimulated 0 ± 0.02

PMA 1.86 ± 0.46

GTvS 1.14 ± 0.3

+NADPH

6.6 ± 1.42

21 ± 1.01

11.7 ± 1.4

+NADPH + SOD 7.41 ± 0.61 11.12 ± 1.11 8.28 ± 0.84 INT reduction was measured in the micro plate assay system. 8x106 cells were permeabilized, and then transferred to the reaction mixture containing 1 mM INT and 2 mM, NADPH. Shown in the table are the rates ± SE of INT reduction (mOD/min) by permeabilized neutrophils activated by PMA (50 ng/ml) or GTP S (330 µM) compared to unstimulated cell in the presence or absence of NADPH. Also shown are the effects of 60 µg/ml SOD on the rate of INT reduction in permeabilized neutrophils.

In results similar to ours, Cross et al have demonstrated that membranes of unstimulated neutrophils possess an INT reductase activity, which is about half of that possessed by membranes of stimulated cells (Cross et al., 1994). In order to determine the effect of superoxides released by the oxidase on INT reduction in permeabilized neutrophils, diaphorase activity was studied in the presence of superoxide dismutase (SOD). The optimal concentration of 60 µg/ml SOD that totally inhibits cytochrome C reduction in intact neutrophils was used. Stimulation of permeabilized neutrophils by either 50 ng/ml PMA or 330 µM GTPγS, in the presence of SOD resulted in a 48% or 51% inhibition in the rate of

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INT reduction, respectively (Table 1). Thus, 60 µg/ml SOD were added to the reaction mixture of all INT reduction assays. In order to prevent any INT reduction by the mitochondrial electron transfer chain, all experiments were carried out in the presence of 0.5 µg/ml rotenone.

4.

ISOLATION OF NADPH OXIDASE SPECIFIC DIAPHORASE ACTIVITY BY THE USE OF DPI

In a cell free system, NADPH oxidase diaphorase activity can be inhibited by diphenylene iodonium (DPI), which is a known NADPH oxidase inhibitor (Cross et al., 1994). The addition of 6.5 µM of DPI to the reaction mixture caused a significant inhibition of diaphorase activity in permeabilized neutrophils stimulated by either 50 ng/ml PMA or 330 µM GTP S, while it had no effect on INT reduction in unstimulated cells. A representative experiment demonstrating the inhibition of diaphorase activity stimulated by GTP S is presented in Figure 1 . The requirement of NADPH as a substrate and of stimuli (PMA or GTP S) for INT reduction and its inhibition by DPI, all performed in the presence of SOD indicates that specific oxidase diaphorase activity may be efficiently determined in permeabilized neutrophils, by this method.

Figure 1.The effect of DPI on INT reduction in permeabilized neutrophils. Stimulated INT reduction was measured in the micro plate assay system, in the presence of 6.5 µM DPI (♦ ) and in its absence ( ■ ). Shown is the time dependency of INT reduction in permeabilized neutrophils activated by 330 µM GT pγ S. The experiments were done in the presence of 60 µg/ml SOD and 0.5 µg/ml rotenone.

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NADPH OXIDASE DIAPHORASE ACTIVITY IN PERMEABILIZED DIFFERENTIATED PLB-985 CELLS

5.

Diaphorase activity in permeabilized differentiated PLB-985 cells was studied and compared to that of permeabilized human neutrophils. Similar to neutrophils, INT reductase activity in permeabilized differentiated PLB-985 cells was significantly higher in cells stimulated with PMA or GTP S than in non-activated cells (p<0.05), was not affected by the addition of 0.5 µg/ml rotenone, and was inhibited by the presence of 60 µg/ml SOD (Table 2). Table 2. INT reductase activity of activated permeabilized differentiated PLB-985 cells

-NADPH

Unstimulated 0 ± 0.04

PMA 1.61 ± 0.39

+NADPH

5.67 ± 0.90

12.28 ± 1.02

+NADPH + SOD

5.97 ± 1.36

8.05 ± 0.95

GTD S 1.29 ± 0.41 8.54 ± 0.97

5.76 ± 0.37 INT reduction was measured in the micro plate assay system. 8x106 cells were permeabilized, and then transferred to the reaction mixture containing 1 mM INT and 2 mM, NADPH. Shown in the table are the rates ± SE of INT reduction (mOD/min) by permeabilized neutrophils activated by PMA (50 ng/ml) or GTPyS (330 µM) compared to unstimulated cell in the presence or absence of NADPH. Also shown are the effects of 60 µg/ml SOD on the rate of INT reduction in permeabilized neutrophils.

Figure 2. The effect of DPI on INT reduction in permeabilized differentiated PLB-985 cells. The time dependency of INT reduction in permeabilized differentiated PLB-985 cells activated by GTPγS (330 µM) in the presence of 6.5 µM DPI (♦ ) or in its absence ( ■ ). The experiments were done in the presence of 60 µg/ml SOD and 0.5 µglml rotenone.

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DPI inhibitable diaphorase activity was determined in permeabilized differentiated PLB-985 cells. 6.5 µM of DPI in the presence of both 60 µg/ml SOD and 0.5 µg/ml rotenone inhibited INT reduction in activated differentiated PLB-985 cells. A* representative experiment demonstrating the inhibition of diaphorase activity in these cells stimulated by GTPγS is presented in Figure 2.

6.

NADPH OXIDASE DIAPHORASE ACTIVITY IN UNDIFFERENTIATED PLB-985 CELLS

DPI inhibitable INT reduction was absent in undifferentiated PLB-985 cells, stimulated by either PMA or GTPγS (Figure 3), further supporting that this activity represents the NADPH oxidase diaphorase activity, which is known to develop only after differentiation.

Figure 3. DPI inhibitable diaphorase activity in undifferentiated PLB-985 cells The time dependency of DPI inhibitable INT reduction in undifferentiated PLB-985 cells activated by 50 ng/ml PMA (0) or 330 µM GTPγS DPI inhibitable diaphorase activity was calculated by the subtraction of the diaphorase activity in the presence of 6.5 µM of DPI from the total diaphorase activity.

7.

CONCLUSION

The results presented in the study show that NADPH oxidase diaphorase activity can be measured in whole human neutrophils and differentiated PLB-985 cells, using the electroporation technique and INT as a substrate. Comparison of diaphorase activity in permeabilized

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neutrophils to that of permeabilized differentiated PLB-985 cells revealed that the fraction of DPI inhibitable diaphorase activity is lower in PLB cells in accordance with the lower level of oxidase activity detected in these cells. This novel system for the detection of NADPH oxidase specific diaphorase activity in whole cells offers a unique tool for studying the oxidase electron transfer chain and the mechanisms resulting in its physiological activation.

ACKNOWLEDGMENT This research was supported by grant no. 97-00178 from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel.

REFERENCES Babior, B. M. (1999). NADPH oxidase: an update. Blood 93, 1464-76. Cross, A. R., and Curnutte, J. T. (1995). The cytosolic activating factors p47phox and p67phox have distinct roles in the regulation of electron flow in NADPH oxidase. J Biol Chem 270, 6543-8. Cross, A. R., Yarchover, J. L., and Curnutte, J. T. (1994). The superoxidegenerating system of human neutrophils possesses a novel diaphorase activity. Evidence for distinct regulation of electron flow within NADPH oxidase by p67phox and p47-phox. J Biol Chem 269, 21448-54. Fujii, H., and Kakinuma, K. (199 1). Electron transfer reactions in the NADPH oxidase system of neutrophils--involvement of an NADPH-cytochrome c reductase in the oxidase system. Biochem Biophys Acta 1095, 201 -9. Henderson, L. M., Thomas, S., Banting, G., and Chappell, J. B. (1997). The arachidonate-activatable, NADPH oxidase-associated H+ channel is contained within the multi-membrane-spanning N-terminal region of gp91-phox. Biochem J 325, 701-5. Knaus, U. G., Heyworth, P. G., Evans, T., Curnutte, J. T., and Bokoch, G. M. (1 99 1). Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2. Science 254, 1512-5. Leto, T. L., Lomax, K. J., Volpp, B. D., Nunoi, H., Sechler, J. M., Nauseef, W. M., Clark, R. A., Gallin, J. I., and Malech, H. L. (1990). Cloning of a 67-kD neutrophil oxidase factor with similarity to a noncatalytic region of p60c-src. Science 248, 727-30. Li, J., and Guillory, R. J. (1 997). Purified leukocyte cytochrome b558 incorporated into liposomes catalyzes a cytosolic factor dependent diaphorase activity. Biochemisty 36, 5529-37. Lu, D. J., and Grinstein, S. (1990). ATP and guanine nucleotide dependence of neutrophil activation. Evidence for the involvement of two distinct GTP-binding proteins. J Biol Chem 265, 1372 1-9.

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Lukacs, G. L., Kapus, A., Nanda, A., Romanek, R., and Grinstein, S. (1993). Proton conductance of the plasma membrane: properties, regulation, and functional role. Am J Physiol 265, C3-14. Nunoi, H., Rotrosen, D., Gallin, J. I., and Malech, H. L. (1988). Two forms of autosomal chronic granulomatous disease lack distinct neutrophil cytosol factors. Science 242, 1298-301. Rotrosen, D., Yeung, C. L., Leto, T. L., Malech, H. L., and Kwong, C. H. (1992). Cytochrome b558: the flavin-binding component of the phagocyte NADPH oxidase. Science 256, 1459-62. Segal, A. W., and Abo, A. (1993). The biochemical basis of the NADPH oxidase of phagocytes. Trends Biochem Sci 18, 43-7. Volpp, B. D., Nauseef, W. M., and Clark, R. A. (1988). Two cytosolic neutrophil oxidase components absent in autosomal chronic granulomatous disease. Science 242, 1295-7. Volpp, B. D., Nauseef, W. M., Donelson, J. E., Moser, D. R., and Clark, R. A. (1989). Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human neutrophil respiratory burst oxidase. Proc Natl Acad Sci USA 86, 7 195-9.

ACTIVATION OF CYTOSOLIC PHOSPHOLIPASE A2 BY OPSONIZED ZYMOSAN IN HUMAN NEUTROPHILS REQUIRES BOTH ERK AND p38 MAP-KINASE

Inbal Hazan-Halevy and Rachel Levy Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev and Soroka Medical Center, Beer-Shew, Israel

1.

SUMMARY

The present study demonstrates that stimulation of human neutrophils with opsonized zymosan (OZ), which binds to Fc gamma receptors (FcγRs) and C3b receptors, activates both ERK and p38 MAPkinase. Thus, the relative role of both types of MAP-kinase, ERK and p38, in activation of cPLA2 by OZ was studied. cPLA2 activation by OZ was detected 15 sec after stimulation, maintained a plateau for 10 min and decreased thereafter. p38 MAP-kinase activation exhibited kinetics similar to that of cPLA2, while ERK activation was detected within 15 sec but decreased significantly in less than 5 min after stimulation. Pretreatment of the cells with the MEK inhibitor, PD-098059, or the p38 MAP-kinase inhibitor, SB-203580 resulted in total inhibition of ERK or p38 MAP-kinase activity, respectively. Each inhibitor caused a partial inhibition during the time course of cPLA2 activity, while their combination caused a total inhibition. Compared to OZ, inactivated OZ, which does not contain the complement proteins, induced an identical time-dependent stimulation of ERK and p38 MAP-kinase as well as a similar cPLA2 activity, suggesting that the role of the C3b receptors in this system is negligible. It is concluded that OZ activates both ERK and The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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p38 MAP-kinase and that the two isotypes are required for the onset and maintenance of cPLA2 activity.

2.

INTRODUCTION

Immune complexes are recognized by specific receptors present on the plasma membrane of phagocytes: Fc gamma receptors (FcγRs) and receptors for complement protein C3b. The binding of immune complexes by neutrophils (PMN) receptors induces essential host defense and inflammatory responses such as adhesion, phagocytosis of antibodycoated microorganisms, degranulation and the respiratory burst (Smith 1994). Mitogen-activated protein kinases (MAP-kinases) are a family of serine/threonine kinases whose activation requires phosphorylation on tyrosine and threonine residues. The MAP-kinase family includes the 4244 kDa extracellular regulated kinases (ERK) and the stress activated protein kinases (SAPK): 38kDa MAP-kinase, c-jun-N-terminal kinase (JNK) and big MAP-kinase (BMK, ERK5) (Seger and Krebs 1995). In various systems, both ERK and p38 MAP-kinases have been shown to phosphorylate p85 cytosolic phospholipase A2 (p85 cPLA2) on serine residue (505) and thus render it active (Borsch Haubold et al 1998, Lin et al 1993). We have previously shown the involvement of ERK1/2 in activating p85 cPLA2 and superoxide production in human neutrophils stimulated with opsonized zymosan (OZ) as a model for the immune complex (Hazan et al 1997). We also provided evidence for the essential requirement of arachidonic acid signals generated by p85 cPLA 2 in activation of the phagocyte NADPH oxidase (Dana et al 1998). The present study was designed to evaluate the relative role of p38 MAPkinase and ERK in cPLA2 activation by OZ.

3.

ACTIVATION OF ERK AND P38 MAP-KINASE BY OZ

In order to differentiate between the role of the two types of MAPkinases, ERK and p38 MAP-kinase in activation of cPLA2, the time course activation of each isotype by OZ was determined and compared to that of cPLA2. As shown in Fig 1A, stimulation of neutrophils with 1 mg/ml OZ caused a temporary and transient phosphorylation of ERK1/2, reaching a maximal level at 1-2 min and decreasing thereafter. ERK1/2 phosphorylation could not be detected 10 min after stimulation. An equal

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amount of ERK2 in each sample was evaluated by immunoblotting with anti ERK2 antibody (Fig 1B). This blot also illustrates, by electrophoretic mobility shift, the same pattern of ERK activation that is demonstrated in Fig 1 A. Phosphorylated ERK2 with reduced electrophoretic mobility was detectable as early as 15 sec after stimulation and showed a maximum activation at 1-2 min. Phosphorylation of p38 MAP-kinase induced by 1mg/ml OZ was detected 15 sec after stimulation, peaked 2-5 min later, stayed elevated for 10 min and slightly decreased at 20 min (Fig 1C). Western blot analysis with anti p38 MAP-kinase antibody confirmed an equal amount of p38 MAP-kinase in each sample (Fig 1D).

Figure 1. Time course of ERK and p38 MAP-kinase activity stimulated by OZ. Representative immunoblots of ERK (A) and p38 MAP-kinase (C) phosphorylation induced by 1mg/ml OZ using phospho-specific ERK or p38 MAP-kinase antibodies, respectively. Equal amounts of ERK2 (B) or p38 MAP-kinase (D) in each sample were evaluated by immunobloting the same blots with anti ERK2 or anti p38 MAP-kinase antibodies, respectively

4.

ACTIVATION OF CYTOSOLIC PLA2 BY OZ

The different temporal patterns of activation of ERK and p38 MAPkinase by 0z would seem to indicate a distinct role for each of these MAP-kinases in cPLA2 activation. Therefore, the time course of cPLA2 activity was determined by measuring arachidonic acid release from radiolabeled arachidonyl-phosphatidylcholine vesicles and was compared

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Activation of Cytosolic Phospholipase

to the time course activation of each MAP-kinase isotype. cPLA2 activity induced by OZ was detected as early as 15 sec after stimulation, maintained a plateau for 5 min and decreasing thereafter (Fig 2). Ten min after stimulation, cPLA2 activity was still elevated, similar to that of p38 MAP-kinase, while ERK activity could not be detected.

Figure 2. Time course of cPLA2 activation by OZ. cPLA2 specific activity was determined in neutrophil lysates using labelled phosphatidylcholine vesicles as a substrate. The results expressed as specific activity.

5.

THE ROLE OF ERK AND P38 MAP-KINASE IN CYTOSOLIC PLA2 ACTIVATION OF BY OZ

Since both ERK and p38 MAP-kinase are capable of phosphorylating and activating cPLA2 active (Borsch Haubold et al 1998, Lin et al 1993), we utilized the MEK and the p38 MAP-kinase inhibitors to evaluate the relative role of these two MAP-kinases in cPLA2 activation. As demonstrated in the representative experiment in Fig 3, preincubation of neutrophils with either 100 µM PD-098059 or with 5 µM SB-203580 caused partial inhibition of cPLA2 activity following stimulation with OZ for the indicated times. Pretreatment of the cells with both inhibitors resulted in total inhibition of cPLA2 activity induced by OZ. These results clearly indicate both types of MAP-kinase, ERK and p38, are

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required for the onset and the maintenance of complete cPLA 2 activity induced by OZ.

Figure 3. Effect of p38 MAP-kinase and/or ERK inhibitors on cPLA2 activity stimulated by OZ. Neutrophils were preincubated for 40 min at 4°C with 100 µM PD-098059 and/or 5 µM SB-203580. Cells were stimulated for 2 min or 10 min with 1 mg/ml OZ at 37°C and cPLA2 activity was determined in neutrophil lysates using labelled phosphatidylcholine vesicles as a substrate.

6.

ACTIVATION OF ERK, P38 MAP-KINASE AND CYTOSOLIC PLA2 BY INACTIVATED OZ

The requirement of two MAP-kinase isotypes for activation of cPLA2 by OZ was queried since various studies have reported that only one type of MAP-kinase is sufficient for cPLA2- stimulation by different agents in human neutrophils. Since OZ is ligated by two kinds of receptors in human neutrophils, FcγRs and C3bR (Smith 1994), the possibility was raised that each of them initiates a response leading to activation of one type of MAP-kinase. In order to evaluate the relative role of C3bR in cPLA2 activity mediated by MAP-kinase, the effect of zymosan opsonized with heat inactivated-pooled human serum (iOZ), which does not contain the complement protein (C3b) was studied. The time course activation of ERK and p38 MAP-kinase by 1 mg/ml iOZ was determined and compared to that of OZ. iOZ induced a time-dependent phosphorylation of ERK (Fig 4A) and p38 MAP-kinase (Fig 4C) identical to that induced by OZ (Figs 1A and IC).

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Activation of Cytosolic Phospholipase

Figure 4. Time course activation of ERK and p38 MAP-kinase stimulated by inactivated OZ. Representative lmmunoblots of ERK (A) or p38 MAP-kinase (C) phosphorylation using phospho-specific ERK or p38 MAP-kinase antibodies, respectively. Equal amounts of ERK (B) or p38 MAP-kinase (D) in each sample were evaluated by immunobloting with anti ERK2 or anti p38 MAP-kinase antibodies, respectively.

In order to compare the effect of iOZ and OZ on the extent of MAPkinase phosphorylation and cPLA2 activation, these activities were simultaneously analyzed in neutrophils stimulated for 2 min with either OZ or iOZ. As demonstrated in Fig 5A and 5C, OZ and iOZ similarly induced a significant phosphorylation of ERK and p38 MAP-kinase. Likewise, a significant and similar stimulation of cPLA2 activity was induced by OZ or iOZ (0.304 and 0.284 pmoles/µg/30 min, respectively) (Fig 5E). Indicating that the complement receptor does not participate in activating ERK, p38 MAP-kinase and cPLA2 in neutrophils.

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Figure 5. Comparison between activation of ERK, p38 MAP-kinase and cPLA2 by OZ and inactivated OZ. Representative immunoblots of ERK (A) and p38 MAP-kinase (C) phosphorylation using phospho-specific ERK or p38 MAP-kinase antibodies, respectively. The blots were stripped and reprobed with anti ERK (B) or anti p38 MAPkinase (D) antibodies, respectively. E - cPLA2 activity was determined in neutrophil lysate using labeled phosphatidylcholine vesicles as a substrate. The results expressed as specific activity.

7.

THE ROLE OF P38 MAP-KINASE AND ERK IN NADPH-OXIDASE ACTIVATION BY OZ

We recently provided evidences indicating the essential requirement for arachidonic acid signals generated by p85 cPLA2 for activation of the phagocyte NADPH oxidase (Dana et al 1998). The results of the present study suggest that both ERK and p38 MAP-kinase are involved in the activation of cPLA 2 associated with OZ stimulation. Therefore, the effect of SB-203580 and PD-098059 on superoxide production was also studied. Pretreatment of neutrophils with PD-098059 or SB-203580 caused a partial inhibition of superoxide production (Fig 6). Preincubation of neutrophils with both 5 µM SB-203580 and 100 µM PD-098059 resulted in complete inhibition of superoxide production in accordance with the effect of these

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Figure 6. Effect of p38 MAP-kinase and/or ERK inhibitors on superoxide production stimulated by OZ. Neutrophils were preincubated for 40 min at 4°C with PD-098059 and/or SB-203580 before stimulation with 1mg/ml OZ. Superoxide production was measured during 20 min and the rate was determined. The results expressed as percentages of control.

8.

CONCLUSION

The present study demonstrates, as summarized in Fig 7, that activation of cPLA2 induced by OZ is mediated mainly by FcγRs. Both ERKs and p38 MAP-kinase are necessary for the onset and the maintenance of cPLA 2 activation induced by OZ.

Figure 7. Proposed is a schematic for OZ signalling to cPLA2 and NADPH-oxidase in human neutrophils. The scheme is based on present and previous studies (Dana et al 1998, Hazan et al 1997).

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ACKNOWLEDGMENT This research was supported by a grant from Ministry of Health, Israel.

REFERENCES Borsch Haubold, A. G., Bartoli, F., Asselin, J., Dudler, T., Kramer, R. M., Apitz Castro, R., Watson, S. P., and Gelb, M. H., 1998, Identification of the phosphorylation sites of cytosolic phospholipase A2 in agonist-stimulated human platelets and HeLa cells. J. Biol. Chem 273: 4449-4458. Dana, R., Leto, T. L., Malech, H. L., and Levy, R., 1998, Essential requirement of cytosolic phospholipase A2 for activation of the phagocyte NADPH oxidase. J. Biol. Chem. 273: 441-445. Hazan, I., Dana, R., Granot, Y., and Levy, R., 1997, Cytosolic phospholipase A2 and its mode of activation in human neutrophils by opsonized zymosan. Correlation between 42/44 kDa mitogen-activated protein kinase, cytosolic phospholipase A 2 and NADPH oxidase. Biochem. J. 326: 867-876. Lin, L. L., Wartmann, M., Lin, A. Y., Knopf, J. L., Seth, A., and Davis, R. J., 1993, cPLA2 is phosphorylated and activated by MAP kinase. Cell 72: 269-278. Seger, R., and Krebs, E. G., 1995, The MAPK signaling cascade. Faseb J. 9: 726-735. Smith, J. A., 1994, Neutrophils, host defense, and inflammation: a double-edged sword. J. Leukoc. Biol. 56: 672-686.

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CYTOSOLIC PHOSPHOLIPASE A2 IS REQUIRED FOR THE ACTIVATION OF THE NADPH OXIDASE ASSOCIATED H+ CHANNEL IN PHAGOCYTE-LIKE CELLS

Rachel Levy, Alexander Lowenthal and Raya Dana Laboraton of Infectious Diseases, Department of Clinical Biochemisty, Faculty of Health Sciences, Ben-Gurion University of the Negev and Soroka Medical Center, Beer-Sheva 84105, Israel

1.

SUMMARY

The NADPH oxidase producing-superoxide is the major mechanism by which phagocytes kill invading pathogens. The human myeloid cell line PLB-985 was transfected to express p85 cytosolic phospholipase A2 (cPLA2) antisense mRNA and stable clones were selected which lack detectable cPLA2. cPLA2-deficient PLB-985 cells differentiate similarly to control PLB-985 cells in response to retinoic acid, DMSO or 1,25 dihydroxyvitamin D3 indicating that cPLA2 is not involved in the differentiation process. Despite the normal synthesis of NADPH oxidase subunits during differentiation of cPLA2-deficient PLB-985 cells, these cells fail to activate NADPH oxidase in response to a variety of soluble and particulate stimuli, but addition of exogenous arachidonic acid (AA) fully restores oxidase activity. This establishes an essential requirement of cPLA2 generated AA for activation of phagocyte NADPH oxidase. In order to elucidate the mechanism by which cPLA2 regulates the oxidase, + the role of cPLA2 in NADPH oxidase associated H channel was studied. Activation of differentiated PLB cells resulted in a Zn+2 sensitive alkalization, indicating H+ channel activity. In contrast, differentiated The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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PLB-D cells failed to activate the H+ channel, but addition of exogenous AA fully restored this activity, indicating an essential and specific physiological requirement of cPLA2-generated AA for activation of the H+ channel. The presence of the H+ channel inhibitor, Zn+2 , caused significant inhibition of NADPH oxidase activity, suggesting a role of the + NADPH oxidase associated H channel in regulating oxidase activity.

2.

INTRODUCTION

The phagocyte NADPH oxidase is a multicomponent transport chain that transfers electrons from NADPH to molecular oxygen and generates superoxide, a precursor of microbicidal oxidants important to host defense (Babior, 1999). NADPH oxidase subunits include three cytoplasmic components, p47phox, p67phox and rac2, and a membrane flavocytochrome b558 composed of gp91 phox and p22phox (Rotrosen et al., 1992). In differentiated phagocytic cells stimulation results in translocation of the cytosolic NADPH oxidase components to the membrane where they interact with the flavocytochrome to form the activated oxidase leading to superoxide generation. The signals responsible for assembly and activation of the oxidase are not clearly defined. It has been demonstrated that the single electron transfer from internal NADPH, through the oxidase complex, to external oxygen is an + electrogenic process and that the efflux of H+ through the H channel is necessary for charge compensation (Henderson et al., 1987). The channel was found to be singularly H+ -selective, voltage-gated outwardly rectifying, regulated by both external and internal protons and sensitive to heavy metals (Cd2+ , Zn2+ ). PLA2 activity has been implicated in a variety of responses by stimulated phagocytes, including degranulation, phagocytosis, adhesion, cell spreading and activation of NADPH oxidase (Cockcroft, 1991; Lefkowith et al., 1992; Lennartz and Brown, 1991). A series of recent studies suggest that AA regulates H+ channel activity in neutrophils (Henderson and Chappell, 1992; Susztak et al., 1997) and in macrophages (Kapus et al., 1994). Transfection studies demonstrating heterologous expression of gp9 1phox have indicated that gp9 1 phox or its N-terminal membrane spanning domain (residues 1-230) also constitutes an AA-activated H+ channel (Henderson et al., 1995; Henderson et al., 1997). The histidine 115 was found to be an amino acid important to the ability of gp9lphox to function as the NADPH oxidase associated H+ channel (Henderson, 1998). All these in vitro studies suggest a role for AA in the regulation of H' channel activity. However, the role of AA in a physiological system and the type of phospholipase A2responsible for its release have not as yet been defined. Furthermore, the effect of AA in these studies is not specific since, apart from AA, a variety of unsaturated fatty acids has been shown to significantly activate

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a proton conductance in both macrophages (Kapus et al., 1994) and neutrophil cytoplasts (Henderson and Chappell, 1992). In the last decade, several secreted and cytosolic mammalian PLA2 isozymes have been described (Clark et al., 1990; Hazen et al., 1993; Kramer et al., 1989; Seilhamer et al., 1989). The existence of several types of PLA2 in phagocytic cells (Bolognese et al., 1995; Rosenthal et al., 1995) complicates delineation of the PLA2 responsible for release of the AA which impacts on NADPH oxidase activation following phagocyte stimulation. In the present study, we used the RNA antisense technique to create in the human phagocyte myeloid cell line, PLB-985, a p85 cPLA2deficient model cell line in order to demonstrate the role of this enzyme in NADPH oxidase activation. In addition, this model was used to + determine whether the H channel is regulated by cPLA2 and whether this channel is involved in the regulation of NADPH oxidase activity.

3.

EXPERIMENTAL PROCEDURES Cell culture and induction of differentiation

PLB-985 cells and selected PLB-D clones were grown in a stationary suspension culture in RPMI 1640 medium. The results presented were found in seven individual clones. Optimal concentrations of 5x10-8 M 1,25(OH)2D3, 10-6 M RA or 1.25% DMSO were added to PLB cells or PLB-D cells (2x10 5 cells/ml) at their logarithmic growth phase to induce differentiation towards monocyte- or granulocyte-like cells, respectively. Differentiation was induced for four days and determined by Mac-1 antigen expression detected by indirect immunofluorescence. Transfection and selection of PLB-985 clones PLB-985 cells (1x107) in logarithmic growth were transfected with 2 0 µ g of plasmid DNA (antisense, plasmid cPLA2 (1-530)-pcDNA3 in antisense orientation, or vector alone) by electroporation and selected in the presence of 0.8 mg/ml G418. The detection of cPLA2 protein was performed using rabbit antibodies raised against a GST fusion with cPLA 2 , as described earlier (Hazan et al., 1997). DTT resistant Phospholipase A2 activity PLA2 activity was determined in the cytosols, using sonicated dispersions of 1-stearoy1-2-[14 C]arachidonyl phosphatidyl choline (30 µM, 50,000 DPM/assay) and sn-l,2-dioleoylglycerol (molar ration 2: 1),

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in an assay mixture containing 5 mM dithiothreitol (DTT), as described earlier (Hazan et al., 1997). Superoxide anion measurements The production of superoxide anion (O2 ) by intact cells was measured as the superoxide dismutase inhibitable reduction of ferricytochrome c as described earlier (Hazan et al., 1997). pHi measurements 7

Cells (5x10 ) were loaded with 2’,7’-bis(carboxyethyl)-5-(6)7 carboxy -fluorescein (BCECF) (3 µg/ml) for 10 min at 37OC. 1x10 cells were suspended in 3 ml of Na+ medium (140 mM NaCl, 5 mM KC1, 10 mM glucose and 10 mM HEPES pH 7.4). Cells were stimulated with 50 ng/ml PMA and the changes in pHi were recorded. H+ channel activity- was monitored by recording changes in pHi under conditions where the contribution of other H+ translocating systems and major acidifying mechanisms were shown to be eliminated (Nanda and Grinstein, 199 1 ).

4.

DEVELOPMENT OF A MODEL OF PHAGOCYTYIC LIKE CELLS LACKING CPLA2 EXPRESSION

PLB-985 cells were transfected with human cPLA2 cDNA engineered in the antisense orientation in the pcDNA3 expression vector which also contains a neomycin resistance element. Cell cytoslic fractions from clones resistant to the neomycin analogue, G418, were screened by Western blot to select those that were cPLA 2 protein deficient. Three pcDNA3-cPLA2 antisense transfected clones were completely deficient of cPLA2 protein (PLB-D) (Fig 1A). Several other clones were shown to be partially deficient, containing residual amounts of cPLA2 protein (PLBR). Both the parent PLB-985 line (PLB) and a G418 resistant clone transfected with the empty pcDNA3 vector (PLB-V) were used as controls. As shown in Figure 1A, PLB-V produced high levels of cPLA2 protein and this amount was indistinguishable from that present in the parent PLB cells. Cytosol fractions of the various PLB derived cell lines were examined for the presence of dithiothreitol (DTT) resistant enzymatic activity 14 characteristic of p85 cPLA2 using 1 -stearoy1-2-[ 1 - C]arachydonyl phosphatidylcholine as a substrate. As shown in Figure 1B, basal cPLA2 activity resistant to inhibition by 5 mM DTT detected in unactivated PLBV cytosol was similar to that seen in the parent PLB cells (1.25 ± 0.2 and

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I .46 ± 0.3 pmol/mg protein/min, respectively). By contrast PLB-D cells which lack cPLA2 protein had no detectable DTT-resistant cPLA2 activity, and PLB-R which express reduced levels of cPLA2 protein demonstrated reduced levels of cPLA2 activity in cytosol (0.71 ± 0.4 pmol/mg protein/min).

Figure 1. Characterization of transfected PLB-985 clones. A. SDS-PAGE immunoblot analysis of cPLA2 in cytosols of the parent PLB-985 cell line (PLB ), a PLB-V clone (V ), PLB-D clones (D1,D2), or a PLB-R clone (R). B. DTT-resistant phospholipase A2 activity measured by [ 14 C ] AA release from PC vesicles, using cell cytosols.

An important characteristic of the PLB cell line is that it can be induced to differentiate toward a mature phagocyte phenotype in response to a variety of differentiating agents (Boron, 1983) As shown in Figure 2A, the percent of parent PLB cells expressing Mac-1 antigen at the cell surface measured by indirect immunofluorescence was less than 8% in undifferentiated cells, but increased to over 70% by day 5 with either RA or D3. The pattern of Mac-1 antigen expression by PLB-V cells or PLB-D cells during induction of differentiation was indistinguishable from that seen with PLB cells. Similarly, the patterns of expression of the subunit components of NADPH oxidase during differentiation of PLB-V, PLB-D or PLB cells in response to RA or D3 were indistinguishable, as shown by the Western blots in Figure 2B, 2C. Rac-2 was detected in all the PLB-derived cell lines and did not change during differentiation (not shown).

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Figure 2. Characterization of differentiated PLB-985 clones. (A) Differentiation determined by Mac-1 antigen was detected by indirect immunofluorescence. Undifferentiated cells (-) or cells differentiated with D 3 (D3 ) or with RA (RA) for 5 days. (B-C) Differentiation assessed by expression of NADPH oxidase components. Shown are SDS-PAGE immunoblots of (B) the cytosolic proteins: p47phox and p67phox and (C) membrane components: endoglycosidase F-deglycosylated gp91 phox, the ß-subunit (indicated by arrow), and the p22phox , a-subunit of cytochrome b558. Undifferentiated cells (-) or cell differentiated with D 3 (D3 ) or with RA (RA) for 5 days.

5.

THE ROLE OF CPLA 2 IN ACTIVATION OF THE NADPH OXIDASE

Differentiated cells generated significant superoxide upon stimulation with various agonists. Figure 3, demonstrates superoxide production stimulated with PMA in differentiated PLB cells. In the differentiated PLB-D cells all oxidase components were expressed (Figure 2) and the

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cytosolic components translocated to the membranes (data not shown), yet they could not produce

Time (min) Figure 3. The role of cPLA2 in stimulation of NADPH oxidase activity. Generation of superoxide stimulated with 100 ng/ml PMA in the differentiated parent PLB cells ( ). G e n e r a t i o n of superoxide stimulated with 100 ng/ml PMA alone in differentiated PLB-D cells (O ) or restoration of superoxide generation by addition of exogenous free AA 10 µM ( or 25 µM ( ❐ ). superoxide after stimulation with various agents. Addition of free AA immediately restored superoxide production of the stimulated PLB-D cells to similar levels produced by parent PLB cells.

6.

THE ROLE OF CPLA2 IN ACTIVATION OF THE NADPH OXIDASE ASSOCIATED H+ CHANNEL

To elucidate the mechanism by which cPLA2 generating AA activates the oxidase we studied whether cPLA 2 is involved in the signaling leading to NADPH oxidase associated H+ channel activation. Addition of PMA triggered a substantial cytoplasmic alkalization in differentiated PLB cells (Figure 4A), indicating a H+efflux. Alkalization was prevented in the presence of the H+ channel inhibitor, Zn2+ (50 µM), further confirming that H+movements take place through the heavymetal sensitive H+ channel. In differentiated PLB-D cells, PMA did not

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induce any cytoplasmic alkalization (Figure 4B) but H channel activity could be restored by the addition of exogenous AA to PMA stimulated or non stimulated cells (Figure 4C). These results clearly indicate that cPLA2 -generated AA is essential for the activation of the NADPH oxidase associated H' channel in differentiated PLB-D cells.

Figure 4. The role of cPLA2 in stimulation of the H+ channel activity. H' channel activity was monitored in differentiated PLB cells (A), and differentiated PLBD cells (B). Restoration of H+ channel activity with addition of 10 µM exogenous AA in differentiated PLB-D cells Stimulated with PMA is shown (C). Stimulation was obtained with PMA (50 ng/ml). Zn2+ (50 µM) were added when noted

7.

THE ROLE OF THE H+ CHANNEL IN ACTIVATION OF THE NADPH OXIDASE

Differentiated PLB-D cells fail to activate both the NADPH oxidase and the H+ channel (Figure 4B) upon stimulation. As demonstrated in Figure 4C, addition of AA restored H+ channel activity in differentiated PLB-D cells in the presence of the oxidase inhibitor, DPI, indicating that oxidase activity is not a prerequisite for activation of this channel. In order to determine the potential role of the H+ channel in promoting or maintaining NADPH oxidase activity, the effect of the H' channel inhibitor, Zn2+, was studied. As shown in Figure 5A, the presence of 50 µM Zn2+ prevented alkalization induced by PMA in granulocytelike PLB cells suspended in a Na+ medium. The presence of 50 µM Zn2+ significantly inhibited the production of superoxide stimulated with PMA, retaining about 40% of the activity (Figure 5B). These results suggest the involvement of the H' channel in the regulation of NADPH oxidase activity.

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Figure 5. The effectofthe H+ channel inhibitor on stimulated pHi changes. (A) and on superoxide production (B) in differentiated PLB cells

Cells were stimulated with PMA (50ng/ml). Zn2+ (50 µM were added when noted.

8.

CONCLUSIONS

The development of differentiated PLB-D cell lines which lack any cPLA2 expression offers a unique tool for determining the physiological role of cPLA2 . The present study demonstrates that cPLA2 and its enzymatic production of AA is an essential requirement for activation of the assembled NADPH oxidase and the oxidase associated H+ channel activation. Stimulation of differentiated PLB-D cells did not induce activation of H+ channel but addition of exogenous AA fully restored this activity. In accordance with our study, previous publications, utilizing the PLA2 inhibitor p-bromophenacyl bromide (Kapus et al., 1993) and the newly developed selective blocker of cPLA2, trifluoromethyl ketone analogue of arachidonic acid (AACOCF3) (Susztak et al., 1997), have proposed that cPLA2 has a possible role in regulation of the electrogenic The similar H+ channel in the plasma membrane of neutrophils. restoration patterns of H+ channel activity (Fig 4C) and of NADPH oxidase activity by AA in differentiated PLB-D cells supports the presence of a tight coupling between these two processes. Thus, the inability of stimulated differentiated PLB-D cells to activate NADPH

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to activate NADPH oxidase may account, in part, to the failure in activating the H+ channel in these cells. The partial inhibition of superoxide production under conditions in which absolute inhibition of the H+ channel was induced suggests that the AA-activatable H+ channel is not the sole mechanism regulating oxidase activity. AA, which caused complete activation of oxidase activity, probably acts at other sites on the oxidase in addition to its effect on the H+ channel.

ACKNOWLEDGMENT This research was supported by grant no. 97-00178 from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel.

REFERENCES Babior, B. M. (1999). NADPH oxidase. an update. Blood 93:1464-76. Bolognese, B., McCord, M., and Marshall, L. A. (1995). Differential regulation of elicited-peritoneal macrophage 14 kDa and 85 kDa phospholipase A2(s) by transforming growth factor-beta. Biochim Biophys Acta 1256:201-9. Boron, W. F. (1983). Transport of H+ and of ionic weak acids and bases. J Membr Biol 72: 1-16. Clark, J. D., Milona, N., and Knopf, J. L. (1990). Purification of a 110-kilodalton cytosolic phospholipase A2 from the human monocytic cell line U937. Proc Nutl Acad Sci USA 87:7708-12. Cockcroft, S. (1991). Relationship between arachidonate release and exocytosis in permeabilized human neutrophils stimulated with formylmethionyl-leucylphenylalanine (fMetLeuPhe), guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and Ca2+. Biochem J .275: 127-3 1. Hazan, I., Dana, R., Granot, Y ., and Levy, R. (1 997). Cytosolic phospholipase A, and its mode of activation in human neutrophils by opsonized zymosan. Correlation between 42/44 kDa mitogen-activated protein kinase, cytosolic phospholipase A2 and NADPH oxidase. Biochem J.326: 867-76. Hazen, S. L., Hall, C. R., Ford, D. A., and Gross, R. W. (1993). Isolation of a human myocardial cytosolic phospholipase A2 isoform. Fast atom bombardment mass spectroscopic and reverse-phase high pressure liquid chromatography identification of choline and ethanolamine glycerophospholipid substrates. J Clin Invest 91. Henderson, L. M. (1998). Role of histidines identified by mutagenesis in the NADPH oxidase-associated H+ channel. J Biol Chem.273:33216-23. Henderson, L. M., Banting, G., and Chappell, J. B. (1995). The arachidonate-activable, NADPH oxidase-associated H+ channel. Evidence that gp91-phox functions as an essential part of the channel. J Biol Chem.270:5909-16. Henderson, L. M., and Chappell, J. B. (1992). The NADPH-oxidase-associated H+ channel is opened by arachidonate. Biochem 5.283: 171-5.

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Henderson, L. M., Chappell, J. B., and Jones, 0. T. (1987). The superoxide-generating NADPH oxidase of human neutrophils is electrogenic and associated with an H+ channel. Biochem J 246, 325-9. Henderson, L. M., Thomas, S., Banting, G., and Chappell, J. B. (1997). The arachidonate-activatable, NADPH oxidase-associated H+ channel is contained within the multi-membrane-spanning N-terminal region of gp91-phox. Biochem J 325, 7015. Kapus, A., Romanek, R., and Grinstein, S. (1994). Arachidonic acid stimulates the plasma membrane H+ conductance of macrophages. J Biol Chem 269, 4736-45. Kapus, A., Susztak, K., and Ligeti, E. (1993). Regulation of the electrogenic H+channel in the plasma membrane of neutrophils: possible role of phospholipase A2, internal and external protons. Biochem J 292, 445-50. Kramer, R. M., Hession, C., Johansen, B., Hayes, G., McGray, P., Chow, E. P., Tizard, R., and Pepinsky, R. B. (1989). Structure and properties of a human non-pancreatic phospholipase A2. J Bioi Chem 264, 5768-75. Lefkowith, J. B., Lennartz, M. R., Rogers, M., Morrison, A. R., and Brown, E. J. (1992). Phospholipase activation during monocyte adherence and spreading. J lmmunol 149, 1729-35. Lennartz, M. R., and Brown, E. J. (1991). Arachidonic acid is essential for IgG Fc receptor-mediated phagocytosis by human monocytes. J Immunol 147, 621-6. Nanda, A., and Grinstein, S. (1991). Protein kinase C activates an H+ (equivalent) conductance in the plasma membrane of human neutrophils. Proc Nutl Acad Sci USA 88, 108 16-20. Rosenthal, M. D., Gordon, M. N., Buescher, E. S., Slusser, J. H., Harris, L. K., and Franson, R. C. (1995). Human neutrophils store type II 14-kDa phospholipase A, in granules and secrete active enzyme in response to soluble stimuli. Biochem Biophys Res Commun 208, 650-6. Rotrosen, D., Yeung, C. L., Leto, T. L., Malech, H. L., and Kwong, C. H. (1992). Cytochrome b558: the flavin-binding component of the phagocyte NADPH oxidase. Science 256, 1459-62. Seilhamer, J. J., Pruzanski, W., Vadas, P., Plant, S., Miller, J. A., Kloss, J., and Johnson, L. K. (1989). Cloning and recombinant expression of phospholipase A, present in rheumatoid arthritic synovial fluid. J Bioi Chem 264, 5335-8. Susztak, K., Mocsai, A., Ligeti, E., and Kapus, A. (1997). Electrogenic H+ pathway contributes to stimulus-induced changes of internal pH and membrane potential in intact neutrophils: role of cytoplasmic phospholipase A2. Biochem J 325, 501-10.

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THE ROLE OF NK CELLS IN INNATE IMMUNITY

Niva Lieberman and Ofer Mandelboim Lautenberg Center for General and Tumor Immunology. The Hebrew University Hadassah Medical School, P.O.Box 12272, Jerusalem 91120, Israel

1.

INTRODUCTION

Natural killer (NK) cells are large lymphocytes with numerous cytoplasmic granules that are capable of lysing tumor and virus infected cells. They do not express T cell or B cell receptors; rather they express the CD56 marker, which enables their specific identification. NK cells are found in the peripheral blood and compose 5-10% of the total lymphocyte population. They can be found in blood and lymphoid tissues especially the spleen ( Abbas et al., 1994). Natural killer cells, as well as cytotoxic T lymphocytes (CTL), are major component of the cellular mechanism by which an immune response leads to the destruction of foreign or infected tissue (Trinchieri, et al., 1989). In contrast to CTL, which are activated in the presence of class I MHC molecules and an appropriate specific peptide, NK cells preferentially lyse target cells deficient in expression of MHC class I proteins. In this manner NK cells carry out immunosurveillance for ‘missing self, rather than for direct detection of foreign antigens (Ljunggren et al., 1990). NK cells are prevented from killing lymphoid cells primarily by NK inhibitory receptors that are specific for HLA-C and HLA-E (Leibson et al., 1998). When activated, these receptors send inhibitory signals to the NK cell that prevent lysis of the target cell. In contrast the majority of The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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CTL are activated by peptides presented by HLA-A and HLA-B proteins (Littaua et al., 1991). In the first part of this review we will describe the work performed by Cohen et al. (1999), showing that the human Immuno Deficiency Virus (HIV) developed mechanisms for abusing this peculiar immune system bias. Using the well characterized lymphoblastoid cell line 72 1.221 (221 cells), that do not express HLA-A, -B or -C. We prepared HIV-1 infected 221 cell lines transfected with genes encoding for specific alleles of HLAA, -B -C and -E. The striking results of this study show that HIV-1 selectively downregulates HLA-A and HLA-B but does not significantly affect HLA-C and HLA-E. This selective downregulation probably allows HIV infected cells to avoid CTL mediated lysis and yet escape the NK immunosurveillance. In the second part of this review we will describe the work of Davis et al. ( 1999). Using fluorescence video imaging technique we demonstrated that, as NK cells survey target cells, inhibitory Killer Immunoglobulinlike Receptor (KIR) induces clustering of HLA-C into an immunological synapse. At the synapse HLA-C distributes into a stable ring around a central adhesion molecule ICAM- 1. We also demonstrated that cells can support multiple synapses simultaneously, and that clusters move as NK cells crawl over target cell surfaces. This organization within the plasma membrane is surely critical for immunological surveillance by NK cells. In the third part of this paper we will discuss the lysis receptors involved in lysis of viral and tumor cells and in particular CD16. By now, three lysis receptors have been identified: CD 16, NKp46 and NKp44, which are all members of the Ig superfamily. Painwise sequence comparisons suggest no significant homology among them (Mandelboim, 1999). CD16 (Fc receptor III has been described as a receptor expressed on NK cells that facilitates antibody dependant cellular cytotoxicity (ADCC) by binding to the Fc portion of various antibodies. Recently, we have shown that CD16 has a broader function and is directly involved in the lysis of some virus-infected cells and tumor cells, independent of antibody binding. We also demonstrated the presence of a still unknown CD 16 ligand, by using CD- 16-Ig fusion protein.

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2.

RESULTS

2.1

The selective downregulation of class I MHC proteins by HIV1 (Cohen et al., 1999)

221 cells stably expressing CD4 and defined class I allotypes were made using retrovirus mediated gene transfer. Effect of HIV infection on cell surface levels of class I MHC proteins was assessed by staining for class I protein with fluorescently conjugated antibodies followed by flow cytometry. Using this method, it was demonstrated that HIV-1 downregulated HLA-A2, HLA-B2705 and B702. In contrast, HIV Infection of 221 cells that expressed HLA-Cw4, -Cw3, or Cw7 did not result in significant downregulation. In order to identify the region on HLA-A2 that accounted for its downregulation, a chimeric molecule was made between Cw4 and HLAA2. This Chimera contained the extracellular region of Cw4 joined to the transmembrane and cytoplasmic tail of region of HLA-A2. Upon HIV infection, the Cw4-HLA-A2 chimera was significantly downregulated. Chimeras containing the HLA-B27 tail were also downregulated. In contrast, chimeras containing the cytoplasmic tail of HLA-E did not undergo downregulation. Therefore, the cytoplasmic tail region of HLAC and HLA-E are responsible for their resistance to downregulation to HIV. Examining the sequence of the cytoplasmic region of class I MHC proteins revealed differences Between HLA-A, HLA-B,HLA-C and HLAE. In order to define the residues that were critical for HLA downregulation, point mutations were progressively made in the B27 tail, so that it gradually became more like the HLA-C tail. This procedure revealed that mutants, which did not contain both Tyr-321 and Asp-328 were not downregulated. Therefore it seems that both these residues are essential for class I downregulation. In addition, although HLA-E contains both Tyr-321 and Asp-328 it is not downregulated by HIV. Further examination revealed that HLA-E contains Lys-325 instead of Ala-325 residue. Downregulation of HLA-E by HIV, was achieved by a single point mutation replacing Lys-325 by Ala-325. Thus, in addition to Tyr-321 and Asp-328, Ala-325 is also critical for downregulation of MHC class I molecules by HIV. The significance of these findings was further demonstrated by “NK-killing assays”, which suggested that HLA-C and HLA-E protect HIV infected cells from NK lysis. Two different sources of NK cells were used in these assays. First, primary NK cells carrying the NK inhibitory

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The Role of NK Cells in Innate Immunity

receptor NKIR1 (which is inhibited by HLA-Cw2, - Cw4, -Cw5, and CW6), were cloned from peripheral blood . Second, we used an immortalized cell line (YTS, which does not express any NKIRs), and infected it with NKIR1 gene by a retroviral vector. The surface level expression of NKIR1 in YTS.NKIR1 and NK.NKIR1 cells was comparable as judged by flow cytometry. Target cells infected with HIV (carrying the gene for green fluorescent protein), were 221 cells that were made to express either HLA-A2 alone, A2 +Cw3, or A2+Cw4. YTS.NKIR1 cells were mixed with targets and incubated together for 6 hours. The mixture was also stained with a mAb specific for HLA-A2 so that class I downregulation could be followed (YTS are HLA-A2 negative). Strikingly, almost all of the HIV infected 221 target cells that expressed HLA-A2 alone or with Cw3 have been lysed. In contrast, those cells that expressed –Cw4 with –A2 were protected from YTS.NKIR1. Cw4 protected HIV infected cells from lysis regardless of the extent of HIV infection or HLA-A2 downregulation. Similar results were seen when primary NK.NKIR1 cells were used as the effector cells. Surface expression of HLA-E requires binding of a peptide from the leader sequence of most HLA-A, -B and -C proteins. In the absence of this peptide HLA-E does not efficiently make its way to the cell surface. The effector cell was chosen to be NK.CD94 cell line. It was derived from peripheral blood, expressed the inhibitory receptor that is specific for HLA-E (a heterodimer of CD94 and NKG2A) and was largely devoid of NK receptors specific for HLA-C. Target cells were of two types. One was 221 cell infected with HIV, and the other was HIV infected 221 cell that have also been transfected with the leader peptide of HLA-A2 (to help HLA-E to reach and appear on the cell surface). While HIV infected 221 cells expressing no class I proteins were efficiently lysed by NK.CD94, target cells that have been made to express HLA-E were hardly lysed. Preincubation of the NK.CD94 cell line with an antibody to CD94, reversed this inhibition. These results suggest that HIV infected cells expressing HLA-E are protected from lysis.

2.2

The human Natural killer cell immunological synapse (Davis et al., 1999)

Inhibitory Killer Immunoglobulin-like Receptor NKIR1 and NKIR2 recognize the class I MHC proteins, HLA-Cw4 or Cw6 and HLA-Cw3 or –Cw7 respectively. Binding kinetics between soluble KIR-MHC proteins are extremely fast (Vales-Gomez et al., 1998). However, video

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microscopy of NK cell immunosurveillaiice shows that intercellular contacts last for minutes. The molecular mechanisms that occur over this frame of time have been partially revealed by this study. Plasmids encoding for green fluorescent protein (GFP) attached to the intarcellular C-terminus of HLA-C were transfected into 221 cells and used to mark the location of HLA-C. These transfectants were then incubated with various NK lines for 20 minutes, after which conjugates of living NK and target cells were imaged by laser scanning confocal fluorescence microscopy. Using this method, an interesting occurrence has been revealed. A single NK cell simultaneously caused clustering of MHC proteins on several target cells forming immunological synapses. Furthermore, one target cell could simultaneously present clusters of MHC proteins at multiple NK cell contacts. Clusters of class I MHC protein follow the intercellular contacts as NK cells move about the surface of the target cell. Such extensive immunosurvellance by NK cells may be required when surveying cells that express class I MHC proteins in patches at the cell surface or at a pole. Using fluorescently labeled monoclonal antibodies, ICAM- 1 (CD-54) was found to concentrate at the center of the class I MHC ring. This distribution of MHU/CAM- 1 is like the transient arrangement seen at the contact between a mouse T cell and lipid bilayer, lasting up to 5 minutes (Grakoui et al., 1999). Here, the arrangement between live human peripheral NK cells and 221 transfectant cells was stable for at least 22 minutes. Further study suggested that HLA-C clustering by NKIR does not require energy nor cytoskeletal movement. Treatment with standard metabolic inhibitors (like rotenone and 2,4 dinitrophenol) that depleted the intracellular ATP in the NK cell/target cell mixtures did not affect the number of immunological synapses formed. Also, treatment with either Cytocholasins B and D (which affect actin filaments), or with colchicine (which inhibits tubulin polimerization), did not affect the number of synapses formed. To follow through the mechanism for the induction of HLA-C clustering by NKIR, 10 µm beads has been prepared as a solid support for NKIR. The beads were coated with a chimeric protein consisting of the extracellular portion of KIR1 attached to the Fc portion of an IgG Ab and incubated with target cells. These beads efficiently attached themselves to 221-Cw4-GFP cells, but did not facilitate MHC clustering.

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The Role of NK Cells in Innate Immunity

Human CD16 as a lysis receptor mediating direct natural killer cell cytotoxicity (Mandelboim et al., 1999)

Initially, The level of CD16 expression was demonstrated to correlate with efficient killing of target cells. Three NK lines that differed in the level of CD16 expression were selected. NK line 62 which did not express CD16, NK line 70 in which 2/3 of the cells expressed the CD16 proteins and NK line m-1, in which all cells expressed CD16. Cytolytic activity of NK lines and clones against the various targets was assayed in 5-hr 35S release assays. The CD16 negative NK62 line moderately lysed 221 target cells, weakly lysed 293 EBNA cells and did not lyse the melanoma cell line 1106 mel at all. In comparison, NK70 line lysed these targets strongly, moderately and weakly respectively. But CD16 expressor, NK line m-I, efficiently lysed all these target cells. A further demonstration of the role of CD16 as a lysis receptor was obtained by blocking CD16 with the Fab fragement of an anti CD16 antibody. These experiments revealed that targets could be ascribed to three groups. Group A, which includes the 221 cell line, was not affected by the addition of the Fab fragements. In contrast, In group B, which includes the 293 EBNA cell line, addition of the Fab fragements resulted in moderate inhibition of lysis (less than 50%). And in group C, which includes the 1106mel cell line, addition of the Fab fragements resulted in efficient blocking of lysis (more than 70%). Thus, blocking by anti CD 16 Fabfragements shows, that CD 16 receptor is directly involved in killing of some target cells (group B and C), yet the lysis of group A targets must employ a different NK lysis receptor. Additional research revealed that group A targets are efficiently lysed by the CD16 negative NK92 tumor line. Furthermore, group B was only moderately lysed and group C could not be killed at all by NK92 tumor line. Therefore, these data suggest the existance of multiple lysis receptors and or multiple mechanisms for killing different targets by NK clones and, therefore multiple ligands on target cells. To assay for the CD16 ligand, a fusion protein have been genetically engineered - containing the extracellular domain of CD16 receptor and the Fc portion of a human IgGl antibody. Various cells were then incubated with CD16-Ig, or CD99-Ig fusion protein as a control. The cells were then washed and incubated with a Phycoerythrin (PE) conjugated anti-human IgG as a second antibody and then analyzed by flow cytometry.

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CD16-Ig specifically bound group B and group C. The CD16 binding was specific, since no binding could be detected with another Ig-fusion protein CD99-Ig. In contrast, little or no binding was observed to group A target cells. These data provide evidence for the existance of unknowk CD16 ligand(s) on some of the target cells.

3.

DISCUSSION

Downregulation of class I MHC proteins by viruses helps them escape CTL response. Yet, this mechanism exposes the infected cells to NK cell mediated lysis. NK killing is inhibited primarily by the presence of either HLA-C or HLA-E and to a lesser extent by some HLA-B alleles (Yokoyama, 1998). Using the 721.221 cell line stably infected with class I alleles, It was found that HIV specifically downregulates HLA-A and –B alleles from the cell surface, but not HLA-C and –E. Direct killing assays presented further evidence that HLA-C and –E left on the surface of HIV infected cells protects cells from NK lysis. This implies that HIV succeeds in defeating the immune system by escaping from both CTL and NK cell response. However, small populations of NK cells and CTL may be uniquely suited for eliminating HIV infected cells. For instance NK cells that express an inhibitory receptor NKB1, specific for a subset of HLA-B molecules, and do not express inhibitory receptors specific for HLA-C and –E. Interestingly, those HLA-B alleles associated with a slower progression of AIDS belong to the subset of alleles that are recognized by NKB1 (Kaslow et al., 1996). Unfortunately, in vivo NKB1 is rarely expressed in the NK cells of the individuals who express the appropriate HLA-B ligand (Valiante et al., 1997). Similiarly, the fact that HIV leaves HLA-C on the cell surface suggests that CTL restricted to HLA-C-presented antigens might be particulary effective at killing HIV infected cells. However, most CTLs are restricted to HLA-A and –B allotypes and only few are HLA-C-restricted. Furthermore, HLA-C is expressed on the cell surface at lower levels than HLA-A and –B (Lawlor et al., 1990). Strikingly, of those few HLA-Cresricted CTLs known, many are directed against HLA-C antigens (Littawa et al., 1991). Also a dominant HLA-C resricted CTLs response have been identified in HIV positive long-term nonprogressors. Thus allotype specific NK ells or CTL may be particulary effective in the clinical setting. Clustering of MHC Class I molecules is likely to be required for the initiation of signal transduction by NKIR. An indication for this

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assumption, is the finding that ions like Zn2+ or Cu2+, which are essential for signal transduction by receptors, are required for the clustering of HLA-C but not for the preliminary binding of NKIR to HLA-C. Pharmaceutical intervention that inhibits specific intercellular clustering, may be used therapeutically e.g. to enhance NK killing of HIVInfected or tumor cells. Finally, CD16 (Fc receptor III was shown to be directly involved in the lytic process mediated by human NK cells, in addition to its role in antibody-dependant cellular cytotoxicity. The nature of the ligand for CD16, that was detected on target cells by the CD16-Ig fusion proteins remains unknown. The expression of a specific ligand which induces lysis, suggests that NK cells have the ability to detect specific antigens expressed on viral infected cells or tumor cells.

REFERENCES Abbas, A. K., Lichtman, A. H., and Pober J.S., Ed., 1994, Cellular and molecular immunology 2nd edition. Cohen, G.B., Gandhi, R.T., Davis, D.M., Mandelboim, O., Chen, B.K., Strominger, J.L., and Baltimore D.. 1999, The selective downregulation of class I Major Histocompatiability Complex proteins by HIV- I protects HIV-Infected cells from NK cells. Immunity, 10: 66 1-67 I. Collins, K.L., Chen, B.K., Kalams, SA., Walker, B.D., and Baltimore, D., 1998, HIV-I Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes. Nature 391 : 397-40 1. Davis, D.M., Chiu, I., Fassett, M., Cohen, G.B., Mandelboim, O., and Srominger, J.L.,1999, The Human Natural Killer Cell Immunological Synapse. Submited for publication. Grakoui, A., Bromley S.K., Sumen, C., Davis, M.M., Shaw A.S., Allen, P.M., Dustin, M.L., The Immunological Synapse: A molecular machine controlling T Cell Activation. Science, 285: 22 1-226. Kaslow, R.A., Carrington, M., Apple, R., Park, L., Munoz, A., Saah, A.J., Goedert, J.J., Winkler, C., O'Brien, S.J., Rinaldo. C., et al, 1996, Influence of combinations of human MHC genes on the course of HIV-I infection. Nat.Med. 2: 405-41 I. Lawlor, D.A., Zemmour, J., Ennis, P.D., and Parham, P., 1990, Evolution of class-I MHC genes and proteins: from natural selection to thymic selection. Annu. Rev. Immunol. 8: 23-63. Leibson, P.J., 1998, Cytotoxic lymphocyte recognition of HLA-E: utilising a nonclassical window to peer into classical MHC. Immunity 9: 289-294. Littaua, R.A., Oldstone, M.B., Takeda, A., Debouck, C., Wong, J.T., Tuazon, C.U., Moss, B., Kievits, F., and Ennis, F.A., 1991, An HLA-C-restricted CD8+ cytotoxic Tlymphocyte clone recognizes a highly conserved epitope on HIV-I gag. J. Virol. 65: 405 1-4056. Ljunggren, H.-G., and Karre, K., 1990, In search of the 'missing self: MHC molecules and NK recognition. Immunol. Today 11: 237.

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Mandelboim, O., Malik, P., Davis, D.M., Jo, C.H., Boyson, J.E., and Strominger J.L., 1999, Human CD16 s a lysis receptor mediating direct natural killer cell cytotoxicity. Imnunol 96: pp. 5640-5644. Trinchieri, G., 1989, Biology of Natural Killer cells. Adv. in Immunol. 47: 187-376. Vales-Gomez, M., Reyburn, H.T., Mandelboim, M., Strominger, J.L., 1998, Kinetics of interaction of HLA-C ligands with natural killer cell inhibitory receptors. Immunity, 9(3): 337-344. Valiante, N.M., Uhrberg, M.. Shilling, H.G., Lienert-Weidenbach . K., Arnett, K.L., D’Andrea, A., Philips. J.H., Lanier. L.L., and Parham, P.. 1997, Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two donors. Immunity 7: 739-75 1. Yokoyama. W.M., 1998, Natural killer cell receptors. Curr. Opin. Immunol. 10: 298-305.

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SIMILARITIES AND DISSIMILARITIES BETWEEN HUMANS AND MICE LOOKING AT ADHESION MOLECULES DEFECTS

¹Amos Etzioni , ²Claire M. Doerschuk, and ³John M. Harlan ¹Department of Pediatrics and Immunology, Rambam Medical Center, B. Rappaport School of Medicine, Technion, Haifa, Israel, ²Physiology Program, Harvard School of Public Health, Boston, MA, USA 021 15, and ³Division of Hematology, University of Washington, Seattle, USA 98195

1.

INTRODUCTION

Important insights into a number of biological processes have come from studies of rare inherited diseases. The Leukocyte Adhesion Deficiency (LAD) syndromes, in which one of several molecules in the adhesion cascade is defective, are such examples. Much has been learned from the study of LAD, yet the puzzle is far from being solved. Animal models have also been pivotal in understanding of biological events and immunology has benefited tremendously from the investigation of various aspects of the immune response in mice. One of the most powerful techniques has been targeted gene deletion (Le., a ‘knockout’) by homologous recombination (Majzoub & Muglia 1996). This approach allows comparison of animals of similar genetic backgrounds that differ only in the absence of a single gene. Phenotypic differences between the knockout and wildtype littermates are presumed then to be due to the targeted gene deletion. Furthermore, various knockout mice can be interbred to produce deletions of several genes, providing animal models for more complex genetic defects. While gene targeting has many advantages, it is important to acknowledge that mice are not humans, and that there may be important The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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differences in biology between the species. Indeed, comparisons between rare human disease and mice with deletion of the same gene have revealed important differences as well as similarities. Similar CD40 L CIITA RAG 1 and 2

Dissimilar BTK JAK3 A-T, ADA

For example, the defect in Bruton’s tyrosine kinase (BTK) in man leads to severe hypogammaglobulinemia with almost no B cells. In contrast, mice with targeted deletion of the BTK gene produce some antibodies with up to 30% of normal B cell number. It is also important to recognize that comparisons may be confounded by the recruitment of alternative pathways in the human or mouse genetic deficiencies. In this regard, it has been reported (Bader 1998) that mice deficient in αvβ3integrin exhibited no deficit in angiogenesis or vasculogenesis, whereas αvβ3-integrin antagonists inhibit angiogenic responses in wildtype animals. Whether such differences result from alternative pathways in the deficient animals or unexplained effects of the antagonists remains to be resolved. In this brief review we will compare the two known human LAD syndromes with gene-targeted mice with various adhesion moleculedeficiencies, highlighting both disparities and similarities.

2.

THE ADHESION CASCADE

Leukocytes must first adhere to the endothelium of blood vessels before they emigrate to tissue. The process of leukocyte emigration is a dynamic one involving multiple steps. (Carlos & Harlan 1994, Springer 1994). Several families of adhesion molecules mediate the adhesive interactions of leukocytes with endothelial cells, each involved in a distinct phase of emigration. The initial, rapidly reversible, adhesion of leukocytes to the vessel wall under condition of flow produces ‘rolling’. This phase is mediated largely by the interaction of selectin receptors [E (CD62E), P (CD62P), and L (CD62L)] and their glycoconjugate ligands. The precise nature of the carbohydrate counter-structures for the various selectins has not been fully defined, but fucosylated, sialylated glycans such as Sialyl Lewis x (SLeX, CD15s) are clearly involved (Varki 1997).

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In post-capillary venules of the systemic microcirculation, rolling is prerequisite for the subsequent steps in adhesion cascade of activation, firm adhesion, and transmigration. These latter events involve leukocyte integrins and their endothelial ligands of the immunoglobulin gene superfamily (IgSF) The 2-subfamily comprises four alpha-subunits (CD1la-d) with the common β 2-subunit (CD18). CD11a/CD18(α Lβ 2) and C D 1 1 b / C D 1 8 ( α Mβ2) are the predominant β2-subunits involved in leukocyte adhesion to endothelium. Other leukocyte integrins involved in emigration are VLA-4(α4β1; CD49d/CD29) and α4β7 The leukocyte integrins interact with IgSF ligands on the endothelial cell. These include intracellular adhesion molecule (1CAM)- l(CD54) and -2 (CD102) for the integrins, vascular cell adhesion molecule- 1 (VCAM-I; CD1 06) for VLA-4 and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) for Table 2 Adhesion Molecule Deficient Mice Integrins a CD18 LFA-I (CD1 la) E-selectin (CD62E) Mac-1 (CD11b) L-selectin (CD62L) ICAM- 1 (CD54) E/P-selectin

While adhesion molecule-deficient mice have been generated for nearly all of the molecules involved in leukocyte emigration , the human LAD syndromes identified to date are restricted to defects in 2-integrin (LAD I and LAD I variant) or selectin ligands (LAD II) (Etzioni & Harlen 1998). However, as LAD I and LAD II affect different phases in the adhesion cascade, much can be learned about leukocyte-endothelial interactions from these rare human diseases.

3. 3.1

-INTEGRINS LAD I

LAD I is a rare disease with only about 200 patients reported. It results from heterogeneous mutations in the gene encoding the common 1988). LAD I was described clinically in 2-subunit, CD18 (8,g)Fisher the late 1970’s and early 1980’s, and is characterized by delayed separation of the umbilical cord, marked neutrophilia, and recurrent

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bacterial infections. Two phenotypes have been reported. In the severe form, there is no detectable expression of CD1 1/CD18 on leukocytes and the patients have a turbulent course with death usually occurring from infection during the first few years of life. Consequently, if feasible, bone marrow transplantation is performed early in life. Of note, bone marrow transplantation has a high success rate in LAD I due to decreased graft rejection, even with haploidentical donors (1 1)Thomas 1995). In the moderate phenotype, cells express 2-5% of the normal level of CD18, and the clinical course is much milder. Recently, two LAD I variants have been described in which the β2-subunits are expressed at adequate levels but are dysfunctional. In one patient the CD18 alleles are normal but may have a signaling defect (12)Kuijpers 1997). The other patient has two mutated CD 18 alleles which are expressed but are non-functional (1 3)Hogg 1999).

3.2

CD18-Deficient Mice

Early studies by Beaudet and colleagues (Wilson 1993) reported a mouse with partial CD 1 8-deficiency, comparable to the mild-moderate LAD 1 phenotype. The CDl8-hypomorphic mice were viable and fertile, without any gross anatomic or histologic abnormalities, and, in contrast to LAD I, exhibited only mild leukocytosis. Unlike LAD I patients (15)Etzioni 1996), these mice did not develop any spontaneous infections in the skin or other organs. However, they did show an impaired inflammatory response to chemical peritonitis and delayed rejection of cardiac transplants. The tolerance to allograft is consistent with the good results obtained after bone marrow transplantation in LAD I. Recently, a murine model with complete absence of CD18 was reported (16,17)Mizgerd 1997). In these animals, as in LAD I, marked neutrophilia was found (1 1- to 30-fold increase over wildtype), and there was almost no emigration of neutrophils into the skin. In contrast, when compared to wildtype, the CD 1 8-deficient animals exhibited comparable neutrophil emigration into inflamed peritoneum and increased emigration into inflamed lung. The persistent emigration of neutrophils into inflamed peritoneum of the CD1 8-deficient mice contrasts with the nearly complete inhibition of peritoneal emigration by a CD18 mAb in rabbits (19,20)Winn & Harlan 1993). This apparent disparity may reflect differences between species, but may also result in part from the marked increase in circulating neutrophils in the CD 1 8-deficient animals. Compared to antibody studies in normal animals, the number of circulating neutrophils in the CD 1 8-deficient animals is many fold higher.

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Since the number of emigrating neutrophils is in part dependent upon the number of circulating neutrophils, the observation that the same number of neutrophils emigrate in the CD18-deficient animals as in the wildtype mice may actually reflect a large inhibition. However, studies in a LAD I patient revealed an absence of neutrophils in infected peritoneum. This discrepancy may reflect a true difference between mice and humans. Alternatively, it may be due to differences in sampling sites (peritoneal fluid in mice versus tissue in humans) or to the duration of the inflammatory response (4 hours in mice versus days in the patient).

3.3

CD11a- and CD11 b-Deficient Mice

The roles of the CD1la and CD1lb subunits in several aspects of neutrophil and lymphocyte function have also been examined in genetically deficient mice. In contrast to LAD 1 or CD18-deficient mice, CD1 la- or CD11 b-deficient mice did not exhibit any leukocytosis or marked increased incidence of bacterial infection (Lu 1997). In vivo studies of CD11 b-deficient mice revealed normal rolling but defective firm adhesion, just as was seen in LAD I (23)von-Adrian 1993). Furthermore, like LAD I, neutrophils of CD11b-deficient mice exhibited in vitro defects of adhesion, iC3b-mediated phagocytosis, phagocytosisinduced respiratory burst, and homotypic aggregation (Lu 1997). However, in contrast to LAD I, neutrophil accumulation in thioglycollate-induced peritonitis was normal or even increased in the CD11b-deficient mice, a surprising result ascribed in part to impaired phagocytosis-induced apoptosis. Of interest, neutrophil emigration into inflamed peritoneum was markedly reduced in these animals when an anti-CD 11 a mAb was administered, suggesting that CDll a/CD18 rather than CD11 b/CD18 was primarily responsible for transendothelial migration of neutrophils (Lu 1997). Consistent with this observation, neutrophil accumulation in thioglycollate-induced peritonitis was modestly reduced in the CD1la-deficient mice (24)Schmits 1996). The reduction in neutrophil emigration into inflamed peritoneum in CD1ladeficient animals or CDll b-deficient animals treated with anti-CD1la mAb, but not in CD18-deficient mice may reflect the relatively normal circulating neutrophil counts in these mutants versus marked increase in circulating neutrophils in the CD 1 8-deficient animals. Based upon studies in vitro and in vivo using blocking CD1la mAbs, the CD11a/CD18 subunit has been implicated in a wide variety of immune functions including delayed-type hypersensitivity (DTH) responses and CD1 la-deficient cytolytic T-lymphocyte (CTL) effector functions. mice exhibited normal CTL responses to systemic virus infection

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(Schmits 1996); LAD I patients also appear to mount adequate immune responses to viral challenge as most infections and deaths have been due to bacterial and fungal infections rather than viruses (9)Fischer 1988). However, CD 11 a-deficient mice failed to develop a DTH response to DNCB immunization and challenge, whereas LAD 1 patients mount normal skin reactions when challenged with antigen to which they have been sensitized. These two mouse models have provided important insights into the role of the CD11a/CD18 and CD11b/CD18 subunits in leukocyte functions. It will be of interest to determine CD11C and CD1Id functions in knockout models and to compare double knockouts for CD11a and CD11 b with CD18-deficient animals.

LAD I Spontaneous Infections

++

CD18deficient ++

Leukocytosis Migration to: Skin Lung Peritoneum NR = not reported

+++

+++

+/-

+/-

++

Absent Present Absent

Absent Increased Present

NR NR Present

NR NR Reduced

NR Present Reduced

3.4

CD1 1bdeficient

CD1 ladeficient

ICAM-1deficient

-

-

-

ICAM-1-Deficient Mice

ICAM-1 is a major ligand for both CD11a/CD18 and CD11b/CD18. As ICAM-2 and 3 are also -integrin ligands, it is expected that the phenotype of ICAM- 1-deficient mice might differ from CD18-deficient mice or LAD I in which multiple ICAM counter-receptors are deficient. ICAM- 1-deficient mice have been reported (25,26)Sligh 1993). The mutant animals generated by Sligh and colleagues were found to express novel isoforms of ICAM-1 due to alternative RNA splicing , although no in vivo function has yet been established for these isoforms. Both ICAM- 1-deficient murine lines develop normally, are fertile, and have a moderate leukocytosis. The mice exhibit multiple abnormalities of inflammatory response, including impaired neutrophil emigration in response to chemical peritonitis, resistance to septic shock, decreased contact hypersensitivity reaction Qin et al (29)1996) showed that Pseudomonas aeruginosa-induced pneumonia did not require ICAM- 1 when studied using ICAM- 1 -deficient mice, while the blocking antiICAM-1 mAb inhibited neutrophil emigration by 70% in wildtype mice

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(but not in the deficient mice). It is not clear whether these marked differences result from compensation in the deficient mouse or are due to effects of mAb or antisense blockade apart from adhesion blockade (e.g., signaling) (30)Ward). Comparisons of ICAM-1-deficient mice with CD1 8-deficient mice should provide insights into the contributions of the other ICAM molecules or other CD18 ligands outside of this family in various inflammatory and immune responses.

4.

SELECTINS

4.1

LAD II

LAD II is a congenital defect in the selectin pathway that was first described in 1992 (40)Etzioni 1992). To date, there are only four known patients. Although there is no consanguinity among the four families, they likely share a common genetic background. Clinically, LAD II is much a milder disease than LAD I. The three LAD II patients did not manifest delayed separation of the umbilical cord- a hallmark of LAD I. Although the children tended to suffer from an increased incidence of infections in early infancy, compared to the severe form of LAD I these episodes were quite mild, not requiring hospitalizations or intravenous antibiotics. Later in life, infections have been rare and the children are not on prophylactic antibiotics. Table 4. Human Leukocyte Adhesion Deficiency Syndromes Clinical Manifestations Recurrent severe infections Neutrophilia basal with infections Periodontitis Skin infections Delayed separation of the umbilical cord Developmental abnormalities Laboratory Findings CD I8 expression SLeX expression Neutrophil rolling Neutrophil firm adherence T and B cell function

LAD I

LAD II

+++

+I-

+ +++ ++ ++ +++

+++ +++ ++ +/+++ or Absent

NL NL

NL Absent

NL

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The only persistent clinical symptom produced by neutrophil dysfunction is chronic, severe periodontitis, similar to that seen in LAD (Etzioni 1998). However, in contrast to LAD I in which all clinical symptoms relate to the leukocyte adhesion defect, multiple other organ systems are affected in LAD II. The children have a rare blood group type – the Bombay phenotypeand suffer from profound mental and severe growth retardation (42)Frydman 1992). The adhesion defect and other abnormalities result from a generalized defect in fucose metabolism (Sturla 1998). Consequently, only 2-3% of normal fucosylated glycoproteins is expressed on the patients' leukocytes. Since fucose is essential for the biosynthesis of E-, P-, and L-selectin ligands such as sialyl Lewis X (SLeX, CD15s), LAD II leukocytes are deficient in binding to endothelial E- and P-selectins (44)Phillips 1995). The endothelium in these patients likely also exhibits reduced expression of fucosylated L-selectin ligands. Thus, although the LAD II defect has no direct effect on the selectin genes themselves, the defect in fucose metabolism produces a deficiency of selectin ligands. Thus, with respect to leukocyte adhesion, the LAD II patients are the human equivalent of mice with combined knockout of Eand P-selectin genes (Bullard 1996, Frenette 1996) and mice with knockout of the fucosyltransferase gene that directs biosynthesis of fucose-containing selectin ligands (Mal 1996){Bullard, Kunkel, et al. 1996 ID: 44). The deficient selectin function accounts for the marked reduction of neutrophil rolling on inflamed mesenteric microvasculature observed by intravital microscopy (23)von-Adrian 1993). Interestingly, the children exhibit no defect in immune function and respond to intradermal antigen with normal numbers of T-cells. Of note, Th1-cells do not home to skin DTH sites in mice treated with anti-P- and anti-E -selectin antibody (Austrup 1997). The growth and mental retardation are most probably due to the defect in fucose metabolism, establishing a heretofore-unknown role for fucose in human growth and development. It is unlikely that deficient selectin function accounts for these abnormalities as no such defects are observed in the selectin-deficient mice (vide infra). Recently, it was found that the primary defect in LAD II is in the biochemical activity of GDP-D-mannose-4,6 dehydratase (GMD), the enzyme which converts mannose to fucose. Cloning of the GMD from a patient and a control revealed normal amino acid sequence of patient GMD, suggesting that the LAD II defect lies in some mutation(s) affecting some yet unidentified GMD-regulating protein(s) (49)Sturla 1998).

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deficient Absent +++ + ++ ++ +++

deficient Absent ++ NR Absent NR Absent

Marked +++ +++ + Absent Absent

NR = not reported

4.2

P-Selectin-Deficient Mice

P-selectin deficient mice are viable, fertile, and without any anatomic abnormalities (50)Mayadas 1993). Circulating neutrophil counts were 23 times higher than in the wildtype. The numbers of progenitors in the bone marrow of the P-deficient mice were similar to the wildtype, suggesting a longer half-life of circulating neutrophils in the mutants. By injecting radiolabelled human neutrophils into the tail vein of mutant and wildtype animals, it was shown that neutrophils indeed survived longer in the P-selectin-deficient mice. These findings in the mouse contrast with results in LAD 1 I . Price et al. (1994) performed kinetic studies in one patient and showed a much reduced circulating half-life (less than 50% of normal) with a markedly increased marrow turnover rate. While the increased turnover in the bone marrow could be explained in part by continuous stimulation (e.g., the severe periodontitis), the reason for the markedly reduced half-life of the circulating neutrophils is not clear. In LAD I, as in the P-selectin mutant mice, the prolonged neutrophil halflife was ascribed to accumulation in the circulation due to the defect in emigration. It would be expected that this would also be the case in LAD 1 I . Possibly, the defect in fucosylation of leukocyte membrane glycoproteins and glycolipids triggers their premature clearance by the reticulo-endothelial system. Intravital microscopy in the P-selectin-deficient mice revealed a marked reduction in the initial rolling phase, confirming the crucial role of P-selectin in the initial interaction of the leukocyte with the blood vessel. Importantly, however, leukocyte rolling was observed at later time-points despite the absence of P-selectin. Using neutrophils from a

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LAD II patient, a similar defect in neutrophil rolling was observed, but this defect persisted for a longer time, indicating that fucosylated glycoconjugates are also involved in later events. Extravasation of neutrophils to the skin was diminished for several hours after insult in the P-selectin-deficient mice (54)Yamada 1995). Using a skin chamber assay, neutrophil accumulation in a LAD II patient was markedly reduced over 24 hours (52)Price 1994). P-selectin-deficient mice were also reported to have a mild defect in hemostasis, but no bleeding tendency has been observed in the LAD II patients. In order to determine the role of P-selectin in lymphocyte emigration, the contact hypersensitivity reaction was investigated in Pselectin-deficient mice. Accumulation of CD4+ lymphocytes, monocytes and neutrophils was reduced significantly, but there was no difference in vascular permeability or edema (56)Subramaniam 1995). In a similar study, a DTH reaction was investigated in a LAD II patient. In contrast to the findings in the P-selectin-deficient mouse, normal numbers of T cells were found, while the clinical signs of redness and swelling were severely depressed compared to normals.

4.3

E-Selectin-Deficient Mice

The E-selectin mutant mouse was viable and exhibited no obvious developmental abnormalities. It displayed no significant change in trafficking of neutrophils in several models of inflammation (57)Labow 1994). More direct studies showed that, while the percent of rolling neutrophils was not reduced in this model, the cells rolled much faster, demonstrating a role for this selectin in the initial phase of the adhesion cascade. Blocking both endothelial selectins by treatment of the Eselectin-deficient mice with anti-P-selectin mAb significantly inhibited neutrophil emigration to the skin and peritoneum, demonstrating that Eand P-selectin are functionally redundant in this regard.

4.4

L-Selectin-Deficient Mice

L-selectin-deficient mice develop normally but exhibit defects in lymphocyte homing and leukocyte rolling (59)Arbon 1994). Lymphocytes from these mice did not bind to peripheral lymph node high endothelial venules, resulting in a marked reduction in the number of lymphocytes localized to peripheral lymph nodes. Other lymph nodes were similarly affected. The DTH reaction was impaired in L-selectin deficient mice with 75% reduction in swelling (60)Tedder 1995), similar

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to that seen in LAD II. Recently, it was shown that the defective DTH reaction in these L-selectin knockout mice was restored by administration of activated platelets into the systemic circulation (6 1)Diacovo 1998). Activated platelets expressing P-selectin can form a bridge between lymphocytes and high endothelial venules, thereby enabling lymphocytes to undergo subsequent β2-integrin-dependent firm adhesion. Interestingly, as in LAD II, T cell-dependent antibody production to keyhole limpet hemocyanin was normal in the L-selectindeficient mice (62)Xu 1996).

4.5

E/P-Selectin-Deficient Mice

The best animal models to compare with LAD II are the double (E/P-deficient) and the knockouts for E- and P-selectin ( 1,3)fucosyltransferase-deficient mice. In contrast to the rather mild phenotypes observed in mice deficient in a single selectin gene, the double-deficient mice present extreme leukocytosis and elevated cytokine levels. These mice develop a severe phenotype characterized by m ucoc utan eo u s infect ions, plasma cell proliferation, hypergammaglobulinemia and severe deficiency of leukocyte rolling in cremaster venules with or without addition of TNF-α (45,46)Frenette 1996). To characterize the role of the endothelial selectins during bacterial sepsis in vivo, Streptococcus pneumoniae was inoculated into wildtype mice and mice with E-, P-, or E/P-selectin deficiency (Munoz 1997). When compared with wildtype mice, all of these selectin-deficient mice showed greater morbidity, a significantly higher mortality associated with persistent bacteremia, and a higher bacterial load. During the first 5 days mortality was higher in the E-selectin-deficient, and only later did the double mutant approach the mortality rate observed in E-selectindeficient mice. It is possible that the presence of persistently high numbers of circulating leukocytes and plasma cells and higher serum levels of immunoglobulins in the E/P-selectin-deficient mice provided an advantage in the initial response to pneumococcal sepsis. Ultimately, however, the defect in emigration was detrimental for prolonged survival after untreated infection. Notably, LAD II patients have had no increase in systemic infections, and the few episodes of localized infection have responded to conventional treatment as in any immunocompetent child. Notably, the phenotype of the E/P-deficient animals appears to be more severe than observed in LAD II. This might simply reflect important differences between man and mouse. Alternatively, it is possible that non-fucosylated ligands participate in selectin interactions

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in vivo, thus the absence of fucosylated ligands in LAD II would not impact all E- and P-selectin-dependent functions whereas the null mice would be severely affected. Deficiency of the receptor proteins (in the deficient mice) would again have a greater impact than absence of their carbohydrate ligands (in the LAD II patients).

4.6

Fuc-TVII-deficient Mice

Synthesis of fucosylated glycans, implicated in E-, P-, and L-selectin ligand activity, is catalyzed by several glycosylation reactions. The final reaction is controlled by ( 1,3)fucosyltransferase Fuc-TVII, and thus knocking out the gene for this enzyme will result in a mouse deficient in SLeX and other fucosylated selectin ligands (47)Mal 1997). Fuc-TVIIdeficient mice yielded normal litter sizes, were vigorous and were free of microbial infection, including the spontaneous bacterial dermatitis exhibited by the double E/P- mutant mice. These mice exhibited a phenotype reminiscent of the human LAD II, including marked leukocytosis, absent binding of leukocytes to E- and P-selectins, and compromised neutrophil trafficking to inflammatory sites. Absence of Fuc-TVII also yielded a deficit in expression of L-selectin ligands by high endothelial venules and a severe alteration in lymphocyte homing. In contrast to LAD II, the Fuc-TVII-deficient mice did not show any gross anatomic abnormalities, implying again that the growth and mental retardation in LAD II is due to the general defect in fucose metabolism and not to the adhesion deficiency.

5.

CONCLUSION

The careful investigation of the LAD syndromes in man and the adhesion molecule-deficient mice has dramatically increased our understanding of the physiology and the cell and molecular biology of leukocyte emigration. These experiments have also generated new questions for future studies. Clearly, William Harvey’s observation of over three hundred years ago holds true even today: “Nature is nowhere accustomed more openly to display her secret mysteries than in cases where she shows traces other workings apart from the beaten path; nor is there any better way to advance the proper practice of medicine than to give our minds to discovery of the usual law of nature by careful investigation of cases of rarer forms of disease.” (I657).

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Kuijpers TW, Van-Lier RA, Hamann D, de BM, Thung LY, Weening RS, Verhoeven AJ, Roos D: Leukocyte adhesion deficiency type 1 (LAD-l)/variant. A novel immunodeficiency syndrome characterized by dysfunctional beta2 integrins. J.Clin.Invest. 100:1725, 1997 Kumasaka T, Quinlan WM, Doyle NA, Condon TP, Sligh J, Takei F, Beaudet A, Bennett CF, Doerschuk CM: Role of the intercellular adhesion molecule-1(ICAM-1) in endotoxin-induced pneumonia evaluated using ICAM-1 antisense oligonucleotides, anti-ICAM- 1 monoclonal antibodies, and ICAM-1 mutant mice. J.Clin.Invest. 97:2362, 1996 Lu H, Smith CW, Perrard J, Bullard D, Tang L, Shappell SB, Entman ML, Beaudet AL, Ballantyne CM: LFA-I is sufficient in mediating neutrophil emigration in Mac-Ideficient mice. J.Clin.Invest. 99: 1340, 1997 Labow MA, Norton CR, Rumberger JM, Lombard-Gillooly KM, Shuster DJ, Hubbard J, Bertko R, Knaack PA, Terry RW, Harbison ML, et a: Characterization of E-selectindeficient mice: demonstration of overlapping function of the endathelial selectins. Immunity 1 :709, 1994 Majzoub JA, Muglia LJ: Knockout mice. N.Engl.J.Med. 334:904, 1996 Mal, ThalI A, Petryniak B, Rogers CE, Smith PL, Marks RM, Kelly RJ, Gersten KM, Cheng G, Saunders TL, Camper SA, Camphausen RT, Sullivan FX, Isogai Y, Hindsgaul 0, von-Andrian UH, Lowe JB: The alpha( 1,3)fucosyltransferase Fuc-TVII controls leukocyte trafficking through an essential role in L-. E-, and P-selectin ligand biosynthesis. Cell 86:643, 1996 Mayadas TN, Johnson RC, Rayburn H, Hynes RO, Wagner DD: Leukocyte rolling and extravasation are severely compromised in P selectin-deficient mice. Cell 74:54 1, 1993 Mizgerd JP, Kubo H, Kutkoski GJ, Bhagwan SD, Scharffetter KK, Beaudet AL, Doerschuk CM: Neutrophil emigration in the skin, lungs, and peritoneum: different requirements for CD1 1/CD 18 revealed by CD1 8-deficient mice. J.Exp.Med. 186: 1357, 1997 Munoz FM, Hawkins EP, Bullard DC, Beaudet AL, Kaplan SL: Host defense against systemic infection with Streptococcus pneumoniae is impaired in E-, P-, and E-/Pselectin-deficient mice. J.Clin.lnvest. 100:2099, 1997 Phillips ML, Schwartz BR, Etzioni A, Bayer R, Ochs HD, Paulson JC, Harlan JM: Neutrophil adhesion in leukocyte adhesion deficiency syndrome type 2. J.Clin.Invest. 96:2898, 1995 Price TH, Ochs HD, Gershoni BR, Harlan JM, Etzioni A: In vivo neutrophil and lymphocyte function studies in a patient with leukocyte adhesion deficiency type II Blood 84:1635, 1994 Schmits R, Kundig TM, Baker DM, Shumaker G, Simard JJ, Duncan G, Wakeham A, Shahinian A, van-der HA, Bachmann MF, Ohashi PS, Mak TW, Hickstein DD: LFA1-deficient mice show normal CTL responses to virus but fail to reject immunogenic tumor. J.Exp.Med. 183:1415, 1996 Sligh JEJ, Ballantyne CM, Rich SS, Hawkins HK, Smith CW, Bradley A, Beaudet AL: Inflammatory and immune responses are impaired in mice deficient in intercellular adhesion molecule 1. Proc.Nat1.Acad.Sci.U.S.A. 90:8529, 1993 Springer TA: Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76:301, 1994 Sturla L, Etzioni A, Bisso A, Zanardi D, De FG, Silengo L, De FA, Tonetti M: Defective intracellular activity of GDP-D-mannose-4,6-dehydratase in leukocyte adhesion deficiency type II syndrome. FEBS Lett. 429:274, 1998

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THE ROLE OF DENDRITIC CELLS AT THE EARLY STAGES OF Leishmania INFECTION

Heidrun Moll Research Center for Infectious Diseases, University of Würzburg, Würzburg, Germany

1.

INTRODUCTION

Human leishmaniasis comprises a spectrum of clinical manifestations ranging from self-healing cutaneous lesions and solid immunity protecting against reinfection to progressive visceral disease with potentially fatal outcome. The symptoms observed in humans can be mimicked after experimental infection of inbred mice with Leishmania major. Mice from the majority of inbred strains, such as C57BL/6, are resistant, but mice from a few strains (e.g., BALB/c) develop progressive lesions and eventually die. This animal model has been extraordinarily valuable for studying the immunological parameters leading to resistance or susceptibility to infection. It has been demonstrated that healing of lesions requires the development of CD4+ T helper cells of type 1 (Th1) producing interferon γ (IFN-γ ), whereas uncontrolled spreading of the parasites is associated with a Th2 cytokine secretion pattern, i.e. interleukin (IL) 4, IL-5 and IL-10 (Reiner and Locksley 1995). This discovery was paradigmatic for many other infections with intracellular pathogens. Leishmania parasites are transmitted by sand flies. In their mammalian host, they exist as obligatory intracellular amastigotes and reside within macrophages and dendritic cells. In the initial phase after infection, natural killer (NK) cells are the primary source of IFN-γ critical for the activation of macrophages and early containment of parasite proliferation. NK cell activation, as well as the development of protective The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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Th1 cells, is regulated by IL-12 (Scharton-Kersten et al 1995, Mattner et al 1996). Whereas IL-12 and IFN- are directing Th1 cell maturation and development of resistance to leishmaniasis, IL-4 has been implicated in the differentiation of Th2 cells and susceptibility to disease (Kopf et al 1996). It is important to note that these regulatory events are launched immediately after infection. Within hours, inoculation of Leishmania parasites modulates the balance of the local cytokine network. A comparison of IL-4 mRNA expression in genetically susceptible BALB/c and resistant C57BL/6 mice revealed that BALB/c mice exhibited a peak of IL-4 in the draining lymph nodes within one day of infection which returned to baseline levels by 48 hours. No IL-4 mRNA expression was seen in resistant mice during the first two days of infection (Launois et al 1995). Together, these observations support the conclusion that the first encounter of Leishmania with cells of the immune system is critical for the course of disease.

2.

INTERACTIONS OF Leishmania AND MACROPHAGES

The two types of host cells harboring Leishmania parasites, macrophages and dendritic cells, also serve as accessory cells that present parasite antigen to specific T cells and regulate the cellular immune response. However, the recognition of infected macrophages by T cells is impaired by several mechanisms of parasite interference with antigen presentation by macrophages: (1) internalization and degradation of major histocompatibility complex (MHC) class II molecules by amastigotes (De Souza-Lão et al 1995), (2) suppression of MHC class II synthesis (Reiner et al 1987), (3) inhibition of antigen processing and MHC class II loading with immunogenic peptides (Fruth et al 1993, Prina et al 1995) and (4) deficient expression of the costimulatory molecules CD80 and heat-stable antigen (HSA) (Kaye et al 1994). Furthermore, it has been shown that Leishmania evade IL-12 induction by macrophages (Reiner et al 1994, Belkaid et al 1998) and actively inhibit macrophage IL-12 production in response to lipopolysaccharide (LPS) (Carrera et al 1996). In this context, it is also noteworthy that macrophages generally lack the ability to induce the primary stimulation of specific T cells. This is an exquisite feature of dendritic cells, the antigen-presenting cells with the most potent accessory functions, which therefore are likely to form an integral component of the initial response to Leishmania and a variety of

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other infectious agents and may represent the link between innate and acquired immunity.

3.

THE DENDRITIC CELL SYSTEM OF ANTIGENPRESENTING CELLS

The role of dendritic cells in the control of immunity has been reviewed in detail recently (Banchereau and Steinman 1998). This article will focus on the features that are relevant for the induction and regulation of immunity to microbial infection. Dendritic cells are characterized by the constitutive expression of MHC class II molecules. Their properties vary with the different stages of their life span. Dendritic cells in nonlymphoid tissues, such as Langerhans cells in the skin, can phagocytose and process particulates but are only weak stimulators of resting T cells. The differentiation of Langerhans cells is triggered in vivo by exposure to pathogens or inflammatory cytokines and is accompanied by migration of the cells to the regional lymph nodes. During this process, there is a loss of endocytic activity and a marked upregulation of the expression of MHC, costimulatory and adhesion molecules which results in the acquisition of the ability to activate naive T cells. Exposure to anti-inflammatory cytokines (such as IL-I0), on the other hand, has been shown to give rise to dendritic cells that induce T cell tolerance (Enk et al 1993) or promote the development of Th2 cell responses (De Smedt et al 1997). The differentiation of dendritic cells can be reproduced in vitro by culture in the presence of growth factors. A stimulus to induce dendritic cell activation in vitro and in vivo is the bacterial cell wall component LPS. Treatment with LPS greatly enhances the ability of dendritic cells to stimulate T cells (De Smedt et al 1996), a pathway that may involve activation of the transcription factors NF-KB and ERK (Rescigno et al 1998). Another bacterial trigger of dendritic cell activation may be carried by the DNA because unmethylated CpG oligonucleotide sequences of bacteria augment the immunostimulatory functions of dendritic cells (Jakob et al 1998, Sparwasser et al 1998).

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4.

INTERACTIONS OF Leishmania AND DENDRITIC CELLS

4.1

Phagocytosis of parasites by Langerhans cells and initiation of the immune response

After cutaneous infection with L. major, Langerhans cells leave the epidermis to take up parasites in the dermal compartment of the skin. The endocytosis of L. major by Langerhans cells is mediated by the receptor for the complement component C3bi, CR3, (Blank et al 1993) which is known to be important also for the internalization of Leishmania by macrophages. The mannose receptor, on the other hand, which contributes to parasite phagocytosis by macrophages and has been suggested to be involved in antigen capture by dendritic cells, does not mediate the endocytosis of L. major by Langerhans cells. The number of Leishmania ingested by Langerhans cells is small compared with macrophages and does not increase during in vitro culture, suggesting that Langerhans cells are able to restrict the intracellular replication of amastigotes. Interestingly, this ability is not based on the production of nitric oxide (NO), due to the lack of expression of the inducible isoform of NO synthase (iNOS or NOS-2). Thus, Langerhans cells seem to control the growth of parasites by an as yet unknown NO-independent mechanism (Blank et al 1996). The observations that infected Langerhans cells carry a low parasite load and constitute only a minority of the cutaneous infiltrate suggested that their functional activities are different from those of macrophages (Moll 1993). Macrophages are avid scavengers of Leishmania and are permissive to infection unless they are stimulated by cytokines such as IFN- and tumor necrosis factor (TNF), enabling them to clear the parasites via expression of iNOS and synthesis of high levels of NO (Stenger et al 1994). Langerhans cells, on the other hand, have the unique ability to transport the ingested parasites from the site of infection in the skin to the T-cell areas of the draining lymph node (Moll et al 1993). This migration occurs within 24 to 48 hours of infection and correlates with the differentiation of Langerhans cells into interdigitating dendritic cells that are capable to stimulate resting T cells with specificity for L. major. Thus, the major function of dendritic cells is the initiation of the T-cell immune response to Leishmania at the early stage of infection. Whereas Leishmania do not elicit IL- 12 production by macrophages (see section 2), there is now compelling evidence that the parasites

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directly stimulate dendritic cell maturation and IL- 12 release by dendritic cells immediately after infection. This has been demonstrated in vitro (Flohé et al 1998, von Stebut et al 1998, Konecny et al 1999) as well as in situ in infected lymphoid tissue (Gorak et al 1998). Therefore, dendritic cells rather than macrophages are responsible for T cell priming and create a microenvironment that favors the early activation of NK cells to produce IFN- and the education of protective Th1 cells (the regulation of both processes is dependent on IL- 12). The observations support the hypothesis that local activation of skin dendritic cells (Langerhans cells) by Leishmania, followed by migration and IL- 12 production by matured dendritic cells in the regional lymph nodes is a requirement for the development of protective immunity to leishmaniasis. Evidence is accumulating that dendritic cells are critical accessory cells for the modulation of the immune response not only to Leishmania but also to other parasites as well as bacteria and viruses. Dendritic cells have been shown to interact with Toxoplasma gondii (Reis e Sousa et al 1997), Mycobacterium tuberculosis (Inaba et al 1993), Bordetella bronchiseptica (Guzman et al 1994), Listeria monocytogenes (Guzman et al 1995), Borrelia burgdorferi (Filgueira et al 1996), Chlamydia (Ojcius et al 1998) and Salmonella (Marriott et al 1999). The role of dendritic cells in antiviral immunity was recently reviewed by Klagge & SchneiderSchaulies (1999).

4.2

Mechanisms of antigen presentation and maintenance of immunity to infection

Characterization of the mechanisms underlying the extraordinary efficiency of dendritic cells in presenting L. major antigen to T cells revealed that MHC class II molecules loaded with immunogenic parasite peptides are unusually long-lived. The MHC class 11-peptide complexes in dendritic cells are much more stable than those of macrophages (Flohé et al 1997). This corresponds with the physiological role of dendritic cells which need to retain the antigen carried from the skin to the lymph nodes for presentation to T cells and triggering of the primary antileishmanial immune reponse. The prolonged retention of Leishmania antigen by dendritic cells may also form the basis for their unique ability to present endogenous parasite antigen to L. major- specific T cells in mice that cured the original skin lesion and are immune to re-infection. Only dendritic cells, but not macrophages, are able to present persistent antigen to T cells and may thus allow the sustained stimulation of parasite-specific T cells that maintain protective immunity against leishmaniasis (Moll et al 1995).

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VACCINATION WITH DENDRITIC CELLS

The findings summarized above support the conclusion that dendritic cells and macrophages fulfill distinct and highly specialized functions in the immunoregulation of leishmaniasis and, as a consequence, the host’s ability to eliminate the parasite and cure the disease (Fig 1). At the very early stage of infection, Langerhans cells are responsible for the transport of parasites from the newly infected skin to the draining lymph nodes and the initial priming of Leishmania-specific T cells. Macrophages are unable to perform these tasks. On the other hand, they are the professional scavenger cells capturing high numbers of parasites and, after appropriate activation with cytokines (e.g., IFN- the most important effector cells for parasite clearance via the production of NO. After spontaneous recovery from cutaneous leishmaniasis, lymph node dendritic cells have the remarkable capacity to present very low amounts of persistent Leishmania antigen to specific T cells and may thus have a key function in the maintenance of protective immunity.

Figure I. lmmunoregulation of cutaneous leishmaniasis. Dendritic cells (DC)/ Langerhans cells (LC) and macrophages (Mac) have specialized functions. (IFN- interferon- NO: nitric oxide).

Considering the intriguing accessory functions of dendritic cells both in the initiation of the Leishmania- specific T cell response and the longterm presentation of parasite antigen, the cells are ideal candidates for vaccine design. Using tumor models, several recent reports documented

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that this is a promising approach. Therapeutic anti-tumor immunity can be induced by immunization with dendritic cells loaded with tumorderived antigens (Mayordomo et al 1995, Hsu et al 1996, Nestlé et al 1998). Analysis of the potency of dendritic cells in vaccination against infectious agents demonstrated that a single application of skin dendritic cells (Langerhans cells) that had been pulsed with L. major antigen in vitro induced a long-lasting protection of susceptible BALB/c mice against subsequent challenges with virulent parasites (Flohé et al 1998). The development of resistance was paralleled by a shift of the cytokine expression from a Th2- towards a Th1-like pattern that is likely to be mediated by production of IL-12 by the dendritic cells. These data demonstrate that dendritic cells are able to serve as a natural adjuvant and to induce a protective immune response to Leishmania. Similar protective effects after immunization with microbe-loaded dendritic cells have been obtained in other models. Dendritic cells pulsed with soluble antigens of the respective organisms were shown to induce protection against Borrelia burgdorferi (Mbow et al 1997), Chlamydia trachomatis (Su et al 1998) and Toxoplasma gondii (Bourguin et al 1998), indicating that they are very potent in the enhancement of immune responses. Dendritic cells are also targets for genetic vaccination. After administration of DNA, dendritic cells isolated from the immunized animals were shown to express the vaccine DNA and to present the corresponding peptides to specific T cells (Akbari et al 1999, Bouloc et al 1999, Condon et al 1996). Furthermore, dendritic cells genetically engineered to secrete IL-12 were able to augment T cell responses to antigens from Leishmania and Histoplasma (Ahuja et al 1998) and may therefore be an important tool for anti-infective therapy.

6.

CONCLUSION

Experimental infection of mice with L. major provides a valuable model to analyze the immunoregulatory events that modulate the course of infection with intracellular micro-organisms. In particular, the study of the functions of dendritic cells in murine cutaneous leishmaniasis revealed that these cells have a pivotal role in the initiation, regulation and maintenance of immune responses to infection. The recent finding that dendritic cells can serve as vehicles for vaccination against leishmaniasis indicates that the cells elicit efficient and long-lasting immunity, mimicking the response induced by natural infection. In this context, many features of dendritic cells need to be understood in more detail to

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allow their use for immunoprophylactic or therapeutic intervention. The molecular mechanisms underlying the processing of parasite antigens by dendritic cells and the loading onto MHC molecules or other restriction elements, such as the CD1 molecule, are largely undefined. The latter aspect is particularly relevant as CD1 molecules have been shown to present lipoglycan antigens to specific T cells (Porcelli and Modlin 1999) and this type of antigen is highly abundant on the surface of Leishmania parasites and other infectious agents. It is also not known whether parasites or parasite products interfere with dendritic cell activation or deviate dendritic cell functions in order to escape the immune response. Further studies will contribute to a better understanding of these issues.

ACKNOWLEDGMENTS The work in my laboratory is supported by grants from the Bundesministerium für Bildung und Forschung (BMBF), the Deutsche Forschungsgemeinschaft (DFG) and the Bayerische Forschungsstiftung, Germany.

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Carrera, L., Gazzinelli, R. T., Badolato, R., Hieny, S., Muller, W., Kuhn, R., and Sacks, D. L., 1996, Leishmania promastigotes selectively inhibit interleukin 12 induction in bone marrow-derived macrophages from susceptible and resistant mice. J. Exp. Med. 183: 515-526. Condon, C., Watkins, S. C., Celluzzi, C. M., Thompson, K., and Falo, Jr., L. D., 1996, DNA-based immunization by in vivo transfection of dendritic cells. Nature Med. 2: 1122-1 128. De Smedt, T., Pajak, B., Muraille, E., Lespagnard, L., Heinen, E., De Baetselier, P., Urbain, J., Leo, O., and Moser, M., 1996, Regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo. J. Exp. Med. 184: 1413-1424. De Smedt, T., Van Mechelen, M., De Becker, G., Urbain, J., Leo, O., and Moser, M., 1997, Effects of interleukin-10 on dendritic cell maturation and function. Eur. J. Immunol. 27: 1229-1235. De Souza-Lão, S., Lang, T., Prina, E., Hellio, R., and Antoine, J.-C., 1995, Intracellular Leishmania amazonensis amastigotes internalize and degrade MHC class II molecules of their host cells. J. Cell Sci. 108: 3219-3231. Enk, A. H., Angeloni, V. L., Udey, M. C., and Katz, S. I., 1993, Inhibition of Langerhans cell antigen-presenting functions by IL-I0: a role for IL-10 in induction of tolerance. J. Immunol. 151: 2390-2398. Filgueira, L., Nestle, F., Rittig, M., Joller, H. I., and Groscurth, P., 1996, Human dendritic cells phagocytose and process Borrelia burgdorferi. J. Immunol. 157: 29983005. Flohé, S. B., Bauer, C., Flohé, S., and Moll. H., 1998, Antigen-pulsed epidermal Langerhans cells protect susceptible mice from infection with the intracellular parasite Leishmania major. Eur. J. Immunol. 28: 3800-381 1. Flohé, S., Lang, T., and Moll, H., 1997, Synthesis, stability, and subcellular distribution of major histocompatibility complex class Ii molecules in Langerhans cells infected with Leishmania major. Infect. Immun. 65: 3444-3450. Fruth, U., Solioz, N., and Louis, J. A., 1993, Leishmania major interferes with antigen presentation by infected macrophages. J. Immunol. 150: 1857-1864. Gorak, P. M. A., Engwerda, C., and Kaye, P. M., 1998, Dendritic cells, but not macrophages, produce IL- 12 immediately following Leishmania donovani infection. Eur. J. Immunol. 28: 687-695. Guzman, C. A., Rohde, M., Bock, M., and Timmis, K. N., 1994, Invasion and intracellular survival of Bordetella bronchiseptica in mouse dendritic cells. Infect. Immun. 62: 5528-5537. Guzman, C. A., Rohde, M., Chakraborty, T., Domann, E., Hudel, M., Wehland, J., and Timmis, K. N., 1995, Interaction of Listeria monocytogenes with mouse dendritic cells. Infect. Immun. 63: 3665-3673. Hsu, F. J., Benike, C., Fagnoni, F., Liles, T. M., Czerwinski, D., Taidi, B., Engleman, E. G., and Levy, R., 1996, Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells. Nature Med. 2: 52-58. Inaba, K., Inaba, M., Naito, M., and Steinman, R. M., 1993, Dendritic cell progenitors phagocytose particulates, including Bacillus Calmette-Guerin organisms, and sensitize mice to mycobacterial antigens in vivo. J. Exp. Med. 178: 479-488. Jakob, T., Walker, P. S., Krieg, A. M., Udey, M. C., and Vogel, J. C., 1998, Activation of cutaneous dendritic cells by CpC-containing oligodeoxynucleotides: a role for dendritic cells in the augmentation of Th1 responses by immunostimulatory DNA. J. Immunol. 161: 3042-3049.

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Kaye, P. M., Rogers, N. J., Curry, A. J., and Scott, J. C., 1994, Deficient expression of co-stimulatory molecules on Leishmania-infected macrophages. Eur. J. Immunol. 24: 2850-2854. Klagge, J. M., and Schneider-Schaulies, S., 1999, Virus interactions with dendritic cells. J. Gen. Virol. 80: 823-833. Konecny, P., Stagg, A. J., Jebbari, H., English, N., Davidson, R. N., and Knight, S. C., 1999, Murine dendritic cells internalize Leishmania major promastigotes, produce IL-12 p40 and stimulate primary T cell proliferation in vitro. Eur. J. Immunol. 29: 1803-181 1. Kopf, M., Brombacher, F., Köhler, G., Kienzle, G., Widmann, K.-H., Lefrang, K, Humborg, C., Ledermann, B., and Solbach, W., 1996, IL-4-deficient BALB/c mice resist infection with Leishmania major. J. Exp. Med. 184: 1127-1 136. Launois, P., Ohteki, T., Swihart, K., MacDonald, H. R., and Louis, J. A., 199.5, In susceptible mice, Leishmania major induce very rapid interleukin-4 production by CD4' T cells that are NK1.1. Eur. J. Immunol. 25: 3298-3307. Marriott, I., Hammond, T. G., Thomas, E. K., and Bost, K. L., 1999, Salmonella efficiently enter and survive within cultured CD1IC+ dendritic cells initiating cytokine expression. Eur. J. Immunol. 29: 1107-1 11 5. Mattner, F., Magram, J., Ferrante, J., Launois, P., Di Padova, K., Behin, R., Gately, M. K., Louis, J. A., and Alber, G., 1996, Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response. Eur. J. Immunol. 26: 1553- 1559. Mayordomo, J. I., Zorina, T., Storkus, W. J., Zitvogel, L.. Celluzzi, C., Falo, L. D., Melief, C. J., Ildstad, S. T., Martin Kast, W., Deleo, A. B., and Lotze, M. T., 1995, Bone marrow-derived denedritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumor immunity. Nature Med. 1: 1297-1302. Mbow, M. L., Zeidner, N., Panella, N., Titus. R. G., and Piesman, J., 1997, Borrelia burgdorferi-pulsed dendritic cells induce a protective immune response against ticktransmitted spirochetes. Infect. Immun. 65: 3386-3390. Moll, H., 1993, Epidermal Langerhans cells are critical for immunoregulation of cutaneous leishmaniasis. Immunol. Today 14: 383-387. Moll, H., Flohé, S., and Rollinghoff, M., 1995, Dendritic cells in Leishmania majorimmune mice harbor persistent parasites and mediate an antigen-specific T cell immune response. Eur. J. Immunol. 25: 693-699. Moll, H., Fuchs, H., Blank, C., and Rollinghoff, M., 1993, Langerhans cells transport Leishmania major from the infected skin to the draining lymph node for presentation to antigen-specific T cells. Eur. J. Immunol. 23: 1595-1601. Nestle, F. O., Alijagic, S., Gilliet, M., Sun, Y., Grabbe, S., Dummer, R., Burg, G., and Schadendorf, D., 1998, Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nature Med. 4: 328-332. Ojcius, D. M., Bravo de Alba, Y.. Kanellopoulos, J. M., Hawkins, R. A., Kelly, K. A., Rank, R. G., and Dautry-Varsat, A., 1998, Internalization of Chlamydia by dendritic cells and stimulation of Chlamydia-specific T cells. J. Immunol. 160: 1297-1303. Porcelli, S. A., and Modlin, R. L., 1999, The CD1 system: antigen-presenting molecules for T cell recognition of lipids and glycolipids. Annu. Rev. Immunol. 17: 297-329. Prina, E., Jouanne, C., de Souza-Lão, S., Szabo, A., Guillet, J.-G., and Antoine, J.-C., 1993, Antigen presentation capacity of murine macrophages infected with Leishmania amazonensis amastigotes. J. Immunol. 151: 2050-206 1.

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Reiner, N. E., Ng, W, and McMaster, W. R., 1987, Parasite-accessory cell interactions in murine leishmanisis. II Leishmania donovani suppresses macrophage expression of class I and class I1 major histocompatibility complex gene products. J. Immunol. 138: 1926- 1932. Reiner. S. L., and Locksley, R. M., 1995, The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13: 151-177. Reiner, S. L., Zeng, S., Wang, S. E.,Stowing, L., and Locksley, R. M., 1994, Leishmania promastigotes evade interleukin 12 (IL- 12) induction by macrophages and stimulate a broad range of cytokines from CD4+ T cells during initiation of infection. J. Exp. Med. 179: 447-456. Reis e Sousa, C., Hieny, S., Scharton-Kersten, T., Charest, H., Jankovic, D., Germain, R. N., and Sher A., 1997, In vivo microbial stimulation induces rapid CD40Lindependent production of 1L-12 by dendritic cells and their re-distribution to T cell areas. J. Exp. Med. 186: 1819-1829. Rescigno, M., Martino. M., Sutherland, C. L., Gold, M. R., and Ricciardi-Castagnoli, P., Dendritic cell survival and maturation are regulated by different signalling pathways. J. Exp. Med. 188: 2175-2180. Scharton-Kersten, T., Afonso, L. C. C., Wysocka, M., Trinchieri, G., and Scott, P., 1995, IL- 12 is required for natural killer cell activation and subsequent T helper 1 cell development in experimental leishmaniasis. J. Immunol. 154: 5320-5330. Sparwasser, T., Koch, E.-s., Vabulas, R. M., Heeg, K., Lipford, G. B., Ellwart, J. W., and H. Wagner, 1998, Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells. Eur. J. Immunol. 28: 20451054. Stenger, S., Thüring, H., Rollinghoff, M., and Bogdan, C., 1994, Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major. J. Exp. Med. 180: 783-793. Su, H., Messer, R., Whitmire, W., Fischer, E., Portis, J. C., and Caldwell, H. D., 1998, Vaccination against chlamydial genital tract infection after immunization with dendritic cells pulsed ex vivo with nonviable chlamydiae. J. Exp. Med 188: 809818. Von Stebut, E., Belkaid, Y., Jakob, T., Sacks, D. L., and Udey, M. C., 1998, Uptake of Leishmania major amastigotes results in activation and interleukin 12 release from murine skin-derived dendritic cells: implications for the initiation of anti-leishmania immunity. J. Exp. Med. 188: 1547-1552.

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DNA-BASED VACCINES: ROLE OF DENDRITIC CELLS IN ANTIGEN PRESENTATION

Lada Paul and Angel Porgador Department of Microbiology and Immunology, Faculty of Health Sciences, and the Cancer Research Center, Ben Gurion University of the Negev, Beer Sheva 84/05, Israel

Since the discovery that plasmid DNA encoding a protein antigen can serve as an effective immunogen, DNA-based vaccination has become an attractive alternative to recombinant live vector immunization strategies. DNA vaccination has several obvious advantages over traditional vaccines (protein and peptide-derived), namely, more safely, easier to manufacture and to manipulate the protein antigenicity in the level of DNA. Moreover, the addition of DNAs encoding various cytokines or costimulatory molecules admixed with DNA encoding antigens serves to enhance the magnitude and type of desired immune responses (Xiang and Ertl 1995). In different animal models, DNA vaccines have been found to be protective against a wide range of diseases, including cancers, infectious, autoimmune and allergic diseases (Tighe et a1 1998). The question arises why is DNA immunization so effective at inducing specific protective immunity? Considering of some features of DNA vaccine provides the answer to this question: (i) it provides an immunogenic antigen; (ii) it is processed via major histocompatibility complex (MHC) class I and II; (iii) it stimulates immunological memory; and (iv) it contains an adjuvant (Tighe et al 1998, Ulmer et al 1996). DNA vaccines may be administered either by direct injection into skeletal muscles or skin; alternatively gold particles coated with DNA that can be physically delivered into living tissues using a helium-powered gene delivery device, the "gene gun" (Wolff et a1 1990, Sato et a1 1996, Williams et a1 1991). All of these routes of DNA delivery have been shown to induce strong and long-lived cellular and humoral immune The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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responses (Ulmer et al 1993, Irvine et al 1996, Ulmer et al 1996). Such plasmid DNA immunization can evoke immune responses dependent on both MHC class I and II molecule-mediated antigen presentation to CD8+ cytotoxic T lymphocytes (CTL) and CD4 T cells, respectively. Following gene gun bombardment of the abdominal epidermis, keratinocytes were found to be the main cell type that expressing the DNA-encoded proteins (Lu et al 1996). Similarly, striated muscle cells are the predominant transfected, antigen-positive cells following i.m. injection (Danko et al 1997). The exact mechanism by which injected or particle-coated DNA leads to antigen presentation capable of eliciting a T cell immune response has yet to be filly defined. There are several possibilities may exist: (i) somatic cells, such as transfected myocytes or keratinocytes present the antigen to immune system; (ii) somatic cells transfected with plasmid DNA produce an antigen, which is then transferred to and presented by professional antigen-presenting cells (APC), so-called crosspriming mechanism of antigen presentation; or (iii) priming of naive T cells depends on transfection of APCs with the introduced DNA, thus presenting the antigen directly to immune system. Here we will overview the current understanding of the role of professional APC in antigen presentation to naive T cells, paying a special attention to the role of DC in priming of CTL response. The experiments using bone-marrow (BM) chimeric mice have shown that BM-derived APCs rather than somatic cells (e.g. myocytes, keratinocytes) are responsible for induction of CTL response to plasmid DNA by either intramuscular (i,m.) or gene gun immunizations (Doe et al 1996, Iwasaki et al 1997a, Con et al 1996, Fu et al 1997). For example, Iwasaki and colleagues constructed BM-chimeric mice by injection of Tcells-depleted BM cells from C57BL/6 (H-2b H-2bxd) or BALB/c (H2d H-2bxd) into X-irradiated F1 (B6 x BALB/c) mice (Figure 1). These chimeric F1 mice were immunized with plasmid DNA encoding influenza nucleoprotein (NP) either i.m. or epidermally using gene gun delivery system. Splenocytes from immunized mice were pulsed with either the H2Kd-restricted NP peptide (147-155) or the H-2Db-restricted NP peptide (366-374) and cell-mediated cytotoxicity was determined. The authors demonstrated that response after i.m. or gene gun immunizations specific CTL to the expressed influenza nucleoprotein is determined by MHC Hhaplotype of transplanted BM cells but not of recipient mice. H-2b 2bxd chimeric mice developed CTL restricted for H-2b, whereas CTLs d d bxd observed in H-2 H-2 were specific for H-2 (Figure 1, bottom). These results clearly demonstrated the key role of BM-derived APC as triggers of immune response to plasmid DNA.

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These BM-derived APC might be dendritic cells (DC); a wealth of evidence suggests that the hostderived cells specializing in antigen presentation to naive T cells are DC of hemopoietic origin (Steinman 1991). Casares et al (1997) have recently demonstrated that the direct uptake of DNA by draining lymph node-derived DC, but not B cells, resulted in effective presentation to MHC class II restricted T cells. Moreover, antigen released from cultured transfected muscle cells can also generate ligands able to activate MHC class 11-restricted T cells when supernatants are pulsed onto DC. It has been demonstrated by Condon et al (1996) that immunization of mice by gene gun bombardment with plasmid DNA encoding green fluorescent protein (GFP) gene resulted in appearance of small number of GFP-containing cells in draining LN. In addition, immediately before immunization with GFP, the abdomens of mice were painted with a red fluorescent sensitizer, rhodamine, and several double positive DC (judged by the expression of both green and red fluorescence) have been observed in draining LNs. These observations provide evidence that gene gun immunization result in direct transfection of DC at the site of injection and their migration to draining LNs. With regard to the role of DC in priming of naive CD8+ T cells after immunization with plasmid DNA, two possible modes of DC antigen presentation might exist: (i) presentation directly by gene-transfected DC trafficking to local LNs and (ii) cross-presentation by untransfected DC of antigen released from or associated with transfected epidermal cells or myocytes. In accordance to study of Condon et al (1996), we also observed that LNs draining the site(s) of gene gun bombardment contain a relatively small number of antigen-carrying cells, We further explored the issue by attempting to quantitate the number of such directly transfected cells and their functional significance. Mice received non-overlapping abdominal deliveries (1, 2, and 5 shots) of gold beads coated with a -galactosidase β-gal)-encoding plasmid. Twenty-four hours after immunization, draining LN were harvested and sectioned for analysis. Frozen sections of LN were stained for β-gal activity. Because β-gal is a cytoplasmic enzyme, only cells directly transfected with the gene and producing the enzyme endogenously have enough β-gal to yield a positive staining signal under these conditions. We demonstrated that there is a linear correlation between the number of non-overlapping abdominal deliveries and the number of β-gal positive cells in the draining inguinal LN. Overall, the numbers are very low and average ~15 β-gal positive cells in each inguinal LN after one abdominal exposure to DNA-coated gold particles (Porgador et al 1998). On average, the total number of directly transfected cells in the skin draining LNs after abdominal delivery did not

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exceed 200 cells per mouse, as assessed by expression of a sufficient amount of active β-gal to yield a highly visible amount of reacted substrate (Porgador et al 1998). Given the likelihood that such cells expressing the plasmid-encoded antigen would be especially effective at processing and presenting antigenic peptides in association with MHC class I molecules, the question arose of whether these cells or the larger number of migrating DC that might present antigen via class I by processing of exogenous antigen from transfected epidermal cells were responsible for sensitization of CD8+ CTL precursors under these immunization conditions. We first tested what type of APC in draining LN 24 h after gene gun delivery of pCMV/β-gal into C57BL/6 mice present a class I-restricted determinant. LN cells were isolated using the collagenase method that allows recovery of CD8+/DEC-205+ interdigitating DC and subjected to depletion of different cell subpopulations using mAbs and magnetic beads. These cells were then incubated with CD8+ T cells from a CTL line b specific for a β-gal derived determinant presented by K and IFNγ secretion by the T cells was measured (Figure 2A). Class II-positive cells were essential for this presentation, but B cells and macrophages were not. Depletion of DEC-205 positive cells eliminated up to 70% of the presentation activity. As only about 50% of CD11c positive DC in the inguinal LN express even moderate levels of cell surface DEC-205 (data not shown), these data suggest that DEC-205-surface positive DC are especially effective at presentation of antigen in association with MHC class I molecules after DNA vaccination by gene gun. These data are consistent with the conclusion that DC in the draining LNs are the primary cell type bearing stimulatory levels of the β-gal determinant associated with Kb after gene gun delivery of β-gal encoding plasmid. We next analyzed whether the DC capable of this presentation acquired antigen from other transfected cells or by direct gene expression. Gold particles were co-coated with plasmid DNA encoding the human CD4 cell surface molecule and with pCMV/β-gal DNA. Thus, directly transfected DC will express human CD4 as a membrane protein and should also present directly processed β-gal determinants. C57BL/6 mice were immunized with 5 abdominal deliveries of these co-coated beads and 24 h later, LN cells or DC-enriched LN cells were specifically depleted of human CD4-positive cells using OKT4 and anti-mouse IgG-coated magnetic beads, or mock depleted using just anti-mouse IgG-coated beads alone. Responses of the Kb-β-gal peptide-specific T cell line to these APC populations were then measured. Mock depleted total or DCenriched cells from mice immunized with either co-coated beads or beads

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singly coated with pCMV/βgal alone, as well as cells from mice immunized with beads coated with pCMV/β-gal alone and treated to deplete human CD4-expressing cells, all retained their full functional activity in stimulating the CD8+ T cells (Figure 2B and data not shown). In contrast, depletion of cells expressing human CD4 from total or DCenriched cell populations obtained from the LN of mice immunized with beads co-coated with pCMV/β-gal and human CD4 plasmids reproducibly reduced presentation by 60 to 70%. Experiments involving in vitro gene gun cotransfection into B 16 melanoma cells with plasmids for GFP and human CD4 demonstrated coexpression in about 70% of the cells expressing GFP (data not shown). Although these in vitro data do not provide a direct measure of the extent of effective in vivo gene coexpression in DC under these DNA delivery conditions, they suggest the coexpression / depletion method will likely underestimate the fraction of cells expressing an intracellular antigen due to direct transfection. Although B cells are also depleted by the anti-mouse IgG coated magnetic beads used to remove hCD4 positive cells, B cells are not the presenting cells as deduced from Figure 2A and from the observation that depletion only with anti-mouse IgG-coated magnetic beads or depletion of LN cells from mice immunized only with β-gal did not affect presentation (Figure 2B). Together, these data imply that directly transfected DC play a predominant role in presentation of antigen via MHC class I molecules after gene gun DNA delivery. To further examine the importance of direct gene transduction of + APC for in vivo CD8 CTL priming, we turned to a previously described model system involving a nonimmunogenic mutant of influenza nucleoprotein (NPo). This mutant protein can elicit CD8+ CTL responses when produced following i.m. injection of a plasmid also encoding B7.2 (Iwasaki et al 1997b). These prior studies did not determine, however, whether expression of transfected B7.2 by antigenpresenting muscle cells, antigen-presenting LN cells, or both were responsible for the observed enhancement of CTL responses. We revisited this issue by using immunization conditions that minimize the effect of direct presentation by transfected tissue cells. I.m. delivery of DNA results in a relatively consistent expression of the transfected gene in muscle cells. In contrast, gene-bombardment of skin with plasmid DNA results in transient expression of transfected gene in the skin, peaking at 24 h post-inoculation and largely lost by 3 days post inoculation (Torres et al 1997). Also, antigen-specific T cells stimulated in the draining LN are initially retained in the LN, with efflux of antigen-stimulated T cells from the LN beginning after 3-4 days (Abbas et al 1994). Hence, following one gene gun immunization of the skin, encounters between

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antigen-specific naive T cells stimulated in the draining LN and directly transfected skin cells are rare. This permitted the design of experiments that compared the effect on CTL priming of coexpression of transfected DNA sequences for NPo and B7.2 in individual cells with that of separate but simultaneous expression of the two proteins. The data obtained showed that priming of anti-mutant influenza nucleoprotein CTL with a single immunization is dependent upon co-expression of the DNAs encoding nucleoprotein and B7-2 in the same cells (Porgador et al 1998). These data support the results of the in vitro presentation studies described above in indicating a predominant role for directly transfected cells in priming CD8+ T cell responses in draining LN after gene gun DNA delivery. This conclusion is consistent with the following ablation studies; despite the fact that after intradermal (i.d.) and i.m. injections of plasmid DNA the antigen-encoded is expressed predominantly by keratinocytes and myocytes, respectively, surgical ablation experiments have suggested that cells from skin or muscle play a minor role in processes of antigen presentation. For example, the muscle bundle can be removed within 10 minutes of DNA injection without any effect on the longevity and magnitude of the humoral and CTL responses (Torres et al 1997). Klinman and collegues showed that gene gun immunization is independent of cells in the bombarded tissue site with regard to induction of memory T cell responses (Klinman et al 1998). Also, Ciernik et al (1996) demonstrated that gene gun inoculation with minigenes encoding class I MHC-restricted T cell epitopes resulted in efficient induction of CTL. The minigenes encoded cytoplasmically-expressed CTL epitopes that are not likely to be secreted and transferred to another cell for class I MHC presentation, although these results do not exclude presentation by DC after apoptotic cell uptake (Albert et a1 1998). To summarize, BM-derived APC are the sole responsible for induction of CD8+ CTL response following DNA immunization. The above results demonstrate that the predominant contribution to priming for CTL after DNA immunization, using the gene gun approach, involves the small number of directly transfected, migrating DC rather than the much larger number of migrating DC that could potentially present antigen via a following antigen expression by epidermal cells.

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CTLs induced after DNA immunization are ristricted to BM-dirived APC

Muscle Iskin bxd bXd bxd

BM-APC T-cell restriction b b d d bXd bxd

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Figure 2. Analysis of APC in draining LN presenting the class I-restricted epitope. Twenty-four hours after bombardment with different plasmids, inguinal LN cells were harvested and pooled. (A) APC and control APC are from mice immunized with pCMV/βgal- and mock lasmid-coated particles, respectively. APC ( l06) were depleted of MQ, B cells, DEC205+ P cells, or class II+ cells using specific mAbs and magnetic beads. Cells then were incubated with l05 T cells specific for a Kb-restricted β -gal epitope and IFN levels in the supernatants were measured and presented as OD). (B) APC are from mice immunized with pCMV/ β -gal-coated gold beads (β -gal) or from mice immunized with pCMV/ β -gal and T4TM (hCD4 plasmid) co-coated gold beads (β -gal+hCD4). APC were either mock-depleted of cells expressing cell surface human CD4 protein (hCD4 dep). Cells were then incubated with T cells and IFN levels were measured. Background OD, obtained from T cells without APC (0.15-0.19), was subtracted from the values in (B). Bars, ±SE. Experiments were repeated three times with similar results.

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REFERENCES Abbas, A.K., A.H. Lichtman, and J.S. Pober, 1994, Functional anatomy of local and systemic immune responses. 2nd ed. In Cellular and molecular immunology. W. B. Saunders Company, Philadelphia, PA., pp. 222-236. Albert, M.L., B. Sauter, and N. Bhardwaj, 1998, Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 392:86-89. Casares, S., K. Inaba, T.D. Brumeanu, R.M. Steinman, and C.A. Bona, 1997, Antigen presentation by dendritic cells after immunization with DNA encoding a major histocompatibility complex class I1-restricted viral epitope. J Exp Med 186: 1481-6. Ciernik, I.F., J.A. Berzofsky, and D.P. Carbone, 1996, Induction of cytotoxic T lymphocytes and antitumor immunity with DNA vaccines expressing single T cell epitopes. J Immunol. 1.56:2369-75. Condon, C., S.C. Watkins, C.M. Celluzzi, K. Thompson, and L.D. Falo, Jr., 1996, DNAbased immunization by in vivo transfection of dendritic cells. Nat Med 2.1 122-8. Corr, M., D.J. Lee, D.A. Carson, and H. Tighe, 1996, Gene vaccination with naked plasmid DNA: Mechanism of CTL priming. J. Exp. Med. 184:1555-1560. Danko, I., P. Williams, H. Herweijer, G. Zhang, J.S. Latendresse, I. Bock, and J.A. Wolff, 1997, High expression of naked plasmid DNA in muscles of young rodents. Hum Mol Genet 6:1435-43. Doe, B., M. Selby, S. Barnett, Baenziger, and C.M. Walker, 1996, Induction of cytotoxic T lymphocytes by intramuscular immunization with plasmid DNA is facilitated by bone marrow-derived cells. Proc. Natl. Acad. Sci. USA 93:8578-8583. Fu, T., J. Ulmer, M. Caulfield, R. Deck, A. Friedman, S. Wang, X. Liu, J. Donnelly, and M. Liu, 1997, Priming of cytotoxic T lymphocytes by DNA vaccines: requirement for professional antigen presenting cells and evidence for antigen transfer from myocytes. Mol. Med. 3:362-371. Irvine, K.R., J.B. Rao, S.A. Rosenberg, and N.P. Restifo, 1996, Cytokine enhancement of DNA immunization leads to effective treatment of established pulmonary metastases. J Immunol 1.56:238-245. Iwasaki, A., C.A. Torres, P.S. Ohashi, H.L. Robinson, and B.H. Barber, 1997a, The dominant role of bone marrow-derived cells in CTL induction following plasmid DNA immunization at different sites. J Immunol 1.59:l1-4. Iwasaki, A., B.J. Stiernholm, A.K. Chan, N.L. Berinstein, and B.H. Barber, 1997b, Enhanced CTL responses mediated by plasmid DNA immunogens encoding costimulatory molecules and cytokines. J Immunol 1 58:459 1-601. Klinman, D.M., J.M.G. Sechler, J. Conover, M. Gu, and A.S. Rosenberg, 1998, Contribution of cell at the site of DNA vaccination to the generation of antigenspecific immunity and memory. J. Immunol. 160:2388-92. Lu, B., G. Scott, and L.A. Goldsmith. 1996. A model for keratinocyte gene therapy: preclinical and therapeutic considerations. Proc Assoc Am Physicians 108: 165-72. Porgador, A., K.R. Irvine, A. Iwasaki, B.H. Barber, N.P. Restifo, and R.N. Germain, 1998, Predominant role for directly transfected dendritic cells in antigen presentation to CD8' T cells after gene gun immunization. J. Exp. Med. 188:1075-1082. Sato, Y., M. Roman, H. Tighe, D. Lee, M. Corr, M.D. Nguyen, G.J. Silverman, M. Lotz, D.A. Carson, and E. Raz, 1996, Immunostimulatory DNA sequences necessary for effective intradermal gene immunization. Science 273:352-4. Steinman, R.M., 1991, The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9:271-279.

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Tighe, H., M. Corr, M. Roman, and E. Raz, 1998, Gene vaccination: plasmid DNA is more than just a blueprint Immunol Today 19(2):89-97. Torres, C.A.T., A. Iwasaki, B.H. Barber, and H.L. Robinson, 1997, Differential dependence on target site tissue for gene gun and i.m. DNA immunizations. J. Immunol. 158:4529-4532. Williams, R.S., S.A. Johnston, M. Reidy, M.J. DeVit, S.G. Mcelligott, and J.C. Sanford, 1991, Introduction of foreign genes into tissues of living mice by DNA-coated microprojectiles. Proc. Natl. Acad, Sci. USA. 88:2726-2730. Wolff, J.A., R.W. Malone, P. Williams, W. Chong, G. Acsadi, A. Jani, and P.L. Felgner, 1990, Direct gene transfer into mouse muscle in vivo. Science 247:1465-1468. Xiang, Z., and H.C. Ertl, 1995, Manipulation of the immune response to a plasmidencoded viral antigen by coinoculation with plasmids expressing cytokines. Immunity 2: 129-135. Ulmer, J.B., J.J. Donnelly, S.E. Parker, G.H. Rhodes, P.L. Felgner, V.J. Dwarki, S.H. Gromkowski, R.R. Deck, C.M. DeWitt, A. Friedman, L.A. Hawe, K.R. Leander, D. Martinez, H.C. Perry, J.W. Shiver, D.L. Montgomery, and M.A. Liu, 1993, Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259:1745-1749. Ulmer, J.B., J.C. Sadoff, and M.A. Liu, 1996, DNA vaccines. Curr Opin Immunol 8:5316.

DISTINCT PATTERNS OF IL-l AND IL-lβ ORGAN DISTRIBUTION-A POSSIBLE BASIS FOR ORGAN MECHANISMS OF INNATE IMMUNITY I

Moshe Hacham, ²Shmuel Argov, 1Rosalyn M.White, ¹Shraga Segal and 1 Ron N. Apte ¹Departnrent of Microbiology and Immunology, ²Department of Pathology, Soroka Medical Center, Faculty of Health Sciences and The Cancer Research Center, Ben-Gurion University of the Negev, Beer-Sheva, Israel

1.

INTRODUCTION

Pro-inflammatory cytokines may play a cardinal role in organ homeostasis, due to their capability to mount and influence inflammatory, immune and metabolic responses. Interleukin-1 (IL-1) is a prototypical such cytokine, being a highly pleiotropic and ubiquitously expressed factor (Dinarello 1989, Dinarello 1996). It is synthesized and secreted by diverse cells, including lymphoid and stromal cells and affects nearly every cell type, often in concert with other cytokines or small protein mediators. As a host-defense promoting factor, IL-1 is apparently involved in stimulation and activation of both innate and adaptive immunity. In the context of innate immunity, IL-1 has been shown to potentiate the activity of NK cells and macrophages, serve as chemoattractant and activator of neutrophils. It is also a strong inducer of chemokines, other cytokines, and many other antimicrobial agents, such as nitric oxide, oxygen radicals, complement molecules and mannose receptors, thus also potentiating phagocytosis. In the context of adaptive immunity, IL- 1 stimulates T and B lymphocytes and strongly induces and potentiates The Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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delayed type hypersensitivity (DTH) responses. As a highly inflammatory cytokine, IL-1 action is characterized by a narrow margin between clinical benefit and toxicity. It has thus been associated with various pathological states, such as autoimmune diseases (type I diabetes, rheumatoid arthritis), atherosclerosis and sepsis, all mediated by excessive or disturbed inflammatory reactions (Dinarello 1996). IL-l and IL-lβ are separate proteins, encoded by distinct genes, that share the same spectrum of biological activities and bind to the same receptors in their recombinant form; the redundancy in the existence of these two similarly acting IL- 1 molecules is not completely understood. Nevertheless, some differences between IL-l and IL-lβ can be pointed out. In monocytes/macrophages, the expression of IL- 1β appears more tightly regulated relatively to IL-lα, as evaluated by parameters such as regulation of gene expression, mRNA stability, rate of translation, intracellular processing, secretion and affinity of binding to the IL1 receptors (Dinarello 1989, Dinarello 1996). When compared to IL- 1 α, IL-lβ was described to be more dominantly involved in some specific inflammatory reactions, such as induction of the acute phase response and collagen-induced arthrtitis (Geiger et al. 1993, van den Berg et al. 1994, Fantuzzi et al. 1995, Zheng et al. 1995). Also, it was shown that IL-1α and IL- 1β differentially regulate tumor-antigen presentation by epidermal cells (Beissert et al. 1998), class II antigen expression in insulinoma cells (Vassililiadis S et al. 1997) and arachidonic acid metabolism (Xiao et al. 1986). IL-1α and 1L-1β differ in the subcellular compartments, in which they are active; 1α is mainly active as a cytosolic precursor (3 1 kD) and a membrane-associated molecule (23kD), being only marginally secreted, while IL- 1 β is solely active in its secreted form. It would probably be only in the context of the producing cell and/or its microenvironment where the differential physiological role of IL-1α and IL-lβ could be elucidated.

1.1 Initial Comments Relatively few studies have been performed on the characterization of tissue distribution of cytokines, an issue which may facilitate the understanding of cytokine-based homeostatic mechanisms in organs. IL1 was demonstrated in most human and murine tissues, mainly at the level of mRNA and in situ expression (Hauser et al. 1986, Takacs et al. 1988, Tovey et al. 1988, Ulich et al. 1990, Clark et al. 1991, Han et al. 1991, Hasegawa et al. 1991, Chensue et al. 1991). The overall impression has been that lymphatic organs, such as the spleen and liver display it at enhanced levels, usually upon stimulation by inflammatory

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inducers, such as lipopolysaccharide (LPS) and cytokines. We have previously shown in this context (Hacham et al. 1996) an inverse pattern of organ IL- 1 versus IL-6/macrophage colony stimulation factor (CSF- 1) expression. This may allow a better understanding of organ homeostatic functioning, thus possibly also leading to an improved classification of organs, reflecting their functional uniqueness. (Figure 1) (Hacham et al. 1996)

Figure 1. Compared relative expression of IL-1 versus IL-6 activity in samples of analyzed organs, obtained from untreated (S) and LPS-stimulated (LPS) mice. The respective activity of each cytokine in conditioned media (CM) and lysates (LYS) of the various organs is expressed as the percentage of activity in relation to that of the organ which displays the highest activity of the relevant cytokine, referred as 100%

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Distinict Patterns of IL- Iα and IL- I β Organ Distribution

Thus, an elevated IL-1 and low IL-6/CSF-1 expression appears typical of the “first-line” lymphoreticular organs (lungs, intestine, spleen and liver), while a low IL-1 and high IL-6/CSF-1 expression is characteristically demonstrated by the internal privileged organs, which perform more specified and vulnerable functions, majorly based on sensitive, electrically-based mechanisms (heart, brain, skeletal muscle and kidney). IL- 1 proposedly mediates and perpetuates the potent defensive inflammatory responses in the lymphoreticular organs, even at the cost of tissue-damage. Conversely, milder and more tightly regulated inflammatory responses, apparently mediated by IL-6/CSF- 1, are operant in privileged organs, thus minimizing the danger of tissue-damage in these organs. In this respect, IL-6 has been shown to mediate the “beneficial” aspects of inflammation, such as induction of fever, hepatic acute phase proteins and Ig production, without being involved in generation of its potential detrimental counterparts (Hirano et al. 1990, Akira et al. 1993, Tilg et al. 1997, Dinarello 1996). Thus, IL-6 is not involved in endothelial and synovial cell activation and does not induce IL-8, platelet-activating factor (PAF) and other pro-inflammatory cytokines or products. Furthermore, and in contrast to IL-1 and tumor necrosis factor (TNF), IL-6 does not induce the expression of adhesion molecules on endothelial cells or the production of metalloproteinases. In fact, IL-6 is a potent inhibitor of these enzymes (Sato et al. 1990). Protection of the vital internal organs can then be provided by shortlived mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC), opsonization and non-immune cytotoxic/apoptotic mechanisms. Indeed, both IL-6 (Borish et al. 1989) and CSF-1 (Mufson et al. 1989) were described as powerful potentiators of ADCC. IL-6 can dominantly affect organ homeostasis through induction of the acute phase response, whereby it promotes generation of anti-microbial agents, such as the C-reactive protein (CRP) (Holzer TJ et al. 1984, Pied et al. 1989), on one hand, and down-regulates inflammatory responses through mechanisms, such as scavenging of oxygen radicals, inhibition of proteases and T cell activation processes, on the other hand (Whisler RL et al. 1986, Steel DM et al. 1994). Moreover, IL-6 inhibits inflammatory responses and IL- 1 activity, either directly or through the acute phase proteins induced by it (Tilg et al. 1997). The findings displayed in this work provide evidence to the existence of differential patterns of IL- 1α versus IL- 1β organ expression in young and old mice, which may significantly pertain to organ-specific innate mechanisms of defense in these two age groups. These findings may also shed light on the possible relevance of organ IL-1 to aberrations in physiological processes typifying old age, such as failing immunity and

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increased rates of degenerative, autoimmune and neoplastic development.

2.

AND IL-1ß ARE INVERSELY EXPRESSEDIN IL-l ORGANS

The patterns of IL-l versus IL-1β organ expression were characterized by ELISA analyses of organ conditioned media (CM), prepared from whole organ cultures in serum free media and organ lysates (LYS), obtained by organ homogenizing. In addition, in situ expression of these cytokines was evaluated by immunohistochemistry (data not shown). It comes out that IL-l is expressed at high levels in lymphoreticular organs and conversely, only in reduced amounts in privileged organs. A reciprocal pattern of expression is displayed for IL1β, which is demonstrated most evidently in organ conditioned media (CM) (Figure2). The relative preferential expression of IL-1β in privileged organs, is most explicitly highlighted in old mice. This demonstrated dichotomy in organ IL- 1 α and IL-1β expression suggests a distinct involvement of these two IL-1 molecules in organ homeostasis.

Figure 2. The expression of IL-1α and IL-1β in organ conditioned media (CM) was assessed by ELISA in young and old mice. The respective IL-1 an IL-1β expression is comparatively depicted for each organ

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Distinct Patterns of IL-1 α and IL-1β Organ Distribution

3. ORGAN IL-1α EXPRESSION The findings presented in this work strongly support the notion, proposed by us before (Hacham M et al. 1996), that IL-1α, expressed at high levels in lymphoreticular organs, may dominantly be involved in their defense. 3.1 High expression of IL-1α in lymphoreticular organs IL-1α is expressed at high levels in lymphoreticular organs, most clearly at old age, as displayed here by the ELISA analyses (Figure 3). IL1α pattern of expression, as characterized by immunohistochemistry, is basically similar, displaying a predominance of lymphoreticular organs in this respect (data not shown).

y

4

Figure 3. Organ IL-1α expression was assessed in young versus old mice by detection of immunoreactive IL-1α in lysates (LYS), obtained from the various organs. This was performed by ELISA analyses and comparatively presented for the two age groups

In lymphoreticular organs, IL- 1α is dominantly detected in lumenfacing epithelial cells (lungs and small intestine), hepatocytes adjacent to venules and mononuclear cells in the spleen. These cells are in immediate exposure to exogenous or circulating microorganisms and/or toxic agents which probably induce IL-1α expression even in the “constitutive” state . Being expressed at such location, IL-1α may play a crucial role in activation and regulation of local defense in these organs. The potential role played by epithelial cells in activation and modulation of local organ defense has increasingly been emphasized (Schonwetter et al. 1995, Martin et al. 1997, Polito and Proud 1998, Reynoso-Paz et al. 1999). It is well recognized that epithelial cells can modulate local immunity through generation of a variety of factors in

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response to stimuli originating from infectious/toxic agents and/or neighbouring cells, e.g macrophages and mast cells. Thus, epithelial cells have been shown to produce cytokines which promote inflammatory responses, such as TNFα colony-stimulating factors (GM-CSF, CSF- 1 and G-CSF) and chemokines, especially IL-8. In addition, epithelial cells can activate both the lipoxygenase and cyclooxygenase pathways, produce peptide mediators, such as endothelins and defensins, and reactive oxygen species, such as nitric oxide. IL-1 expressed by epithelial cells, possibly acting in a autocrine and/or paracrine manner and activating adjacent cells, such as endothelial cells, fibroblasts, macrophages and other immune cells, may thus prove pivotal in the context of “first-line immunity”; its outstanding capability to induce and perpetuate the inflammatory response is well accepted and include the mechanisms enumerated above. In this context, cell-associated or locally secreted IL-1 seems to play a dominant role in activation of endothelial cells, leading to recruitment and activation of inflammatory cells. The role of the latter is vital in first-line confrontation with prolonged and sometimes massive microbial/toxic insults. Thus, it has been shown that membrane-expressed IL- 1 a can induce local expression of IL-8 by juxtacrine stimulation of endothelial cells (Kaplanski et al 1994). Also, IL-1 stimulates synthesis and expression of IL-8 (Denning et al. 1998) and complement factors (Berge et al. 1996) in endothelial cells. Apparently, hepatocyte-expressed IL- 1α, possibly acting in an autocrinic and/or paracrinic manner, may similarly attract and activate inflammatory cells through stimulation of chemokines, such as IL-8 and epithelial neutrophil activating peptide-78 (ENA-78), and cytokines, such as TNFα and CSF-1 (Rowell et al 1997). By directly activating antigen presenting cells (i.e. dendritic cells and macrophages) and T cells, IL-1 may also play a fundamental role in adaptive cellular responses, including DTH. The efficiency of this cellmediated response entails however, a considerable risk of tissue-damage, deriving from the marked production by these cells of agents potentially damaging the host, such as proteolytic enzymes and reactive oxygen radicals. The intracellular elevation of IL- 1α appears essential in “first-line” lymphoreticular organs, as it precedes the expression of membraneassociated IL- la and enhanced secretion of the mature IL-1α molecule. Except for immunoregulatory functions, intracellular IL- 1 α may also be involved in the regulation of other homeostatic functions in the producing cell itself. Thus, endogenously generated IL- 1α was shown to be invoved in cell senescence (Maier et al. 1990, Kumar et al. 1992, Rinchart et al. 1999), differentiation (Hammerberg et al. 1998) and

192 Distinct Patterns of IL- Iα and IL-1β Organ Distribution

potentially in apoptosis (Yomada et al. 1996, Wang and Sinha 1996, Xie et al. 1997), effects that are possibly mediated, at least in part, by binding of cytosolic (Wessendorf JHM et al. 1993, Maier JAM et al. 1994) or exogenous (translocated through receptor-mediated endocytosis) (Curtis BM et al 1990) IL-1α to receptors on the nucleus. The marked staining for IL-1α in the intestinal apical region at old age, as found in this work, provides additional support to the linkage of cell-associated IL-1 and apoptosis, as enhanced apoposis is typical of this site (Westcarr et a l 1999). The existence of an intracellular, nonsecretable form of the IL1Ra (icIL-1Ra), which possibly down-regulates the activity of cytosolic IL-1 may further attest to the importance of tightly regulating expression of intracellular IL- 1 α and the possible danger deriving from failure of such regulation (Muzio et al. 1995, Muzio et a l 1999). Thus, under steady-steady conditions, increased expression of intracellular IL1α would be feasible, without severely endangering homeostasis of the producing cell. Increased IL- 1α expression in lymphoreticular organs at old age, may denote a compensatory defensive attempt. Ultimately, however, this may alter cellular function, possibly resulting in cell damage and apoptosis.

3.2

Low expression of IL-1 in privileged organs

As demonstrated in Figure 3, IL-1α is detected in minute amounts in privileged organs, as evaluated by ELISA. The immunohistochemical stainings (data not shown) demonstrate that, except for the kidneys, where it is most clearly demonstrated in the tubular cells, IL1 a expresion in the heart, skeletal muscle and brain is not lumen-oriented but rather diffusely cytoplasinatic (heart, skeletal muscle) or localized to specific cells (macrophage-like cells and neurons in the brain). IL-1 level of expression is equally low in young and old mice (Figure 3), thus exhibiting a tightly regulated pattern of expression. It may be envisioned that the low expression of IL-1α in privileged organs signifies a protective modality of defense, This may appear especially relevant in view of the potential tissue-damage, which can be inflicted on these organs by even relatively “restrained” mechanisms of defense, as elaborated below. Based on IL-1α ability to specifically modulate factors involved in regulation of cell homeostasis, it may contribute to safeguarding of critical processes, such as DNA repair and inhibition of apoptosis, when expressed so restrictively. Under these conditions, IL-1 α may modulate factors involved in apoptosis, such as Fas, cyclin-dependent kinases (Cdks)/Cdk inhibitors, Bcl-2 and Topoisomerase I (Yomada et al. 1996,

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Wang and Sinha 1996, Xie et al. 1997, Zeki et al. 1999). Some of these factors may promote apoptosis in cells stimulated by relatively high concentrations of IL- 1 α, as demonstrated by these investigators. Ultimately, the specific effect exerted by these factors on cell homeostasis may depend on the different timing, context and levels of IL-1α expression, integrating with other locally acting cytokines. It was indeed demonstrated that endogenous IL- 1 α can bidirectionally regulate cell growth (Cozzolino et al. 1990, Garfinkel et al. 1992), an effect that can be mediated by IL-1α -modulated Cdks/Cdk inhibitors, as recently shown (Zeki et al. 1999). The tightly controlled expression of cellexpressed IL-1α which thus appears essential for homeostasis of privileged organs, can be assured by controlling either IL- 1 α generation and secretion or/and its cytosolic activity via modification of structure (i.e. glycosylation), binding to inhibitory molecules or neutralization of its intracellular receptors (i.e. by icIL-1Ra).

4. ORGAN IL-1ß EXPRESSION In contrast to organ IL-1α expression, the expression of IL-1 β is overall more characteristic of privileged organs and increases at old age (Figure 4). Considering the differential homeostatic/defensive behavior typifying privileged and lymphoreticular organs, IL-1β may thus emerge as mediating more restrained inflammatory responses.

Figure 4. Organ IL-1β expression was assessed in young versus old mice by detection of immunoreactive IL1β in lysates (LYS), obtained from the various organs. This was performed by ELISA analyses and comparatively presented for the two age groups.

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4.1

Distinct Patterns of IL-1 α and IL-1 β Organ Distribution

Generalized enhanced expression of intracellular IL1β in old mice

A widespread elevation of intracellular IL- 1β expression is demonstrated in both privileged and lymphoreticular organs of old mice, as assessed by ELISA (Figure 4) and immunohistochemistry (data not shown). The basic histological distribution of IL- 1 β in the various organs resembles that observed for IL-1 (data not shown). This and the outstanding predominance of expression at old age may suggest that ILlβ possibly mediates inflammatory responses that are with lower risk for tissue-damage. The marked relative increase in IL- 1 β intracellular expression at old age may derive from increased rate of IL-1β generation, delayed cleavage of the IL-1β precursor and export of the mature IL-1β molecule and other control mechanisms; in this context, cytosolic IL- 1β molecules may also be biologically functional. The latter assumption may thus expand the existing notion, which relates biological activity only to the mature forms of IL-1β molecules, readily secreted from macrophages. Indeed, cell-associated IL-1β was reported to be minimally active in lysates of macrophages (Jobling SA et al. 1988). In this context, cell-associated IL- 1β may regulate and perhaps curb apoptosis. This is mainly supported by recent findings which show that intracellularly expressed IL- 1β can counteract the apoptotic process by “diverting” IL- 1 converting enzyme (ICE) activity from apoptosis promotion to engagement in cleaving of the IL- 1β precursor molecules (Tatsuta T et al. 1996, Watson RW et al. 1998).

4.2

Relative dominance of IL-1ß in privileged organs of old mice

Aiming to highlight the effect of aging in IL-1α and IL-1β organ expression at the two age groups, the old/young ratio of cytokine expression was calculated, based on IL-1α and IL-1β values, as respectively detected in young and old mice by the ELISA analyses (Figure 5) and immunohistochemistry (data not shown). For better illustration, the sequence of the “privileged” organs was arranged according to decreasing values of old/young ratio of IL-1β expression, respectively calculated in the various organs. As shown in Figure 5, aging appears to enhance the inverse relationship between IL-1α and IL-1β in organs, emphasizing the relative dominance of IL-1β expression in privileged organs.

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Figure 5. Relative organ expression of IL- 1α and IL-1β in old compared to young mice is presented by calculating the oldyoung ratio of organ cytokine expression, based on ILlα and IL-1β values, as detected in young and old mice by ELISA analyses of organ lysates(LYS) and conditioned media (CM). The respective values, obtained by ELISA for organ CM (A) and LYS (B), are comparatively presented for IL-1α and IL-1β.

IL- 1β apparent “restrained” inflammatory potential, as emerges from recent works, is compatible with its marked expression in the highly vulnerable privileged organs. Indeed, IL- 1β appears to be more involved in stimulation/activation of defensins (Singh et al. 1998, Schroder and Harder 1999) and NK cells (Peppoloni 1989, Conti et al. 1991), which exert immediate but relatively short-term defense responses. Also, recombinant IL- 1β has beeen shown to increase expression of mannose receptors in endothelial cells (Asumendi et al. 1996), thus possibly contributing to trapping and subsequent elimination of invading microorganims, e.g.,By locally acting anti-microbial agents and/or phagocytic cells (surface phagocytosis).

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Distinct Patterns of IL-1α and IL- 1β Organ Distribution

Most interestingly, it was demonstrated in this respect that IL-1β differentially induces expression of nitric oxide and down regulates expression of platelet activating factor (PAF) in mast cells (Hogaboam et al. 1993), thus pointing to a reduced potential for leukocyte infiltration. IL-1β may complement the action of IL-6 at the local and systemic level, being more relevant in activation of the acute response, considering its “hormonal” nature. Due to its anti-inflammatory/anti-IL1 effects, the prominent presence of IL-6 in privileged organs may provide an additional mechanism for “restraining” potential IL- 1mediated tissue damage. In addition to its defensive inflammatory roles, the role of IL-1β may possibly include other homeostatic functions. Thus, the suggested antiapoptotic role of IL-1β, as elaborated above, may appear relatively more influential in privileged organs. Moreover, autocrinic and/or paracrinic pathways of IL-1β activity may also pertain to counteracting of apoptosis in this context, as suggested by the apoptosis-inhibiting action of exogenous IL-lβ (Chun et al. 1995, Rodriguez et al. 1996, Tsuboi et al. 1996, Kothny-Wilkest G et al. 1999). The latter was shown to operate via different mechanisms, including generation of nitric oxide and cGMP (Chun et al. 1995), production of endogenous GM-CSF (Rodriguez et al. 1996) and down-regulation of Fas expression (Tsuboi et al. 1996). In addition, the elevated levels of secreted IL-1β in CM of privileged organs may be aimed to preserve the normal function of critical and highly vulnerable organs, such as the heart; exogenous IL-1β was indeed shown to promote growth of cardiomyocytes and inhibit cardiac fibroblast proliferation (Palmer et al. 1995).

5.

POTENTIAL IN VIVO INTERACTIONS BETWEEN ORGAN IL-1 AND IL-1β

Organ expressed IL-1α and IL-1β may potentially interact in various ways. Organ IL-1α and IL-1β may thus interact in a cooperative manner. Thus, in lymphoreticular organs, immediate, basic defense can be induced and activated by IL-1β; if an additional sustained cellular response is needed, IL-1 α may be “mobilized”, proposedly complementing the action of IL-1β in this context. Likewise, IL-1α (expressed at low levels) and IL-1β (expressed at high levels) may cooperate in preservation of defense and other homeostatic functions in privileged organs, as separately elaborated above for each cytokine.

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The high intracellular expression of both IL-1α and IL-1β in lymphoreticular organs of old mice can be interpreted as a mutually regulating state, representing perhaps a steady-state equilibrium imposed by the unique conditions characterizing these organs. In the extreme, an unbalanced expression of 1L- 1 α and/or IL-1 1β may cause apoptotic death or uncontrolled cell proliferation, respectively. Exaggeration of the suggested anti-apoptotic trend at old age, may also result in autoimmune and/or neoplastic deterioration, as shown in several experimental models and by the increased incidence of these processes with aging. Indeed, a marked IL-1 1β gene upregulation was demonstrated in kidneys, liver, lymph nodes and spleen of diseasedMRL/lpr mice (Lemay et al. 1996) and overexpression of IL- 1 1β was demonstrated to suppress apoptosis and enhance proliferation in leukemic cells (Furukawa et al. 1995). The inflammatory potential of IL- 1 1β which according to the above mentioned considerations, may appear relatively self-limited, should be reconciled, however, with the ample evidence which associates IL- 1 1β with destructive inflammatory responses. In many of the studies that ascribed a pro-inflammatory role to IL-1β the comparative role of IL-1 was not explicitly addressed. This may be of crucial importance, as IL-1 may appear as a major perpetuator of inflammation, possibly induced by IL-1β. Thus, an earlier and limited expression of IL-1β compared to the more delayed and prolonged expression of IL-1 was demonstrated in inflammatory responses (Chensue et al. 1991). Actually, this sequence of events may also occur in lymphoreticular organs under physiological conditions, where the lower levels of secreted IL-1β may also induce expression of IL-1α Finally, as is the rule with other cytokines, IL-1β may turn into a dangerous factor when exceeding critical quantitative thresholds or acting under drastic alterations of the microenvironment, such as hypoxia, trophic factor withdrawl and changing of locally acting cytokine networks (Friedlander et al. 1996, Troy et al. 1996). Under these circumstances it may turn into a destructive, pro-apoptotic agent (Friedlander et al. 1996, Troy et al. 1996, Kothny-Wilkes et al. 1999).

6.

CONCLUSION

The findings presented in this work show that IL-1α and IL-1β display differential and even inverse patterns of organ expression, demonstrated most clearly at old age and beyond the realm of immunology. Based on these IL- 1α /IL- 1β specific patterns of organ expression and considering the distinct physiological function of various organs, IL- 1 α may be highlighted more as a potent anti-infectious/toxic

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agent, which can initiate and potentiate prolonged inflammatory responses, apparently mainly based on activation of cellular immunity. In contrast, it possibly appears that IL-1β may be involved in more restricted inflammatory responses, than conceived before. Other homeostatic functions of both IL-1 molecules can also be envisioned, including for example control of the cell cycle and apoptosis. In this context, diverse and even opposing biological effects of IL-la/IL-lb may be anticipated. The “net” activity of IL-1 in a certain organ may depend on the type of expressed IL-1 and IL-lreceptors, as well as on the locally operant cytokine network. The in vivo interactions between the two IL-1 forms may be of crucial importance to organ homeostasis, especially under the stressful conditions, characterizing old age. Further studies are needed to clarify the full perspective of IL-la and IL-lb roles in organ homeostasis, the understanding and manipulation of which may hopefully lead to development of beneficial therapeutic interventions.

ACKNOWLEDGMENTS R.N. Apte is supported by the Israel Ministry of Science (MOS) jointly with the Deutsches Krebsforshungscentrum (DFKZ), Heidelberg, Germany), The Chief Scientist’s Office, The Israel Ministry of Health, The Israel Cancer Research Fund, the Israel Cancer Association, The United States-Israel Binational Foundation (BSF) and the Israel Science Foundation (The George and Eva Klein Fund), which was founded by the Israel Academy of Sciences and Humanities. The authors also wish to thank Mrs. Vera Hirsh and Ms. Parvin Zrin for dedicated technical assistance in performing the immunohisto-chemical stainings.

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STRUCTURE AND BIOLOGY OF CATHELICIDINS

¹, ²Margherita Zanetti, ³Renato Gennaro, ¹, ²Marco Scocchi, ¹Barbara Skerlavaj

1

Department of Sciences arid Medical Technologies, University of Udine, 1-33100 Udine; ²National Laboratory C.I.B., AREA Scienze Park, Padriciano, 1-34012 Trieste; ³Department of Biochemistry, Biophysics and Macromolecular. Chemistry, University of Trieste, I34127 Trieste, Italy.

1.

INTRODUCTION

1.1

Endogenous Antimicrobial Peptides

The initial response to microbial invasion is sustained by defense mechanisms that are triggered by differences in the structure of microbial components relative to the host, and are active on contact irrespective of prior exposure. These mechanisms provide a first line of defense that may rapidly clear most of the infectious organisms. Recent studies have focused on one mechanism of innate immunity that is based on an array of small antimicrobial peptides of fewer than one hundred residues. These peptides are widespread in nature. They are an ancient host defense mechanism and exert their activity by disrupting the membrane integrity of target microorganisms (Boman 1995, Ganz and Weiss 1997, Ganz and Lehrer 1998). Despite a considerable variety of sizes and structures, two broad groups of antimicrobial peptides can be defined, based on chemical-structural criteria, i.e., linear and cyclic peptides. Within the first group are linear peptides that can adopt an amphipathic, a-helical conformation and non a-helical linear peptides with one or two predominant amino acids. The second group includes cysteineThe Biology and Pathology of Innate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer AcademidPlenum Publishers, 2000

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containing peptides and can also be divided into two subsets based on the presence of a single or multiple disulfide bonds. Common feature of these peptides is a cationic and amphipathic character which accounts for their functioning as membrane-permeabilizing agents.

1.2

Cathelicidins

In mammals, antimicrobial peptides are components of epithelia and phagocyte host defenses (Ganz and Weiss 1997). Two evolutionary distinct groups of these peptides have been recognized, i.e., the alpha and beta defensins (Ganz and Lehrer 1998), and the cathelicidin peptides (Zanetti et al 1995). The latter group includes a variety of recently discovered antimicrobial peptides that are synthesized at the C-terminus of propeptides showing a highly conserved N-terminal propiece (Zanetti et al 1995). This propiece is approximately 100 residues long and is the hallmark of all cathelicidins (Fig 1). Propeptides of this family were first identified in cattle as deduced from myeloid bone marrow cells mRNA (Zanetti et al 1993). They were denoted after cathelin, a protein that was purified from pig leukocytes (Ritonja et al 1989) prior to identification of the cow cathelicidins. The cow propeptides were found to share higher than 70% sequence identity to pig cathelin, and it was clear that the latter protein was the propiece of a split propeptide of this family. Novel sequences rapidly added to the family. The presence of a conserved 5' region spanning the 5' UTR and the sequence coding for the signal peptide and propiece in the mRNAs, allowed the identification of many congeners in other species, as deduced from bone marrow cells mRNA (Zanetti et al 1995). These were identified by using a reverse transcription-PCR based approach that allows amplification of transcripts containing the conserved propiece. Members of the cathelicidin family show molecular masses of 16-26 kDa and have been found in humans, cow, pigs, rabbits, sheep (Zanetti et al 1997), mice (Popsueva et al 1996, Gallo et al 1997), guinea pigs (Nagaoka et al 1997), horses (Scocchi et al 1999). Interestingly, the number of different cathelicidins varies substantially among species. They are most abundant in artiodactyl species, whereas only one is expressed in humans.

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Figure 1: Gene (A) and protein (B) structure of cathelicidins

2.

PROTEIN AND GENE STRUCTURE

'antimicrobial' features, i.e., a net positive charge at neutral pH, and the possibility to assume an amphipathic conformation. The C-terminal domain and the cathelin-like propiece are joined by a residue (Val is most frequent, but Ala, Ile or Thr have also been found) that is a common cleavage site for elastase and is present at corresponding positions in most congeners (Zanetti et al 1995). Some more distantly related members of this family, like the rabbit p15s (Levy et al 1993), do not display elastase cleavage sites at these positions and have been isolated only in the uncleaved form (Ooi et al 1990, Zarember et al 1997). The prosequence is anionic at neutral pH, and displays four cysteine residues. These form two disulfide bonds in a 1-2, 3-4 arrangement that likely impose structural constraints on the molecule (Storici et al 1996). Interestingly, similarity searching and topology-based structure prediction indicate

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significant sequential and structural similarity of the propiece to members of the cystatin superfamily of cysteine proteinase inhibitors (Storici et al 1996). This common evolutionary origin is also supported by the inhibitory effects exerted by the bovine congeners on the in vitro activity of cathepsin L (Verbanac et al 1993, Storici et al 1996). Cathelicidins are encoded by multiple genes that lie in close proximity and generate a variety of different peptides. Up to eleven genes are present in cow and cluster at a CATHL@ locus on bovine chromosome 22q24 (Castiglioni et al 1996, Scocchi et al 1997). Several potential regulatory motifs, including the consensus sites for nuclear factors involved in hemopoiesis, inflammation and acute phase reaction, such as NF-IL6, NF-κB, acute phase-response factor (APRF) and gammainterferon response element (γIRE) in the 5' flanking sequences, suggests that the expression of these genes may be coordinately controlled. The identification of binding sites for transcription factors involved in haematopoiesis, such as NF-IL6, is in keeping with the expression of these genes during the maturation stages of neutrophils in the bone marrow. The cathelicidin genes are organized into four exons (Fig 1). The preproregion is specified in exons 1 to 3, the region encoding the elastase cleavage site and the varied antimicrobial domain is comprised in exon 4. This structural organization is conserved among species (Zhao et al 1995 a, b, Gudmundsson et al 1995, Gudmundsson et al 1996, Larrick et al 1996, Scocchi et al 1997, Huttner et al 1998), and the sizes of exons 1 to 3 have remained remarkably stable. Repeated gene conversion events may have contributed to the homogenization of the 5' portion of the gene, accounting for the high level of homology of this region both at the exon and intron level. The similarity of the propiece with the cystatins also extends to the gene structure. Each of the three tandem cystatin-like repeats that precede the C-terminal kinine sequence in the kininogen molecule is specified by a set of three exons (Kitamura et al 1985) which are similar in length to the corresponding exons I, 2, 3 of the cathelicidin genes. The sequence identity between cathelicidin and kininogen genes at corresponding exons is 40-50%, and the positions and spacings of the cysteine codons (two in the second, and two in the third exons) are almost identical. The evolutionary relationship of these two gene families can be traced back to an ancient two-disulfide cystatin precursor thought to have appeared about a billion years ago (Rawlings and Barrett 1990). Repeated duplications and sequence divergence of this ancient gene, and acquisition of 3'-terminal appendages unrelated to the cystatin sequence, likely contributed to the appearance of separate families of related members with new functions. The structural heterogeneity of the

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antimicrobial domain reflects a rapid evolutionary divergence of the cathelicidin genes. In fact, a much higher variability is observed in the peptide coding sequence than in the 3' UTR, indicating that within exon 4, only the antimicrobial sequence is a primary target for diversification. While the diversity of this sequence finds an obvious explanation in the need for keeping up with different and rapidly evolving microorganisms, understanding of the molecular mechanisms that drive variability in this region of the gene requires further investigation.

3,

BIOSYNTHESIS AND PROCESSING, AND TISSUE DISTRIBUTION

Thus far, cathelicidin genes have only been described in mammals (Zanetti et al 1997), and myeloid bone marrow cells are the primary site of their expression. Cathelicidins have been shown to be synthesized at a myelocyte and metamyelocyte stage of myeloid cell maturation in cow, guinea pig, man, rabbit (Zanetti et al 1990, Nagaoka et al 1997, Sorensen et al 1997a, Zarember et al 1997). After the signal peptide has been removed, the propeptides are targeted to the secretory granules. The human congener is present in equimolar ratio with lactoferrin in the specific granules (Sorensen et al 1997a), and both cathelicidins and lactoferrin are stored in the secretory 'large granules' of neutrophils in cattle (Gennaro et al 1983, Zanetti et al 1990). Once secreted, most propeptides can undergo limited proteolysis that liberates the C-terminal antimicrobial peptides (Zanetti et al 1991, Gudmundsson et al 1996, Panyutich et al 1997). Elastase cleaves the propeptides under conditions that favour concomitant exocytosis of the secretory and the azurophil granules (i.e., degranulation of activated neutrophils). This has been shown for the bovine (Zanetti et al 1991, Scocchi et al 1992) and pig (A. Panyutich et al 1997) congeners. Cathelicidins can also be released extracellularly in uncleaved proforms (Zanetti et al 1991, Frohm et al 1996, Zarember et al 1997, Sorensen et al 1997b). The human congener has also been found associated to the plasma lipoproteins which may serve as a reservoir of this protein in plasma (Sorensen et al 1999). The propeptides are not microbicidal (Scocchi et al 1992, Panyutich et al 1997) most likely because the cationic C-terminal peptide is shielded by the anionic propiece. The propiece may thus be a means to protect the host from potential detrimental effects of the peptide. The secretion of unprocessed forms however suggests that they may play other functions. In addition to myeloid-derived cells, the human congener has also been identified in testis (Agerberth et al 1995), in a variety of human

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squamous epithelia (Bals et al 1998, Frohm et al 1999) and in keratinocytes during inflammatory skin diseaseas (Frohm et al 1997). Its presence on the airway surface in particular suggests it plays an important role in the pulmonary host defense. The mouse congener is also expressed in various adult tissues and during embryogenesis (Gallo et al 1997). By contrast, the cow (unpublished) and pig (Wu et al 1999) congeners have been found expressed only in myeloid-derived cells, in myeloid/lymphoid organs such as spleen, thymus, lymph nodes.

4.

STRUCTURE AND FUNCTION OF CATHELICIDIN-DERIVED PEPTIDES

The processing of precursors of the cathelicidin family liberates antimicrobial peptides that are representative of all the structural groups described in the Introduction section and will be grouped accordingly. These peptides display antibacterial, antiendotoxic and antifungal properties, and appear to substantially contribute to the systemic and local host defense, as suggested by their presence in neutrophils and in a variety of epithelia (Bals et al 1998, Frohm et al 1999). They also have stimulated a great interest for their potential to develop a novel class of antimicrobial agents against antibiotic-resistant pathogens, and some of these studies will be described.

Figure 2: Helical wheel representation of cathelicidin peptides. Charged residues are i boldface (negatively charged are circled), hydrophobic residues are boxed, all othe residues are in italics. am: C-terminal amidation.

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209

Peptides with a-Helical Conformation

Cathelicidin-derived peptides with a-helical conformation have been found in every mammalian species where cathelicidins have been identified. One is present in man (Agerberth et al 1995, Larrick et al 1995) and mouse (Gallo et al 1997), and up to three in artiodactyls (Zanetti et al 1995). These peptides can be assigned to two subgroups according to the presence (e.g., SMAP-29 from sheep, Bagella et al 1995) or the absence (e.g., BMAP-34 from cattle, Gennaro et al 1998) of a hydrophobic C-terminal tail (Fig 2). When arranged according to the Edmundson wheel plot, sequences corresponding to these peptides form an amphipathic helix spanning at least part of the molecule. This structural prediction has been confirmed by CD spectroscopy for most of these peptides (Tossi et al 1995, Agerberth et al 1995, Skerlavaj et al 1996), and also by 2D-NMR for rabbit CAP18(106-142) (Chen et al 1995). Alpha helical peptides are in general active against both Gram-positive and Gram-negative bacteria, and some also against fungi (Table I). Targets of these peptides also include clinical isolates multiresistant to conventional antibiotics, such as methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecalis (VREF), as well as P. aeruginosa isolates from patients with cystic fibrosis (Table I). They act by rapidly permeabilizing the membranes of susceptible bacteria in a dose-dependent manner, at concentrations comparable to those antibacterial (Tossi et al 1995, Skerlavaj et al 1996). A study performed with over 100 antibiotic-resistant clinical isolates of 12 different species indicates that SMAP-29 is the most potent among cathelicidin-derived peptides (unpublished). Table 1. MIC values (uM)of cathelicidin peptides against clinical isolates of bacteria and fungi, as obtained by the microdilution susceptibility assay. PROTEGRIN 1 INDOLICIDIN Organism BMAP-27 SMAP-29 E.coli O18K1H7 0.5 0.25 1 8 A. baumannii 2 0.5 2 1 P.aeruginosa (CF patient) 0.5 0.25 2 8 S. marcescens 1 0.25 2 4 S. aureus (MRSA) 8 1 1 4 S. epide rin idis 1 0.25 1 2 E. feacalis (VREF) 4 0.5 0.5 2 S. agalactiae 4 0.5 0.5 1 C. albicans 8 4 4 8 C. neoformans 4 1 2 4 CFpatient: Cystic Fibrosis; MRSA: Methicillin-resistant S. aureus; VREF: vancomycin-resistant E. fauclis.

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Assays aimed at evaluating the tendency to select resistant mutants show that the MIC values are unchanged after 20 passages when E. coli, S. aureus or P. aeruginosa strains are subcultured in the presence of sublethal concentrations of peptide. Under the same conditions, conventional antibiotics such as gentamicin and erythromycin, rapidly lead to selection of highly resistant-mutants. This property is of great advantage with respect to the commercial antibiotics. Studies on the in vivo efficacy of SMAP-29 in a peritonitis model induced in mice by i.p. injection of P. aeruginosa or S. aureus (MRSA strain) show that 0.4-0.8 mg/Kg of i.p. injected peptide protects mice from infections that cause over 90% mortality of control animals (unpublished). Peptides with a C-terminal hydrophobic tail usually show a relevant cytotoxicity to eukaryotic cells, including erythrocytes and tumor cell lines (Skerlavaj et al 1996, Risso et al 1998). This activity largely depends on the presence of the tail, as shown by structure/activity relationship studies with BMAP-27 and BMAP-28 (Skerlavaj et al 1996). These peptides also cause membrane permeabilization and programmed death of human transformed cell lines and of activated lymphocytes at bactericidal concentrations (1-3 uM) (Risso et al 1998). A therapeutic potential in the treatment of the septic shock is suggested for rabbit CAP18( 106-142) and human LL-37/CAP18(104-140) that have been shown to bind LPS, and neutralize some of its effects in vitro, protecting mice from LPS lethality (Larrick et al 1994, Larrick et al 1995).

4.2

Pro- And Arg-Rich Peptides

Two Pro- and Arg-rich cathelicidin-derived peptides were first isolated from bovine neutrophils and named Bac5 and Bac7 (Gennaro et al 1989). One member, named PR-39, was later isolated from pig (Agerberth et al 1991), and four were deduced from sheep genes or myeloid cDNA (Bagella et al 1995, Huttner et al 1998). Despite a similar amino acid composition, these peptides display completely different sequences (Frank et al 1990). Structural studies have shown that these peptides assume a poly-L-proline II conformation in acqueous solution (Raj et al 1996). The Pro- and Arg-rich peptides are mainly active against Gram-negative bacteria and their lethal action is exerted without membrane permeabilization (Boman et al 1993, Skerlavaj et al 1999). SAR studies using synthetic fragments of Bac5 and Bac7 have indicated that the highly cationic N-terminal region is essential for the antibacterial activity of both peptides and that a minimum length of 16-18 residues is required (Skerlavaj et al 1999). An important feature of PR-39 is the ability to induce the expression of cell surface heparan sulphate

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proteoglycans (syndecan-1 and -4), as part of the wound repair process (Gallo et al 1994). In an attempt to determine how this peptide can alter cell gene expression, it was found that PR-39 rapidly enters cells and binds a number of cytoplasmic proteins, including p130(Cas) (Chan and Gallo 1998). The binding to this SH3 domain-containing signal transduction molecule might explain the inducing effects of this peptide on mammalian cells. This multifunctional peptide is also chemoattractant for neutrophils, but not for mononuclear cells (Huang et al 1997), and inhibits the phagocyte NADPH oxidase activity by binding to SH3 domain of the cytosolic oxidase component p47Phox (Shi et al 1996).

4.3

Tryptophan-Rich Peptides

A tridecapeptide amide was isolated from the cytoplasmic granules of bovine neutrophils (Selsted et al 1992) and named indolicidin after the unusual abundance of Trp residues, 5 out of 13. Indolicidin rapidly neutralizes Gram-positive and Gram-negative bacteria, as well as fungi (Selsted et al 1992, Van Abel et al 1995) by permeabilizing the membranes of susceptible microorganisms (Falla et al 1996). These effects are substantially reduced after pretreatment of bacteria with an uncoupler, indicating the requirement of transmembrane potential for full activity of the peptide. Indolicidin is cytotoxic to rat and human lymphocytes and lyses erythrocytes (Schluesener et al 1993). Systemic injection of free peptide in mice is highly toxic. Conversely, the liposomal formulation shows a dramatically reduced toxicity and has therapeutic effect on mice challenged with A. funzigatus spores, suggesting the potential of this peptide as a systemic agent in the treatment of opportunistic fungal infections (Ahmad et al 1995). In an attempt to decrease indolicidin cytotoxicity, analogs have been synthesize that maintain or improve the antibacterial, while greatly reducing the hemolytic activity (Subbalakshmi et al 1996, Falla and Hancock 1997).

4.4

Peptides With A Single Disulfide Bond

The presence of two cysteines is a distinctive feature of a cathelicidinderived dodecapeptide from cattle and its sheep homologue. This peptide was isolated from extracts of bovine neutrophil granules (Romeo et al 1988), and then identified in ovine myeloid cells as deduced from cDNA (Bagella et al 1995). The peptide was formerly thought to be maintained in a cyclic structure by an intramolecular disulfide bond. Later mass studies indicate that the mature peptide may exist as a covalent dimer formed via intermolecular disulfide bonds (Storici et al 1996). It remains

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to be established whether the dimer is arranged in parallel or antiparallel form. The native peptide exhibits bactericidal activity against E. coli and S. aureus at µmolar concentrations (Romeo et al 1988). The synthetic monomeric cyclic dodecapeptide shows a selective toxicity to neuronal and glial cells not exerted by other antibiotic peptides (Radermacher et al 1993). Relevant changes of the spectrum of activity have been observed on linearization of the cyclic monomeric peptide. The cyclic peptide is more active against Gram-negative bacteria, whereas only the linear peptide and other linear analogues are active against Gram-positive species such as Staphylococcus epidemidis and Enterococcus faecalis (Wu and Hancock 1999). These small size and broad spectrum analogs show the potential for clinical use.

4.5.

Peptides With Two Disulfide Bonds

Five highly similar (>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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REFERENCES Agerberth, B., Lee, J.-Y., Bergman, T., Carlquist, M., Boman, H.G., Mutt, V. and Jornvall, H. 1991, Amino acid sequence of PR-39. Isolation from pig intestine of a new member of the family of proline-arginine-rich antibacterial peptides. Eur. J. Biochem. 202:849-854. Agerberth, B., Gunne, H., Oderberg, J., Kogner, P., Boman, H. G., Gudmundsson, G. H., 1995, FALL-39, a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis. Proc Natl Acad Sci U S A. 92:195-199. Ahmad, I., Perkins, W. R., Lupan, D. M., Selsted, M. E., and Janoff, A. S. 1995, Liposomal entrapment of the neutrophil-derived peptide indolicidin endows it with in vivo antifungal activity. Biochim Biophys. Acta 1237: 109-1 14. Aumelas, A., Mangoni, M., Roumestand, C., Chiche, L., Despaux, E., Grassy, G., Calas, B., and Chavanieu, A., 1996, Synthesis and solution structure of the antimicrobial peptide protegrin- 1. Eur. J. Biochem. 237:575-583. Bagella, L., Scocchi, M., and Zanetti, M., 1995, cDNA sequence of three sheep myeloid cathelicidins. FEBS Lett. 376:225-228. Bals, R., Wang, X., Zasloff, M., and Wilson, J. M., 1998, The peptide antibiotic LL37/hCAP- 18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface. Proc. Natl. Acad. Sci. USA. 95:95419546. Boman, H. G., 1995, Peptide antibiotic and their role in innate immunity. Annu. Rev. Immunol. 13:61-92. Boman H. G., Agerberth, B. and Boman, A., 1993, Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine. Infect. Immun. 61 :2978-2984. Castiglioni, B., Scocchi, M., Zanetti, M. and Ferretti, L., 1996, Six antimicrobial peptide genes of the cathelicidin family map to bovine chromosome 22q24 by fluorescence in situ hybridization. Cytogen. Cell Genet. 75:240-242. Chan, Y. R. and Gallo, R.L., 1998, PR-39, a syndecan-inducing antimicrobial peptide, binds and affects pl30(Cas). J. Biol. Chem. 273:28978-28985. Chen, C., Brock, R., Luh, F., Chou, P.-J., Larrick, J., Huang, R.-F. and Huang, T.-H., 1995, The solution structure of the active domain of CAP18 a lipopolysaccharide binding protein from rabbit leukocytes. FEBS Lett. 370:46-52. Cho, Y., Turner, J. S., Dinh, N.-N., and Lehrer, R. I., 1998, Activity of protegrins against yeast-phase Candida albicans. Infect. Immun. 66:2486-2493. Falla T. J., and Hancock, R. E. W., 1997, Improved activity of a synthetic indolicidin analog. Antitnicrob. Agents Chemother. 41 :77 1-775. Falla T. J., Karunaratrne, D. N., and Hancock, R. E. W., 1996, Mode of action of the antimicrobial peptide indolicidin. J. Biol. Chem. 271: 19298- 19303. Frank, R., Gennaro, R., Schneider, K., Przybylski, M., and Romeo, D., 1990, Amino acid sequences of two proline-rich bactenecins, antimicrobial peptides of bovine neutrophils. J Biol Chem 265: 1887 1-1 8874. Frohm, M., Gunne, H., Bergman, A., Agerberth, B., Bergman, T., Boman, A., Liden, S., Jornvall, H., and Boman, H. G., 1996, Biochemical and antibacterial analysis of human wound and blister fluid. Eur. J. Biochem. 237: 86-92. Frohm, M., Agerberth, B., Ahangari, G., Stahle-Backdahl, M., Liden, S., Wigzell, H., and Gudmundsson, G. H., 1997, The expression of the gene coding for the antibacterial peptide LL-37 is induced in human keratinocytes during inflammatory disorders. J. Biol. Chem. 272: 15258- 15263.

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Frohm Nilsson, M., Sandstedt, B., Sorensen, O., Weber, G., Borregaard, N., and StahleBackdahl, M., 1999, The human cationic antimicrobial protein (hCAP18), a peptide antibiotic, is widely expressed in human squamous epithelia and colocalizes with interleukin-6. Infect. Immun. 67:2561-2566. Gallo, R.L., Ono, M., Povsic, T., Page, C., Eriksson, E., Klagsbrun, M., and Bernfield, M., 1994, Syndecans, cell surface heparan sulfate proteoglycans, are induced by a proline-rich antimicrobial peptide from wounds. Proc. Natl. Acad. Sci. USA 91:1103511039. Gallo, R.L., Kim, K., Bernfield, M., Kozak, C., Zanetti. M., Merluzzi, L. and Gennaro, R., 1997, Identification of CRAMP, a cathelin related antimicrobial peptide expressed in the embryonic and adult mouse. J. Biol. Chem. 272:13088-13093 Ganz, T., and Weiss, J., 1997, Antimicrobial peptides of phagocytes and epithelia. Seminars Hematology 34:343-354. Ganz, T., and Lehrer, R., 1998, Antimicrobial peptides of vertebrates. Curr. Opin. Immunol. 10:41-44. Gennaro, R., Dewald, B., Horisberger, U., Gubler, H. U., and Baggiolini, M., 1983, A novel type of cytoplasmic granule in bovine neutrophils. J. Cell Biol. 96:1651-1661. Gennaro R., Skerlavaj B., and Romeo, D., 1989, Purification, composition, and activity of two bactenecins, antibacterial peptides of bovine neutrophils. Infect Immun 57:3 142-3146. Gennaro R., Scocchi, M., Merluzzi, L., and Zanetti, M., 1998, Biological characterization of a novel mammalian antimicrobial peptide. Biochim. Biophys. Acta 2468 1 : 1-8. Gudmundsson, G.H., Magnusson, K.P., Chowdhary, B.P., Johansson, M., Anderson, L. and Boman, H.G., 1995, Structure of the gene for porcine peptide antibiotic PR-39, a cathelin genefamily member: Caomparative mapping of the locus for the human peptide antibiotic FALL-39 Proc. Natl. Acad. Sci. USA 92:7085-7089. Gudmundsson, G.H., Agerberth, B., Odeberg, J., Bergman, T., Olsson, B. and Salcedo, R., 1996, The human gene FALL-39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes. Eur. J. Biochem. 238:325-332. Huang, H.-A., ROSS, C. R., and Blecha, F., 1997, A novel type of cytoplasmic granule in bovine neutrophils. J. Leukoc. Biol. 61 :624-629. Huttner, K. M., Lambeth, M. R., Burkin, D. J., and Broad, T.E., 1998, Localization and genomic organization of sheep antimicrobial peptide genes. Gene 206:85-91. Kitamura, N., Kitagawa, H., Fukushima, D., Takagaki, Y., Miyata, T., and Nakanishi, S., 1985, Structural organization of the human kininogen gene and a model of its evolution. J. Biol. Chem. 260:8610-8617 Kokryakov, V. N., Harwig, S. S. L., Panyutich, E. A., Shevchenko, A. A., Aleshina, G. M., Shamova, 0. V., Korneva, H. A., and Lehrer, R. I,.1993, Protegrins: leukocyte antimicrobial peptides that combine features of corticostatic defensins and tachiplesins. FEES Lett. 327:23 1-236. Larrick, J.W., Hirata, M., Zengh, H., Zhong. J., Bolin, D., Cavaillon, J-M., Shaw Warren, H., and Wright, S., 1994, A novel granulocyte-derived peptide with lipopolysaccharide-neutralizing activity. J. Immunol. 152:23 1-240. Larrick, J. W., Hirata, M., Balint, R.F., Lee, J., Zhong, J., and Wright, S., 1995, Human CAP] 8: a novel antimicrobial lipopolysaccaride-binding protein. Infect. Immun. 63: I291 - 1297. Larrick, J.W., Lee, J., Ma, S., Li, X., Francke, U., Wright, S.C., and Balint, R.F., 1996, Structural, functional analysis and localization of the human CAP18 gene. FEBS Lett. 398:74-80.

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Levy, O., Weiss, J., Zarember, K., Ooi, C.E., and Elsbach, P., 1993, Antibacterial 15kDa protein from rabbit polymorphonuclear leukocytes that modulate the antibacterial actions of other leukocytes proteins. J. Biol. Chem. 268:6058-6063. Mangoni, M. E., Aumelas, A., Charnet, P., Roumestand, C., Chiche, L., Despaux, E., Grassy, G., Calas, B., and Chavanieu, A., 1996, Change in membrane permeability induced by protegrin 1: implication of disulphide bridges for pore formation. FEBS Lett. 383:93-98. Miyakawa. Y., Ratnakar, P., Rao, A. G., Costello, M. L., Mathieu-Costello, O., Lehrer, R. I., and Catanzaro, A., 1996, In vitro activity of the antimicrobial peptides human and rabbit defensins and porcine leukocyte protegrin against Mycobacterium tuberculosis. Infect. Immun. 64:926-932. Nagaoka, I., Yomogida, T-I S.. and Yamashita, T., 1997, Isolation of cDNA encoding guinea pig neutrophil cationic antibacterial polypeptide of 11 kDa (CAP1 1) and evaluation of CAP1 I mRNA expression during neutrophil maturation. J. Biol. Chem. 272:22742-22750. Ooi, C. E., Weiss, J., Levy, O., and Elsbach, P., 1990,. Isolation of two isoforms of a novel 15-kDa protein from rabbit polymorphonuclear leukocytes that modulate the antibacterial actions of other leukocyte proteins. J. Biol. Chem. 26.5: 15956- 15962 Panyutich, A., Shi, J., Boutz, P.L., Zhao, C., and Ganz, T., 1997, Porcine polymorphonuclear leukocytes generate extracellular microbicidal activity by elastase-mediated activation of secreted proprotegrins. Infect. lmmun. 65:978-985. Popsueva, A. E., Zinovjeva, M. V., Visser, J.W., Zijlmans, J.M., Fibbe, W.E., and Belyavsky, A.V., 1996, A novel murine cathelin-like protein expressed in bone marrow. FEBS Lett. 391:5-8. Qu, X.-D., Harwig, S. S. L., Oren, A., Shafer, W. M., and Lehrer, R. I., 1996, Susceptibility of Neisseria gonorrhoeae to protegrins. Infect. Immun. 64: 1240-1245. Radermacher, S. W., Schoop, V. M., and Schluesener, H. J., 1993, Bactenecin, a leukocytic antimicrobial peptide, is cytotoxic to neuronal and glial cells. J. Neurosci. Res. 36:657-662. Raj, P. A., Marcus, E., and Edgerton, M., 1996, Delineation of an active fragment and poly(L-proline) II conformation for candidacidal activity of bactenecin 5. Biochemistry 35:43 14-4325. Rawlings, N. D., and Barrett, A. J., 1990, Evolution of proteins of Cystatin superfamily. J. Mol. Evol. 30:60-7 1. Risso, A., Zanetti, M., and Gennaro, R., 1998, Cytotoxicity and apoptosis mediated by two peptides of innate immunity. Cellular. Immunol. 189:107-115. Ritonja, A., Kopitar, M., Jerala. R., and Turk, V., 1989, Primary structure of a new cysteine proteinase inhibitor from pig leucocytes. FEBS Lett. 255:211-214. Romeo, D., Skerlavaj, B., Bolognesi, M., and Gennaro, R., 1988, Structure and bactericidal activity of an antibiotic dodecapeptide purified from bovine neutrophilis J. Biol. Chem 263:9573-9575. Roumestand, C., Louis, V., Aumelas, A., Grassy, G., Calas, B., and Chavanieu, A., 1998, Oligomerization of protegrin-] in the presence of DPC micelles. A proton highresolution NMR study. FEBS Lett. 42 1:263-267. Schluesener, H. J., Radermarcher, S., Melms, A., and Jung, S., 1993, Leukocytic antimicrobial peptides kill autoimmune T cells. J. Neuroimmunol. 47: 199-202. Scocchi, M., Skerlavaj, B., Romeo, D., and Gennaro, R., 1992, Proteolytic cleavage by neutrophil elastase converts inactive storage proform to antibacterial bactenecins. Eur. J. Biochem. 209:589-595.

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Scocchi, M., Wang, S., and Zanetti, M., 1997, Structural organization of the bovine cathelicidin gene family and identification of a novel member. FEBS Lett. 417:311315. Scocchi, M., Bontempo, D., Boscolo, S., Tomasinsig, L., Giulotto, E., and Zanetti, M., 1999, Novel cathelicidins in horse leukocytes. FEBS Lett. 457:459-464. Selsted, M. E., Novotny, M.J., Morris, W.L., Tang, Y-Q, Smith, W., and Cullor, J. S., 1992, Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils. J. Biol. Chem. 267:4292-4295. Shi, J., Ross, C. R., Leto, T. L., and Blecha, F., 1996, PR-39, a proline-rich antibacterial peptide that inhibits phagocyte NADPH oxidase activity by binding to Src homology 3 domains of p47 phox. Proc. Natl. Acad. Sci. USA 93:6014-6018. Skerlavaj, B., Gennaro, R., Bagella, L., Merluzzi. L., Risso, A. and Zanetti, M., 1996, Biological characterization of two novel cathelicidin-derived peptides and identification of structural requirements for their antimicrobial and cell lytic activities. J. Biol. Chem 27 1 :28375-2838 I. Skerlavaj, B., Scocchi, M., Tossi, A., Romeo, D., and Gennaro, R., 1999, A synthetic approach for a SAR study of the Pro- and Arg-rich bactenecin Bac7. In Innovation and Perspectives in Solid Phase Synthesis & Combinatorial Libreries (R. Epton. Ed.) Mayflower Scientific Limited, Birmingham, pp. 395-398. Sorensen, 0., Arnljots, K., Cowland, J.B., Bainton, D. F., and Borregaard, N., 1997a, The human antibacterial cathelicidin, hCAP- 18. is synthesized in myelocytes and metamyelocytes and localized to specific granules in neutrophils. Blood 90:27962803. Sorensen, O., Cowland, J.B., Askaa, J., Borregaard, N., 1997b, An ELISA for hCAP-18, the cathelicidin present in human neutrophils and plasma. J. Immunol. Methods 206: 53-59. Sorensen, O., Bratt, T., Johansen, A. H., Madsen, M. T., and Borregaard, N., 1999, The human antibacterial cathelicidin, hCAP- 18, is bound to lipoproteins in plasma. J. Biol. Chem. 274:22445-2245 1. Steinberg, D. A., Hurst, M. A., Fujii, C. A., Kung, A. H., Ho, J. F., Cheng, F.-C., Loury, D. J., and Fiddes, J. C., 1997, Protegrin-1: a broad-spectrum, rapidly microbicidal peptide with in vivo activity. Antimicrob. Agents Chemother. 41: 1738-1742. Storici, P., Tossi, A., Lenarcic, B., and Romeo, D., 1996, Purification and structural characterization of bovine cathelicidins, precursors of antimicrobial peptides. Eur. J. B io ch em . 2 3 8 : 7 69 -77 6. Subbalakshmi, C., Krishnakumari, V., Nagaraj, R., and Sitaram, N., 1996, Requirements for antibacterial and hemolytic activities in the bovine neutrophil derived 13-residue peptide indolicidin. FEBS Lett. 395:48-52. Tossi, A., Scocchi, M., Zanetti, M., Storici, P., and Gennaro, R., 1995, PMAP-37, a novel antibacterial peptide from pig myeloid cell. cDNA cloning, chemical syntesis and activity. Eur. J. Biochem. 228:941-946. Van Abel, R. J., Tang, Y.-Q., Dobbs, C.H., Tran, D., Barany, G., and Selsted, M. E., 1995, Synthesis and characterization of indolicidin, a tryptophan-rich antimicrobial peptide from bovine neutrophils. Int. J. Pept. Protein Res. 45:401-9. Verbanac, D., Zanetti, M., and Romeo, D., 1993, Chemotactic and protease-inhibiting activities of antibiotic peptide precursors. FEBS Lett. 3 17:255-258. Wu, M. and Hancock, R. E. W., 1999, Interaction of the cyclic antimicrobial cationic peptide bactenecin with the outer and cytoplasmic membrane. J. Biol. Chem. 274:2935.

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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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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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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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Klebsiella Infections in the Immunocompromised Host

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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Urinary tract infections Pneumonia Septicemia Wound infections Infections in ICU

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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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Klebsiella Infections in the Imnunocompromised Host

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.

REFERENCES Arakava, Y., Ohta, M.. Wacharotayankun, R., and Mori, M. e. a. (1991). Biosynthesis of Klebsiella K2 capsule polysaccharide in Esherichia coli HB 101 requieres the functions of rmpA and the chromosomal cps gene cluster of the virulent strain Klebsiella pneumoniae Chedid (O1:K2). Infect. Immun. 59, 2043-50. Ayars, G. H., Altman, L. C., and Fretwell, M. D. (1982). Effect of decreased salivation and pH on the adherence of Klebsiella species to human buccal epithelial cells. Infect lmmun 38, 179-182. Casewell, M., and Talsania, H. G. (1979). Predominance of certain klebsiella capsular types in hospitals in the United Kingdom. J Infect 1, 77-79. Christensen, S. C., and Korner, B. (1972). An endemic caused by multiresistant Klebsiella in an urological unit. Scand J Urol Nephrol 6, 232-238. Ciurana, B.. and Tomás, J. M. (1987). Role of lipopolysaccharide and complement in susceptibility of Klebsiella pneumoniae to nonimmune serum. Infect Immun 55, 274 1-2746. Clegg, S., and Gerlach, G. F. (1987). Enterobacterial fimbriae. J Bacteriol 169, 934-938. Cryz, S. J., Furer, E., and Germanier, R. (1984). Experimental Klebsiella pneumoniae burn wound sepsis: role of capsular polysaccharide. Infect Immun 43, 440-441.

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Cryz, S. J., Jr., Fürer, E., and Germanier, R. (1985). Safety and immunogenicity of Klebsiella pneumoniae K1 capsular polysaccharide vaccine in humans. J Infect Dis 151, 665-671. Cryz, S. J., Mortimer, P. M., Mansfield, V., and Germanier, R. (1986). Seroepidemiology of Klebsiella bacteremic isolates and implications for vaccine development. J Clin Microbiol 23, 687-690. Darfeuille-Michaud, A., Jallat, C., Aubel, D., Sirot, D., Rich, C., Sirot, J., and Joly, B. (1 992). R-plasmid-encoded adhesive factor in Klebsiella pneumoniae strains responsible for human nosocomial infections. Infect Immun 60, 44-45. De Champ, C., Sauvant, M. P., Chanal, C., and al., e. (1989). Prospective survey of colonization and infection caused by extended spectrum-beta-lactamse-producing members of the family Enterobacteriacae in intensive care unit. J. Clin. Microbiol. 27, 2887-2890. Di Martino, P., Livrelli, V., Sirot, D., Joly, B., and Darfeuille-Michaud, A. (1996). A new fimbrial antigen harbored by CAZ-5/SHV-4-producing Klebsiella pneumoniae strains involved in nosocomial infections. Infect Immun 64, 2266-2273. Doebbeling, B. N. (1 993). Epidemics: identification and management. In “Prevention and control of nosocomial infections” (R. P. Wenzel, ed.), pp. 177-206. Williams & Wilkins, Baltimore. Domenico, P., Johanson, W. G., and Straus, D. C. (1982). Lobar pneumonia in rats produced by clinical isolates of Klebsiella pneumoniae Infect Immun 37, 327-335. Duguid, J. P., and Old, D. C. (1980). Adhesive propertien in Enterobacteriaceae. In: Beachy, E.H. (ed.), Bactrerial Adherence (Receptors and Recognition, Vol 6). Chapman and Hall, London, pp 185-217. Ehrenwort, L., and Baer, H. (1956). The pathogenicity of Klebsiella pneumoniae for mice: the relationship to the quantity and rate of production of type-specific capsular polysaccharide. J Bacteriol 72, 713-717. Eisenstein, B. (1990). Enterobacteriaceae. In: MandellG. L., Douglas R.G, and Bennet J. E. (Hrsg): Principlesand practice of infectious diseaes. 4. Aufl. Churchill Livingstone, New York 1995, S 1964-1980. . Fader, R. C., and Davis, C. P. (1980). Effect of piliation on Klebsiella pneumoniae infection in rat bladders. Infect Immun 30, 554-561, Fader, R. C., and Davis, C. P. (1982). Klebsiella pneumoniae-induced experimental pyelitis: the effect of piliation on infectivity. J Urol 128, 197-201. Favre-Bonte, S., Darfeuille-Michaud, A., and Forestier, C. (1995). Aggregative adherence of Klebsiella pneumoniae to human Intestine-407 cells. Infect Immun 63, I3 18-1328. Favre-Bonte, S., Joly, B., and Forestier, C. (1999). Consequences of reduction of Klebsiella pneumoniae capsule expression on interaction of this bacterium with epithelial cells. Infect Immun. 67, 554-561. Fumagalli, O., Tall, B., Schipper, C., and Oelschlaeger , T. A. (1997). N-Glycosylated proteins are involved in efficient internalization of Klebsiella pneumoniae by cultured human epithelial cells. Infect. Immun. 65, 4445-4451. Gikas, A., Samonis, G., Christidou, A., Papadakis, J., Kofteridis, D., Tselentis, Y., and Tsaparas, N. (1 998). Gram-negative bacteremia in non-neutropenic patients: a 3-year review. Infection 26, 155-9. Griffiths, E. (1 987). The iron-uptake systems of pathogenic bacteria. In “Iron and infection” (J. J. Bullen and E. Griffiths, eds.), pp. 69-137. John Wiley & Sons, New York. Highsmith, A. K., and Jarvis, W. R. (1985). Klebsiella pneumoniae: selected virulence factors that contribute to pathogenicity. Infect Control 6, 75-77.

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Horan, T., Culver, D., Jarvis, W., Emori, G., Banerjee, S., Martone, W., and Thornsberry, C. (1 988). Pathogens causing nosocomial infections. Antimicrobic Newsletter 5, 6567. Hornick, D. B., Allen, B. L., Horn, M. A., and Clegg, S. (1992). Adherence to respiratory epithelia by recombinant Escherichia coli expressing Klebsiella pneumoniae type 3 fimbrial gene products. Infect Immun 60, 1577-1588. Hughes, T. W., Armstrong, D., Bodey, G. P., Brown, E. A., Edwards, J. E., Feld, R., Pizzo, P., Rolston, K. V. I., Shenep, J. L., and Young, L. S. (1997). 1997 Giudlines for the use of antimicrobial agents in neutropenic patients with unexplaned Fever. Clin Infect Dis 25, 551-73. Ishida, T., Hashimoto, T., Arita, M., Ito, I., and Osawa, M. (1998). Etiology of community-acquired pneumonia in hospitalized patients: a 3-year prospective study in Japan. Chest 1588-93, 114. Jacoby, G. (1996). Antimicrobial-resistant pathogens in the 1990s. Annu Rev Med 47, 169-7. Jacoby, G. A., and Medeiros, A. A. (1991). More extended spectrum B-lactamases. Antimicrob Agents Chemother 35, 1697- 1704. Joiner, K. A. (1988). Complement evasion by bacteria and parasites. Ann Rev Microbiol 42, 201-230. Khimji, P. L., and Miles, A. A. (1978). Microbial iron-chelators and their action on Klebsiella infections in the skin of guinea-pigs. Br J Exp Path 59, 137-147. Kiihn, I., Ayling-Smith, B., Tullus, K., and Burman, L. G. (1993). The use of colonization rate and epidemic index as tools to illustrate the epidemiology of faecal Enterobacreriaceae strains in Swedish neonatal wards. J Hosp Infect 23, 287-297. Maayan, M. C., Ofek, I., Medalia, O., and Aronson, M. (1985). Population shift in mannose-specific fimbriated phase of Klebsiella pneurnoniae during experimental urinary tract infection in mice. Infect Immun 49, 785-789. Maschmeyer, G. (1999). lnterventional anmtimicrobial therapy in febrile neutropenic patients. Diagn Microbial Infect Dis 34, 205-12. Matatov, R., Sechter, I., Perry, R., Kabha, K., Sahly, H., Podschun , R., Ofek, I., and Godhar, J. (1995). Expression of type 1 fimbriae and capsule in Klebsiella pneurnoniae K21. .Adv. Exper. Med. Biol. 408, 275. McCallum, K. I., and Witfield, C. (1991). The rcsA gene of O1:K20 is involved in expression of serotype- specific K (capsular) antigen. Infect. Immun. 59, 494- 502. Merino, S., Camprubi, S., Alberti, S., Benedi, V. J., and Tomás J. M. (1992). Mechanisms of Klebsiella pneumoniae resistance to complement-mediated killing. Infect Immun 60, 2529-2535. Miles, A. A., and Khimji, P. L. (1975). Enterobacterial chelators of iron: their occurrence, detection, and relation to pathogenicity. J Med Microbiol 8, 477-490. Mizuta, K., Ohta, M., Mori, M., Hasegawa, T., Nakashima, I., and Kato, N. (1983). Virulence for mice of Klebsiella strains belonging to the O1 group: relationship to their capsule (K) types. Infect Immun 40, 56-61. Mobley, H. L. T., Chippendale, G. R., Tenney, J. H., Mayrer, A. R., Crisp, L. J., Penner, J. L., and Warren, J. W. (1988). MR/K hemagglutination of Providencia stuartii correlates with adherence to catheters and with persistence in catheter-associated bacteriuria. J Infect Dis 157, 264-271. Montgomerie, J. Z. (1 979). Epidemiology of Klebsiella and hospital-associated infections. Rev Infect Dis 1, 736-753.

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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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Sahly, H., and Podschun, R. (1997). Clinical, Serological, and Epidemiological aspects of Klebsiella infections and their spondylarthropathic sequelae. Clin Diag Lab Immunol 4 (4). Simoons-Smit, A. M., Verweij-van Vught, A. M. J. J., Kanis, I. Y. R., and MacLaren, D. M. (1985). Chemiluminescence of human leucocytes stimulated by clinical isolates of Klebsiella. J Med Microbiol 19, 333-338. Simoons-Smit, A. M., Verweij-van Vught, A. M. J. J., and MacLaren, D. M. (1986). The role of K antigens as virulence factors in Klebsiella. J Med Microbiol 21, 133-137. Tarkkanen, A. M., Virkola, R., Clegg, S., and Korhonen, T. K. (1997). Binding of the type 3 fimbriae of Klebsiella pneumoniae to human endothelial and urinary bladder cells. Infect Immun 65, 1546-1549. Tomás, J. M., Benedí, V. J., Ciurana, B., and Jofre, J. (1986). Role of capsule and 0 antigen in resistance of Klebsiella pneumoniae to serum bactericidal activity. Infect Immun 54, 85-89. Torres, A., Serra-Batlles, J., Ferrer, A., and al., e. (1991). Severe community acquired pneumonia; epidemiology and prognostic factors. Am Rev Respir Dis 144, 312-18. Ullmann, U. (1986). Pattern of microbial isolates in hospitalized patients. Zbl Bakt Hyg B 183, 103-113. Williams, P., Lambert, P. A., Brown, M. R. W., and Jones, R. J. (1983). The role of the 0 and K antigens in determining the resistance of Klebsiella aerogenes to serum killing and phagocytosis. J Gen Microbiol 129, 2181-2191. Williams, P., and Tomas, J. M. (1990). The pathogenicity of Klebsiella pneumoniae Rev Med Microbiol 1, 196-204. Wurker. M., Beuth, J., Ko, H. L., Przondo-Modarska, A., and Pulverer, G. (1990). Type of fimbriation determines adherence of Klebsiella bacteria to human epithelial cells. Zbl Bakt 274, 239-245.

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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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Macrophage-Recognized Molecules of Apoptotic Cells

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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Macrophage-Recognized Molecules of Apoptotic Cells

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

.

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.

255

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.

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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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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 (p<0 .0005).

Figure 4. Cell sorting of the TAU-44-MT AKR lymphoma according to low- (LB) and high-binding (HB) of Maakia amurensis lectin (MAL-I) and determination of the biological behavior of the two cell subpopulations Tumor cells were treated with MAL-1 at a concentration of 1µg/l06ceIIs/mI for 30 minutes at 4oC. After cell sorting, 2x105 tumor cells of the low- and high-MAL-I binding cells were inoculated subcutaneously to two groups of AKR/J mice. Each group consisted in 5 mice. a.Binding of fluorescent MAL-1 to the TAU-44-MT AKR lymphoma cells; b. Cell sorting according to degree of MAL-1 binding; c. Determination of the biological behavior of the two MAL-1 binding subpopulations of the AKR lymphoma TAU-44 variant.

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Figure 5. Western blot analysis of the cell membrane proteins of the PT and MT cells of the AKR lymphoma malignancy variants after identification by different lectins For the identification by MAL-I lectin was used TAU-44 malignancy variant and by PNA lectin was used TAU-33 malignancy variant.

The Western blot analysis of the membranal proteins of PT and MT cells with MAL-I and PNA is presented in Figure 5. In the case of the MAL-1, a membrane glycoprotein of MW=130 kDa was found to be sialylated at a higher level in metastatic as compared to primary tumor cells. This sialylated glycoprotein has a sialic acid residue linked at the 3 position of galactose. As for the PNA binding, the glycoprotein of MW=130 kDa was found to contain a much lower amount of galactose in MT as compared to PT cells. Figure 6 presents the effect of treatment by WGA on AKR lymphoma growth. A marked, statistically highly significant, inhibition of tumor development was seen 'in the WGA-treated mice as compared to the nontreated ones (p<0.0005 for days 14 and 18).

Figure 6. Effect of in vitro treatment with WGA on the tumorigenicity of the TAU-44 variant of AKR lymphoma The concentration of the lectin was 100 mg/106 cells/ml. The inoculum consisted in 2x10 5 tumor cells/0.2 ml medium.

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DISCUSSION

Tumor progression may proceed via development of resistance to host immune response, innate or acquired. Exploration of mechanisms of the evolution of such resistance of the tumor in its advanced stages, at the cellular and molecular level, are necessary for the understanding of the process of increase in malignancy of tumors during their development. Macrophages have been shown to possess antitumoral activities, exerted via different mechanisms, such as secretion of lysosomal hydrolytic enzymes (Hibbs 1974) or of neutral proteases (Adams 1980) or attack by oxygen burst products (Adams 1980, Keisari et al 1984). Immunoprotection has been achieved against murine melanoma (Dranoff et al 1993) and recently against human melanoma (Leong et al 1999) by using granulocyte-macrophage colony-stimulating factor (GM-CSF). The sensitivity to macrophages during tumor progression has been subject to controversy. While some authors claim that macrophages recognize tumor cells as such, with no distinction between different degrees of malignancy (Killion & Fidler 1998, Xie & Fidler 1998), other authors (Migita et al 1999) including ourselves (Leibovici et al 1986) did find a differential sensitivity to macrophages between tumors of high and low-malignancy. Macrophage number was markedly decreased in the invasive margin (tumor-host interface) of the diffuse-type than in the less malignant intestinal type of gastric cancer (Migita et al 1999). In previous studies we have shown a lower sensitivity to macrophages of high-malignancy AKR lymphoma variants as compared to lowmalignancy ones (Leibovici et al 1986). In the present study we have shown that in the AKR lymphoma, during the tumor progression process, tumor cells lose cell surface properties which make them recognizable by macrophages. A gain in sialic acid cell surface content concomitent with a loss in galactose residues during tumor progression may explain the decreased sensitivity to macrophages of the high-malignancy lymphoma cells. Tumor progression may therefore involve a reduced sensitivity to innate immunity. Information concerning the changing role of macrophages during the process, may suggest new appropriate therapeutic modalities with relevance to the stage of tumor development.

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ACKNOWLEDGMENTS This study was performed in partial fulfillment of the requirements for a Ph.D. degree of Mrs. Tatiana Kaptzan, Sackler Faculty of Medicine, Tel-Aviv University, Israel. The study was partially supported by the Mayerbaum Grant for Hematological Research and by the Hylda Portnoy Grant for Medical Research.

REFERENCES Adams D.O. Effector mechanisms of cytolytically activated macrophages. J Immunol. 124:286-292. 1980. Bardos H., Juhasz A., Repassy G., Adany R. Fibrin deposition in squamous cell carcinomas of the larynx and hypopharynx. Thromb Haemost. 80(5):767-72, 1998. Bosslet K., Schirrmacher V. Escape of metastasizing clonal tumor cell variants from tumorspecific cytolytic T lymphocytes. J Exp Med. 154557-562: 1981. Crawford H.C., Matrisian L.M., Liaw L. Distinct roles of osteopontin in host defense activity and tumor survival during squamous cell carcinoma progression in vivo. Cancer Res. 58(22):5206-15. 1998. Dong G.. Chen Z., Kato T. Van Waes C. The host environment promotes the constitutive activation of nuclear factor-kappaB and proinflammatory cytokine expression during metastatic tumor progression of murine squamous cell carcinoma. Cancer Res. 59( 14):3495-504, 1999. Dranoff G., Jaffee E., Lazenby A. et al. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci USA. 90:3539-43, 1993. Dvorak H.F., Dvorak A.M., Simpson B.A., Richerson H.B., Leskowitz S. and Karnovsky M. Cutaneous basophil hypersensitivity II A light and electron microscopic description. J Exp Med. 132:558-582, 1970. Gorelik E.M., Fogel M.F., Segal S. Differences in resistance of metastatic tumor cells and cells from local tumor growth to cytotoxicity of natural killer cells. J Natl Cancer Inst. 63:1397-1404, 1979. Gregory C.D., Murray R.J., Edwards C.F., Rickinsen A.B. Downregulation of cell adhesion molecules LFA-3 and ICAM-I in Epstein-Barr virus-positive Burkitt lymphoma underlies tumor cell escape from virus specific T cell surveillance. J Exp Med. 167: 18 1 1 -1824, 1988. Hanna N., Fidler J.I. Role of natural killer cells in the destruction of circulating tumor emboli. J Natl Cancer Inst. 65:801-809, 1980. Hibbs J.B. Heterocytolysis by macrophages activated by Bacillus Calmette-Guerin: lysosome exocytosis into tumor cells. Science. 184:468-471, 1974. Hildenbrand R., Jansen C., Wolf G., Bohme B., Berger S., von Minckwitz G., Horlin A., Kaufmann M., Stutte H.J. Transforming growth factor-beta stimulates urokinase expression in tumor-associated macrophages of the breast. Lab Invest. 78( 1):59-7 1, 1998. Husby G.. Hoagland P.M., Strickland R.G., Williams R.C. Tissue T and B cell infiltration of primary and metastatic cancer. J Clin Invest. 57:1471-1482, 1976.

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Joachim H.L. The stromal reaction of tumors: an expression of immune surveillance. J Natl Cancer Inst. 57:465-475, 1976. Keisari Y., Flescher E., Geva I. Macrophage oxidative burst and related cytotoxicity. Differential activation by tumor-promoting and non-tumor-promoting phorbol esters. Int J Cancer. 34(6):845-8, 1984. Killion J.J., Fidler I.J. Therapy of cancer metastasis by tumoricidal activation of tissue macrophages using liposome-encapsulated immunomodulators. Pharmacol Ther. 78(3):141-54, 1998. Klein 0, Savion N, Staroselsky A, Leibovici J. Cellular functions related to metastasis differing between low- and high-malignancy variants of AKR lymphoma. Pathobiology. 60: 157-162, 1992. Klein 0, Staroselsky A. N., Savion N, Donin N., Michowitz M. and Leibovici J. Metastasis associated cell functions in AKR lymphoma malignancy variants. Invasion Metastasis. 15: 21 1-221, 1996. Klein O., Staroselsky A., Huszar M., Hiss J., Kay S., Donin N., Zeidel L., Michowitz M., Leibovici J. Biological behavior and cell properties of new AKR lymphoma malignancy variants. Tissue & Cell. 30:95-103, 1998. Leibovici J, Klein 0, Argaman H, Klorin G. Michowitz M: Differential metastatic capacity of three AKR lymphoma variants. Int. J. Exp. Pathol.73:273-82, 1992. Leibovici J, Stark Y, Kopel S. Different biological behavior of AKR lymphoma cells from primary and metastatic tumors. Experientia .4 1 :404-407, 1985 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., Michowitz M., Argaman H., Klein O., Klorin G. and Hoenig S. A model for cancer therapy in advanced compared to early cancer. Anticancer Res. 6: 1225-30, 1986. 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. 65:39 1-396, 1980. Leong S.P., Enders-Zohr P., Zhou Y.M., Stuntebeck S., Habib F.A., Allen R.E.Jr., Sagebiel R.W., Glassberg A.B., Lowenberg D. W., Hayes F.A. Recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) and autologous melanoma vaccine mediate tumor regression in patients with metastatic melanoma. J Immunother. 22(2): 166-74, 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. Migita T., Sato E., Saito K., Mizoi T., Shiiba K., Matsuno S., Nagura H., Ohtani H. Differing expression of MMPs-1 and -9 and urokinase receptor between diffuse- and intestinal-type gastric carcinoma. Int J Cancer. 84( 1):74-9, 1999. Nakashima E., Kubota Y., Matsushita R., Ozaki E., Ichimura F., Kawahara S., Nakanishi I., Kuno K., Matsushima K. Synergistic antitumor interaction of human monocyte chemotactant protein-I gene transfer and modulator for tumor-infiltrating macrophages. Pharm Res. 15(5):685-9, 1998. North S.M., Nicolson G.L. Heterogeneity in the sensitivities of the 13762 NF rat mammary adenocarcinoma cell clone to cytolysis mediated by extra and intratumoral macrophages. Cancer Res. 45:1453-1458, 1985. Ohtani H. Host reactions affect cancer progression. Nippon Geka Gakkai Zasshi. 99(7):452-6, 1998. Ran M., Witz J.P. Tumor-associated immunoglobulins. Enhancement of syngeneic tumors by IgG2-containing tumor eluates. Int J Cancer. 9:242-247, 1972.

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OPPOSING EFFECTS OF IL-lα AND IL-1β ON MALIGNANCY PATTERNS

Tumor cell-associated IL- I α potentiates anti-tumor immune responses and tumor regression, whereas IL- 1 β potentiates invasiveness Ron N. Apte¹, Tatyana Dvorkin¹, Xiaoping Song¹, Eyal Fima¹, Yakov Krelin¹, Alon Yulevitch¹, Reuven Gurfinkel¹, Ariel Werman¹, Rosalyn M. White¹, Shmuel Argov², Yacob Shendler², Olle Bjorkdahl³, Mikael 3 Dohlsten , Margot Zoller4, Shraga Segal¹ and Elena Voronov¹ Department of Microbiology and Immunology, ¹Faculty of Health Sciences and The Cancer Research Center, Ben-Gurion University of the Negev, 2Department of Pathology, Soroka Medical Center, Beer-Sheva, Israel, 3University of Lund Luind, Sweden and the 4 German Cancer Center, Heidelberg, Germany

1.

INTRODUCTION

Cytokines, expressed in the microenvironment of a developing tumor, are important modulatory molecules of the malignant process; they can affect tumor growth or angiogenesis and also modulate in-situ anti-tumor immune responses. In the tumor arena, cytokines can be generated by leukocytes or stromal cells, as a response to tumor development, and also by the tumor cells themselves. Overexpression of cytokines by tumor cells, by gene-transfer approaches, represents an important experimental tool for studying their effects of the cytokine on tumor-host interactions. Till now, most of the studies have been aimed to potentiate the immunogenicity of malignant cells, in order to develop tumor cell vaccines, while other effects of the cytokine on the malignant process were quite neglected. Thus, experimental models of cytokine genetransfer into malignant cells, aimed to potentiate anti-tumor immune responses at the site of tumor development, include cytokines of both The Biology and Pathology of lnnate Immunity Mechanisms Edited by Yona Keisari and Itzhak Ofek, Kluwer Academic/Plenum Publishers, 2000

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TH cell subsets and of accessory cell origin (reviewed in Apte et al 1994a, 1994b, Dranoff & Mulligan 1998). Interleukin- 1 (IL-1) is a pleiotropic cytokine that mainly affects inflammatory and immune responses, but also other homeostatic functions of the body (reviewed in 7). IL-1 consists of a family of two proteins, namely IL-lα and -1β, which overlap in their biological activities, at least when tested in their recombinant form, and bind to the same receptors. However, IL- and IL-lβ differ dramatically in the subcellular compartments in which they are active. IL-1β is active in its secreted form (17.5 kD), whereas its cytosolic precursor is inactive; ILla is mainly active as an intracellular precursor (31 kD) or as a membrane-associated form (23 kD) and only marginally active in the secreted mature form (17.5 kD). It is not yet understood why two IL-1 genes exist; extensive studies have been focused on the characterization of differential effects of the IL-1 molecules in various biological systems with no definite results. IL-1 differs from most other cytokines by the lack of a signal sequence, thus not passing through the endoplasmic reticulum-Golgi pathway. A cysteine protease, termed the IL- 1 Pconverting enzyme (ICE) or caspase- 1, cleaves the inactive precursor of IL-1β to its active secreted form, while calpain is the protease which processes the IL- 1 a precursor (Dinarello 1996). IL-1 has multiple anti-tumor activities that are broader than those of some cytokines used in immunotherapy (Dinarello 1996). IL- 1 potentiates the function of all immune-effector cells and exerts direct cytostatic/cytotoxic effects on some tumor cells. Thus, IL- 1 activates non-adaptive immune-surveillance cells (i.e. NK cells and macrophages) that have the potential to limit tumor growth before specific immunity develops and it also facilitates the development of anti-tumor specific immune responses, mainly by affecting IL-2 secretion, thus serving as a co-stimulatory signal for T cell activation. In addition, IL-1 is one of the strongest inducers of cytokine production in diverse cells; its expression may induce a cytokine network to amplify and sustain antitumor immune responses. IL- 1 possesses myeloprotective/myelorestorative features, which may be beneficial to cancer patients who suffer from infections and leukopenias, especially following chemotherapy/irradiation. Anti-tumor effects of IL- 1 have been described in experimental tumor systems and in phase I and II clinical trials (Dinarello 1996). Till now, immunotherapeutic protocols using IL-1 have consisted of repeated bolus injections of large amounts of the cytokine, which frequently exert side-effects to patients (i.e. hypotension, fever, etc.) and have to be terminated before beneficial anti-tumor effects can be observed. This has hindered the exploitation of

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IL- 1 in immunotherapy. Tumor cell-associated IL-1, focused on the producing cell and in its microenvironment, may represent an efficient On the other hand, form to use IL-1 in tumor immunotherapy. potentiating effects of recombinant IL- 1 on tumorigenicity patterns and metastasis have been described (Dinarello 1996, Anasagasti et al 1997). We have overexpressed the biologically active forms of IL-la and ILlβ in tumor cells in order to assess their differential effects on the malignant process.

2.

RESULTS AND DISCUSSION

2.1

IL- la expression by tumor cells exerts effective antitumor immune responses

We were the first to demonstrate the anti-tumor effects of IL-lα expression by malignant cells (Apte et al 1994a, 1994b, Douvdevani et al 1992, 1991, Apte et al 1992, Zoller et al 1992a,b, Apte 1995). Constitutive expression of IL- lα has been observed in some oncogenetransformed fibroblasts, possibly due to alterations in the control of ILlα expression as a result of the transformation process, or following transfection of violent fibrosarcoma cells with the cDNA of the precursor of IL-lα (Douvdevani et al 1991, Zoller et al 1992a, 1992b). IL-1 expression was assessed on the levels of mRNA, protein (ELISA) and biological function (stimulation of T cell proliferation or IL-6 induction in fibroblasts). IL- 1α -transduced fibrosarcoma cells, which express the cytokine in the cytosol and on the cell membrane, lose their tumorigenicity; they either do not grow in mice or start to grow and subsequently regress, 1421 days after tumor cell inoculation (Figure 1) (Douvdevani et al 1992). In immunohistochemical studies, a dense infiltrate of mononuclear cells, composed mainly of CD8+ T cells, NK cells and macrophages, and only a few CD4 + T cells, was shown to invade IL-lα-positive fibrosarcomas, starting from the first week after inoculation of the malignant cells, and ultimately replacing the tumor's mass. In contrast, in violent cells and in IL- 1β transfectants, which induce progressively tumors (see below), an infiltrate composed mainly of neutrophils was observed only at the periphery of the tumor.

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Days

Figure I: Tumorigenicity patterns of IL-lα and IL-1β-transduced

fibrosarcomas.

Mice were injected i.f.p. with 2x105 tumor cells of an IL-lα-positive cell line (clone 2, cl. 2), which constitutively expresses the cytokine, and various transfectants of a violent fibrosarcoma cell line (clone 5, cl. 5) and tumor growth was measured twice a week using a caliper. Shown are tumorigenicity patterns of violent cells and clones transfected with the empty vector (LXN) or with vectors bearing the following cDNAs: the precursor of IL-lα, the mature formof IL-1β and the mature form of IL-β linked to a signal peptide (ssIL-lβ). Shown is an average tumor diameter (5 mice); individual diameters did not vary more than IO-20% from the average.

Regression of IL-la-positive fibrosarcomas is mainly mediated by T cells and involves the induction of a long-term specific immune memory, which protects against a challenge of violent parental cells (IL-lαnegative). In vivo depletion of CD8+ T cells, possibly cytotoxic T lymphocytes (CTLs), abrogates regression, while CD4+ T cell depletion does not alter tumor growth. Thus, tumor cell-associated IL-la may directly activate CD8+ CTL precursors in the absence of CD4+ TH cells. TH1 -type cytokines, i.e. large amounts of IFNγ and moderate amounts

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of I L-2, characterize the regression of IL- 1α-positive fibrosarcomas, however, the nature of cells which secrete them has still to be elucidated. The contribution of tumor cell-associated IL-lα to the presentation of tumor peptides was also documented (Zoller et al 1992b). Thus, IL-lapositive fibrosarcoma cells, when treated in culture with IFNγ to express MHC class II molecules, can present exogenous antigens and also tumorderived peptides to T cells. IFNγ may be present at the site of the tumor development, since large amounts of IFNγ are secreted by spleen cells from mice which have rejected IL-lα-transduced tumors. In addition, we have shown that IL- lα -positive fibrosarcoma cells can stimulate the function of tissue-resident APCs, possibly via the secretion of stimulatory cytokines, such as GM-CSF, IL-6 etc. Co-stimulatory signals for the activation of anti-tumor T cell-mediated immune responses include the concomitant expression of surface B7.1 on IL- 1α-positive fibrosarcoma cells, possibly induced by endogenous IL- la expressed by the tumor cells, and secretion of IL- 12 by lymphoid cells. Non-adaptive effector cells, such as NK cells and activated macrophages, also play a role in the eradication of IL-I α-positive fibrosarcomas, especially in the early stages of tumor development before specific immunity develops (Zoller et al 1992a). We have emphasized the importance of potentiation of early tumorspecific immune responses by tumor cell-associated IL-lα . Briefly, violent fibrosarcomas are weakly immunogenic; upon inoculation into mice, a weak and transient anti-tumor immune response develops, peaking on days 5-10 and subsequently sharply declining, resulting in the progressive growth of the tumors and death of the mice (Figure 2). In contrast, in mice injected with IL- lα transfectants, the specific anti-tumor immune responses are amplified and sustained, even after regression is terminated (Figure 2). These anti-tumor immune responses are characterized by the development of proliferating T cells, the secretion of large amounts of IFNβ and the development of specific CTLs. In the spleens of mice injected with violent fibrosarcoma cells, transient activity of proliferating T cells and cytokine production occurs, but not the differentiation of anti-tumor cell specific CTLs. Small existing tumors of violent fibrosarcomas have been cured following a single treatment with syngeneic IL- lα-positive cells (2,9). Successful immunotherapeutic intervention occurs at a critical time (“therapeutic window”) after tumor cell inoculation (5- 10 days) (Figure 3).

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Figure 2: CTL activity and' IFNγ secretion in spleen cell cultures from mice bearing IL1α-positive or violent fibrosarcomas. 5x105 violent tumor cells (cl. 5) or IL-lα-positive cells (cl. 2) were injected into mice. At different intervals thereafter mice were sacrificed and spleens were removed. CTLs and IFNγ secretion was assessed in spleen cell cultures in response to the sensitizing tumor cells (mixed lymphocyte-tumor cell reaction, MLTR). IFNγ was assessed in supernatants (harvested on day 5) by a commercial ELISA kit (Pharmingen), while CTLs were assessed by 51Cr release from labeled tumor cells.

The described therapeutic window for treatment with IL-lα-positive tumor cells possibly reflects the kinetics of development of anti-tumor immunity in response to violet fibrosarcomas. As shown in Figure 2, upon the inoculation of violent fibrosarcomas, a transient anti-tumor immune response develops, however, it is not adequately amplified and thus the tumors develop progressively (Figure 2).

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Days Figure 3: Immunotherapeutic effects of IL-lα-positive cells on the growth of violent fibrosarcomas. I05 violent tumor cells (cl. 5) were injected into the footpad of mice and at different intervals thereafter IL-lα-positive cells (5x105 ) (cl. 2) were injected into the tumorbearing footpad. Shown is an average tumor diameter (5 mice); individual diameters did not vary more than 10-20% from the average.

If at that critical “therapeutic window”, IL-lα is supplied by the treating cells, such an inefficient immune response is converted into an efficient one, leading to tumor regression. At earlier times, when no or a sub-threshold anti-tumor immune response develops, no effects of tumor cell associated IL-lα, at this treating regimen, are observed. Indeed, in mixing experiments, when the violent and IL-lα-positive tumor cells are injected together, or when the treating cells are injected on days 1 and 3 after the inoculation of violent cells, no effects on tumorigenicity patterns of violent fibrosarcoma cells are observed. At late intervals, day 14 and onwards, there are no immunotherapeutic effects to treatment with IL-lα-positive tumor cells, either because the inability of the immune system to cope with established tumors or due to the development of tumor-mediated suppression. Multiple treatments of existing violent tumors with IL-1α-positive cells may broaden the "immunotherapeutic window" for achieving positive therapeutic effects. If of clinical relevance, in patients, it will be possible to apply immunotherapeutic treatments with IL-lα -positive cells after debulking the mass of the primary tumor, to avoid metastasis.

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Figure 4:

Opposing effects of IL-1 α and IL-1β on Malignancy Patterns

Reduced tumorigencity of IL-lα -transfected TS/A cells.

2x104 violent tumor cells (TS/A) or cells of clones of IL-lα -transfected TS/A cells (clones 8 and 22; TS/A-8 and TS/A-22) were injected itrafoorpad and tumor growth and mortality were scored. TS/A-8 cells generate 200 pg/106 celld24 hrs, whereas TS/A-22 cells generate 80pg/106 cells/24 hrs. Shown is an average tumor diameter (5 mice); individual diameters did not vary more than 10-20% from the average.

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We have recently shown that the expression of IL-lα in highly metastatic cells of low immunogenicity reduces their malignancy patterns. Thus, we have used the TS/A breast carcinoma cell line that spontaneously metastasizes to the lung (Forni et al 1995). Upon transfection of the tumor cells with the precursor of IL-lα, reduced tumorigenicity patterns were observed, as manifested on the levels of growth of the primary tumor and mortality, due to lung metastases. (Figure 4). As can be seen in Figure 4, the tumorigenicity of TS/A-8 cells was completely abolished, whereas the growth of TS/A-22 cells was only retarded, as compared to the violent tumor cells. These differences were also manifested on the level of survival rate; effects on tumorigenicity were related to the amount of IL-lα that was expressed by the transfected tumor cells. Further studies on the immunotherapeutic potential of tumor cell vaccines, consisting of IL-1α-transfected TS/A cells, on the development of post-operative metastases and survival rates are in progress. If of clinical relevance, in patients, it will be possible to apply immunotherapeutic treatments with IL- lα-positive cells after debulking the mass of the primary tumor, to avoid metastasis.

2.2

IL-lβ expression by tumor cells potentiates their invasive potential

In order to elucidate whether the two IL-1 genes exert similar effects on tumorigenicity patterns, we transfected violent fibrosarcoma cell lines, - the same as those previously transfected with IL-lα, with constructs bearing cDNAs of active forms of IL-1β: the mature form of IL-lβ and the mature form of IL-Iβ linked to the signal peptide of the human IL-I receptor antagonist (IL-1Ra) (Windren et al 1996), to enable active secretion of IL- 1 β through the endoplasmic reticulum-Golgi pathway. To our surprise, IL- 1β transfectants grow progressively and are even more tumorigenic than the parental violent tumor cells, as manifested by quicker tumor growth (Figure 1) and earlier mortality of the mice. Tumors of IL-lβ transfectants developed into large and necrotic tumors. In accordance, some reports have shown that ILlβ may enhance the tumorigenicity of malignant cells and also promote metastasis, mainly by affecting interactions between the tumor cells and endothelial cells (Dinarello 1996, Anasagasti et al 1997). It was demonstrated that no leukocytic infiltrate was observed in the inner part of tumors induced by IL- 1β transfectants, except of neutrophils in necrotic areas. In the periphery of such tumors, a sparse mononuclear infiltrate was occasionally observed. In contrast, in

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regressing IL- 1 α transfectants a dense mononuclear infiltrate invaded all areas of the tumor and ultimately replaced the tumor's mass. Thus, it appears that effective eradication of the tumor by immune effector cells depends on the ability of leukocytes to invade the inner part of the tumor. Studies on the mechanisms that enable the access of infiltrating cells into tumors induced by in IL-lα transfected fibrosarcoma cells, and those that inhibit it in tumors of IL-lβ transfectants, are in progress. Studies on the immunity of tumors induced by IL-lβ transfected tumor cells have revealed that these cells fail to induce effective anti-tumor immune responses, as manifested by the inability of lymphoid cells from mice injected with IL- 1β transfectants to induce IFNγ secretion in MLTR cultures. In addition to impaired induction of anti-tumor immunity, IL1β transfectants were shown to express molecules that promote tumor invasiveness. Metalloproteinases (MMPs) are matrix-degrading enzymes that are crucial for invasiveness and metastasis. In IL-1 B transfectants, an upregulation of MMP-2 and MMP-9, compared to the violent tumor cells was observed, while in IL-la transfected fibrosarcomas cells partial downregulation of MMP-2 and complete downregulation of MMP-9 was observed. Thus, in IL- 1 transfectants, expression of the cytokine by the tumor cells affects the patterns of interaction of the malignant cells with the host's immune system and also controls the endogenous expression of molecules that affect invasiveness and metastasis.

3.

CONCLUSIONS

From our results it appears that the compartmentalization of IL-1 within the producing cell or its microenvironment determines its scope of biological functions. It is also suggested that only within the producing cell, the physiological roles of both IL-1 molecules can be assessed accurately. It is notable that IL-la and IL-lb exert the same biological functions, when tested in their recombinant form, and bind to the same receptors. Our results substantiate the importance of the membrane-associated form of IL- lα for fibrosarcoma regression. Active secreted and cytosolic forms of IL-1 are also expressed in the IL-lβ transfectants, which grow progressively in mice, however, the IL-lβ molecule is not displayed on the surface membrane. Membrane-associated IL-1α may serve as an adhesion-molecule, promoting efficient cell-to-cell interactions between the malignant cells and immune effector cells, and as a focused cytokine with a strong adjuvant activity. Indeed, we have shown that tumor cells

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expressing relatively small amounts of IL-I α, ~50-250 pg/l06 fibrosarcoma cells/24 hr, exert very potent immunotherapeutic effects. These expression levels are far below those which are toxic to the host. Studies in our lab also indicate the immunotherapeutic efficiency of tumor cell-associated IL- 1 α in experimental metastatic tumor systems of T cell lymphomas and a breast carcinoma. These results provide new insights on the use of IL-I in tumor cell vaccines. On the other hand, IL-1β may be released from the tumor cells either actively or following cell death and by this exert pronounced effects on the microenvironment, that in this experimental system are manifested by enhanced tumorigenicity patterns. It seems that within the producing tumor cells or it microenvironment IL- lα represents an immunostimulatory cytokine, while IL-1β represents an inflammatory cytokine. The autocrine/intracrine effects of ectopic IL- lα and IL- 1β expression in fibrosarcoma cells, especially those affecting gene expression, control of proliferation and susceptibility to immune surveillance mechanisms are being studied in our lab.

ACKNOWLEDGMENTS E. Voronov is supported by the Gileadi and Kamea Programs of The Israel Ministry of Immigrant Absorption, The Chief Scientist’s Office, The Israel Ministry of Health, The Israel Cancer Research Fund and The Israel Cancer Association. R.N. Apte is supported by the Israel Ministry of Science (MOS) jointly with The Deutsches Krebsforschungscentrum (DKFZ), Heidelberg, Germany, The Chief Scientist’s Office, The Israel Ministry of Health, The Israel Cancer Research Fund, The Israel Cancer Association. The United States-Israel Binational Foundation (BSF) and THE ISRAEL SCIENCE FOUNDATION founded by The Israel Academy of Sciences and Humanities (The Eva and George Klein Fund).

REFERENCES ANASAGASTI, M.J., OLASO. E., CALVO, F., MENDOZA, L., MARTIN, J.J., BIDAURRAZAGA, J.and VIDAL-VANACLOCHA, F. Interleukin 1 -dependent and independent mouse melanoma metastases. J. Natl. Cancer Inst., 89:645-65 1, 1997. APTE, R. N. Mechanisms of cytokine production by fibroblasts-implications for normal connective tissue homeostasis and pathological conditions. Folia Microbiologica, 1995.. 40:392-404, APTE, R.N., DOUVDEVANI, A., HULEIHEL, M., FIMA, E., HACHAM, M., SHIMONI, N and SEGAL, S. IL-1 and pro-inflammatory cytokines produced by primary and

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transformed fibroblasts abrogate the tumorigenic potential of fibrosarcomas. Folia Biologica (Praha), 38:240-257, 1992. APTE, R.N., DOUVDEVANI, A., ZOLLER, M., WHITE, R.M., DVORKIN, T., SHIMONI, N., FIMA, E., HACHAM, M., HULEIHEL, M., BENHARROCH, D., VORONOV, E. and SEGAL., S. Cytokine-induced tumor immunogenicity: Interleukin-1 alpha expressed by fibrosarcoma cells confers reduced tumorigenicity. Immunol. Lett., 39:45-52, 1994a. APTE, R.N., DOUVDEVANI, A., ZOLLER, M., WHITE, R.M., DVORKIN, T., SHIMONI, N., HULEIHEL, M., FIMA, E., HACHAM, M., VORONOV, E., BENHARROCH, D., and SEGAL., S. Involvement of immune responses in the eradication of IL-1 alpha gene-transduced tumour cells: mechanisms of tumour rejection and immunotherapeutical implications. Folia Biologica (Praha), 40: I- 18, 1994b. COLOMBO, M.P. and FORNI, G. Immunotherapy 1 : Cytokine gene transfer strategies. Cancer Metastasis Rev., 15: 3 17-328, 1996. DINARELLO, C.A. Biologic basis for interleukin- 1 in disease. Blood, 87:2095-2147, 1996. DOUVDEVANI. A., HULEIHEL, M., SEGAL, S. and APTE, R.N. Regulation of interleukin- I generation in immune-activated fibroblasts. Eur. Cytokine Net., 2:257264, 1991. DOUVDEVANI, A., HULEIHEL, M., ZOLLER, M., SEGAL, S. and APTE, R.N. Reduced tumorigenicity of fibrosarcomas which constitutively generate IL-1 alpha either spontaneously or following IL-1 alpha gene transfer. Int. J. Cancer, 52:822830, 1992. DRANOFF, G. and MULLIGAN, R.C. Gene transfer as cancer therapy. Adv. Immunol., 58:417-454, 1998. FORNI, G., CAVALLO, F., CONSALVO, M., ALLIONE, A., DELLABONA, P., CASORATI, G. AND GIOVARELLI, M. Molecular approaches to cancer immunotherapy. Cytok. Molec. Ther., 1; 225-248, 1995. PARDOLL, D.M. Cancer vaccines: a road map for the next decade. Curr. Opin. Immunol., 8:619-621, 1996. QIN, Z. and BLANKENSTEIN, T. Influence of local cytokines on tumor metastases: using cytokine gene-transfected tumor cells as experimental models. Curr. Top. Microbiol. Immunol., 213: 55-64, 1996. WINGREN, A. G., BJORKDAHL, O., LABUDA, T., BJORK, L., ANDERSSON, U., GULLBERG, U., HEDLUND, G., SJOGFEN, H. O., KALLAND, T., WIDEGREN, B.and DOHLSTEN, M. Fusion of a signal sequence to the interleukin-I beta gene directs the protein from cytoplasmic accumulation to extracellular release. CellImmunol. 169:226-37, 1996.. ZOLLER, M., DOUVDEVANI, A., SEGAL, S. and APTE, R.N. Interleukin-I produced by tumorigenic fibroblasts influences tumor rejection. Int. J. Cancer, 50:443-449, 1992a. ZOLLER, M., DOUVDEVANI, A., SEGAL, S. and APTE, R.N. Interleukin-I production by transformed fibroblasts. II Influence on antigen presentation and T-cell-mediated anti-tumor response. Int. J. Cancer, 50: -457. 1992b.

ABSTRACTS

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Abstracts

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CLEARANCE OF APOPTOTIC CELLS IS MEDIATED BY THE COMPLEMENT SYSTEM Dror Mevorach The Laboratory for Cellular and Molecular Immunology, Division of Medicine, TelAviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv, Israel

Apoptotic cells are rapidly engulfed by phagocytes, but the receptors and ligands responsible for this phenomenon are incompletely characterized. The previously described receptors on blood-derived macrophages have been characterized in the absence of serum and show a relatively low uptake of apoptotic cells. Addition of serum to the phagocytosis assays increased the uptake of apoptotic cells by >3-fold. The serum factors responsible for enhanced uptake were identified as complement components that required activation of both the classical pathway and alternative pathway amplification loop. Exposure of phosphatidylserine on the apoptotic cell surface was partially responsible for complement activation and resulted in coating the apoptotic cell surface with C3bi. In the presence of serum, the macrophage receptors for C3bi, CR3 (CD11b/CD18) and CR4 (CD11c/CD18), were significantly more efficient in the uptake of apoptotic cells compared to previously described receptors implicated in clearance. Complement activation is likely to be required for efficient uptake of apoptotic cells within the systemic circulation and early component deficiencies could predispose to systemic autoimmunity by enhanced exposure to and / or aberrant deposition of apoptotic cells.

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Abstracts

PULMONARY COLLECTINS AND INNATE IMMUNITY Erika C. Crouch Department of Pathology, Washington University School of Medicine, St. Louis, MO

The lungs are constantly exposed to inhaled infectious agents and toxic particles. To meet this challenge, the upper airways, bronchioles, and alveolar regions of the mammalian lung have evolved a complex and multilayered system of defense that includes locally-synthesized and constitutively secreted or readily inducible defense molecules that accumulate at the air-lung interface. The surfactant-associated proteins, SP-A and SP-D, are members of a family of collagenous C-type lectins, designated collectins. There is growing evidence that these proteins, which are synthesized by epithelial cells lining the alveoli and airways, are important components of the innate immune response to microbial challenge and participate in immune and inflammatory regulation within the airspace of the lung. The collectins are synthesized as multimers of trimeric subunits each consisting of an amino-terminal crosslinking domain, a triple helical collagen domain, and a terminal, high affinity, trimeric, carbohydrate recognition domain. SP-A and SP-D bind to distinct ligands associated with conserved saccharide components integral to bacterial, fungal, and viral cell walls, and to certain surfactantassociated lipids in vitro. Although binding may facilitate microbial clearance through agglutination or other direct effects on the organism, the lung collectins can also modulate the host defense functions of leukocytes, alter leukocyte interactions with biologically active bacterial products such as bacterial lipopolysaccharide, and inhibit the stimulated proliferation of T-lymphocytes. In vitro studies suggest SP-A and SP-D interact with organisms during distinct phases of the infectious process, and their interactions with leukocytes involve distinct or only partially overlapping binding mechanisms. Acquired deficiencies in lung collectins may alter surfactant homeostasis, increase the susceptibility to respiratory infection, modify the pulmonary inflammatory response in the setting of injury or microbial challenge, and influence the development of clonal immunity.

Abstracts

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STIMULATION OF NO AND INTERLEUKINS PRODUCTION IN MACROPHAGES BY Klebsiella pneumonaie AND LUNG COLLECTIN SURFACTANT PROTEIN D (SPD) A. Mesika¹, I. Ofekl, Y. Keisari¹, H-B. Wang¹,S. Riklis E.C2. Crouch3 and M. Kalina² Departments of Human Microbiology and Cell Biology, Sackler Faculty of Medicine, Tel-Aviv University, Israel and Department of Pathology at Jewish hospital, Washington University St. Louis, USA

Encapsulated bacteria which invade the lung and cause pneumonia, encounter in distal airways the alveolar macrophages and lung collectins SPA and SPD, which are responsible for eradicating the pathogen in this serum poor environment. In the present study we examined the role of Klebsiella pneumoniae (KP) capsule and SPD in stimulating NO production by a cell line NR-8383 of alveolar macrophage and transcription of mRNA encoding for TNFalpha , IL-1ß, IL-6, IL-12 and IL- 10 by human monocyte-derived macrophages (HuMoDM). We employed encapsulated and a non-encapsulated derivative of K50 serotype and human recombinant SPD whose carbohydrate recognition domain reacts with the core region of the KP lipopolysaccharide. The capsulated strain stimulated both production of NO and transcription of IL/TNF-mRNA, whereas the non-encapsulated derivative was not active. This activity was inhibited by mannan, suggesting that it was mediated by the macrophage mannose receptor interacting with dimannose residues of the capsular polysaccharide of K50 and K21a . SPD at concentrations > 50 ng/ml stimulated NO production and agglutinated the nonencapsulated but not the encapsulated Kp. NO production was markedly enhanced in macrophages exposed to non-encapsulated bacteria pretreated with SPD and washed free of SPD excess. Because the amount of SPD remaining bound on bacterial surface is estimated to be < 5 ng assuming that each LPS molecule is bound to one SPD molecule and lysate of sonicated bacteria pretreated with SPD was inactive, we conclude that presentation of SPD on the bacterial surface to its cognate receptor on macrophages was responsible for triggering enhanced NO production.

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A HIGH IgG1 to IgG2 ANTI LPS ANTIBODY RATIO FACILITATES THE KILLING OF Shigella Spp. BY HUMAN PHAGOCYTES Guy Robin, Dani Cohen, Mark Katzenellenbogen, Yona Keisari Israel Defense Force, Medical Corps; Depts. of Epidemiology and Preventive Medicine, and Human Microbiology, Sackler Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel

Dysentery and diarrhea caused by Shigella are major public health problems, especially in developing countries. It has been estimated that shigellosis or bacillary dysentery results in more than 200 million cases, with approximately 650,000 deaths per year. Shigella infection confers serotype-specific protection against recurrent infection. The relative importance of locally secreted antibodies, serum antibodies, and cellmediated immunity in protection against shigellosis is not fully understood. Previous results have indicated that serum anti-Shigella IgG are associated with protection against homologous infection. This study examined the efficacy of phagocytes to bind and kill Shigella organisms, in the presence or absence of specific homologous antibodies. It was found that binding of phagocytes and Shigella increased in dependence with the concentration of specific purified antibodies. Heterologous antibodies had no impact on the binding of Shigella to phagocytes. Tests performed in the presence of specific antibodies and monocytes/macrophages showed a massive attachment of Shigella to the cells, that a short time later led to cell death. Analysis of the polymorphonuclear cells capability to kill Shigella in the presence of specific antibodies revealed that the ratio between IgG1/IgG2 was important to this function. When IgG2 was the dominant component of the examined sample (ratio of 7:1 - IgG2/IgG1) the antibacterial activity measured was of 25%, while in the presence of a sample with higher levels of IgGl (ratio of 1:1.44 IgG2/IgG1) the antibacterial activity revealed was 79%. Thus, differences in the host production of IgG antiShigella LPS subclass antibodies are associated with different capability to kill Shigella. Analysis of IgG subclass distribution of anti-Shigella LPS antibodies in serum samples showed distinct patterns of IgG subclass response after S. sonnei or S. flexneri infection. In the IgG response to S. sonnei, IgG1 was the major component produced, and in contrast the ant i-S. flexneri response was marked by a predominant increase in IgG2. Epidemiological studies showed that S. flexneri 2a infection induced a

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significantly longer chronic infection, which caused more cases of dysentery and bacteremia as opposed to infections induced by S. sonnei. It is suggested that anti-Shigella LPS antibodies of the IgGl subclass are associated with the capability of the host to over come more easily-on infection caused by S. sonnei as compared to S. flexneri.

296

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PROFILE OF ANTIBODY RESPONSE TO Streptococcus pneumoniae (PNC) SURFACE PROTEINS DURING HEALTH, DISEASE AND CONVALESCENCE Sarit Lifshitz¹; Michal Shani-Seckler¹, Iris Hillel¹; Ron Dagan¹; Gideon Fleminger2; Yaffa Mizrachi¹,² ¹Pediatric Infect. Dis. Unit. Soroka Med. Center;²Dept. Microbiol. & Immunol., Fac. Health Sciences: Ben Gurion Univ., Beer Sheva. ³Dept. Biotechnol. Tel Aviv

The currently used Streptococcus pneumoniae (Pnc) vaccines are bot covering the full range of Pnc serotypes. Further studies are needed for the development of the next generations of Pnc vaccines. The importance of Pnc surface and membrane proteins in health, disease and convalescence is not yet fully explored. Using a mutant unencapsulated type 3 Pnc mutant we analyzed the humoral immune response to Pnc lectin and non-lectin cell wall and membrane proteins. The proteins were separated by fetuin affinity chromatography, and analyzed by western blot. Strong and weak antibody profiles were defined as the number of proteins detected by the tested sera, and their intensity, measured by densitometer. Sera from randomly chosen healthy adults (N=8), healthy children from Day Care Center (DCC; N=7) and children during bacteremia and convalescence (N=7) were studied. Five adults with anti polysaccharides (PS) antibodies ” 5µg/ml IgG to all 7 antigens tested) demonstrated high levels of antibodies to 10- 15 lectin and non-lectin proteins, respectively. In contrast, 3 adults with anti PS antibodies <5µg/ml IgG to all 7 antigens tested, recognized only 3-7 proteins with lower intensity. 6/7 DCC children had high levels antibodies to lectin and non-lectin (10 and 15 proteins, respectively). 5/7 children, aged 11 days to 12 years, from whom paired sera were obtained during acute Pnc disease and convalescence, demonstrated anti protein antibodies responses. The 2 children with no response were younger than 1 month old. 3 proteins with MW around 30 KDa were common to all groups analyzed. Further characteristics of the most immunogenic proteins and their relation to protection against Pnc infection are currently being studied.

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297

MYCOBACTERIA BIND TO CR1 USING HSP 65 P. Accardo, G. Amodeo, M. Brai Inst. of General Pathology, University of Palerom, Palermo, Italy

Mycobacteria (M) are intracellular pathogens that reside almost exclusively within macrophages of infected individuals. The initial contact between inhaled M and host cells takes places in the lung, where bacteria are phagocytosed by alveolar macrophages. Complement receptors CR1 and CR3 and mannose receptors play an important role in the adhesion of M to the cell targets, with or without serum opsonins. We studied the binding of recombinant soluble CR1 (rsCR1 to M bovis BCG ( 2 x 1 0 6 cells) by an ELISA system based on bacteria coated microwells. The non opsonic binding of rsCR1 to BCG was of high affinity (Kd ˜= 10-¹º, 38,550 site/cell) and saturable. Several other M strains were studied and gave similar results. Non opsonic binding of rsCR1 to other intracellular pathogens (L. monocytogenes, C. xerosis, N meningitidis) was also detectable, although to a lower level than for M, whereas a strain of E. coli (InvaF') showed no binding at all. Inhibition studies of the direct binding of rsCR1 to BCG, performed using C3i, C4i, Clq and 3 different anti-CR1 moAbs (3D9, J3D3, Ell) demonstrated that C3i and 3D9 induce 60% inhibition of binding, C4i and Ell inhibit only 10-20% and Clq, a recently demonstrated CR1 ligand, inhibit 100%. No inhibition was obtained with J3D3. Using PPD (20 µg/ml) as a specific competitor, more than 70% of inhibition was observed. Western blot of sonicated M with rsCR1 followed by peroxidase-conjugated antiCR1, showed two bands in the range of 60-65 kDa. Recombinant M heat shock protein 65 (Hsp65, MA5C), but not M 38 kDa protein, inhibited, in a dose-dependent manner, rsCR1 binding to M. A panel of 11 peptides derived from Hsp65 sequence were used in inhibition experiments. Only peptide 277-293 (100 µg/ml) induced 66% inhibition. In conclusion, our study has shown that M are able to bind CR1 directly and with high affinity. Other studies have reported that CR1 is one of several receptors utilized by M to enter host cells but have not reported data on the affinity of such interactions. Furthermore, we have identified Hsp 65 from M as a ligand of CR1, thus as a possible candidate for entering the target cells via CR1. This is the second report of the involvement of Hsps in the attachment of M to macrophage receptors. We propose they might represent recognition structures of different host cell receptors, each receptor possibly specific for a certain M strain.

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298

Abstracts

ASSEMBLY OF THE SUPEROXIDE GENERATING NADPH OXIDASE - TAKING PEPTIDES FOR A WALK Iris Dahan, Irina Issaieva, Igor Morozov, Gili Joseph, Natalia Sigal, Amir Toporik, Yara Gorzalczany, Ofra Lotan & Edgar Pick The Julius Friedrich Cohnheim - Minerva Center for Phagocyte Research and the Ela Kodesz Institute of Host Defense against Infectious Diseases, Sackler School of Medicine, Tel Aviv University

The enzymatic complex responsible for the generation by phagocytes of the primordial oxygen radical, the superoxide anion (O2-), is composed of a membrane-embedded compone t flavoc ochrome b559 (cyt b), and four cytosolic proteins, p47phox , p67phox, the small GTPase Rac1, and p40phox Cyt b is a heterodimer composed of a larger glycosylated subunit (gp91 phox ) and a smaller subunit (p22phox). O2- is generated by the NADPH-derived one-electron reduction of molecular oxygen. Electrons are transported from NADPH to oxygen via two redox stations (one molecule of FAD and temes), all elements involved in transport being located on gp91phox. The conversion of gp91phox from its “resting” state to the O2 producing conformation is achieved by the assembly of the cyt b dimer with some or all cytosolic components, a process involving the translocation of these from the cytosol to the membrane. The fully-assembled structure is known as the NADPH oxidase complex and it is assumed that assembly of the complex is required and, perhaps, sufficient for the initiation of electron transport. Assembly of cyt b with the cytosolic components and additional affinities between the cytosolic components are governed by a complex and finely orchestrated set of protein - protein interactions. NADPH oxidase assembly can be induced in a cell-free system consisting of purified or recombinant components exposed to a critical concentration of anionic amphiphiles, such as arachidonate or Na or Li dodecyl sulfate; in the presence of NADPH, the system generates O2- . We have introduced a novel approach to the study of protein-protein interactions in NADPH oxidase assembly, termed “peptide walking”. This involves the synthesis of overlapping 15-mer peptides corresponding to the entire length of a particular oxidase component or its subunits. The peptides overlap by 11 to 13 residues and are modified by biotinylation of the N-terminus and amidation of the C-terminus. The peptides are used in two assays: a. Inhibition by peptides of the activation

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(assembly) of the NADPH oxidase complex in a semirecombinant cellfree system , and b. Binding by peptides derived from one component of another component, presented in the form of intact protein. In this technique, the biotinylated peptides are attached to the surface of streptavidin-coated 96-well plates and the interacting proteins are presented in the fluid phase. Detection of bound proteins is by specific antibodies, followed by peroxidase-labeled second antibodies. So far, we analyzed th ee NADPH oxidase components by “peptide walking” : phox Rac1, p47 , and the p22phox subunit of cyt b. The most recent results obtained by usin this system concern the ability of p22phox peptides to bind p47phox and p67phox. p47phox was found to bind to a domain in p22phox (residues 56-63), forming part of a cytosolic loop extending from residues 56 to 91, an to phox the proline-rich region 151-160, located in the cytosolic tail. p67 bound to domain 81-91, at the distal part of the cytosolic loop 56-91, and to a domain 11-115) located at the beginning of the cytosolic tail. Surprisingly, p67phox was also found to attach to the proline-rich region three ( 15 1 - 160), which also binds p47 phox These fi dings contai phox phox phox also novel element . 1. p67 binds directly to p22 ; 2. p47 phox binds to p22 at a site other than the proline-rich region, and 3. phox Apparently, th same proline-rich region of p22 serves as an anchor for both p47phox and p67phox. When peptides, corresponding to the major p47phox - and p67phox- binding domains were scrambled, their binding abilities were lost, indicating that these are strictly sequencespecific i teractions. These data also provide support for the proposal that p67 phox is the key component interacting with the cyt b dimer and phox phox that p47 might merely serve as shuttle for p67

300

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REGULATION OF CHEMOKINE AND CHEMOKINE RECEPTOR EXPRESSION IN INNATE IMMUNITY Alberto Mantovani lstituto di Ricerche Farmacologiche Mario Negri, Milan, and Dept. Biotechnology, Section of General Pathology, University of Brescia, Italy

Chemokines are a superfamily of cytokines which includes about 50 members in humans. They are characterized from the point of view of structure by a "chemokine scaffold", from the point of view of function by the fact that most of them induce directional migration (chemotaxis) of leukocytes and from the point of view of the receptors by the fact that they use seven transmembrane domain receptors. The chemokine scaffold consists of an N-terminal loop connected via Cys bonds to the more structured core of the molecule (three ß sheets with a C terminal a helix). The chemokine field has long been dominated by the interest in the regulation of the recruitment and function of cells involved in innate immunity. More recently, it has become clear that chemokines play a fundamental role in the activation and orientation of specific immunity. Based on a cysteine motif, chemokines have been classified into a CXC, CC, C and CX3C molecules (Baggiolini 1998, Hedrick and Zlotnik 1996, Mantovani 1999, Rollins 1997). It is useful to classify chemokines based on their mode of production. Constitutively produced chemokines, the prototype of which is a molecule called SDF1, are made either by a variety of tissues or in a more restricted way in lymphoid organs, and play an important role in regulating the normal trafficking of leukocytes. Inducible or inflammatory chemokines, the prototype of which are IL-8 (CXC) and MCP-1 (CC), are made in response to microbial, inflammatory or immune stimuli. Their general significance is that of regulating recruitment "on demand". While this classification has been and is useful, it is an oversimplification. It is more and more apparent that the realms of inducible and constitutive chemokines overlap. Molecules such as LARC, MDC, DC-CK1, which were originally identified as constitutively expressed chemokines, have subsequently been shown to be produced in a regulated way. The two realms also overlap in terms of pathology because in neoplastic disorders inducible chemokines are made by transformed cells in a constitutive way, and this is particularly common for MCP-1. In this context it was shown recently that MDC, originally described as a chemokine constitutively expressed by dendritic

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301

cells and mature macrophages, is regulated by Th1 and Th2 chemokines (see below). The borders of the chemokine world are still not in sight, as new molecules keep popping up, in particular as a result of search in specialized tissues. Tuning of the system The chemokine system is tuned either by regulating agonist production or by changing the level of receptor expression. For instance, MDC, a chemokine which interacts with CCR4 and probably with other undefined receptors, is a preferential attractant for polarized Th2 cells. It is induced in monocytes and T cells by IL-4 and IL-13. In addition, it is also regulated at the level of mature protein. The dipeptidyl peptidase IV CD26 processes the N-terminus of MDC, which looses activity on CCR4 and Th2 cells, but retains the capacity to attract monocytes. Gene transfer technology reveals that chemokines and chemokine receptors are part of the genetic program differentially expressed in polarized Th1 and Th2 cells. The function of chemokines is regulated by changing levels of receptor expression. In monocytes it is now apparent that proand antinflammatory signals have reciprocal and divergent effects on agonist production versus receptor expression. Downregulation of receptor expression by primary proinflammatory signals such as LPS may serve as a means to arrest and focus host defense cells at sites of inflammation and infection whereas, under certain conditions, upregulation of receptor expression by anti-inflammatory signals such as IL-10 and glucocorticoids may serve as means to scavenge the agonists. Differential regulation of receptor expression during T cell activation may be instrumental to the appropriate positioning of T cells during an immune response. In general, the microenvironmental context and in particular cytokines, dictate the real in vivo spectrum of action of chemokines. Various components of the chemokine system have been studied using gene targeting technology. Moreover, a considerable fraction of normal human individuals bear the –32 mutation of the CCR5 gene. The mild phenotype observed with most of these genetic alterations may be accounted for by the apparent redundancy of the system. It has been argued that redundancy may render certain outputs of the system (e.g. a minimum of phagocyte recruitment for tissue remodeling in ontogeny and for innate resistance) robust, i.e. minimally affected by quantitative or qualitative alterations of individual components. Chemokine receptors are 7 transmembrane, G protein coupled receptors. In addition to conventional signal transducers typically coupled to this class of receptors, JAK/STAT components have been suggested to be involved. Internalization and recycling are crucial steps

3 02

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which dictate receptor availability. These properties are both a function of the receptor and agonist examined. Rantes analogues modified at the N-terminus (AOP-Rantes) are poor agonists, but cause R internalization and block recycling. The search for simple chemicals capable of blocking chemokine receptors or downstream events is the subject of intense efforts and represents a holy grail for industrial research in the field. References Baggiolini, M. 1998, Chemokines and leukocyte traffic. Nature 392, 565-568. Hedrick, J. A. , Zlotnik, A. 1996, Chemokines and lymphocyte biology. Curr. Opin. Immunol. 8, 343-347. Mantovani, A. 1999, The chemokine system: redundancy for robust outputs. Immunol. Today 20, 254-257. Rollins, B. J. 1997, Chemokines. Blood 90, 909-928.

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SIGNAL TRANSDUCTION AND THE INTERPLAY BETWEEN THE EARLY ENDOCYTIC AND PHAGOCYTIC PATHWAYS Philip D. Stahl, Manuel A. Barbieri, Carmen Alvarez-Dominguez and Rick Roberts Washington Univ. School of Medicine, St. Louis, MO., USA

Phagocytosis, phagosome maturation and phagosome-lysosome fusion are key elements of a successful host defense program against bacterial pathogens. A substantial body of work indicates that phagocytosis and endocytosis and the processes leading to phagosomelysosome and endosome-lysosome fusion, respectively, utilize common membrane trafficking regulatory factors. The identification of low molecular weight GTPases and other factors required for membrane fusion and membrane budding on phagosomes and the reconstitution of phagosome-endosome and phagosome-lysosome fusion in vitro have aided in the development of models for phagosome maturation. A hypothesis supported by work from several groups but not yet fully tested is that phagosome maturation is a highly ordered sequential process. Initial events following phagosome formation (i.e., interaction of phagosomes with the early endocytic compartment) set into motion a process of membrane exchange whose completion is required for subsequent trafficking events leading to fusion with the lysosomal compartment. Activation of phagosome-endosome fusion and endosome-endosome fusion appear to require the activation of a receptor mediated cascade leading to guanine nucleotide exchange on Rab5a. EGF stimulated endocytosis is Rab5a isoform specific and is blocked by dominant negative Rab5a (S34N). EGF activation of endocytosis provides a good model for the study of stimulated phagosome endosome fusion. Pathogens appear to exploit the sequential nature of the process by producing factors that block one or more steps thereby preventing subsequent maturation steps. Listeria monocytogenes and Salmonella typhimurium are among the best studied pathogens that interfere with proper maturation of early phagosomes, however via different mechanisms. Listeria monocytogenes interferes with maturation by altering the function of Rab5a whereas Salmonella produces factors such as SpicC which interfere with phagosomeendosome fusion. The interplay between the early endocytic and early phagocytic pathways will be discussed.

3 04

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MAST CELL-T CELL INTERACTIONS: BIDIRECTIONAL FUNCTIONAL RELATIONSHIPS Yoseph A. Mekori Meir General Hospital, Kfar-Saba and Sackler School of Medicine, Tel-Aviv University, Israel

Mast cells are known to be essential resident effector cells in the elicitation of the allergic response. IgE sensitised mast cells, upon encounter with specific antigen that is recognised by surface receptor (FcεRI)-bound IgE, secrete and generate bioactive mediators which facilitate the development of allergic inflammation. Apart from activation via FcεRI, mast cells can also be activated via nonimmunologic routes by substances such as neuropeptides, complement components and bacterial products. In addition to being a major effector cell in the elicitation of allergic inflammation, mast cells have been found to be activated in various T cell-mediated inflammatory processes, and to reside in close physical proximity to T cells. Such observations, and the wide spectrum of mediators produced and secreted by mast cells, have led investigators to propose a functional relationship between these two cell populations. Indeed, mast cell activation has been reported to induce T cell migration either directly by the release of chemotactic factors such as lymphotactin or IL-16, or indirectly by the induction of adhesion molecule expression on endothelial cells. Mast cells are also able to present antigens to T cells, resulting in their activation, in either an MHC class I or class 11-restricted and co-stimulatory molecule-dependent fashion. Adhesion molecule-dependent intercellular contact, or MHC class II cognate interactions between T cells and mast cells result in the release of both granule-associated mediators and cytokines from the latter. Also, T cell derived mediators such as β-chemokines directly induce mast cell degranulation. On the other hand, mast cell derived cytokines such as IL-4 have been found to polarise T cells to preferentially differentiate into the Th2 subset. Thus, T cell-mast cell interactions are bi-directional, fulfilling regulatory and/or modulatory roles affecting various aspects of the immune response.

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305

SIGNALLING BY G PROTEIN-COUPLED PHOSPHOINOSITIDE 3-KINASE G NEUTROPHIL RESPONSES IN PI3KG KNOCKOUT MICE Vladimir L. Katanaev, Emilio Hirsch#, Genevieve Bulgarelli-Leva, and Matthias P. Wymann* Institute of Biochemistry, University of Fribourg, Switzerland, #Department of Genetics, Biology and Biochemistry, University of Torino, Italy

PI 3-kinase γ is predominantly expressed in differentiated hematopoietic cells and transduces signals from trimeric G proteincoupled receptors. To elucidate the physiologic role of PI3Kγ, the gen was disrupted in mice by homologous recombination. Resulting PI3Kγ animals are viable and fertile, and have normal numbers of white blood cells. Neutrophils without PI3Kγ still responded to opsonized zymosan or the protein kinase C activator phorbol myristate acetate with a respiratory burst, while the fMLP-induced response was impaired under certain conditions. Similarly, chemotaxis of PI3Kγ -/- neutrophils towards gradients of fMLP, interleukin-8 (IL-8), or C5a was decreased, and fMLP- and IL-8- induced adhesion to fibronectin and actin polymerization were reduced as well. These results underline an important role of PI3Kγ in neutrophil activation and inflammatory reactions, and validate an important pharmacological target.

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306

Abstracts

H+ CHANNELS AND NADPH OXIDASES : FROM PROFESSIONAL PHAGOCYTES TO NON-PHAGOCYTIC CELLS Karl-Heinz Krause University of Geneva Medical School, Switzerland

The phagocyte NADPH oxidase is a key enzyme in the host defense. In my talk, I will address three connected issues : 1. Electron currents generated by the phagocyte NADPH oxidase. Using the patch-clamp technique we were able to demonstrate that the NADPH oxidase generates of electron currents across the plasma membrane (Nature 392: 734-737, 1998). This is a unique type of current is the source of electrons that reduce oxygen, but might also play other roles in phagocyte biology. 2. H+ currents through NADPH-oxidase-dependent and -independent pathways. During NADPH oxidase activation, large quantities of H+ ions are generated, therefore requiring H+ removal mechanisms. Recent results suggest that in addition to is electron transport function, the NADPH oxidase can also function as an H+ channel. Indeed, we can demonstrate that granulocytes posses two types of H+ channels, one directly occurring through the NADPH oxidase gp9 1 phox subunit, another occurring through separate pathways (J Exp Med. 190 : 183-194, 1999). 3. NADPH oxidase homologues in non-phagocytic cells. Over the last years it has become clear that the ability to actively generate superoxide is not limited to phagocytes. Indeed, superoxide generation by nonphagocytic cells has been implicated in a variety of cellular functions from the host defense and regulation of cell growth, to oxygen sensing and regulation of blood pressure. We have identified NOH-1, a homologue of the large subunit of the phagocyte NADPH oxidase flavocytochrome b. The gene is expressed in heart, pancreas, prostate and colon, but absent various other tissues including spleen, thymus and leukocytes. The NOH-1 is a full length homologue of gp91phox and has a fully conserved heme-spanning domain, FAD binding site and NADPH binding site. Taken together our studies demonstrate a complex function of the NADPH oxidase including an active generation of electron currents, as well as a passive flux of H+ ions through the enzyme. Novel NADPH oxidase homologues are expressed in non-phagocytic cells, where they are likely to play an important role in various aspects of cell function.

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307

REGULATION OF MONOCYTE SECRETION OF METALLOPROTEASES BY EXTRACELLULAR MATRIX MOIETIES AND TNFαAlpha Ofer Lider Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel

Tissue injury caused by infection or physical damage evokes inflammatory reactions and events which are necessary for regaining homeostasis. Central to these events is the translocation of from the vascular system into the extracellular matrix (ECM) surrounding the injured tissue. This transition elicits remarkable changes in leukocyte behaviour, as leukocytes adhere to and migrate across ECM before carrying out their effector functions. Growing evidence suggests that, through its interactions with cytokines and degradative enzymes, the ECM microenvironment have a specialized role in providing intrinsic signals for coordinating leukocyte actions. Recent advances also reveal that enzymatic modifications to ECM moieties and cytokines induce distinctive cellular responses, and are likely part of the mechanisms regulating the perpetuation or arrest of inflammation. To test this notions, we have elucidated the interactions of TNFα with ECM and intact ECM glycoproteins, such as fibronectin, on monocyte secretion of metalloproteases (MMP) and found that TNFa binds avidly to the ECM substrates. The bound, and to a lesser degree soluble TNFα, induced the expression, secretion, and activities of MMP from resting human monocytes in an integrin-independent fashion. Thus, the context of the inflammatory milieu appears to affect immune cell behaviour which results in the chemical modification of the matrix.

308

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TRANSEPITHELIAL NEUTROPHIL MIGRATION IS CXCRl DEPENDANT IN VITRO AND IS DEFECTIVE IN IL-8 RECEPTOR MUTANT MICE Gabriela Godaly, Long Hang, Björn Frendéus and Catlarina Svanborg Department of Medical Microbiology, Division of Clinical Immunology, Lund University, Lund, Sweden

This study examined the role of epithelial chemokine receptors for neutrophil migration across infected mucosal sites in vitro and in vivo. Infection of the human kidney A498 cell line with uropathogenic E. coli in vitro increased the expression of CXCRl and CXCR2 as shown by FACS and confocal microscopy after staining with monoclonal antibodies to the CXCRl and CXCR2 receptors. The CXCRl was involved in the IL-8 dependant neutrophil migration across the epithelial cell layers, and anti-CXCR1 antibodies inhibited neutrophil transmigration by >60% (p<0.004). Anti-CXCR2 antibodies had little or no effect. The role of IL-8 receptors for neutrophil migration across Escherichia coli (E. coli) infected urinary tract mucosa was studied in mIL-8Rh knock-out mice. There was a drastic difference between the IL-8R deficient and control mice. In these mice, neutrophil migration into the tissues was delayed and the neutrophils were unable to cross the epithelium into the urinary tract of the mutant mice. These results demonstrate that CXC receptors are required for neutrophil migration across infected epithelial cell layers in vitro and across the urinary tract mucosa in vivo.

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309

EFFECT OF ANTI POLYGLYCEROL PHOSPHATE ANTIBODIES ON IL-6 SECRETION BY MACROPHAGES SENSITIZED WITH LIPOTEICHOIC ACID

+ Ar i Gargir*, Itzhak Ofek*, Haim Tsubery,-Yona Keisari*, Ahuva § Nissim *Dept. Israel; Wolfson

of Human Microbiology, Sackler School of Medicine, Tel Aviv University, ± Dept. of Organic Chemistry, Weizmann Inst. of Science, Rehovot, Israel; §The Centre for Applied Structural Biology, The Hebrew Univ. of Jerusalem, Israel

The pathophysiology of Lipoteichoic acids (LTA) in septic shock caused by gram positive bacteria is controversial because there are conflicting results on its ability t o elicit proinflammatory agents in macrophages. In the present study we selected a clone from a library o f M13 phage, displaying a peptide that corresponds t o a human single chain antibodies (scFv) against the polyglycerol phosphate (PGP) backbone of L T A from S. pyogenes. Two batches were isolated from the anti-PGP clone, one of which was considered multivalent because it displayed about three times the amount of anti-PGP than the other which mas considered monovalent. Non of the LTA preparations employed (S. pyogenes, S. aureus and E. hirae) caused secretion of IL-6 when added t o human monocyte derived macrophages (hMoDM). However, in contrast, when macrophages were sensitized with either LTA preparation and reacted with multivalent phage, they secreted significant amounts of IL-6 while secretion was t o a great extent lower when the LTA sensitized macrophages were sensitized with monovalent phage. Furthermore, the monovalent anti PGP phage inhibited the stimulation caused by the multivalent anti PGP phage. When LTAsensitized macrophages were reacted both with monovalent and multivalent anti PGP phages followed by anti phage antibodies, a marked increase in IL-6 secretion was obtained reaching twice the level o f that obtained with lipopolysaccharide (LPS). W e hypothesize that one mechanism through which Gram positive bacteria cause shock may be via sensitization of macrophages with LTA, followed by crosslinking of the LTA receptors with multivalent anti PGP antibody which rise during gram positive infection. Sensitization and crosslinking is followed by signal transduction, macrophage stimulation and finally, the release of proinflammatory agents ,

310

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EXTRAHEPATIC SYNTHESIS OF COMPLEMENT PROTEINS IN FIBROBLASTS: A CONVENIENT CELLULAR SOURCE TO STUDY COMPLEMENT DEFICIENCY STATES Yitzhak Katz Assaf-Harofeh Medical Center, Zerifin and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

The major site of complement biosynthesis in man is the liver. Many extracellular sources of complement biosynthesis have been identified, including monocytes/macrophages, epithelial cells, amnion and fibroblasts. Using metabolic labeling followed by immunoprecipitation and SDS-PAGE, constitutive fibroblasts synthesized significant amounts of C 1 r, C Is and C 1 inhibitor. IFN-γ induced increased synthesis of factor H and C2 while stimulation with LPS, IL-1 or TNF induced increased synthesis of C3 and factor B. We described a family with two members, AGI and AVG, suffering from C3 deficiency type II The synthesis and regulation of C3 was 1705 normal but due to a mutation in exon 13(G AC to AAC) the mutant product was not secreted. A kindred with lethal hemolytic uremic syndrome (HUS) and low serum levels of C3. Actually it was found that the primary defect is in factor H, a defected that was expressed as reduced levels of secretion of factor H. The functional activities of the mutant factor H, and the relation to the clinical syndrome are still under investigation. Two kindred with C1 inhibitor deficiency type II were also characterized and a reduced ability of the mutant C1 inhibitor from fibroblasts to bind activated Cls, Clr, kallikrein and Hageman factor aXIIa. Currently we are studying a case of recurrent infections and reduced levels factor I.

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311

IL-8 RECEPTOR DEFICIENCY CONFERS SUSCEPTIBILITY TO ACUTE PYELONEPHRITIS 1 1 Björn Frendéus¹, Gabriela Godaly , Long Hang , Diana Karpman¹,² and Catharina Svyborg¹ Departments of ¹Microbiology, Immunology and Glycobiology, Institute of Laboratory medicine, Pediatrics, Lund University, Lund, Sweden

The susceptibility to acute experimental Escherichia coli urinary tract infection (UTI) was compared between knockout (KO) mice lacking the IL-8 receptor (IL-8R), and mutant mice lacking functional alpha/beta T cells (alpha/beta TCR KO), gamma/delta T cells (gamma/delta TCR KO) or B and T cells altogether (RAG-1 KO). Experimental UTI was established in female mice by intraurethral inoculation with E. coli 1177, a human pyelonephritis isolate. A specific loss of resistance was found in the IL-8R KO mice. These mice were unable to clear bacteria from kidney and bladder tissue and showed abnormal neutrophil recruitment to the mucosa. They eventually developed bacteremia and symptoms of systemic disease. These findings in the IL-8R KO mice were in marked contrast to the lymphocyte KO mice; alpha/beta TCR KO, gamma/delta TCR KO and RAG-1 KO mice were equally resistant to UTI compared to controls. The expression of the two high affinity IL-8 receptors CXCRl and CXCR2 was examined on neutrophils from patients and controls. Children with a history of recurrent UTI and episodes of acute pyelonephritis (n= 12) showed reduced expression of the CXCRl receptor compared to resistant controls (n=12), as determined both by flowcytometry (p>0.045) 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