Workshop G: Cytokines and Their Receptors

WORKSHOP G

Cytokines and Their Receptors

The Picower Institute for Medical Research, NY, USA; 1Dept. of Anatomy and Cell Biology, Philipps-University Marburg, Marburg, Germany

G.l Immunohistochemical localization of MIF suggests a role linking the immune and endocrine systems M. BACHER, A. MEINHARDT 1,]. SEITZ 1, and R. BUCALA Macrophage migration inhibitory factor (MIF), one of the first cytokine activities to be described, was originally reported to be associated with delayed-type hypersensitivity reactions and activated T cells. More recent studies have identified MIF to be present preformed in macrophages and in anterior pituitary cells, to be released by endotoxin (LPS) stimulation, and to play an essential role in endotoxic shock. We performed an immunohistochemical survey of MIF in normal rats using affinity-purified anti-MU; antibody. As expected, MIF was localized within the tissue macrophages present in many organs, including the spleen, lung, liver, intestine and skin. Interestingly, large quantities of MIF also were observed within a variety of endocrine cells. These included the glucocorticoid and aldosterone producing cells of the adrenals, the testosterone producing Leydig cells of the testes, and the insulin and the glucagon producing cells of the islets of Langerhans. Additional sites of pre-formed MIF protein included the epithelium of the lung, the prostate, and the uterus. These data provide further evidence that MIF serves to link together a variety of immune and endocrine responses and complements recent data showing that physiological levels of glucocorticoids induce MIF release from different cell types. We are presently examining the whole body distribution of MIF during periods of extreme hormonal imbalance as induced by hypophysectomy or endotoxic shock.

Biochem. Pharmacology, Faculty of Biology, University of Konstanz, Konstanz, Germany

G.2 Granulocyte-macrophage colony-stimulating factor (GMCSF) as a modulator in endotoxic shock in mice: Potentiation of mortality and release of proinflammatory cytokines ]. BARSIG, G. TIEGS, and A. WENDEL GM-CSF stimulates the proliferation and differentiation of bone marrow progenitor cells. In addition the hemopoietic cytokine primes inflammatory responses in mature leukocytes. This is documented in a variety of in vitro studies, but there is little

172 . 25th Meeting of the Society of Immunology information about priming effects of GM-CSF in vivo. We detected GM-CSF in plasma of lipopolysaccharide (LPS) challenged mice with kinetics similar to tumor necrosis factor (TNF), reaching peak levels 2 h after LPS administration. GM-CSF pretreatment induced 100 % mortality in mice challenged by a sublethal dose of LPS. Plasma levels of TNF and interleukin-6 (IL-6) were significantly enhanced. Neutralizing anti-GM-CSF antibodies rendered mice less sensitive towards LPS. TNF- and IL-6-levels were reduced in these mice compared to control animals without antibody treatment. In vitro, a several-fold potentiation of LPS-induced cytokine release by GM-CSF was observed in murine bone marrow cell cultures. These data demonstrate the pro inflammatory capacity of GM-CSF and suggest that the hemopoietic cytokine is an endogenous up-modulator of LPS toxicity.

Sportmedizinisches Institut, Universitat-GH Paderborn, Paderborn, Germany

G.3 Activation of the immune system by acute and repeated physical exercise M. BAUM and H. LJESEN Severe physical exercise is followed by an acute phase reaction comparable to an infectious disease. The granulocytosis reaches its maximum some hours after the exercise; the lymphocytes show a different pattern of change. After heavy exercise first especially the NK-cells increase, some hours later NK-cells decrease below the counts before exercise. In our study about the acute reaction we saw a 5-fold increase of NK-cells (CD 16). There are also changes in the specific immune system. Soluble interleukin-2 receptor (SIL2R) increases significantly 24 hours after exercise. Also interleukin-6 and s-ICAM-l increase after exercise. In repeated exercise (under the conditions of top sports), we found chronically increased activation parameters. The soluble interleukin-2-receptor was as high as in patients with active sarcoidosis and the ICAM-l on monocytes (measured semi-quantitatively) was also increased. Neopterin and the soluble ICAM-l were not increased. The expression of the CD23-antigen on B-lymphocytes was also statistically significantly elevated.

Tumorimmunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany

G.4 Structural analysis of the human APO-l gene I. BEHRMANN, H. WALCZAK, and P. H. KRAMMER APO-1/Fas (CD95), the antigen recognized by the apoptosis inducing monoclonal antibody anti-APO-l, is a cysteine-rich membrane protein belonging to the tumor necrosis factor/nerve growth factor receptor family. Reduced expression of a functional APO-1/Fas (CD95) receptor in lpr mutant mice leads to severe lymphadenopathy and an

25th Meeting of the Society of Immunology . 173 autoimmune syndrome similar to systemic lupus erythematosus in man. A cosmid cAPO-1, isolated from a human genomic library using an APO-l cDNA probe, was used to assign the human APO-l gene to a subregion of chromosome 10 band 10q23 (LICHTER et aI., Genomics 14: 179-180), a region with synteny to mouse chromosome 19 where the lpr mutation is located. The entire gene is present on this cosmid. It spans about 25 kb and consists of nine exons and eight introns. Exon-intron boundaries had splice sites in agreement with consensus sequences. We are currently mapping the transcriptional start site by primer extension and RNAse protection analysis. A genomic fragment including 1.8 kb of the putative promoter region was further cloned into a plasmid containing the reporter gene encoding chloramphenicol acetyl transferase. This plasmid is currently being used to analyze promoter activity in HeLa cells that can be induced to upregulate APO-l surface expression upon treatment with y-interferon. The structure of the promotor with potential regulatory elements will be discussed.

Dept. of !Medicine, 2Immunology and 3 Anatomy, University of Mainz, Mainz; 4Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany

G.S Differential expression of mRNA encoding interleukin-12 p3S and p40 subunits in situ M. BETTE!, S.-c. ]IN 2, T. GERI\li\NN2 , M. K.-H. SCHAFER3, E. WEIHE 3, E. RUDE 2 , and B. FLEISCHER !,4 For biological activity of interleukin-12 (IL-12) the expression of both subunits of IL-12, p35 and p40 within one cell, is required. Moreover, in the mouse the isolated p40-chain specifically inhibits the effects of the IL-12 heterodimer. In the present study we have analyzed by in situ hybridization the expression of the p35 and p40 mRNA in spleens of BALB/c and mutant (Scid, nude) mice, unstimulated and after in vivo stimulation with LPS or SEB. In unstimulated spleens of BALB/c mice p35 and p40 mRNA were only detectable in a few strongly stained single cells, p35 mRNA was expressed in addition weakly in the B cell areas. After injection of LPS or SEB, p40 mRNA was strongly induced in the T cell areas all over the spleen, whereas expression of p35 mRNA increased only slightly and its distribution pattern did not change. Surprisingly, most of the mRNA for p35 and p40 was localized in different areas of the spleen and were produced apparently by different cells. In macrophage-depleted spleens the increased expression of p40 mRNA in response to LPS was reduced but still detectable, demonstrating that other cells besides macrophages can upregulate IL-12 p40 mRNA. Nude mice showed a stronger expression of p35 mRNA, Scid mice lacked the weak p35 staining of the B cell areas but showed a strong basal expression of both p35 and p40 mRNA and a focal response to LPS. These data demonstrate a spatial dissociation of expression of the two chains of IL-12 and are compatible with a regulatory role of the isolated IL-12 p40 chain in vivo. In addition, they indicate that the demonstration of mRNA for both chains of IL-12 in whole tissues or cell mixtures is of little value in the study of functional IL-12.

174 . 25th Meeting of the Society of Immunology Dept. of Immunology and Cell Biology, Forschungsinstitut Borstel, Institut fi.ir Experimentelle Biologie und Medizin, Borstel, Germany

G.6 Platelet-derived interleukin 1 activates vascular smooth muscle cell cytokine production R. BIL, u. SCH0NBECK, E. BRANDT, H.-D. FLAD, and H. LOPPNOW Human vascular smooth muscle cells (SMC) probably contribute to regulation of the immune response, since these cells can produce and respond to cytokines. During vascular injury platelet-derived activators, such as PDGF or interleukin 1 (Ill) may activate SMC. We thus investigated IL6 and IL8 production of SMC in coincubation experiments with platelets. We show here that platelets themselves did not release detectable IL6 or ILS. Unstimulated platelets induced little IL6 or IL8 production of unstimulated SMC. Thrombin-treated platelets increased the IL6 and IL8 release of unstimulated SMC. Preactivation of SMC with TNF for 4 h enhanced the response of the SMC potently. The activation of SMC by platelets was blocked by III receptor antagonist, but not by antibodies directed against PDGF. Furthermore, platelet lysates stimulated fibroblast proliferation. This proliferation was inhibited by polyclonal monospecific ILla and/or ILl~ antibodies. In PCR experiments we also showed that platelets expressed ILla and ILl~ mRNA. No contaminant leukocyte mRNA was present in the preparation as indicated by the lack of IL8 receptor type I mRNA expression. These data suggest that in pathological situations platelets may activate SMC by production of cytokines such as ILL Partially supported by grant Lo 385/4-1 (H. LOPPNOW) and by SFB 367, Projekt C4 (E. BRANDT, H.-D. FLAD) of the Deutsche Forschungsgemeinschaft.

Biochem. Pharmacology, Faculty of Biology, University of Konstanz, Konstanz, Germany

G.7 IL-l induces tolerance against LPS- and TNF-mediated hepatotoxicity. Characterization of TNF-induced liver damage 1. BOHLINGER, M. LEIST, F. GANTNER, P. GERMANN, G. TIEGS, and A. WENDEL We studied endotoxin (LPS-) tolerance in a liver failure model in galactosamine (GaIN)sensitized mice. There, either sublethal doses of LPS, or of its distal pathogenic mediators TNFa or IL-l ~ protect against a second, otherwise lethal dose of LPS. Our experiments showed that IL-l is the most potent inducer of tolerance. The mechanism of IL-l mediated protection involved hepatic protein synthesis and was blocked by coadministration of GaIN with IL-l. Since IL-1 pretreatment induced tolerance not only against LPS- but also against TNF-toxicity, we conclude that the protection conferred by IL-l is confined to events distal to the release of TNF. Liver damage induced by GaIN /TNF was characterized by apoptotic changes which were seen some hours prior to the rise of liver specific enzymes in the plasma. Apoptosis was identified by measurements of cytosolic

25th Meeting of the Society of Immunology . 175 oligonucleosomes via ELISA and agarose gel and by histological examination. Apoptotic cell death was prevented by pretreatment of mice with IL-l, suggesting a relation between apoptosis and subsequent liver damage.

Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Hannover, Germany

G.8 Human C3a-receptor (C3aR) and C5a-receptor (C5aR) expression and functional coupling in y-interferon (y-IFN) induced U937-cells M. BURG, C. RHEINHEIMER, U. MARTIN,J. K('mL, E. C. BOTTGER, W. BAUTSCH, andA. KLos The anaphylatoxic peptides C3a and CSa play a major role in host defense. Both receptors are not expressed on the surface of the native, myelo-monocytic U937 cell-line. Dibutyryl-cAMP (Bt2cAMP) differentiates these cells to a more macrophage-like status, including C3aR- and CSa-expression. We studied the ability of the potent cytokine yIFN to induce expression/coupling of these receptors. In binding studies we were able to show that y- IFN induces the C3aR and CSaR in a time and dose dependent fashion. We determined 20800 ± 7300 CSaRs/cell with a dissociation constant (Kd) of 0.64 ± 0.6 nM instead of 1200000 ± 440000 receptors with a Kd of 16.1 ± 10.9 nM for Bt2cAMP (n = 3). Surprisingly, we found the EDSOs for the CSa-dependent free cytosolic Ca2+-response using a Fura2 fluorescence-assay in reverse order: approx. 0.5 nM for y-IFN versus 0.1 nM for Bt2cAMP. We speculate that: 1. the observed low affinity of the CSaR for Bt2cAMP-induced U937 cells can be explained by a relative lack of G-proteins due to the non-physiologically high receptor number. 2. The different EDSOs can be explained by the expression and coupling to different G-proteins in both induction-schemes. In contrast, no Ca2+-response could be observed for C3a in y-IFN induced U937 cells. Further analyses of the physiological link of y-IFN and anaphylatoxic receptor expression and response will be given at the meeting.

1Arbeitsgruppe fur Infektabwehrmechanismen, Lehrstuhl fur Medizinische Mikrobiologie und Immunologie, Ruhr-Universitat, Bochum; 2Theodor-Boveri-Institut fur Biowissenschaften, Wurzburg; 3Behringwerke, Marburg, Germany; 4Dept. of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati Medical Center, Cincinnati, Ohio, USA

G.9 The role of IL-4, IL-4 mutant protein (Y1240) A. Soluble IL-4 receptor on proinflammatory cytokines in a TSST-1 infection model A. DRYNDA 1, B. KONIC!, W. SEIIAL])2, H. ENSSLE3, F. SEILER 3, P. F. BONVENTRE\ and W. KONIG! The staphylococcal superantigen TSST -1 is involved in several disease processes and is a potent inducer of proinflammatory cytokines. Cytokines have supportive as well as counteracting effects. In this regard IL-4 suppresses proinflammatory cytokine release.

176 . 25th Meeting of the Society of Immunology We studied the effect of IL-4 in the absence and presence of TSST -1, with regard to cytokine expression (IL-l, TNF-u, IL-6, IL-S) and cytokine release (IL-S). We show that IL-4 suppressed cytokine expression (IL-l, TNF-u, IL-6, IL-S) by PCR and cytokine release (IL-S), alone and in cell stimulations of IL-4 and TSST -1 treated cells. Addition of sIL-4R (0.1-1 [tg) as well as IL-4M (YI24D) (0.5-10 nM) showed differential effects. With sIL-4R the IL-4 induced suppression is abolished for both systems, while with IL-4M (YI24D) negligible effects are observed. The data suggest a potential therapeutic application of sIL-4R in IL-4 induced immunosuppression.

Institute of Clinical Immunology, FSU Jena, Jena, Germany

G.10 On time of culture, changes of levels of interleukins 6 and 10, and production of immunoglobulins G and M in cultures of humanPBMNC A. FRITSCH, U.JUNKER, H. VOGELSANG, and L. JAGER In the literature there is a lot of controversial information about how cells are cultured to measure e.g. immunoglobulin (Ig) production, differentiation or proliferation in response to growth factors. However there is nearly no information about the stability andlor consumption of growth factors in the culture medium. To establish which timecourses and re-feeding regimen might be useful and sensible, we cultured peripheral blood mononuclear cells (PBMNC) of several healthy donors with IL6 (1000 U/ml) and ILlO (10 ng/ml) for up to ten days and determined levels of IL6, ILlO, IgG and IgM as well as cell viability for each successive day. Our results show that a) IL6 is output by PBMNC at begin of the culture period in significant amounts and it is not consumed during culture; b) III 0 levels decrease over time so re-feeding seems desirable when stable levels are to be obtained; c) Ig-production is independent of at least IL6- and ILlO re-feeding and useful levels of IgM and IgG are reached between day 5 and day 7 whereafter d) cell viability rapidly decreases to levels that make any measurement senseless. We conclude that to obtain comparable results, culture conditions must be very accurately tested for each individual system.

University of Konstanz, Biochemical Pharmacology, Konstanz, Germany

G.ll The cytokine response of human whole blood T. HARTUNG, A. SAUER, and A. WENDEL Human whole blood incubations are an easily accessible source to measure human leukocyte cytokine release. This approach minimizes preparation artefacts and allows interactions between different leukocyte populations. Here, cytokine release was initi-

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ated by lipopolysaccharide, lipoteichoid acid, muramyl dipeptide, Streptococcus enterotoxin A and B, streptolysin 0, heat killed Staphylococcus aureus, phytohemagglutinin, complement factor Sa and phorbol myristate acetate. Kinetics and concentration dependence of stimulated interleukin-I (IL-I), IL-2, IL-3, IL-4, IL-6, IL-8, IL-I 0, tumor necrosis factor (TNF) a and ~, interferon a and y, MIP-Ia, leukemia inhibitory factor, granulocyte colony-stimulating factor as well as granulocyte macrophage colonystimulating factor were assessed. In addition, soluble receptors of TNF, IL-2 and IL-6 as well as IL-I receptor antagonist were determined. Each stimulus led to a unique cytokine pattern with an individual concentration dependence and kinetics. This system is suitable to assess the immunocompetence of patients.

Institute of Molecular Pharmacology, Medical School Hannover, Hannover, Germany

G.12 Regulation by phosphoprotein phosphatases of interleukinl-induced activation of MAP kinases in Hela cells A. HEINER, K. RESCH, and M. SZAMEL Interleukin-I (IL-I) exerts its biological effects via cell surface receptors. Human HeLa cells express Type I IL-I receptors as revealed by reverse transcriptase-polymerase chain reaction. The specific activity of MAP kinase(s), partially purified by ion exchange chromatography, was increased 7-fold by epidermal growth factor (EGF) and 2.s-fold by IL-I-~, respectively. In sharp contrast, no activation by phorbol-12-myristate-13-acetate (PMA) of MAP kinase(s) was detectable, as measured by phosphorylation of myelin basic protein (MBP). These data suggest that protein kinase(s) C are not involved in the IL-I induced protein kinase cascade in HeLa cells. The specific inhibitor of phosphoprotein phosphatases, PPI and 2A, okadaic acid enhanced IL-I-induced activation of MAP kinase(s), suggesting the involvement of SerlThr-specific phosphatases in the regulation of the activation of MAP kinase(s) in HeLa cells. The results suggest that both protein kinases and phosphoprotein phosphatases are acti vated via the type I IL-I receptor.

Universitatskinderklinik, Gottingen, Germany

G.13 Immunocytochemical studies of cytokine production in an in vitro granuloma model D. HEINEMANN and M. GAHR The aim of our study was to elucidate the role of cytokines in granuloma-formation. We studied granuloma formation using an in vitro model with human monocytes depleted of lymphocytes exposed to heat-killed Candida albicans which were spotwise fixed on the surface of culture wells. In this system the cells accumulated, proliferated (as shown by incorporation of BrdU following immunochemical detection) and formed multinucleated

178 . 25th Meeting of the Society of Immunology giant cells (MGC). In previous studies in supernatants of the whole population of MNL cultivated as mentioned above IL-2, -4 and IFN-y were not detectable in contrast to ILla/~, and TNF a. We now examined the local distribution of cytokine production immunocytochemically using cytokine specific antibodies. We found an almost locally restricted production of IL-l~, IL-6, M-CSF and TNF-a in the initial phase (first 3-4 days) of granuloma formation mainly by phagocytosing monocytes. In some cases TNFa and M-CSF were also found in MGC. IFN-y could not be found in cells cultivated in the presence of Candida albicans. However, in MNL stimulated with ConA IFN-y was found immunocytochemically. These results confirm our idea that IFN -y is not critically involved in this kind of granuloma formation.

Biochem. Pharmacology, Faculty of Biology, University of Konstanz, Konstanz, Germany

G.14 Protection from LPS shock by xanthine derivatives is accompanied by a modulated cytokine response S. JILG and A. WENDEL Since specific strategies of sepsis therapy (e.g. anti TNF antibodies) have failed to improve sepsis outcome, multifunctional drugs such as xanthine derivatives, which inhibit cAMP and cGMP specific phosphodiesterases, have gained importance in drug development. Here we show that A 802715 (1-[5-hydroxy-5-methyl] hexyl-3-methyl-7propyl-xanthine, Hoechst AG), which protects against LPS and its distal mediator TNF in various in vivo and in vitro models, modulates LPS induced cytokine release in a murine shock model by attenuating TNFa and IFNy in plasma and IL-l in peritoneal lavage. On the contrary, the antiinflammatory cytokine IL-IO as well as IL-6 were increased 2 h after LPS challenge. G-CSF levels in plasma were diminished 4 h after challenge. The xanthine derivative Pentoxifylline did not inhibit IFNy production nor did it increase IL-6 release. Our findings suggest that the different PDE inhibition patterns of drugs may be the molecular basis of the differential modulation of cytokine responses to septic stimuli.

Medizinische Klinik I, Universitatskliniken des Saarlandes, Homburg, Germany

G.1S The soluble form of C030, sC030, is released from activated lymphocytes W. JUNG, B. MEROTH, N. FADLE, S. KREITER, A. GAUSE, and M. PI'REUNDSCHUH Expression of the membrane form of CD30, a member of the NGFR family, has been found on activated or virally transformed lymphocytes of either T or B cell origin and on tumor cells of a restricted number of lymphomas. The membrane form of CD30 (120

25th Meeting of the Society of Immunology . 179 kDa) gives rise to a soluble form of the receptor, sCD30 (88 kDa), that seems to originate from the extracellular part of the antigen. Release of sCD30 from CD30 positive cells has only been observed following viral or malignant transformation, so far. Recently, we have established an improved ELISA for the detection of sCD30. The detection limit of this assay has been shown to be at least 250 pg CD30/mi. Use of this ELISA has enabled us to demonstrate that sCD30 is released from PBL following stimulation with PHA. sCD30 is detected after 3 to 4 days of culture and the amount of sCD30 in the supernatant correlates with the fraction of CD30 positive cells as revealed by F ACS analysis. Addition of the PKC activator TPA to PHA blasts on day 4 of culture resulted in further enhancement of both formation of sCD30 and cell surface expression of CD30. TP A also mediated an increase in the formation of sCD30 by a variety of CD30 positive cell lines. In these cells however, the increased formation of sCD30 was accompanied by a rapid decrease of cell surface expression which could be detected within one hour. Addition of the PKA activator forskolin mediated an increase of CD30 surface expression in CD30 positive cell lines and PHA blasts. This increase of cell surface expression however did not lead to an increased formation of sCD30. Possible mechanisms responsible for the different regulation of expression of CD30 and release of sCD30 in these cells are being studied.

University of California, San Diego; Dept. of Medicine, La Jolla, CA, USA

G.16 Expression of ILA (induced by lymphocyte activation), a novel member of the NGFITNF receptor family and human 4-1 BB homologue, in mesenchymal cells is differentiation-dependent

J. VAN KEMPIS, H. SCHWARZ, and M. LOTZ This study reports on the expression of ILA, a novel member of the human NGF/TNF receptor family and probable human homologue of the murine 4-lEB, in cells of mesenchymal origin. Resting or activated human synovial and skin fibroblasts did not express ILA mRNA as measured by a sensitive RT-PCR. In contrast, in primary cultures of human articular chondrocytes ILA was inducible by IL-1~, TNFa, LIF, IFNy and LPS but not by TGF~. IL-l stimulated chondrocytes expressed the 4.8, 4.0 and 1.9 kb isoforms of ILA mRNA which had been observed in human lymphocytes and additional isoforms at 3.2, 1.5 and 1.2 kb. ILA mRNA was detectable as early as 4 h and for> 80 h after IL-l stimulation. Low levels of ILA mRNA were induced by the second messenger agonists PMA, cAMP and the calcium ionophore A23187. TGF~ and dexamethasone inhibited the IL-l induction of ILA. Cyclohexamide alone lead to accumulation of ILA mRNA in primary chondrocytes while the combination of IL-l and cyclohexamide resulted in superinduction. This suggests that expression of ILA mRNA is under control by a constitutively synthesized labile protein and that the induction by IL-l is independent on de novo protein synthesis. ILA mRNA is translated and expression of the membrane protein is increased by IL-1 and TNFa but not by TGF~. Further analysis of the cell type specific expression of ILA revealed that with prolonged subculture and dedifferentiation to a fibroblastoid phenotype ILA was no longer inducible by IL-l in chondrocytes. ILA mRNA was however induced in fibroblasts and subcultured, dedifferentiated chondrocytes by cyclohexamide and at higher levels by the combination with

180 . 25th Meeting of the Society of Immunology IL-l. In conclusion, ILA is not only expressed in the immune system but also in mesenchymal cells. ILA expression is induced by specific stimuli and is modulated by the cellular differentiation status. ILA expression in chondrocytes and fibroblasts is at least in part controlled by a negative regulator which may contribute to cell-type specific gene expression during mesenchymal cell differentiation.

Med. Mikrobiologie Immunologie, AG Infektabwehr, Ruhr-Universitat Bochum, Bochum, Germany

G.17 The role of cytokines, hematopoietic growth factors and heat-shock proteins in mechanisms of cellular protection M. KOLLER, P. WACHTLER, T. HENSLER, and W. KONIG The radioprotective effect of endotoxin, distinct lipid mediators, IL-l and TNF-a has been described in murine models. Due to their specific properties distinct heat-shock proteins (hsps) are known as protective cellular proteins, therefore, we studied the possible involvement of hsps in cellular protection towards radiation injury or challenge with cytolytic bacterial toxins in human leukocytes using 35S-methionine-uptake, Western blot, Northern blot and polymerase-chain-reaction (PCR). Expression of hsp72 was observed in trauma and radiotherapy patients. Incubation of leukocytes with lipid mediators (HETEs), cytokines and hematopoietic growth factors (IL-6, TNF-a, GMCSF, and IL-3) led to the induction of hsp72. In contrast, IL-8 and IL-4 did not induce the cellular hsp-response. Preincubation of leukocytes with IL-6, TNF-a, or GM-CSF led to an enhanced resistance towards ionizing radiation or cytolytic toxins. These data provide evidence for the protective role of heat-shock proteins during inflammatory reactIOns.

lMed. Mikrobiologie Immunologie, AG Infektabwehr, Ruhr-Universitat Bochum, Bochum, Germany; 2Sanofi Elf Bio Recherches, France; 3Universitiit Wiirzburg, Wiirzburg; 4Behring Werke Marburg, Marburg, Germany

G.1S Comparison of Il-4 and Il-13 effects on cytokine, and IgE regulation interaction with the staphylococcal enterotoxin B

IL-4 and IL-13 have been described as Th2 cell derived cytokines which playa key role in IgE synthesis and cytokine expression. Here, we compared the effects of recombinant human IL-13 and IL-4 on immune responses (proliferation, cytokine production, CD23 expression, IgE synthesis) from peripheral blood mononuclear cells (PBMC) to the staphylococcal enterotoxin B (SEB). Our results show that IL-4 as well as IL-13 induced a significant increase in CD23 expression (up to 41 % binding), IgG, IgA, and IgE synthesis as compared to unstimulated cells (0.5 % binding) which were downregulated

25th Meeting of the Society of Immunology . 181 by SEB below baseline levels (untreated PBMC). Concomitant to an increase in CD23 expression the soluble CD23 receptor (sCD23) was up regulated by IL-4 and by IL-13. In contrast to IL-4 the IL-13 induced sCD23 was not diminished by SEB indicating different mechanisms for sCD23 cleavage. Both IL-4 and IL-13 downregulated IL-8 mRNA expression and protein secretion in untreated as well as SEB treated PBMe. PCR analysis revealed for IL-4 and IL-13 a stimulatory effect on CD40 ligand mRNA expression in untreated as well as in SEB treated PBMe. Soluble IL-4 receptor and an IL4 mutant protein had antagonistic activities only on IL-4 induced biological effects. Moreover, in the presence of soluble IL-4 receptor the IL-13 induced effects were significantly augmented. These results indicate that IL-13 shares important immunoregulatory activities with IL-4; however IL-13 activities are not mediated by IL-4R.

Dept. of PathologyIT umorimmunology and IDept. of Internal Medicine I, University of Regensburg, Regensburg, Germany

G.19 Comparison of in vivo efficacy of anti-mouse TNF antibodies versus TNF receptor-Ig constructs H. KRAHT, M. HAFNER, W. FALKI and D. N. MANNEL The extracellular domains of mouse p55 and p75 TNF receptors were cloned by RT-PCR and PCR, respectively. Plasmids were constructed that allowed the expression into the supernatant of transfected cells of chimeric proteins consisting of the respective receptors covalently linked to a murine IgG 1 Fc heavy chain. Stably transfected mouse L 929 cells were cloned by limiting dilution and the expressed fusion protein was purified by protein A affinity chromatography. The protein preparations were adjusted to equal concentration of the fusion protein by ELISA using the Fe part as common parameter. The neutralizing capacity of the two fusion proteins was compared in the L 929 cytotoxicity assay in the presence of actinomycin D. The fusion proteins exhibited similar TNF neutralizing capacity when rmTNF or natural mouse TNF from LPS-stimulated RA W264.7 cells was used for L cell killing. Furthermore, the protective activity of the two fusion proteins was compared to the protection conferred by neutralizing monoclonal antibodies to mouse TNF in vivo in LPS-treated mice. LPS-induced hypothermia and survival were used as reliable and quantitative readout for the protection in this endotoxin shock model. Results and possible implications for the usage of the equivalent human fusion proteins in therapy of septic patients will be discussed.

182 . 25th Meeting of the Society of Immunology lNeurologische Universitatsklinik, 2Medizinische Poliklinik, and 3Theodor-BoveriInstitut fur Biowissenschaften (Biozentrum) der Universitat Wurzburg, Wurzburg, Germany

G.2D Mutational analysis of human interleukin-4: Identification of crucial amino acids for receptor binding and signal generation N. KRUSE!, H.-P. TONy2, and W. SEBALD 3 Interleukin-(IL-)4 is a cytokine involved in growth and differentiation of cells of various lineages. It induces the expression of several cell surface markers on target cells, such as CD23, MHC II complex and IL-4 receptor. The mature protein consists of 129 amino acid residues and binds to its Receptor (IL-4R) with a Kl) of about 100 pM. A set of IL-4 mutants was constructed by site-directed mutagenesis and expressed in E. coli. Receptor binding affinities and biological activities of the IL-4 variants were analyzed by a binding/competition assay using the Raji B cell line or by performing a eH]thymidine incorporation test employing peripheral human T cells. Finally, we quantitatively assayed induction of CD23 expression on splenic human B cells. We found that amino acids E9 (helix A) and R88 (helix C) are part of the binding site for the cloned IL-4R. Amino acids within helix D are crucial the signal generation. Substitution of residues R12l, Y124, and S125 by aspartic acid reduces or abolishes biological activity without affecting receptor binding to a major extent. IL-4 variant Y124D showed the characteristics of an IL-4 antagonist in the T cell-proliferation test, while exhibiting some residual activity in the CD23 expression system. Our results show that the structural requirements for receptor binding and signal generation by IL-4 are not identical. This may have implications for the as yet poorly understood mechanisms involved in the activation of receptors of the hematopoietin receptor superfamily.

Biochem. Pharmacology, Faculty of Biology, University of Konstanz, Konstanz, Germany

G.21 The cytokine response in macrophage and T cell-dependent inflammatory mouse models S. KOSTERS and G. TIEGS Cytokines play an important role as mediators of inflammatory reactions. We have employed an ELISA technique to investigate the time dependency of circulating cytokines such as interleukin-4 (IL-4), IL-6, IL-lO, interferon y (IFNy) and tumor necrosis factor (TNF) in four different mouse models; Con A induced T cell-mediated liver injury, lipopolysaccharide (LPS) induced septic shock, liver injury caused by LPS in Dgalactosamine-(GaIN-)sensitized mice, as well as T cell-mediated shock triggered by staphylococcal enterotoxin B (SEB) in GaiN-treated mice. IL-6, IFNy and TNF were detectable in all four models with various time courses and at different concentrations. Only very low concentrations of IFNy were measured in GaIN/LPS liver failure. IL-4 was only found in the T cell-dependent systems as opposed to IL-lO, which was detectable only in LPS-induced shock and GaIN/LPS liver injury.

25th Meeting of the Society of Immunology . 183 Institut fur Immunologie, Johannes-Gutenberg-Universitat, Mainz, Germany

G.22 Production of IL-12 by macrophages in vitro and by spleen cells ex vivo D. LAUBERT, S. FISCHER, s.-c. JIN, J. SZELIGA, E. RODE, and T. GERMANN IL-12 is a recently discovered heterodimeric cytokine with multiple effects on T cells such as mediating Th1-development and providing costimulatory signals for Th1 cells. It enhances proliferation and cytokine production, for instance IFN -y secretion by T and NK cells. One possible source of IL-12 are macrophages. Macrophages were generated by culturing bone-marrow cells for two to three weeks in the presence of GM-CSF. They release IL-12 when stimulated for 24-48 hours with bacteria/bacterial products such as heat-killed Listeria, Tuberculin and LPS or, alternatively, in the interaction with Th 1-cells but not Th2-cells. Highest concentrations of IL-12 were obtained by macrophages activated with a combination of LPS and Listeria or by a coculture of Th1 with macrophages and LPS. IL-4 or TGF-B inhibited bacteria-induced IL-12 slightly. In contrast IL-10 could abrogate IL-12 production almost completely. Moreover, ex vivo experiments were performed with spleen cells of BALB/c and C3H/He mice pretreated in vivo with LPS. These cells were incubated with bacteria/bacterial products. Herein Listeria were most effective in promoting IL-12-production. The regulation of IL-12synthesis was monitored 1. by RT-PCR for the mRNA of both chains of the heterodimeric IL-12-molecule and 2. by an IL-12-bioassay: a Th1-line produces IFN-y induced by IL-12 in a dose-dependent manner. IfN-y is determined in a sandwich-ELISA.

Biochem. Pharmacology, Faculty of Biology, University of Konstanz, Konstanz, Germany

G.23 Tumor necrosis factor induces programmed cell death in murine hepatocytes in vivo and in vitro under transcriptional arrest M. LEIST, F. GANTNER, 1. BOHLINGER, G. TIEGS, P. GERMANN, and A.

WENDEL

Tumor necrosis factors (TNf) not only are powerful immunomodulators but also considered as distal mediators of septic shock. A convenient model of septic organ failure is liver injury, caused by LPS or TNF in galactosamine-sensitized mice. Here we describe the metabolic conditions required for a direct toxicity of TNF towards murine hepatocytes as well as the characteristics of this cell death. Only after treatment with transcriptional inhibitors TNF induced a concentration-dependent toxicity and morphological changes typical of apoptosis in cultured hepatocytes. DNA-fragmentation preceded cell death as characterized by LDH-release. In corroboration of these in vitro findings formation of apoptotic bodies, chromatin condensation and DNA-fragmentation into oligonucleosomal fragments also preceded hepatocyte death in vivo in mice following an injection of actinomycin D plus TNF. Thus the cytokine TNf may directly trigger programmed cell death under the metabolic conditions of transcriptional arrest and thus initiate organ damage by this mechanism.

184 . 25th Meeting of the Society of Immunology Institute of Clinical Immunology and Rheumatology, Dept. of Medicine III, University Erlangen-Niirnberg, Erlangen, Germany

G.24 Anti-TNFa-antibodies inhibit mixed lymphocyte reaction in synergism with cyclosporine A. H.-M. LORENZ, W. WOITH, M. HERRMANN, T. GEILER,J. STELL, C. ANTONI, J. R. KALDEN, and M. MANGER TNF-a is a potent proinflammatory cytokine expressed by a variety of immunocompetent cells. In the present study we opted to investigate its role in the initiation of mixed lymphocyte reaction (MLR). PBMC of healthy donors were irradiated (3000 rad) and added to non-irradiated PBMC of another healthy donor along with medium, a neutralizing, humanized TNF-a antibody (10 ~g/ml), or human IgGI as control. DNAsynthesis was measured after 8 days, supernatant collected after 7 days, and RNA isolated after 5 days for RT-PCR based semiquantification of specific cytokine transcripts. We found a potent inhibition of MLR under conditions with TNF-a blockade, but no comparable inhibition in control experiments. Washing experiments revealed that TNF-a-antibodies were effective mainly in the first four days after initiation of the MLR. Adding the antibody later than 4-5 days after initiation of the MLR showed a decreasing efficacy in the inhibition of MLR. Adding both cyclosporine A and TNF-a showed synergistic effects of TNF-a-blockade with cyclosporine A. Thus TNF-a expression seems to be important for the initiation of MLR.

Institute of Immunology, Philipps-University, Marburg, Germany

G.25 Chemokines and viral infection: preferential induction of ~-chemokines by Influenza A-infected macrophages R. G. MEYER, E. RISCHKOWSKY, D. GEMSA, and H. SPRENGER Chemokines are chemoattractant cytokines that are divided into two subfamilies. Members of the a-subfamily, such as IL-8 or GRO-a, act primarily on neutrophils, whereas members of the ~-subfamily, e.g. MIP-la and RANTES, preferentially attract monocytes and T cells. Typically, an infiltrate within virally-infected tissue consists of mononuclear leukocytes, such as macrophages and T lymphocytes while polymorphonuclear neutrophils seem to be of minor importance. To understand the underlying mechanisms which selectively attract mononuclear effector cells, we infected the human monocytic cell line MonoMac 6 with Influenza A virus strain APR8 (2 MOl) and determined the pattern of chemokine production. After 2, 4, 8, 16 and 24 hours of infection, release of the a-chemokines IL-8 and GRO-a, and of the ~-chemokines MIPla and RANTES was analyzed by specific ELISA techniques. Most interestingly, our results suggest that an influenza A virus infection primarily stimulated the production of ~-chemokines, whereas a-chemokine release remained unaffected and did not differ from that one of non-infected control cells. Thus, specific release of MIP-1a and RANTES, which both preferentially attract monocytes and T lymphocytes, could help to explain the predominant development of a mononuclear infiltrate in virally infected tissue.

25th Meeting of the Society of Immunology . 185 !Institute of Medical Immunology, lDept. of Nephrology, and 3Institute of Virology, Charite, Humboldt University Berlin, Berlin, Germany

G.26 Association between CMV-related graft injury and circulating cytokine-producing C08+ memory cells S. ODEHAK!M!, F. KFRN 2, H. NUGat, P. REINKE2, and H.-D. YOLK!

s. PROSCH 3, R. EWERT2,

Several groups reported on the association between CMV infection and graft injury. Cytofluorometric analysis of PBMC from> 400 long-term renal allograft recipients demonstrated an increased proportion of CD3+8+57+LFA-l(CDlla/18)-«bright>, T lymphocytes in 20 % of these patients. They also showed a significantly higher incidence of CMV infection at both DNA and RNA levels using PCR technique and a four-fold increase in graft lost. To study the mechanism of this phenomenon, we investigated the cytokine gene expression pattern (IL-l to IL-I0, IFN-y, TNF-u, granzyme A) of PBMC from transplant patients with CMV-related graft injury, acute rejection, and without complications as well as from healthy donors by a novel semiquantitative PCR technique. We noticed 50 to 1000 times higher levels of IFN-y transcripts in PBMC from allograft recipients with CMV-related graft injury as compared with control patients. In contrast to all other studied cytokines the IFN-y mRNA was restricted to the «memory»-typ CD8+ T cell subset as shown by separation experiments. This subset could be further separated by three-color cytofluorometric analysis into CD57+ and CD57- cells. However, we found no differences in cytokine gene expression between these two subsets. The increased levels of IPN-y transcripts of CD8+ «memory»-type T cells from patients with persistent clinically latent CMV infection may explain the poor prognosis of CMV-related graft injury.

!Institute of Medical Immunology, 2Dept. of Nephrology, Charite, Humboldt U niversity Berlin, Berlin, Germany

G.27 Elevated IL-2 gene expression in core biopsies from renal allograft recipients with late acute rejection S. ODE HAKIMt, P. REINKE 2 , E. FIETZELt, K. VOGTt, and H. D. YOLK I Cytokines have long been recognized to be involved in the pathogenesis of acute graft rejection. Several groups reported a correlation between IL-2 gene expression and acute graft rejection in the early phase after transplantation. However, very little is known about the cytokine profile in acute rejection of the late phase. Using a semiquantitative RT-PCR technique we investigated the cytokine gene expression pattern in PBMCs (n = 15) and core biopsies (n = 11) from long-term renal allograft recipients with histologically proven acute rejection. IL-4, IFN-y and IL-I0 mRNAs were expressed in most patients at high levels in both peripheral blood and the graft. In contrast to this, IL2 mRNA was only detectable in core biopsies but not in PBMCs of all but 2 patients who had neglected to take their cyclosporine A persistently. We assume that the restimulation of infiltrating effector T lymphocytes and the probably lower cyclosporine A concentration might be responsible for the higher levels of IL-2 message within the graft.

186 . 25th Meeting of the Society of Immunology DSM - Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany

G.28 IL-4 inhibits the LPS-induced expression of C014 and monocyte specific esterase mRNA in MONO-MAC-6 cells H. QUENTMEIER, K. KOLSDORf, M. ZABORSKI, and H. G. DREXLER The human MONO-MAC-6 cell line expresses the monocyte-associated differentiation markers CD14 and monocyte specific esterase (MSE) and can be stimulated by lipopolysaccharide (LPS) to produce high mRNA levels of monocyte-related cytokines. This similarity to human peripheral blood monocytes (PBMo) renders this cell line a promising model for studies of monocyte activation and differentiation. Here we compared the effects of interleukin-4 (IL-4) and LPS on the expression of CD14, MSE and tumor necrosis factor a (TNFa) mRNA on PBMo and the MONO-MAC-6 cells. IL-4 inhibited the LPS-induced expression of TNFa mRNA and downregulated the LPS receptor CD14 in PBMo, but not in the cell line. Up regulation of CD14 and MSE mRNA expression in MONO-MAC-6 cells by a two-day incubation with LPS were inhibited by IL-4. Obviously long-term treatment with LPS made the cells responsive to IL-4. The increase in responsiveness was not due to IL-4 receptor (IL-4R) upregulation, as neither LPS nor IL-4 influenced the constitutive expression of the IL-4R.

1Institut fur Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Hannover; 2Institut fur Biochemie, RWTH Aachen, Aachen, Germany

G.29 Epitope-tagged C5a-receptor (C5aR) mutants identify intramembraneous amino acid residues important for recep-

tor folding

u. RAFFETSEDER\ C. LEHFELDTl, D. ROPER2, A. KLOS1,J. KOHL\ A. WOLLMER2, and W. BAUTSCH 1

The human C5aR belongs to the family of G-protein coupled receptors which are characterized by a common motif of seven transmembrane helices. Intramembraneous amino acid residues are supposed to be important determinants of receptor folding, to form part of the ligand binding site and to participate in the signal transduction pathway. We have analyzed several intramembraneous amino acid positions deduced from computer modelling studies for altered ligand binding and/or expression by site-directed mutagenesis experiments of a C5aR eDNA clone, transient expression of the mutants in COS cells and competitive binding experiments. Correct localization of the C5aR mutants on the cell surface was checked by immunofluorescence with a monoclonal antibody recognizing an epitope which had been introduced into the N-terminus of the C5aR eDNA clone. Introduction of this epitope tag did not alter expression and binding behavior of the wild type C5aR. Epitope-tagged C5aR mutants (e.g. Asp282 --> Ala) were identified which exhibited only a residual ligand binding capacity (with a wild type) Kd of ca. 2 nM!) while immunofluorescence analysis revealed full expression on the cell surface. Replacement by other residues, however, restored full ligand binding capacity (e.g. Asp282 --> Asn), thus indicating a major impact on receptor folding.

25th Meeting of the Society of Immunology . 187

lZ entrum fur Infektionsforschung, Universitat Wurzburg, 2Hautklinik der Universitat Wurzburg, Wurzburg, Germany; 3UNAM Medizinische Fakultat, Mexico City, Mexico; 4Institut fur Klinische Mikrobiologie, Universitat Erlangen, Erlangen, Germany

G.30 Chemokine expression in lesions of patients with the localized and the diffuse form of American cutaneous leishmaniaSIS

Leishmaniasis is caused by intracellular protozoan parasites of the genus Leishmania. Dermal macrophages playa pivotal role in the disease process because they serve as host cells for the parasites and as antigen-presenting cells for induction of the immune response. The high abundance of macrophages (40-70°/,) of dermal cells) suggests that active processes of chemotaxis may be involved. We have studied the expression of chemokines of the C-C subfamily in localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL). Using in situ hybridization and immunhistochemistry we investigated the expression of the chemokines macrophage chemoattractant protein I (MCP-I), the factor «Regulated on Activation, Normal T expressed and Secreted» (RANTES), 1-309, macrophage inflammatory protein Ia (MIP-Ia) in lesions of mexican patients with LCL (n=II), and DCL (n=4). We could detect high levels of MCP-I mRNA and protein particularly in LCL. MCP-l mRNA was localized mainly in the area of dermal infiltration, but also in the basal keratinocytes above the dermal infiltrate. In addition, MIP-l a was detected in clusters within the dense cell infiltration. In contrast, RANTES and 1-309 m-RNA expression was minimal. Our data suggest that high levels of MCP-I and MIP-la are responsible for the high density of dermal macrophage which may critically influence the disease processes in LCL and DCL. In addition, our data indicate that keratinocytes are also actively involved in inflammatory processes of cutaneous leishmaniasis.

Universitats-Hautklinik, Kaln, Germany

G.3l IL-13 induces type I collagen synthesis in human dermal fibroblasts M. Raux, S. SCHACHTSCHNEIDER, B. ECKES, T. SCHAHER, H. SMOLA, S. SOLLBFRG, and T. KRIEG The interaction between T lymphocytes and fibroblasts is critical in the development of fibrosis in sclerotic diseases. There is evidence that the deposition of collagen type I (Col I) in scleroderma is regulated by T cell derived cytokines. Thus, transforming growth factor f3I (TGF~I) and interleukin-4 (lL-4) could be shown to induce Col I synthesis in human dermal fibroblasts. Interleukin-13 (IL-13) is secreted by activated T lymphocytes and shares some biological activities with IL-4 in the modulation of B cells and monocytes. Moreover, it could be shown that the receptors for IL-13 and IL-4 probably share a signal transducing element. We investigated whether IL-13 is able to induce Col I synthesis in human fibroblasts. Primary fibroblasts were derived from skin biopsies and grown in

188 . 25th Meeting of the Society of Immunology monolayer cultures. At near confluence they were incubated in DMEM, 1 % FCS, 50 ftgl ml ascorbic acid in the presence of various concentrations of E. coli derived recombinant human IL-13. At 24 h IL-13 induced Col I mRNA transcription as determined by Northern hybridization with a Col ul(I) cDNA probe. It could be demonstrated by densitometrical analysis that at a concentration of 10 ng/ml, IL-13 is at least as effective in the induction of Col I mRNA as TGF~1 or IL-4, respectively. This was substantiated on the protein level by the determination of incorporated L-(2,3)-3H -proline. IL-13 did not influence the proliferation of fibroblasts. Given the relative abundance of IL-13 transcripts in activated T cells, IL-13 could be a major regulator of Col I synthesis in vivo and might also participate in the dysregulation of Col I in fibrotic diseases.

Institut fur Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Hannover, Germany

G.32 Cloning and expression of recombinant human C3a in Escherichia coli C. SCHIEBL, M. GROVE, J. KOHL, A. KLOS, and W. BAUTSCH The human C3a anaphylatoxin is an important inflammation promoting agent. In order to study structure-function relationships of this protein by site-directed mutagenesis and to probe the human C3a receptor by appropriately labelled derivatives we have started to produce recombinant human C3a (rhC3a). The coding sequence was amplified from total RNA of the U937 cell line by a combined reverse transcription-polymerase chain reaction approach and cloned into several expression vectors. The sequence was successfully expressed as a fusion protein with the maltose-binding protein as detected by C3aspecific mAb. Further optimizations of this construct included insertion of an additional cysteine residue and an epitope tag flanked by thrombin and enterokinase cleavage sites, respectively, at the N-terminus of the C3a sequence. RhC3a could be successfully cleaved off from this fusion protein by thrombin. In contrast to a previous report (1), however, we were not able to produce any rhC3a when the sequence was directly expressed from an ATG initiation codon. 1. FUKUOKA et al. 1991. Biochem. Biophys. Res. Comm. 175: 1131.

1 Institute for Genetics, University of Cologne, Cologne; Institute for Immunology, University of Mainz, Mainz, Germany

G.33 IL9 production of naive C04+ T cells depends on IL2 and is synergistically enhanced by a combination of TGF~ and IL4 E. SCHMI1T, T. GERMANN, S. GODERT, P. HOHN, C. HOLS, S. KOLSCH, R. KOHN!, W. MOLLER 1, N. PALM, and E. RODE Dense CD4+ T cells isolated from naive mice produce only trace amounts of IL9 in response to stimulation by immobilized anti-CD3 in combination with anti-CD28 antibodies. IL9 production of such T cells is significantly stimulated by TGF~ and

25th Meeting of the Society of Immunology . 189 further enhanced by the addition of IL4 which by itself has only a minimal influence. The application of CD4+ T cells isolated from IL2-«knock out» (KO) mice unequivocally revealed that IL2 is essential for the production of IL 9 by T cells. In addition, the use of T cells from IL4- KO mice elucidated that the basic IL2 + TGF~-mediated IL 9 production is independent from IL4. Our results therefore demonstrate that optimal IL9 production of naive dense CD4+ T cells is positively regulated at different levels: a) by IL2 that is essential for IL9 secretion, b) followed by TGF~ which promotes a considerable increase in IL9 production above the level induced by IL2, and c) finally by IL4 that requires the presence of IL2 and TGF~ to strongly enhance the production of IL9. Supported by the Deutsche Forschungsgemeinschaft (SFB 311).

Dept. of Immunology and Cell Biology, Forschungsinstitut Borste!, Institut fUr Experimentelle Biologie und Medizin, Borstel, Germany

G.34 Human vascular endothelial (EC) and smooth muscle cells produce interleukin 8, but only EC express IL8 binding sites U. SCHONBECK, E. BRANDT, F. PETERSEN, H.-D. FLAD> and H. LOPPNOW Interleukin 8 (ILS) is an important regulator of chemotaxis and migration. Vascular endothelial (EC), as well as smooth muscle cells (SMC) produce this cytokine. However, little is known regarding the isoform of ILS produced by SMC. Furthermore, the expression of IL8 receptors on EC and SMC was not determined. We show in Western blot analysis that SMC produce two isoforms of IL8. Although these cells produced ILS, they did not express IL8 binding sites as measured in competition assays with radiolabeled IL8. In contrast, EC or fibroblasts expressed specific IL8 binding sites. As described previously for PMN, neutrophil activating peptide-2 (NAP-2), another member of the f:l-thromboglobulin superfamily, also interfered with ILS binding to EC. Both EC and fibroblasts expressed IL8 receptor type I mRNA, but not type II mRNA. In line with the binding data SMC did not express mRNA for both receptors. The differential expression of ILS activity and IL8 receptors by vascular EC and SMC may contribute to regulation of the immune response in the vessel wall. Partially supported by grant Lo 385/4-1 (H. LOPPNOW) and by SFB 367, Projekt C4 (E. BRANDT, H.-D. FLAIl) of Deutsche Forschungsgemeinschaft.

190 . 25th Meeting of the Society of Immunology IDept. of Gastroenterology, 2Dept. of Abdominal Surgery, Hannover Medical School, Hannover, Germany

G.35 Effects of c-kit ligand on human intestinal mast cells S. SCHWENGBERG!, s. c. BISCHOFF!, K. WORDELMANN!, H. R. RAAB 2 , H. DRALLE 2 , H.J. MEYER2, and M. P. MANNS I Although mast cells are one of the dominant effector cells in both allergic reactions and inflammatorylregenerative processes, the regulation of mast cell function is mostly unknown. Previous studies have shown that c-kit ligand, or stem cell factor (SCF), is a potent regulator of human lung mast cells G. Exp. Med. 175: 237-244, 1992). In the present study we tested the effects of c-kit ligand on histamine release of human intestinal mast cells (IMC). We isolated human IMC from 54 macroscopically normal surgery specimen (37X carcinoma, lOx benign tumors, 8x CIBD, 6x diverticulitis, 3x others) by mechanical dissection and enzymatic dispersion. We obtained 18.5 Mio cells/g mucosa containing 1-12 Mio IMC (mean = 3). The cells were stimulated in a shaking water bath at 37°C. After a warm-up period of 10 min the cells were preincubated with or without c-kit ligand (100 ng/ml) for 15 min and then stimulated with an anti-IgE receptor-antibody (mAb 29C6, 100 ng/ml) for 35 min. The histamine-content of the supernatant was measured in an immunoassay (Dianova, Hamburg) and expressed as % histamine of the cellular total histamine-content before stimulation. IgE-receptor crosslinking induced only in one of 13 cases significant degranulation, whereas after preincubation with c-kit ligand mAb 29C6 always induced a significant histamine-release (about 50 % of the histamine-release induced by ionomycin). These results show that IMC mediator release is strongly enhanced by c-kit ligand and therefore this cytokine seems to be an important regulator of IMC-function.

LMI, BRMP, National Cancer Institute, FCRDC, Frederick, MD, USA; IInstitute of Immunology, Philipps-University, Marburg, Germany

G.36 Genomic cloning and promoter analysis of the human interleukin-8 receptor genes, IL-8RA and-B H. SPRENGER I, A. R. LLOYD, R. G. MEYER I, J. A. JOHNSTON, and D. J. KELVIN Two unique but homologous receptors for the neutrophil chemoattractant IL-8 have been cloned (designated IL-8RA and -B), each of which binds IL-8 with high affinity. Both receptors are abundantly expressed on mature neutrophils. By Northern blot analyses and Nuclear run-on assays we found the mRNAs for both IL-8Rs were upregulated by G-CSF at the transcriptional level. On the other hand LPS and TNF-a downregulated IL-8R expression predominantly by a posttranscriptional mechanism. To understand the tissue specific expression and to identify gene-regulatory elements we have cloned, sequenced and characterized both human IL-SR genes, IL-SRA and -B. Full length cDNAs were cloned by a modified RACE-technique (rapid amplification of cDNA-ends). For both IL-SR genes we identified extensive 5'-untranslated regions (UTR). After comparison with the genomic sequence of A-DASH- and pWE15 cosmid

25th Meeting of the Society of Immunology . 191 clones, obtained by screening of human genomic libraries, we found the IL-SRA-gene consisting of 2 exons interrupted by an intron of 1.7 kb. The IL-SRB gene consisted of 3 exons with the promoter region separated from the ATG-initiation codon by S.75 kb. Commonly, the open reading frames and 3' - UTRs were entirely encoded by the last exon. The promoters of both IL-SR-genes showed a high degree of similarity: Besides numerous homologous elements the immediate GCrich 5' -flanking region of both genes could serve as a constitutively active promoter in CAT-expression assays. Expression analysis of additional upstream regions suggested the presence of silencer elements up to position -SOo. For the IL-SRB promoter we could demonstrate inducibility by G-CSF. In conclusion, cloning of full length cDNAs permitted us to clone the human IL-SR genes, to identify their genomic structures and characterize the promoter regions.