Workshop G: Lymphocyte subsets and immune regulation

Institute of Medical Microbiology, Immunology and Hygiene, Technische Universitat Munchen, and llnstitute of Medical Microbiology and Hygiene, Department of Immunology, University of Freiburg, Germany

G. 1 Analysis of self-veto mechanism of C08+ T cells A. BERGENTHAL, H. PIRCHER1, H. WAGNER, and K. HEEG CD8+ cytotoxic T cells (CTL) are paralyzed in vitro when they recognize their specific peptide presented by MHC class I on the surface of another activated CTL. The antigen specificity of the peptide-presenting CTL is irrelevant. Here we have analysed peptide-induced paralysis of CD8+ T cells from LCMV-TCR transgenic mice. Addition of peptide paralyzed the cytotoxicity of these CTL as well as the antigeninduced produktion ofTNF, IL-10, IL-12, IL-2, and IL-4. However, IFN-yproduction was not affected yet even enhanced. Paralysis could neither be blocked by cycloheximide nor is transfered by supernatants of paralyzed CTL, demonstrating that soluble factors do not induce/ maintain CTL paralysis. To exclude that exhaustion of lytic granules accounts for paralysis we incubated normal and paralyzed CTL with cold targets and added later at different time points 51Cr-Iabeled target cells. The lytic activity of normal and paralyzed CTL was unaltered after a 8h-preincubation with cold targets, demonstrating that lytic granules were not exhausted. Thus paralysis requires cell-cell contact and can be ·characterized by selective suppression of CTL function.

Department of Functional and Applied Anatomy, Hannover Medical School, Germany

G. 2 Mechanisms of leukocyte mobilization: Evidence for ~-receptor mediation revealed by determination of in vivo E050-dosage after intravenously applied catecholamines in freely behaving rats

J. DRUBE, H. NAVE, S. VON HORSTEN, S. KUHLMANN, and

R. PABST

Leukocytosis is often observed in patients and the term refers to an increased concentration of circulating white blood cells. This increase can be due to an increased production of leukocytes or more often due to a mobilization from the marginal pool into the circulating blood. Among other factors also catecholamines are known to induce leukocytosis. However, under in vivo circumstances dose- and time-dependent characteristics of this catecholamine-induced leukocytosis are largely unknown and systematically collected data are not available. Only multiple samples of the same animal enable a real kinetic study. Therefore we developed a modelfor chronic intravenous (i.v.) catheterization of the superior vena cava in freely moving rats. This procedure allows repeated blood sampling without additional stress for the animal. In male Lewis rats (20 ± 5 weeks of age; 355 ± 30 g/body weight (BW); n = 47) single injections (350 plover 60 sec.) of adrenaline and noradrenaline were applied at eight different dosages 10-4.-3,-2, 0.1, 1, 10, 50, 100 j.1g/kg/BW, respectively. At -15, +1, +3, +5, +10, +15, +30 mins, +1, +2.5, +6, +46 h time points cell numbers were determined using coulter counter. Results obtained using these methods revealed at 1 min post i.v. application and ED50 dosage for adrenaline of 1 pg/kg/BW (D+3x10 6 cells/pI) and for noradrenale of 10 pg/kg/BW (D+3x10 6 cells/pI). Cetacholamine-

494 . 29th Annual Meeting 1998 induced leukocytosis was observed over a period of 5 min. At 15 min post i.v. application cell numbers were normalized. Interestingly at 1 h after catecholamine application a second peak in leukocyte counts of yet unknown origin was observed. The lo-fold higher ED50 for noradrenaline provides evidence for ~-adrenoceptor-mediated mechanism, since adrenaline has a higher ~ potency. Presently, receptor-specificity of the phenomenon is determined using selective adrenoceptor-agonists and antagonists and furthermore leukocyte subset specificity is investigated using FACScan analysis. The support of this study by a grant of the Volkswagen Foundation (11/72029) is acknowledged.

Institute of Immunology, University of Heidelberg, Germany

G. 3 Increased reactivity of cord blood lymphocytes to C02-mediated stimulation T. GRADER, S. MEUER, and T. GIESE The reduced incidence of severe graft versus host reaction observed in recipients of umbilical cord blood (CB) stem cells is attributed to the immaturity of the neonatal immune response. Experimental data suggest defective responses in T lymphocytes. We compared the proliferative response of unseparated mononuclear cells (PBMC) and purified CD4-positive lymphocytes of healthy full-term neonates to adult healthy volunteers after anti-CD3 or anti-CD2 stimulation and detected intracellular interleukin-2 (IL-2) after shortterm stimulation with PMA and Ionomycin by flowcytometry. CB PBMC and also CD4-positive T cells showed a markedly increased proliferative response after stimulation with the mitogenic CD2-antibodies AICD2.Ml and AICD2.M2. This response was more sensitive to IL-2 receptor blocking than in adult cells. Staining for intracellular IL-2 revealed however an increased frequency of IL2-secreting CD4+ lymphocytes in CB after PMA and Ionomycin stimulation. Remarkably, the proliferative response after stimulation with the anti-CD3 antibody OKT3 was not elevated in CB cells. We conclude that cord blood lymphocytes are not defective in their proliferative response to different T cell activation pathways compared with adult lymphocytes. The increased sensitivity to CD2-mediated activation in vitro resembles the phenotype of ex vivo isolated lamina propria T lymphocytes. Both populations are located at interfaces between self and foreign antigen without in vivo evidence for increased activation. In this context the increased IL-2 production in cord blood could be a factor that promotes hyporesponsiveness in agreement with data of the IL-2 knock-out mouse showing that lack of IL-2 leads to T cell hyperreactivity in vivo.

University of California, Berkeley, USA

G.4 Identification and characterization of the mouse "MAFA" homolog as an NK cell receptor TH. HANKE, L. CORRAL, and D. H. RAULET

The "mast cell function-associated antigen" (MAFA) is known to be an immune-receptor tyrosine-based inhibitory motif containing inhibitory C-type lectin like transmembrane protein expressed on the surface of rat mast cells. By using a novel monoclonal antibody for expressioncloning and flow cytometric analyses, we show that the mouse homolog of MAFA (mMAFA) is an NK cell specific antigen which is not found on mouse mast cells. Interestingly, mMAFA

29th Annual Meeting 1998 . 495 expression on NK cells is influenced by the presence of MHC class I. Northern and Southern blot analyses suggest that mMAFA is non-polymorphic and encoded by a single gene. The high degree of conservation between MAFA, mMAFA and a newly identified human MAFA homolog argue for an important and evolutionary conserved function of this molecule.

Institut fur Klinische Mikrobiologie, Immunologie und Hygiene, Friedrich-Alexander-Universitat Erlangen-Nurnberg, Erlangen, Germany

G. 5 Simultaneous analysis of proliferation and cytokine production at the single-cell level in Th cells M. HUBER, M. RbLLINGHOFF, and M. LOHOFF So far, cytokine production and proliferation at the single T cell level could be detected only separately by using two different techniques::The method of bromodesoxyuridine (BrdU)-staining allowed to analyze the proliferative beh~viour of cells, the method of intracellular cytokine staining enabled to detect the cytokine profile. Here, we report a new method to simultaneously measure proliferation and cytokine producti~)ll of T cells at the single cell level by flow cytometry. With this new method we analyzed the :expression of cytokines during the proliferation of Th1- and Th2-cell clones after antigen-specif~c stimulation in vitro. So far, our data indicate that the cytokine expression and proliferation of Th1- and Th2-cells peak at different time points and are mutually exclusive in most cell clones. These results suggest a defined sequence of events in individual Th cells after antigen-specific s~imulation, in which the cytokine production is followed by proliferation. This method will bejapplied to study proliferation and cytokine profile of CD+ T cells in the infectious model of Imurine cutaneous Leishmaniasis. In addition, the method will be used to elucidate the relation~hip between the different published types of T cell anergy.

1 Molecular Immunology, Robert Koch-Institute, and 2 Protein Chemistry, Max-Delbriick-Center, Berlin, Germany

G. 6 Cloning of a new inducible T cell surface molecule with potent co-stimulatory properties A. HUTLOFF!, A.-M. DITTRICH!, K. BEIER!, R. KRAFT2, and R. A. KROCZEK 1 Using the previously generated mAb 8F4, we purified and cloned the 8F4 antigen, a disulfidelinked dimer with an apparent Mr of 27 and 29 kDa. Flow cytometry and Northern Blot analysis revealed that the 8F4 protein, a member of the Ig-superfamily, is an early activation antigen exclusively expressed on human T cells. Immunohistological analysis of different lymphoid organs showed that the 8F4 antigen is expressed on T cells in germinal centers of lymph nodes and tonsillar tissue. Ex vivo analysis of lymphoid cells demonstrated that 60-80% of tonsillar T cells express 8F4 and that these cells belong to the CD45RO subset. In terms of function, 8F4 is a co-stimulator of T cell proliferation, with a potency comparable to CD28. When crosslinked together with anti-CD3 mAb OKT3 at optimal concentrations, the 8F4 antigen enhances the secretion of various lymphokines in a pattern which clearly differs from the pattern obtained with the CD28-specific mAb 9.3. Most rem<}rkably, the 8F4 antigen does not enhance the secretion of IL-2, but significantly increases the production of IL-lO to levels not observed with CD28 co-stimulation. Our data suggest that the new cell surface molecule 8F4 plays a significant role in the T-B cooperation in lymphoid tissues.

496 . 29th Annual Meeting 1998 Deutsches Rheumaforschungszentrum, Berlin, Germany

G. 7 Induction and stability of cytokine expression of naive and antigenexperienced human Th helper cells isolated from adult peripheral blood S. KIMMIG, S. NITSCH, A. RICHTER, A. SCHEFFOLD, J. BRAUN, J. SIEPER, A. RADBRUCH, and A. THIEL We have classified CD4+ Th cells from adult human peripheral blood according to the expression of CD45 isoforms, CD27, CD28, and CD31 into naive and memory/effector Th cells. CD3i+ and CD31- CD45RN CD45RO-Th cell populations, containing exclusively and/or predominantly naive Th cells, as was evident from lack of short term response within 5hrs to PMA/ionomycin stimulation, were primed in vitro with aCD3 and aCD28 and analysed for polarisation into Th1 versus Th2 cells in the absense of antigen-presenting cells. Kinetics of induction and pattern of cytokines (IFN-y/TNF-a versus IL-4/IL-10) induced were analysed by intracellular multiparameter immunoflourescence. A considerable degree of interindividual variation was observed, indicating either genetic or ontogenetic predispositions of naive Th cells to the expression of particular cytokines. Memory/effector Th1 cells, as identified by phenotype (CD45RO+CD27+1CD45RO+CD27-,CD45RO+CD28+/CD45RO+CD28-) and rapid reaction to PMAlionomycin with IFN-y expression, were isolated alive according to the expression of surface IFN-y, for analysis of the stability of IFN-y expression after Th1 preserving (TNF-a, IL12) or converting (IL-4) restimulations. The results of this experiment will be presented.

Institute for Immunology, Johannes Gutenberg University, Mainz, Germany

G. 8 Th 1 development induced on endogenous IL-2

by a combination of TGF-~ and IL-4 depends

K. LINGNAU, S. KOLSCH, C. NEUDORFL, E. RUDE, and E. SCHMITT Polyclonal activation of naive CD4+Mel14 high Th cells by immobilized anti-'Y13 TCR mAb in the presence of IL-4 and TGF-~ leads to the development of Th1 cells. This alternative Th1 development was found to be independent of IL-12 but to be dependent on the presence of endogenous IFN-y. Herein, we demonstrate that, in addition to IFN-y, endogenous IL-2 is also essential for an optimal Th1 development induced by a combination of TGF-~ and IL-4. Priming naive Th cells in the presence of TGF-~ strongly inhibited the development of Th1 cells and simultaneously suppressed the production of IL-2 by the developing Th cells. The addition of IL-4 and TGF-~ to such a priming culture compensated for the TGF-~-mediated inhibition of primary IL-2 production and promoted in parallel the development of Th1 cells. Thus, it was possible that the Th1 promoting effect of IL-4 is mediated by endogenous IL-2. This assumption was further supported by the fact that neutralization of IL-2 completely inhibited this alternative Th1 development. However, priming naive Th cells by a combination of saturating amounts of IL-2 and TGF-~ in the presence or absence of IL-4 revealed that a) IL-2 and TGF-~ are essential but not sufficient for an optimal alternative Th1 development and that b) the Th1-promoting effect of IL-4 depends on the coordinate action of TGF-~, IL-2 and IFN-y. Since polyclonal activation of naive CD4+ Th cells by immobilized anti-TCR mAb is a rather artificial approach, we also used an antigenspecific system. Naive OVA 323 _339-specific Th cells, which were activated by APCs and the OVA323 _339 peptide, differentiated towards Thl cells in the presence of a combination of IL-4 and TGF-~. Hence, this finding confirmed the results obtained by polyclonal activation of naive CD4+ Th cells and implicates that this alternative Thl development may also occur in vivo under the influence of TGF-~ and IL-4 independently of the Th1-promoting effect of IL-12.

29th Annual Meeting 1998 . 497 !Deutsches Rheumaforschungszentrum, Berlin, Germany 2Millennium Pharmaceuticals, Cambridge, MA, USA 3Medizinische Klinik, Rheumatologie/Immunologie, Universittsklinikum Charit, Berlin, Germany

G. 9 T1 /5T2 is preferentially expressed on murine Th2 cells, independent of IL-4, IL-5, IL-6, and IL-10, and critical for Th2 effector function M. LHNING\ J. GROGAN!, T. COYLE 2, A. STROEHMANN!, C. MEISEL!, A. RICHTER!, D. LEVINSON2, A. RADBRUCH!, and T. KAMRADT!,3 T helper (Th) cells can be categorized according to their cytokine expression. The differential induction of Th cells expressing Thl and/or Th2 cytokines is key to the regulation of both protective and pathological immune responses. Cytokines are expressed transiently and there is a lack of stably expressed surface molecules, significant for functionally different types of Th cells. Such molecules are of utmost importance for the analysis and selective functional modulation of Th subsets and will provide new therapeutic strategies for the treatment of allergic or autoimmune diseases. To this end, we have identified potential target genes preferentially expressed in Th2 cells, expressing IL-4, IL-5 and/or IL-lO, but not IFN-y. One such gene, TlIST2 is expressed stably on both Th2 clones and Th2-polarized cells activated in vivo or in vitro. Tl/ST2 expression is independent of induction by IL-4, IL-5, IL-6, or IL-lO as demonstrated by use of the respective cytokine-deficient mice. Expression of Tl/ST2 is markedly upregulated in mice infected with Schistosoma mansoni. Four-colour FACS analysis allowed us to analyze the co-expression of Th2 cytokines in T1/ST2+ cells at the single-cell level. TlIST2 plays a critical role in Th2 effector function. Administration of either a monoclonal antibody against Tl/ST2 or recombinant Tl/ST2 fusion protein attenuates eosinophilic inflammation of the airways and suppresses IL-4 and IL-5 production in vivo, following adoptive transfer of Th2 cells.

Departments of Immunology, Pathology, and Surgery, University of Heidelberg, Germany

G. 10 Insensitivity towards inhibition by Cyclosporin A, Rapamycin, and Tacrolimus in human lamina propria T lymphocytes

J. BRAUNSTEIN, F. AUTSCHBACH, B. Smo, G. NEBL, A. SCHRODER, Y. SAMSTAG, and S. C. MEUER Intestinal lamina propria T lymphocytes (LP-T) possess functional properties which are distinct from peripheral blood lymphocytes (PB-T) reflecting a special function of the intestinal microenvironment. While responsiveness of LP-T towards antigen receptor / CD3 stimulation is reduced when compared to PB-T, their sensitivity to CD2 / CD28 activation is enhanced. To further address the molecular basis of CD2-hyperresponsiveness in LP-T their susceptability to immunosuppressive agents like Cyclosporin A, Tacrolimus (FK506) and Sirolimus (Rapamycin) was analysed. Under identical experimental conditions CD2-induced proliferation of LP-T is rather resistant to inhibition by these drugs, while cell growth of PB-T is significantly reduced. Moreover, interleukin 2 (IL2) production of CD2-stimulated LP-T, which exceeds that of PB-T by more than hundred fold, is hardly affected by low concentrations of Cyclosporin A which abolish IL2 production in PBL- T. At higher concentrations of Cyclosporin A, IL-2 secretion by LP-T still remains considerably high and may therefore account for cell growth in the presence of this drug. On the molecular level, the increased Cyclosporin A / FK506 resistance of LP-T could be explained by the constitutive nuclear translocation of the transcription factor NFATc in the majority of these cells. Dephosphorylation and subsequent migration to the nucleus of NFATc is controlled by the Cyclosporin A-sensitive protein phosphatase calcineurin. Furthermore,

498 . 29th Annual Meeting 1998 cofilin, a 19 kD phospho-protein exclusively involved in costimulatory signalling pathways in T cells, is constitutively dephosphorylated in LP- T. Dephosphorylation of cofilin in activated T cells is known to be Cyclosporin A-insensitive. Hence, the Cyclosporin A / FK506-resistance of LP-T may result from both the up-regulation of Cyclosporin A-insensitive pathways as well as Cyclosporin A-sensitive pathways beyond the stage at which these substances can exert inhibitory influences. Given the large amount of xenobiotic substances with potential immunosuppressive effects that may be encountered at the bodys largest interface with ist exogenous environment the resistance of LP-T to such agents may be essential for maintaining a functional mucosal immune system.

Department of Pneumology, University Medical Clinic, Freiburg, Germany

G. 11 Identification of intracellular cytokine production in BAL-Iymphocytes of patients with pulmonary sarcoidosis A. PRASSE, C. GEORGIS, H. BILLER, H. MATTHYS, W. LUTTMANN, and J. C. VIRCHOW JR. Background: Sarcoidosis has been associated with a Th1-like cytokine pattern with increased concentrations of IFN-y and IL-2 as mRNA production for these cytokines. In addition, expression of TNF-a has been reported in BAL-supernatants of these patients. However, at present, cytokine production in sarcoiodosis has not been analysed at the single cell level on bronchoalveolar lavage (BAL) lymphocytes. Methods: To address this question BAL was performed in 7 untreated patients in whom sarcoidosis had been diagnosed by histology. Furthermore, all patients had pulmonary involvement as evidenced by chest X-ray corresponding to a radiological classification of stage II or III carcoidosis. BAL cells were stimulated with phorbolester (PMA) and Ca-Ionophor A23187 for 4 hand cytokine release was blocked by adding brefeldine. Intracellular cytokines were then detected in CDY, CD4+, and CD8+ cells by specific mAb using flow cytometry following fixation of cells with paraformaldehyde and permeabilisation with saponin. Results: Following stimulation with PMA and A23187 a high percentage of CDY T lymphocytes were shown to contain INF-y as well as TNF-a and IL-2 compared to unstimulated control cells. No significant difference in the cytokine-profile were observed between CD4+ and CD8+ subpopulations (CD3+ lymphocytes: IFN-y: 79.4%, TNF-a: 72.4%, IL-2: 53.7%, CD4+: IFN-y: 78.6%, TNF-a: 72.4%, IL-2: 56.4%, CD8+: IFN-y: 87.1 %, TNF-a: 72.6%, IL-2: 51.8%). There was a close correlation between the percentage of IL-2 producing" lymphocytes in BAL fluid and the degree of lymphocytic alveolitis (p = 0.042, r = 0.72) as assessed by the total number of lymphocytes recovered from BAL-fluid of these patients. This relationship was not observed for IFN-y and TNF-a. Conclusion: In conclusion, our results are compatible with the hypothesis of a predominant Th1-like cytokine pattern in CD4+ as well as CD8+ T lymphocytes in sarcoidosis with up to 90% IFN-r T lymphocytes. In addition, the observed correlation between IL-2+ cells and the total number of lymphocytes suggests a causal relationship between these parameters in the pathogenesis of pulmonary sarcoidosis.

29th Annual Meeting 1998 . 499 Institute of Clinical Molecular Pharmacology, Medical School, Hannover, Germany, and lPathology, Case Western Reserve University, Cleveland, USA

G. 12 C04+ T cells recognizing sl?ecific antigen deposited in glomeruli cause glomerulonephritis-like kiCiney injury· H. H. RADEKE, P. V. LEHMANN!, K. RESCH, and M. TARY-LEHMANN1 Recently we presented preliminary data on a new model of immune-mediated glomerular injury using immune deficient SCID mice. This model is suitable to test both the classical concept of GN starting with immune complex deposition, complement fixation and downstream, non-specific inflammatory reactions and the concept of T cell mediated GN with initial glomerular antigen presentation to proinflammatory T cells leading to a DTH-like local injury. We are currently investigating the latter hypothesis. First, we targeted crosslinked ovalbumin polymers (OVA-XL) at a defined size of 150-300 kDa specifically and exclusively to the glomerular mesangium. These polymers alone did not cause any local alteration in the absence of IgG and/or complement. Subsequently, T helper clone cells characterized by the ELISPOT assay as pure Th-l-like (IFN-yand IL-2) were adoptively transferred 4 hrs after antigen. Histological examination at days 1, 2, 5 and 21 showed major infiltrates in the proximal tubular region (PTR) at day 5 accompanied by significant proteinuria. No injury was observed after deposition of irrelevant antigen. Detailed histological analysis revealed that T cell numbers peaked early in the glomeruli (2.1 ± 0.6 vs. zero /gcs). Macrophages however, were hardly detectable in glomeruli (0.5 ± OJ/gcs) at this time point, while they formed the major constituent of the PTR infiltrates at day 5 (83 ± 18). Interestingly, we observed a "MHC class II gap", i.e. macrophage numbers did not account for the total number of cells staining positive for MHC class II. These data with a newly developed GN model indicate that memory T helper 1 cells in cooperation with local MHC class II positive cells may be sufficient to trigger renal injury. '- supported by DFG-Grant SFB244/Bl, and Ra 525/5-1

Institute for Virology and Immunobiology, University of Wiirzburg, Wiirzburg, Germany

G. 13 C028 stimulation without coengagement of the T cell receptor induces a Th2 differentiation both in vitro and in vivo M. RODRfGUEZ-PALMERO, and T. HONIG Full T cell activation is only achieved when both the T cell receptor (TCR) and a costimulatory molecule are ligated. CD28 is the most potent costimulatory molecule on T cells. MAbs to rat CD28 developed in our group fall into two categories: Classical anti-CD28 mAbs (prototype: JJ319) induce proliferation only when the TCR is coengaged. In contrast, "directly stimulatory" mAbs (prototype: ]]316) activate resting T cells in vitro and in vivo without the need for TCRengagement. The present study shows that this direct stimulation also induces differentiation towards a Th2 phenotype. In vitro, LN CD4+ T cells from adult BN rats were stimulated and IL-4 production was measured by intracellular staining. IL-4 production was more pronounced and long-lasting in directly than in costimulated cultures, and was strongly increased by the addition of IL-4 in directly stimulated cells only. These results correlated with the mRNA levels for the cytokines, detected by RNase protection assay. In vivo, inoculation of J]316, but not of ]]319, in 3-week old Lewis rats led to the production of mRNA for IL-4 and IL-I0 in LN and spleen. Strongly increased IL-4 production was also detected by intracellular staining, and the biological effect of enhanced IL-4 production was evidept from increased serum IgE levels. These data show that the stimulation via CD28, without the signal through the TCR, induces a Th2 differentiation. The possibility of skewing CD4 cells in vivo towards Th2 could provide a novel immunotherapeutic strategy for the prevention and control of autoimmune and inflammatory diseases.

500 . 29th Annual Meeting 1998 Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Germany

G. 14 Dose·dependent costimulation and inhibition of Tcell responses by G-rich phosphorothioate oligodeoxynucleotides U. SALZER, R. LANG, G. B. LIPFORD, H. WAGNER, and K. HEEG Synthetic oligodeoxynucleotides (ODN) containing CpG motifs stimulate immune cells. While macrophages and dendritic cells are activated by CpG-ODN in a direct fashion, T cells become sensitive to CpG-ODN after crosslinking of their T cell receptor. CpG-ODN mediated costimulation of T cells results in IL-2R expression, IL-2 production and thus T cell proliferation. We report here, that ODN containing G-rich sequences were also active as costimulatory ODN on T cells while these ODN lacked macrophage stimulatory activity. Surprisingly, G-rich ODN did not require CpG-motifs for T cell costimulation suggesting that distinct signal pathways might exist in macrophages and T cells. While G-rich ODN showed strong costimulatory activity at concentrations of 0.1 to 3 pM, the same ODN were inhibitory at higher concentrations (> 5 pM). Inhibition included mixed lymphocyte reactions (MLR), T cell stimulation with antiCD3 plus anti-CD28 and anti-CD3 and PMA. Furthermore, IL-2 driven growth of ConA-activated T cells was also inhibited by G-rich ODN. Experiments with various murine and human cell lines suggested that growth inhibition by G-rich ODN is selective for cells of the T cell lineage. Taken together, our data suggest that the sequence requirements for T cell costimulation of ODN is less stringent than those for direct APC activation. Therefore it might be possible to design ODN that primarily act on T cells while lacking APC activating activity. Furthermore Grich ODN could be a tool to selectively inhibit undesired T cell responses. lInstitute of Immunology and Transfusion Medicine, University of Liibeck School of Medicine, Lubeck, 2German Cancer Research Center, Heidelberg, Germany

G. 15 Variability of interferon (IFN)-y production is independent of differences in IFN-y producing lymphocyte subsets CH. SCHEFFER l, L. RINK!, R. ZAWATZKy 2, and H. KIRCHNER! IFN-y is a potent modulator of immune function produced by T cells and NK cells. In this study we investigated if IFN-y is a more sensitive parameter for determining the immune status than leukocyte subtyping, which at present is commonly used for this purpose. Leukocyte subpopulations and IFNy production in a whole blood assay of healthy blood donors were tested two to four times over a period of one year. The time interval between the tests was at least ten weeks. All persons tested showed stable quantities of white blood cell counts over the whole study. Analyses of the lymphocyte subpopulations of two time points resulted in a strong correlation with high statistical significance for the percentage of CD3, CD4, CD8, CD19, CD45-subtypes and CD56/16 positive cells. IFN-y production by the individuals correlated between these time points also, but only if an ELISA strongly correlating to IFN-y bioactivity was used instead of other commercial IFN-y ELISA. Interestingly, there were individuals constantly above the 75 th centile and below the 25 th centile. However, there were no correlations between white blood cell counts or any lymphocyte subpopulation and the IFN-y production even in these high- and low-responders. The high- and low-responder group did not show any differences in leukocyte subpopulations. In contrast, TNF-y production strongly correlated to IFN-y secretion. IFN-y production by males was less variable than by females. Furthermore, intraindividual differences in IFN-y secretion were minimal after the age of forty. In conclusion, our data demonstrate that IFN-y is a more sensitive parameter for the actual status of the immune system since its titer shows alterations faster than leukocyte subtyping. Furthermore, leukocyte subtypes did not correspond to the production of the regulatory cytokine IFN-y.

29th Annual Meeting 1998 . 501 Department of Internal Medicine III, University of Erlangen, Erlangen, Germany; and UT Southwestern Medical Center, Dallas,TX, USA

G. 16 Regulation of T helper type 1 effector cell differentiation from resting human peripheral blooCl memory Tcells H. SCHULZE-Koops, A. L. SKAPENKO, S. D. JOERG, P. E. LIPSKY, and]. R. KALDEN In a variety of organ specific autoimmune diseases, the inappropriate differentiation of polarized T helper type 1 effector cells has been implemented in the initiation and perpetuation of chronic immunity. As the mechanisms determining the differentiation of memory T cells into Thl effectors in man is not completely understood, we investigated the regulation of Thl effector cell differentiation from human CD4+ memory cells in healthy individuals in vitro. A cell culture system was established that permitted the differentiation of polarized Th effectors from memory T cells by short term priming. Highly purified resting CD4+ peripheral blood memory T cells were stimulated for five days with different concentrations of a mAb to CD3 in the presence or absence of anti-CD28, rIL-4 or rIL-12. After resting, cytokine production was determined in individual effectors by intracellular flow cytometry and was compared to the cytokine secretion profile from the starting population. Freshly isolated memory T cells produced primarily IL-2 with little IFN-yand IL-4. Generation of IFN-y producing Thl effectors was dependent on CD3 stimulation and could be inhibited by recombinant IL-4. Costimulation with anti-CD28 significantly increased the amount of IFN-y produced per individual Thl cell, however, it did not result in Thl effector cell differentiation. In contrast, CD28 costimulation yielded increased IL-4 producing Th2 effectors. Priming in the presence of rIL-12 significantly increased both, the amount of IFN-y produced by individual cells and the frequency of Thl effectors. In the presence of IL-12, the generation of Thl effectors correlated with the degree of TCR engagement during priming. Most interestingly, the combination of CD28 and rIL-12 had an additive effect on the generation of Thl effectors and on the increase of IFN-y production per individual cell. The results indicate that the differentiation of Thl effectors from memory T cells was dependent on IL-12 and could be inhibited by exogenous IL-4 and by IL-4 producing Th2 cells. However, the data further suggest that the inhibitory potential of IL-4 was inferior to the Th1 inducing IL-12 effects. The results might have implications on the development of new therapeutic strategies for chronic (auto)immune diseases.

Medical Department IV, University Homburg, Germany

G. 17 Tcell activation follows Th 1 rather than Th2 pattern in hemodialysis patients M. SESTER, U. SESTER, M. GIRNDT, M. HAUK, H. KAUL, and H. KOHLER Patients with end stage renal failure are treated with hemodialysis (HD) and show an impaired response to vaccinations (e.g. Hepatitis B) which strongly correlates with a reduced activation of T cells. In vitro proliferative responses as well as IL-2 production are low. The defect was localized in antigen presenting cells, which show a decreased expression of the costimulatory B7-2 molecule. In response to primary activation CD4 positive T cells mainly differentiate into either Thl- or Th2 cells, which are characterized by the pattern of cytokines they express. Thl cells (marker cytokine IFN-y) support cell mediated immunity whereas Th2 cells (marker cytokine IL-4) enhance humoral immune responses. Since both types of response mutually inhibit each other the impaired humoral immune response seen in HD patients could either be due to a

502 . 29th Annual Meeting 1998 reduced number of Th2 cells or to a predominant Th1 response. To distinguish between these possibilities we determined cytokine expression of CD4 positive T cells using FACS analysis. Peripheral blood leucocytes (PBL) were isolated from 12 HD patients as well as 6 healthy controls, and stimulated with PMA/ionomycin. After 4 hours of incubation the percentage of IFN-yand IL-4 positive Th cells was determined. We found that HD patients have significantly more IFN-y positive cells than controls (17%, SD 8.4% vs. 9.2%; SD 3.2%; p=0.046). In contrast, both groups show equal levels of IL-4 producing cells (2.5%; SD 0.7% vs. 2.3%; SD 1.4%; p=O.7). Interestingly, while the percentage of positive cells from patients and controls was different, we found equal amounts of IFN-y per single cell. Due to the limited number of cytokine producing cells upon primary stimulation we further analysed the cytokine pattern after restimulation of T cells derived from primary stimulation of PBL with PHA. Using these conditions, the effect of an increased percentage of IFN-y positive cells in HD patients was even more pronounced (28%; SD 9.5% vs. 13.5%; SD 3.8%, p=0.0026). The number of Th2 cells did not differ (6.8%; SD 2.5% vs 5.5%; SD 2.3%, p=0.31). We conclude that the predominant Th1 response is in line with the reduced response to vaccinations in HD patients.

Department of Surgery and !Institute of Immunology, University of Heidelberg, Germany

G. 18 Thiol-mediated redox-regulation of intestinal human lamina propria I~mphocytes by blood monocytes. Relevance for inflammatory I>owel disease B. Smo, J. BRAUNSTEIN!, C. HERFARTH, and S. C. MEUER! Chronic inflammatory bowel disease like Crohn's disease and ulcerative colitis is characterized by an overproduction of reactive oxygen species and a decreased antioxidative capacity of the gut mucosa resulting in a pro-oxidant microenvironment of lamina propria T lymphocytes (LPL-T). In lymphocytes, activation and proliferation are known to be influenced by redox-regulated processes. In this study, we investigated the influence of oxidative and reducing culture conditions on the proliferative response of human LPL-T. LPL-T were isolated from normal colon mucosa removed from resection specimens and were stimulated in vitro via CD3 ± IL-2 or by a combination of CD2 monoclonal antibodies. Proliferation of LPL-T was shown to be strongly influenced by redox-active substances: oxidants like non-lethal micromolar concentrations of HzO z, BCNU (a specific inhibitor of glutathione reductase) or BSO (an inhibitor of glutathione synthesis) inhibited proliferation, while reducing agents like 2-ME and dithiothreitol were potent stimulants. Addition of 2-ME was able to reverse the suppressive effect of HzO z' Similarly, co-cultivation of LPL-T with blood monocytes from the same patient (PBMO) completely abrogated the HzOz-induced suppression of proliferation, while lamina propria macrophages (LPMO) were uneffective, suggesting an antioxidative metabolic activity of PBMO. PBMO provided a potent co-stimulus for CD3-mediated activation of LPL-T in the absence IL-2 in contrast to LPMO. Again, the addition of 2-ME was able to fully substitute for the requirement of PBMO or IL-2 for CD3-mediated LPL-T cell proliferation. 2-ME acts by increasing the cellular uptake of cysteine for glutathione synthesis, which is prerequisite for cell proliferation. We therefore speculated that PBMO could serve to supply LPL-T with cysteine, which is the reduced form of cystine and not present in standard culture medium. We could show that large amounts of cysteine, measured as acid-soluble thiol, were released from stimulated and unstimulated PBMO into the supernatant as compared with LPMO. Lymphocytes usually have a low transport activity for cystine and a high transport activity for cysteine. PBMO as opposed to LPMO act as a cysteine pump and, therefore, are able to restore the proliferation of LPL-T in cystine-deficient culture medium in the same way

29th Annual Meeting 1998 . 503 as 2-ME does.We first demonstrate that cell proliferation of LPL-T is subject to redox control. As large amounts of PBMO infiltrate into the gut mucosa in inflammatory bowel disease, PBMO in contrast to LPMO could trigger activation and support proliferation of LPL-T in the pro-oxidant microenvironment by the release of reduced thiol compounds.

Department of Internal Medicine III, University of Erlangen-Nurnberg, Erlangen, Germany, and UT Southwestern Medical Center, Dallas, TX, USA

G. 19 Impaired Th2 cell differentiation in rheumatoid arthritis A. L. SKAPENKO, S. D. JOERG,]. WENDLER, P. E. LIPSKY,]. R. KALDEN, and H. SCHULZE-Koops It has been suggested that the chronic immune response in rheumatoid arthritis (RA) might be driven by activated Th1 cells without sufficient Th2 cell differentiation to down-modulate inflammation. To test the hypothesis that in RA T cells express an impaired ability to differentiate into Th2 effectors we investigated Th2 cell differentiation of resting CD4+ memory T cells in patients with active RA and in age and sex matched healthy controls in vitro. All patients fulfilled the ACR criteria for the classification of RA. To minimize the effects of drug treatment, only patients were included who had never been treated with DMARDs or corticosteroids. A cell culture system was established that permitted the differentiation of Th2 effectors from resting memory T cells by short term priming. Highly purified resting CD4+ peripheral blood memory T cells were primed for five days with a mAb to CD3 in the presence of anti-CD28 and the presence or absence of rIL-4. After resting, cytokine production was determined in individual effector cells by intracellular flow cytometry and was compared to the cytokine secretion profile from the starting population. Th2 cells could be induced in all healthy controls and in Sill RA patients by priming with anti-CD3 in the presence of anti-CD28. By contrast, priming under those conditions did not result in Th2 differentiation in 6/11 RA patients tested. The addition of high dose exogenous IL-4 during priming could overcome the apparent Th2 differentiation defect in 4 of those 6 patients, however, was without any effect in the remaining patients. Interestingly, in contrast to Th2 cell differentiation, an increase of IL-4 produced per individual Th2 effector cell could be induced in all RA patients and healthy controls. This effect was comparable in all individuals and was inversely correlated with the extent of CD3 engagement during priming. The data suggest that in some patients with RA CD4+ memory T cells might express an intrinsic deficiency to differentiate into Th2 effectors after appropriate stimulation. Impaired Th2 cell differentiation might contribute to the characteristic chronic (auto)immune inflammation in RA.

Institute of Immunology!, Organic Chemistry Institute 2, University of Munster, Munster, Germany

G. 20 Cognate T/B interaction and involvement of C028 in the suppression of the IgG antibody response against the TI-2 antigen all ~3) dextran C. SPECHT!, A. RADEMAEKERS!, A. KRUGER2, and E. KOLSCH 1 Analysis of the humoral immune response to a(l--73) dextran (Dex) in BALB/c mice reveals novel aspects of T cell-mediated control of "type 2 thymus-independent" responses against polysaccharide antigens. The formation of IgM and IgG antibodies (Ab), dominated by the J558

504 . 29th Annual Meeting 1998 idiotype (Id), is controlled by Id-specific T cells. These regulatory T cells, for which the T cell clone 178-4 Ts with characterized TCR a and ~ chain sequences is the prototype, expand in all BALB/c mice upon immunization with Dex (1). They suppress in cognate interaction the expansion of J558 Id bearing B cells committed for production of IgG Ab. Furthermore they provide a gate which precludes variability in the VH CDR3 region of IgG Ab appearing occasionally in the periphery. The VH CDR3 region is the recognition element of 178-4 Ts analogous T cells but contributes little to affinity for the antigen. For recognition by 178-4 Ts cells not even minimal sequence deviations of the J558 Id peptide are allowed. Additional analysis shows that only conventional B-2 lymphocytes produce IgG whereas B-1 cells do not participate in the production of this isotype. Using an all a(I-?3)-D-gluco configurated tetrasaccharide the Dexspecific B cells can for the first time be analysed in FACS. The data show that T cell mediated suppression does not result in B cell apoptosis. Thus it must be a true silencing of persisting Bg memory cells. In addition to Id recognition by TCR, a CD28 involvement is required in the suppression of this Dex-specific IgG response. The tight germline programmed complementarity between J558 Id-bearing Dex-specific Band J558 Id-specific 178-4 Ts analogous T cells leaves on both sides little room for ontogenetic variability in this immune response against bacterial antigens. (1) Rademaekers, A. and K6lsch, E. Eur. J. Immunol. 25: 623-626 (1995). Supported by DFG, SFB 310.

Institut fur Virologie und Immunbiologie, Universitat Wurzburg, Germany

G.21 Activation induced downmodulation of the CDSp chain on rat "I/o but not alP T cells F. STRAUBE and T. HERRMANN

The "(Ib T cells in blood, spleen and lymph nodes of rats - like in mice and men - represent a minor subpopulation of T cells. About 90% of human and mouse "(Ib T cells in peripherallymphoid organs are double negative for CD4 and CD8, and most of the remaining cells express CD8 (the aa homodimer). In contrast the respective "(Ib T cells of rats express CD8a~ (74 ± 4% in Lewis rat spleen), some bear the aa homodimer (14 ± 4% in spleen) and some lack CD8 (12 ± 3% in spleen). Only about 2-3% express CD4. LEW and F344 rat "(Ib T cells and purified CD8+ a/~ T cells were stimulated in vitro using immobilized anti TCR mAb (monoclonal antibody) and IL-2 or anti CD28 mAb as cosignal. After this activation the proportion of CD8~ expressing cells decreases in the "(Ib T cell population while expression of the ~ chain on CD8+ a/~ T cells remains nearly unchanged. By activating purified CD8W T cells we excluded the possibility that the effect could be due to different growth rates of "(Ib T cell subpopulations: In these experiments less than half of the "(Ib population retained normal expression of the CD8~ chain while the remaining cells became CD8~ negative at cell surface and mRNA level, but still expressed the CD8aa homodimer. Preliminary characterization of functional properties of CD8aa and CD8a~ expressing "(Ib T cells by intracellular FACS staining and RT-PCR showed no differences in the expression of IFN-"(, IL-2 and IL-4. Also no difference in lytic activity was found for redirected lysis. This report shows that the b chain of CD8, which is stably expressed on a/~ T cells can be modulated on rat "(Ib T cells. This plasticity of coreceptor expression represents a lineage difference between a/~ and "(Ib T cells. Moreover, exclusive expression of the CD8aa homodimer is no suitable marker for extrathymic origin of rat "(Ib T cells. Email: [email protected]

29th Annual Meeting 1998 . 505 Department of Neuropharmacology, Division of Virology, IMM6, the Scripps Research Institute, La Jolla, CA, USA

G.22 Influence of cytokines on autoimmune diabetes in a virally induced transgenic model: The role of the STAT 4 and STAT 6 signaling pathways A. HOLZ, A. BOT, B. COON, M. GRUSBY, and M. G. VON HERRATH RIP-LCMV transgenic lines show different kinetics in the onset of diabetes after viral infection. Fast-onset IDDM develops in lines without thymic expression of the self/viral-transgene. These lines generate high levels (1/100) of anti-self/viral cytotoxic T-lymphocytes (CTLs). In contrast, slow-onset IDDM occurs in transgenic lines that have only low-affinity CTL (H-2 d ) or low levels of CTL (about 1/3000; H-2 b) in the periphery. The slow-onset lines are the more relevant models for IDDM since they show participation of CD8+ as well as CD4+ T-Iymphocytes and antigen spreading in the autoimmune process. In the RIP-LCMV model, the kinetics and severity of IDDM have been associated with enhanced expression of T HI over TH2 cytokines. In order to dissect direct effects .of T HIlT H2 cytokines on the induction and progression of IDDM, we used stat-4 (IL-12 pathway) and stat-6 (IL-4 pathway) transcription factor deficient mice. Interestingly, deficiency in stat-4 did not abrogate rapid-onset IDDM, but completely prevented slow-onset IDDM. Generation of LCMV-specific autoreactive lymphocytes was not affected by the stat-4 deficiency and they were still capable to produce significant amounts of interferon-y in an IL-12 independent manner. Thus, a slowly progressive autoimmune process as present in pre-diabetic human individuals might depend on IL-12 mediated signaling likely through APCs in order to progress and lead to disease. In contrast, absence of stat-6 did neither affect the progression nor the incidence of IDDM in any of our RIP-LCMV lines. However, prevention of diabetes by oral insulin administration was not achieved in stat-6 deficient mice, although it was very efficient in stat-6 competent littermates (80-100% prevention of IDDM). Therefore, mediation of "oral tolerance" putatively through "bystander suppressor" lymphocytes depends on an intact IL-4 signaling pathway. Our study defines the profound regulatory involvement of the IL-12 and IL-4 signaling pathways during the pathogenesis of autoimmune diabetes and should further the development of selective immune-based intervention.

Department of Internal Medicine, Philipps-University, Marburg, and lDepartment of Internal Medicine, Charite University Hospital, Humboldt-University, Berlin, Germany

G. 23 Melatonin promotes Th 1-/ Tc 1-Differentiation of human T cells in vitro M. WAHLE, A. KRAUSE), P. V. WICHERT, and C. BAERWALD Background: Melatonin is a neurohormone produced by the pineal gland. Blood levels are increased during night. Radioligand binding studies demonstrated that receptors for melatonin are expressed on lymphocytes. Nuclear receptors have been described as well. Previous studies suggest that melatonin has immunomodulationg properties and it has been reported that melatonin stimulates Thl-differentiation in human T-helper-cells in vitro. Therefore a role in the circadian rhythmicity of the immun system has been proposed for melatonin. Methods: PBMC of healthy volu:nteers were isolated from heparinzed venous peripheral blood, incubated with melatonin (il0 or 30 ngl ml) for 3 or 24 h and then stimulated with PMA and Ionomycin for 6 h. Cultures without melatonin served as controls. Monensin (2 pM) was added to inhibit protein transport. Intracellular Interleukin-4 (IL-4) and Interferon-gamma (IFN-y) were determined in CD4+ and CD8+ lymphocytes using three-colour flow cytometry.

506 . 29th Annual Meeting 1998 Results: Mainly IFN-y was produced by human CD4+ and CD8+ T cells upon stimulation with PMA and Ionomycin in vitro. Compared to IFN-,¥, the number of IL-4 positive cells remained low. Incubation with melatonin increased the number of intracellular IFN-y positive CD4+ and CD8+ T cells significantly. The number of IL-4 positive CD4+ and CD8+ T cells was not affected after incubation with melatonin. Conclusion: Our results further underline the immunomodulating properties of melatonin. Physiological concentrations of melatonin promote IFN-y production in CD4+ and CD8+ human T cells in vitro. In contrast, IL-4 production is not influenced by melatonin. The effects of melatonin may be mediated by cell surface receptors expressed by lymphocytes. Nuclear receptors may be also involved.

Department of Clinical Immunology, Medical School Hannover, Hannover, Germany

G. 24 Expression of CD8aa and CD8a~ on Tcell populations T. WITTE, B. DRESCHER, and R.E. SCHMIDT The CD8 coreceptor can be expressed on the cell surface as a CD8aa homodimer or as a CD8ap heterodimer. CD8aa is found on NK cells and intraepithelial T lymphocytes. So far it has been believed that T cells in peripheral blood expressed predominantly the CDSap heterodimer. In previous experiments we and others have shown that the TCR can interact with both CDaa and CD8ap. However, the CDSap heterodimer is an approximately 100 fold more efficient coreceptor than CD8aa. We analyzed T cell populations obtained from peripheral blood using monoclonal antibodies against CD8a and CDSp. Two CD8+ T cell populations could be defined: Approximately SO% of these cells expressed predominantly CD8a13 whereas approximately 20% expressed more CDSaa than CDSap. Both populations were CD3+ and CD16-. The two populations could not be distinguished with antibodies against activation markers such as HLA-DR, CD25 and CD45RA and CD45RO. The population expressing predominantly CD8aa appears to be increased in peripheral blood of patients suffering from multiple myeloma. Currently we are trying to characterize the function of both T cell populations and determine if multiple myeloma can downregulate CDSap. Supported by SFB 2441 A09 and BMFT-Projekt 01 VMS60S/9

IGerman Cancer Research Center, Heidelberg, Germany; 2Basel Institute of Immunology, Basel, Switzerland

G. 25 CD44v7 is involved in the regulation of Th l-mediated autoimmune diseases B. M. WITTIG l , D. S. SCHMIDT!, U. GUNTHERT2, and M. ZOLLER! TNBS (trinitrobenzenesulfonic acid) induced colitis is considered as an animal model of chronic inflammatory bowel diseases with an overshooting Th1 cytokine production. It has been shown that silencing of costimulatory molecules abrogates the inflammatory activity in this model. Knowing that CD44, the standard as well as some variant isoforms functions as costimulatory molecules we evaluated whether blockade or deletion of the molecule might influence progression of TNBS-induced colitis. In the experimental model a single rectal application of TNBS in

29th Annual Meeting 1998 . 507 50% ethanol induces a severe colitis within 5 days. We have recently shown that treatment of mice with anti-CD44v7 prevented a death-promoting colitis in 71 % of mice and efficiently cured an established colitis in 90% of mice. The specificity of these observed effects was further strengthened by the observation that CD44v7 knockout mice did not develop experimental colitis. Resistance towards TNBSinduced colitis could be transfered to susceptibel mice strains (balb/c) by reconstituting them with bone marrow cells of CD44v7-ko mice. Investigation of the underlying mechanism revealed that anti-CD44v7 treated mice developed early signs of inflammation in the lamina propria with an increased interferon-g production. However, as compared to the control group, the intestine fully regenerated whereby in the lamina propria lymphocytes as well as in spleen cells a strong upregulation of IL-I0 production and a downregulation of IL-12 was noted with a delay of 3 to 4 days. There was no change in the expression of IL-4, indicating that there was no shift towards a Th2-cytokine response. Since CD44v7 is mainly expressed on activated T cells and antigen presenting cells (APC) whereby anti-CD44v7 treatment stimulates IL-I0 production of APC, it is tempting to speculate that CD44v7 is part of a regulatory circuit. We are currently in the process of evaluating our working hypothesis that CD44v7 actively triggers IL-I0 production by antigen presenting cells. We so far suggest that this trigger may require crosslinking of CD44v7 with e.g. the Fc receptor as signal transducing molecule.

Institute of Immunology and lDepartment of Nephrology at the University of Heidelberg, Germany

G. 26 Binding of neutrophil elastase to the cell surface of polymorphonuclear neutrophils (PMN): ~2-integrins might act as major binding sites F. ZIMMERMANN, K. ANDRASSY!, and G. M. HANSCH

Neutrophil elastase (HNE) is the major lysosomal enzyme of human PMN. Upon stimulation elastase is released from PMN; a part of the enzyme, however, remains attached to the cell surface. HNE can be found on the surface of PMN of patients with active Wegener's disease in vivo, but not on PMN of healthy donors. Expression can be induced by chemotactic peptides. Surface bound elastase is catalytically active, the catalytic activity is not inhibited by the physiological inhibitor acproteinase inhibitor. Scatchard plot analysis, competitive binding assays with . radiolabeled and unlabeled elastase, ligand plots as well as cross-linking of elastase to the cell surface pointed to the presence of multiple binding sites. Monoclonal antibodies (moAbs) to the I-domains of the ~2-integrin a-chains (CDlla or CDllb), an antibody to the complement receptor 1 (CRl, CD35) or an moAb against leukosialin (CD43) inhibited the binding of labeled elastase. Antibodies against the ~-chain of the ~2-integrins (CDI8) as well as antibodies to different other surface molecules and mouse-IgG were not inhibitory. Binding of exogenous HNE was also inhibited by a complement C3b-derived peptide containig the RGD sequence. Immunoprecipitation of membrane proteins from stimulated PMN with moAbs against CDII or a polyclonal antibody against HNE coprecipitated elastase and a small protein fragment with total molecular weight of about 40 to 50 kD. CDII was not precipitated as whole molecule suggesting protein cleavage during stimulation or membrane isolation. In summary our data suggest that CDII and CD35 serve as binding sites for elastase. This finding might be of particular interest with regard to the mechanism of cell activation by autoantibodies to elastase.