Workshop L: Cell traffic and adhesion

Workshop L: Cell traffic and adhesion

WORKSHOPL Cell TraHic and Adhesion A. Hamman; W. Zimmermann Centre of Anatomy, Medical School of Hannover, Germany L. 1 Continuous migration of activ...

5MB Sizes 0 Downloads 87 Views

WORKSHOPL Cell TraHic and Adhesion A. Hamman; W. Zimmermann Centre of Anatomy, Medical School of Hannover, Germany

L. 1 Continuous migration of activated T lymphocytes from blood into lymphoid organs and their subsequent organ- and compartment-specific proliferation within the tissue

U. BODE, C. DUDA, R. PABST, and J. WESTERMANN Lymphocytes continuously migrate from blood to all parts of the body. Lymphoid tissues offer specialized endothelia for entry of lymphocytes, the so called high endothelial venules (HEV). However, it is not clear whether activated T lymphocytes use this entry site or migrate via afferent lymphatics into lymphoid tissues. Once migrated into lymphoid organs, the fate (proliferation, return to the blood) of activated T lymphocytes within the tissue in vivo is still poorly understood. To study this, LEW RT.7B rat cells were activated via the T cell receptor (TCR) and CD28. For labeling, these cells were cultured in the presence of 5 }1M BromodesoxyUridine (BrdU). After injection into LEW RT.7A rats the migration of these cells was followed from the injection into the blood to lymphoid organs (peripheral- (pLN), mesenteric lymph node (mLN) and Peyer's patches (PP)) over time. To study the proliferation of activated T lymphocytes within the tissue (pLN, mLN, PP) by immunohistology, activated LEW RT.7B mLN T lymphocytes were injected into LEW RT.7A rats, which received one dose of BrdU lh before exsanguination (72 h after injection). Activated T lymphocytes were found in the wall of HEV of pLN, mLN and PP at all time points after injection (in pLN 1 h = 1.7 ± 0.8,24 h = 2.3 ± 1, 96 h = 4 ± 1.5 cells/mm2 HEV, n = 4-5). Activated mLN T lymphocytes proliferated highest in PP (T cell area: 5 ± 1%, B cell area: 12 ± 4%, n = 6) and in the medulla of mLN (18 ± 10, n = 6). While progeny of activated T lymphocytes was rarely seen at early time points after injection in the blood (2 ± 0.5%, n = 5), these cells increased in the blood at late time points (11 ± 1%, n = 5). The data clearly demonstrate, that activated T lymphocytes continuously enter lymphoid tissues from blood via HEV in vivo. Within the tissue these cells start to proliferate to a large extent irrespective of a special antigen, but in dependence on the organ and the compartment they enter, suggesting a function for establishing memory after an immune response. Furthermore, the data show that progeny of activated T lymphocytes, generated within the tissue, is released into the blood stream again.

Research Group Immunobiology, Biomedical Research Center, and Department of Dermatology, Heinrich-Heine-University of Diisseldorf, Diisseldorf, Germany

L. 2 Ultraviolet induction of nitric oxide synthase in endothelial cells: Different molecular mechanisms for UVA and UVB irradiation

D. BRUCH-GERHARZ, C. SUSCHEK, V. KRISCHEL, T. RUZICKA, and V. KOLB-BACHOFEN Nitric oxide (NO), a molecular mediator with vasodilative and immunoregulatory pro-perties, is a candidate molecule that may play a key role in sunburn erythema and inflammation. The mechanism and cellular source of NO production in these processes, however, is not yet known.

548 . 29th Annual Meeting 1998 In the present study, we demonstrate for the first time the ex-pression of inducible NO synthase (iNOS) mRNA and protein in endothelia of human skin organ cultures after UVA (50 J/cm 2 ) or UVB (200 mJ/cm2) irradiation. To elucidate the molecular mechanisms of iNOS induction in response to UV radiation, cultured rat aortic endothelial cells (AEC) were treated with UVA or UVB either alone or in the pre-sence of IL-lb, a potent inducer of iNOS expression. UVA (6 J/cm2) or UVB (50 mJ/cm2) irradiation of resting AEC resulted in iNOS mRNA expression but no induced NO pro-duction. The preincubation of AEC with IL-lP (200 U/ml) for 24 h prior to UVA irradiation did not increase iNOS mRNA expression or NO production. However, the incubation of AEC with IL-lP immediately after UVA irradiation led to an additive enhancement of iNOS mRNA expression and NO production when compared to non-irradiated cells. In contrast, preincubation of AEC with IL-lP for 24 h prior to UVB irradiation led to an additive enhancement of iNOS mRNA expression and NO production, which could not be observed when cells were incubated with IL-l P immediately after UVB irradiation. Strikingly, incubation of AEC with IL-lP 24 h prior and immediately after UV irradiation led to a synergistic increase in iNOS mRNA expression and NO production only in UVB- irradiated cells, but not in UVA-irradiated cells. We conclude that the expression of iNOS is a key component of the response of endothelial cells to UV radiation. UVA appears to enhance the iNOS-inducing activity of IL-l P, whereas UVB may induce cellular media-tors that synergize with IL-l P in inducing iNOS expression.

lLudwig Institute for Cancer Research, Lausanne Branch, 2Institute for Biochemistry, University of Lausanne, Epalinges, 4Institute of Microbiology, University of Lausanne, Switzerland, 3Complurense University, Madrid, Spain

L. 3 Homing and survival of Ag-primed Band C04+ T lymphocytes D. FINKEl, C. ARDAVIN3, F. LUTHI\ and H. ACHA-ORBEA 1,2 Studies on transfer of in vitro stimulated lymphocytes have shown that migration and persistance is determined by several factors including expression of adhesion molecules. To investigate the fate of Band T lymphocytes that had been activated in an in vivo environment we used BALB/c mice infected with mouse mammary tumor virus (MMTV). After subcutaneous injection of MMTV(SW) B lymphocytes of the draining lymph node become infected and present a retroviral superantigen (SAg) to CD4+ T lymphocytes expressing T cell receptor Vp6. As viremia is not observed after MMTV injection and residual re-infection can be blocked by AZT treatment, detection of viral DNA in other organs reflects migration of infected cells exported from the draining lymph node. Furthermore, this model allows to study the distribution of MMTV SAg-reactive Vb6+ T lymphocytes by flow cytometry. Our data show that extrafollicular plasmablast B cells migrate to peripheral lymphoid and non-lymphoid organs in a particular order before germinal centers are formed. The infected plasmablasts seem to be long-lived and have lost Ag presentation capacity. Ag-primed B cells highly upregulate expression of a4-integrin but the levels of any known associated b chain are unchanged implicating a novel b chain linked to a4-integrin. The migration of lymphocytes to the mammary gland is abrogated by blocking of a4 in vivo. SAg-reactive CD4+ T cells become deleted in the course of infection but persist in the primary responding lymph node.

29th Annual Meeting 1998 . 549 Department of Pneumology, Medical University Clinic, Freiburg, Germany

L. 4 Modulation of chemotaxis, viability and activation of human eosinophils

by transforming-growth-factor-~

P. FRANZ, W. LUTIMAN, H. MATTHYS, and J. C. VIRCHOW JR. Eosinophilic granulocytes have been implicated in the pathogenesis of asthma, parasitic infestations and several other immunological disease. Chemotaxis, degranulation, production of lipid mediators and expression of cell surface proteins can be induced following eosinophil activation. TGF-~, a multifunctional protein, which inhibits the growth and activity of inflammatory cells, has been suggested to lay a role in the regulation of chronic inflammation. Since TGF-~ has recently been found in increased concentrations in asthmatic sputum we investigated whether TGF-~ could influence eosinophil migration, activation and eosinophil viability. Peripheral blood eosinophils from normal donors were isolated by Percoll gradient centrifugation and negative MACS separation. Chemotaxis was measured with a modified Boyden chamber technique, activation by analysing HLA-DR expression on the cell surface and eosinophil viability was analysed flowcytometrically by propidium iodide exclusion. Eosinophil chemotaxis was significantly increased in the presence of TGF-~ in concentrations between 10-9 and 10-4 ]lg/ml. The optimal concentration of TGF-~ in this assay was 10-9 and 10-4 ]lg/ml TGF-~. The chemotactic effect of TGF-~ diminished when higher as well as lower concentrations (between 10-12 and 10-3 ]lg/ml) were employed. In contrast, inhibition of eosinophil survival induced by IL-3, IL-5 and GM-CSF reached its maximum at concentrations of TGF-~ between 10-4 and 10-3 ]lg/ml. While isolated eosinophils express low levels of surface HLA-DR, incubation with GM-CSF and IL-3 increased HLA-DR expression on eosinophils significantly. However, coincubation of TGF-~ together with either GM-CSF or IL-3 decreased the expression of HLA-DR. This effect of TGF-~ on IL-3 induced HLA-DR expression was attenuated dosedependently in the presence of monoclonal anti- TGF-~ antibodies. Furthermore, similar to the results observed on HLA-DR expression, TGF-~ reduced viability of activated eosinophils. From these data we conclude that TGF-~ in low concentrations can induce eosinophil chemotaxis whereas higher concentrations reduce HLA-DR expression and eosinophil survival mediated by IL-3, IL-5 and GM-CSF.

Molecular Immunology, Robert Koch-Institute, Berlin, Germany

L. 5 C040 on human platelets induces down-regulation of co-expressed TRAP (C040 ligand) V. HENN, and R. A. KROCZEK We have recently shown that platelets, apart from their central role in hemostasis, are involved in the initiation of an inflammatory response of the vessel wall. Following stimulation/aggregation platelets rapidly express membrane-bound TRAP (TNF-related activation protein/CD40 ligand/CDI54) for a short period of time. TRAP on platelets can interact with CD40 on endothelial cells and thus induce these cells to express adhesion molecules and to release chemokines. Here we show that platelets, in addition to TRAP, also co-express CD40. In contrast to TRAP, CD40 is present constitutively on the platelet surface and is not downregulated following platelet stimulation. In the course of platelet aggregation the interaction of TRAP and CD40 molecules leads to the activation of an as yet unidentified platelet-bound protease which quamitatively cleaves off membrane TRAP within 1 h from the cell surface. The resultant 18 kDa and 17 kDa soluble TRAP molecules have no inflammatory effects on CD40 expressing endothelial cells. Neither blocking of the TRAP/CD40 interaction nor crosslinking of CD40 has

550 . 29th Annual Meeting 1998 an effect on the aggregation of platelets. We thus could identify the mechanism responsible for the downregulation of platelet TRAP. The rapid removal of TRAP from the platelet surface limits the inflammatory potential of this molecule and prevents a prolonged and unspecific activation of other CD40 expressing cells such as B cells and monocytes in the vascular system. At present we investigate whether CD40 transduces additional signals into platelets.

Institute of Pathology, Friedrich Schiller Universitat Jena, lUniversity Hospital, Med. Clinic, Department of Immunology, Hamburg, Germany

L. 6 Antigen-induced arthritis is reduced in LFA- 1 and ICAM-l deficient mice G. H. W. HERSMANN, A. HAMANN l , S. HENZGEN, J. SIMON, K. THoss, and R. BRAUER In antigen-induced arthritis (AlA), an experimental model of rheumatoid arthritis, various adhesion molecules are highly expressed in the arthritic knee joint. Recently, we have shown therapeutic effects of antibodies against adhesion molecules on arthritis development. Now we determined the incidence and development of AlA in LFA-l and ICAM-l knockout (KO)-mice (Imperial Cancer Research Fund, London). Arthritis was induced by i.a. injection of methylated bovine serum albumin (mBSA) into the knee joint cavity of mice preimmunized with the same antigen in complete Freund's adjuvant. Flare-up was provoked by i.v. injection of mBSA 6 weeks after induction of primary arthritis. Arthritis development was determined by measurements of joint swelling and histological scoring. Delayed type hypersensitivity (DTH) was measured as ear swelling. Antibody formation against mBSA, collagens and proteoglycans as well as immunoglobulin subclasses were measured by ELISA. Joint swelling was reduced in ICAM-l KO-mice compared to wild type mice and control group of C57B1I6 mice. Histological arthritis score was ameliorated in both LFA-1 and ICAM-l KO-mice compared to control mice. DTH was reduced in both LFA-l and ICAM-l KOmice. During flare-up reaction joint swelling was diminished in ICAM-l KO-mice and histological score was significantly reduced in KO mice. Cartilage and bone destruction was significantly reduced in LFA-l KO-mice. Differences in DTH and antibody formation were not found during this time. However, IgG2a production in LFA-l KO-mice was significantly reduced compared to wild type mice in primary arthritis as well
IDivision of Clinical Immunology, 2Division of Clinical Psychology, Hannover Medical School, Hannover, 3Division of Medical Psychology, University of Essen, Germany 4lmmunology, University Hospital for Children and Youth, Utrecht, the Netherlands

L. 7 Lymphocrte subsets and imm.nune function during stress in patients

suffering from systemic lupus erythematosus or rheumatoid arthritis

R.JACOBS l , C. PAWLAK l ,2, E. MIKESKA l , S. OCHSMANN l , D. MEYER l , C. HEI]NEN\ M. SCHEDLOWSKI3, and R.E. SCHMIDTl In systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA) an association between psychological stress and the course of these inflammatory diseases has been assumed for a long time. In order to elucidate the role of stress in this context patients (pts) and normal controls

29th Annual Meeting 1998 . 551 were asked to perform a public speech to induce acute psychlogical stress. Cardiovascular, endocrinological and immunological variables were recorded before, directly after the speech, and one hour later. At these time points blood samples were drawn to determine catecholamine levels, lymphocyte subsets (CD2+, CD3+, CD4+, CD8+, CD16+, CD20+, and CD56+), NK activity (vs K562), intracellular cytokines (IL-2, IL-4, IL-6, IL-lO, IFN-y), and cytokine contents collected from supernatants of PHA or LPS stimulated mononucleated cells. The expression of adrenergic receptors on lymphocytes was assessed. Cardiovascular parameters (frequency of the heart and blood pressure) were monitored continuously during the whole experiment. The experiments revealed clearly stress-induced increases of heart frequency and blood pressure in all three groups. Changes in lymphocyte counts in particular of NK cells (CDI6+/CD56+) were observed in normal controls and RA pts, but not in SLE pts. The increases in NK cells were paralleled by enhanced cytotoxicity only in normal controls but not in RA pts. Enhanced IFN-y and IL-2 production under stress was only seen in controls, but not in pts. Although all SLE pts were lymphopenic absolute numbers of IFN-Y cells were comparable to normal controls due to increased percentage of these particular cells that double after stress in all three groups. In addition, percentages of IL-4+ cells were also increased in SLE pts (4% vs 2%) resulting in absolute numbers comparable to the other groups. These IL-4+ cells doubled due to the stress event only in SLE pts. The expression of adrenoceptors did increase in normal controls but decreased in SLE and RA pts. These data suggest a different immunological reponse to stress in normal controls and pts with inflammatory diseases. This study was supported by Volkswagen Stiftung 1/72 031

lInstitute of Immunology and Transfusion Medicine, University of Lubeck School of Medicine, Lubeck, Germany; Departments of 2Immunology and 3Internal Medicine, University of Tartu, Estonia

L. 8 Polymorphisms in the tumor necrosis factor (TNF), human leukocyte anti-

gen (HLA) and adhesion molecule genes in estonian patients wit~ inflammatory bowel disease

K. HIRV!, M. SEYFARTH!, R. UIB0 2, K. KULL3, R. SALUPERE3, and 1. RINK! There is some evidence that genetically determined susceptibility to inflammatory bowel disease (IBD) exists, but genes decisively associated with this disorder, have not been found. Regarding the crucial role of the immune system in mediating the tissue damage in IBD, genes involved in initiation and regulation of the immune response are potential candidates for investigating genetic susceptibility. Recent studies have shown that even within different clinical forms of IBD, ulcerative colitis (UC) and Crohn's disease (CD), heterogeneous subgroups of diseases may be found. In our study, specific clinical markers of these diseases, antineutrophil cytoplasmic antibodies (ANCA), were taken into consideration by dividing UC and CD into probably more homogeneous subgroups. We believe that the group of patients and controls from the small and homogeneous Estonian population gave us a gC!0d opportunity to investigate the genetic heterogeneity of IBD. TNFII2, TNFB':-1I2 and eight polymorphisms in ICAM-l, E- and L-selectin genes have been analysed. In addition HLA-DR, -DQ typing was performed. There were significant differences in allele frequencies between control group (n=70) and patients (n=51), after dividing the patients into subgroups concerning the clinical form of IBD (UC, n=31 or CD, n=20) or the presence of ANCA. In the genes encoding adhesion molecules, in patients with CD the less frequent allele of the E-selectin EGF-like domain was detected in higher frequency in comparison with controls (20% vs. 14%) and conversely the less frequent allele of the L-selectin EGF-like domain was found in lower frequency (15% vs. 22%). Functionally important polymorphism in the TNFa promotor region at position -308 (TNFII2) seems to be associated with ANCA-posi-

552 . 29th Annual Meeting 1998 tivity, especially in patients suffering from DC (TNF2 allele frequency 22% vs. 0% in the ANCA-negative group with DC). Additionally, we can confirm the previously described results, that HLA-DRBP15 is associated with ANCA-positive subgroup of patients with DC (35% vs. 13% in the ANCA-negative group with DC). Interestingly 91 % of the DC patients with TNF2 or HLA-DRBP15 were ANCA-positive, in comparison with 33% of the DC patients without TNF2 or HLA-DRBP15. Recent studies have shown that ANCA is not only an epiphenomenon of IBD, although exact molecular mechanism of its involvement in the pathogenesis of IBD have not been found. Our findings may be helpful in clarifying the role of the ANCAs and molecules encoded in the major histocompatibility complex region for the pathogenesis of IBD. We suggest that dividing DC and CD patients into further subgroups on the basis of genetic or clinical markers is necessary in order to elucidate the pathogenesis of IBD.

Research Group Immunobiology, MED-Heinrich-Heine-Dniversity of Dusseldorf, Dusseldorf, Germany

L. 9 Inhibition of ICAM- 1 synthesis in mouse endothelial cells by nitric oxide at the transcriptional level K.-D. KRONCKE, D. BEREND]I, and V. KOLB-BACHOFEN Intercellular adhesion molecule-1 (ICAM-1) is an inducible cell surface glycoprotein expressed on the surface of activated endothelium and serves as a receptor for the leukocyte integrins lymphocyte function-associated antigen-1 (LFA-1), Mac-I, and CD43. Adhesion of circulating polymorphonuclear leukocytes to the vascular endothelium via ICAM-1 is a critical step in the inflammatory response. An antiadhesive activity of nitric oxide (NO) has been repeatedly observed. Recently NO has been found to inhibit ICAM-l protein expression on endothelial cells. In addition" inducible NO synthasecdeficient mice show enhanced leukocyte-endothelium interactions during endotoxemia. We now have studied the molecular mechanism of the NOmediated inhibition of ICAM-l expression on EC. Therefore, primary mouse endothelial cells were activated with IL-l~ in the presence or absence of the spontaneous physiological NOdonor S-nitrosocysteine (SNOC). By RT-PCR we found, that SNOC at untoxic concentrations inhibits IL-l~-induced ICAM-l mRNA expression in a concentration-dependent manner. In contrast, HzO z at subtoxic concentrations does not inhibit ICAM-l mRNA expression. The ICAM-l promoter contains binding sites for the transcription factors Spl, AP-l, and NF-lCB. Data will be presented that show interactions of NO with the DNA binding activities of these transcription factors. We conclude that nitrosative in contrast to oxidative stress inhibits ICAM1 expression at the transcriptional level and that NO acts as a gene-regulatory radical.

Max-Planck Institut fur physiologische und klinische Forschung, WG. Kerckhoff-Institut, Abt. Molekulare Zellbiologie, Bad Nauheim, Germany

L. 10 Differential involvement of a4-integrin/VCAM-l interaction during

adhesion and transendothelial migration of autoagressive T cell blasts with brain endothelium in vitro

M. LASCHINGER, and B. ENGELHARDT Migration of encephalitogenic T cells across the blood-brain-barrier (BBB) endothelium is a crucial step in autoimmune inflammatory diseases of the CNS such as experimental autoimmune encephalomyelitis (EAE). Based on the observation that antibodies directed against a4-integrin

29th Annual Meeting 1998 . 553 and its counterreceptor VCAM-l have been shown to inhibit the T cell infiltration of the CNS during EAE, it has been suggested that a4-integrinIVCAM-1 interaction plays a critical role in T cell recruitment into the CNS. In order to define the role of a4-integrin and VCAM-l in T cell migration across the BBB we set up in vitro studies where we investigated the interaction of autoagressive T cell blasts with the brain endothelial cell line bEndS. By using a large panel of mAbs against the a4-, ~1-, ~7-integrin subunit and the a4~7-heterodimer, we could demonstrate that the different antibodies reduced adhesion of T cell blasts to bEndS to a dramatic degree (73±S%). A panel of anti-VCAM-l mAbs also inhibited adhesion of T cell blasts to activated bEndS, however, to a slightly lesser degree than did the anti-integrin mAbs. In contrast, none of the above antibodies showed any significant effect on transendothelial migration of T cell blasts across the bEndS monolayer. At the same time mAbs directed against LFA-l and ICAM-l reduced transmigration of T cell blasts across activated bEndS by 72±4% and 47±13%, respectively. Based on our in vitro observations, a4-integrins mediate adhesion of T cell blasts to brain endothelium mainly via VCAM-l, however, a4/VCAM-l mediated interaction is not critically involved in transendothelial migration of T cell blasts. Forschungszentrum Borstel, Abteilung Immunologie und Zellbiologie, Borstel, Germany

L. 11 Intracellular and surface expression of differentially glycosylated CXCchemokine receptor 2*

A. LUDWIG, J. E. EHLERT, H.-D. FLAD, and E. BRANDT In neutrophils the CXC-chemokine receptor 2 (CXCR-2) mediates chemotaxis in response to the CXC-chemokines IL-8 and NAP-2. Thereby the receptor undergoes a regulatory process which involves its internalization and reexpression. However, nothing is known about the existence of extracellular and intracellular pools of CXCR-2 and their molecular properties. In this study we demonstrate and separately characterise these two receptor populations by means of immunoprecipitation using one of our monoclonal antibodies to CXCR-2 (moAb RIIllS). Surface expressed CXCR-2 was analysed following its crosslinking to radiolabeled chemokines on intact neutrophils. After subsequent cell lysis and immunoprecipitation a single 62 kDa receptor-ligand complex was identified by electrophoresis and autoradiography. However, the determined molecular weight of the crosslinked receptor was considerably higher than the value (37 kDa) predicted from its amino acid sequence. This discrepancy was due to glycosylation of the receptor since treatment of precipitates with N-glycosidase F shifted its apparent weight from 62 kDa to 44 kDa. In a different approach, using biotinylated moAb RIIllS the immunoprecipitated receptor was detected in Western blots without previous crosslinking to labeled ligands. A 55 kDa protein, and several additional proteins, one of which migrated at 37 kDa, were detected in immunoprecipitates from lysed cells. All these proteins represented CXCR-2 since (i) they were also recognized by another antibody reactive to a different region of CXCR-2 and (ii) they were also present in a murine fibroblast cell line (3T3) transfelYted with CXCR-2 (but not in CXCR-l transfectants). Upon treatment with N-glycosidase F all precipitated proteins migrated at 37 kDa indicating that the 55 kDa receptor is N-glycosylated, while the 37 kDa protein does not carry N-linked sugars. However, in contrast to the unglycosylatyd 37 kDa protein, only the glycosylated 55 kDa receptor is expressed on the cell surface. This was shown by precipitation of exclusively the latter molecule following in~ubation of intact cells with moAb RII115, a method that allowed the selective precipitation of s~rface expressed receptors. This glycosylated form of CXCR-2 completely disappeared from the cell surface upon stimulation with NAP-2. Moreover, after recovery from NAP-2 treatment reexpressed extracellular CXCR-2 consisted only of the glycosylated form while no unglycosylated receptors were found. Since we, have observed, that enzymatic removal of sialic acids on neutrophil~ reduces chemokine binding and chemokine-induced degranulation of the cells, it appears likely that the glycosylation of extracellular CXCR-2 is of functional relevance. e:·supported in part by Deutsche Forschungsgemeinschaft SFB 367).

554 . 29th Annual Meeting 1998 Department of Immunology, Vilnius University Heart Surgery clinic; lClinic of Pulmonology and Allergology, Vilnius University, Vilnius, Lithuania

L. 12 Lymphocyte subsets of lung lavage cells from patients with interstitial lung diseases

L. JURGAUSKIENE, R. MALICKAITE, and E. DANILA! In order to analyse relative contribution of bronchoalveolar lavage (BAL) fluid investigation in differential diagnosis of lung diseases, 31 subjects (8 healthy non-smokers, 10 healthy smokers, 12 patients with the suspicion of sarcoidosis as well as one patient with the suspicion of idiopathic fibrosing alveolitis) were examined by fibrobronchoscopy. Cell differentials and lymphocyte surface markers investigation were performed from bronchoalveolar lavage fluid obtained during this procedure. The cell smears of native BAL preparations have been stained with MayGrunvald-Giemsa. In order to reach a good reproducibility, differential cell count was performed by counting at least 600 cells. Phenotyping of lymphocyte subsets using monoclonal FITC and PE conjugated antibodies (Becton Dickinson, DAKO) has been performed by FACSCalibur flow cytometer (Becton Dickinson). In 6 cases diagnosis of sarcoidosis was accepted relaying on high cellularity with the lymphocyte predominance (lymphocytes constitute 42.3±13.1 % of total recovered BAL cells in sarcoid patients, 9.4±6.1 % in healthy nonsmokers and 17.5±2.2% in healthy smokers). T cells constitute up to 92.4±4.9% of totallymphocytes in sarcoid group comparing to 82.5±8.9% in healthy non-smokers and 84.0±9.3% in healthy smokers. Exogenous allergic alveolitis of unclear etiology has been diagnosed in one case examined in suspicion for idiopathic fibrosing alveolitis. In this case a marked lymphocytosis (94%) with CD8+ lymphocyte predominans up to 79% has been found. 41 % of CD8+CD57+ and 73.6% of CD54+ positive cells were found in exogenous allergic alveolitis patient comparing to 7.1±2.5% and 31.6±3.6% accordingly in healthy control. BAL fluid investigation had shown diagnostic significance and good correlation with transbronchiallung biopsy specimens examination in interstitial lung diseases.

!University of Leipzig, Institute of Zoology, Department of Immunbiology, Leipzig; 2University of Erlangen, Institute of Biochemistry, Erlangen, Germany

L. 13 NAO-degrading activity on human monocytes: A role for C038? M. PFISTER!, A. OGILVIE2, and S. HAUSCHILDT! CD38 is a type II transmembrane glycoprotein prevalently expressed by immature and activated T and B lymphocytes. Two main functions of this protein have been reported. First it has multifunctional ectoenzyme activities (NAD-glycohydrolase, ADP-ribosylcyclase and cyclic ADP-ribose [cADPR] hydrolase) catalyzing the formation of ADP-ribose (ADPR), nicotinamide and cADPR from NAD. Second it is involved in cell-cell interaction. CD38 acts as an adhesion molecule mediating selectin-like binding to endothelial cells via its counterreceptor CD31.We have shown that during the differentiation of human monocytes (61-96% CD38+) to macrophages the expression of CD38 declines (2-17% CD38+). The loss of CD38 is paralleled by a decrease of extracelluar NAD-glycohydrolase activity of the cells, suggesting a role of CD38 in extracellular NAD degradation. The main product of NAD degradation was identified as ADPR which might subsequently be transfered to cytosolic and membrane proteins (mono-ADP-ribosylation). To test whether monocytes also possess intracellular NAD glycohydrolase and ADP-ribosylcyclase activity we applied a fluorimetric assay using £-NAD and NHD respectively as substrates. We could demonstrate that both enzyme activities are measur-

29th Annual Meeting 1998 . 555 able in lysates and cytosols of monocytes. The monocytic cyclase activity (cells and lysates) amounts to 2 to 3 percent of the hydrolase activity. By Western blot analysis we detected a 38 to 40 kDa protein band which might depict an intracellular (soluble?) isoform of the CD38 molecule. The importance of the CD38-like enzymatic function on and in human monocytes will be discussed with respect to NAD-glycohydrolase activity seen in other cell types.

Institute of Immunology, University of Muenchen, Munich, Germany

L. 14 Preferential localization of CO 14+CO16+ monocytes in the marginal pool

B. STEPPICH, and H. W. 1. ZIEGLER-HEITBROCK In human blood there are 2 monocyte populations: the strongly CD14 positive (CD14++) cells and the CD14+CD16+ monocytes that express CD14 at low density together with the CD16 Fe receptor. The CD14+CD16+ monocytes exhibit higher levels of adhesion molecules suggesting that they may interact more avidly with the vascular endothelium which then may lead to a preferential localization of these monocytes to the marginal pool. We have therefore asked whether the CD14+CD16+ monocytes can be mobilized by stress in the form of physical exercise. When healthy young males exercised for 1 min at 100 or 200 W then there was no change in any of the monocyte populations. An exercise of 1 min at 400 W resulted, however, in a selective 2fold increase of the CD14+CD16+ monocytes, while the CD14++ monocytes were hardly affected (1.2fold). After 2 min at 400 W the CD14+CD16+ monocytes increase by up to factor 3. Expression of adhesion molecules on the mobilized CD14+CD16+ cells compared to the CD14++ monocytes was 3 and 4fold higher for CD11d and VLA-4, respectively. The selective increase of the CD14+CD16+ monocytes was evident within seconds after the 1 min exercise, cells remained high for 5 min and returned to the initial value after 20 min. The data suggest that the majority of the CD14+CD16+ blood monocytes resides in the marginal pool.

University of Leipzig, Institute of Zoology, Department of Immunbiology, Leipzig, Germany

L. 15 A possible role of N-cadherin in monocyte function A. THIELE and S. HAUSCHILDT Thalidomide known as a highly potent teratogen has been readdressed as an immunomodulating and angiogenesis-inhibiting drug. The molecular mechanism underlying the action of thalidomide is still unknown. Its immunomodulatory property has partly been ascribed to its ability to inhibit the production of TNF-a. Since modulation of the adhesion cascade has also been claimed as a possible mode of the action of thalidomide we focussed our attention on the adhesion molecule N-cadherin as a possible target for thalidomide. N-cadherin plays a significant role in early embryonic development and its expression on endothelial cells is well documented. Here we show that N-cadherin is present on human monocytes and monocytic cell lines. Ligation with a monoclonal antibody, which is believed to prevent homophilic binding of two Ncadherins by binding to the the adhesion face, results in an increased TNF-a production in stimulated cells. The interaction between thalidomide and N-cadherin is presently under investigation.

556 . 29th Annual Meeting 1998 1 Institute of Experimental and Clinical Pharmacology and Toxicology, 2 Institute of Experimental Medicine, 3 Institute of Anatomy, University of Erlangen-Nurnberg, Erlangen, Germany

L. 16 Expression and functional role of adhesion molecules in T cell-mediated hepatitis in mice

D.WOLF 1, R. HALLMANN2 , S. KDsTERSl, W. NEUHUBER3, and G. TIEGs l The role of selectins for sticking and rolling of lymphocytes along endothelial cells, and of ICAM-1/LFA-1-interaction for their tight adhesion and subsequent transendothelial migration is well documented. However, it rem~ins obscure whether these adhesion molecules take part in the accumulation of T cells in hepatic sinusoids and subsequent transmigration into the hepatic parenchyma. In our present study we investigated the expression and the functional role of these m?lecules in a well established model of CD4+ T cell-, TNF- and IFN-y-mediated hepatitis in mlce. We demonstrated with immunofluorescent staining and confocal laser imaging that ConAapplication results in CD4+ T cell accumulation throughout the hepatic parenchyma 6 hours after challenge. 24 hours after ConA most of the CD4+ T cells were detectable in the periportal area. Time-course analysis of adhesion-molecule expression revealed strong induction of ICAM-1 staining on sinusoids, central and portal veins as early as 6 hours after ConA. 24 hours after ConA the same distribution was detectable. VCAM-1 expression also increases 6h hours after challenge with strong staining of portal vessels, whereas the staining of the sinusoids was less prominent. 24 hours after ConA only portal veins were stained. E-selectin was detectable on Kupffer cells, sinusoids, central and portal veins 6, 8 and 24 hours after ConA but staining was discontinuous. We provide evidence that the expression of these adhesion molecules did not play any functional role for the onset of acute liver injury. Neither application of blocking mAb against Eselectin (UZ4) or P-selectin (RB40) or a combination of both, nor anti-ICAM-1 (YN 1/1.7.4.) together with anti-LFA-1 (MK 17/4) were able to prevent acute liver injury as detected of plasma ALT- and AST-measurement. Furthermore ICAM-1 knockout-mice failed to be protected against acute hepatitis. CD4+ T cell accumulation, as well as plasma cytokine levels (TNF, IFN-y, IL2) and oligonuc1eosomal DNA-fragmentation in liver homogenates of mAb-pretreated mice were unaltered. Hence ConA-induced hepatocellular damage is unlikely to depend on selectin or ICAM1/LFA-1-mediated lymphocyte accumulation in hepatic sinusoids and subsequent transendothelial migration.