Workshop E Inflammation

WORKSHOP E Inflammation

Department of Internal Medicine I, Division of Hematology, Department of Surgery, Department of Anaesthesiology B, Sandoz Research Institute, Vienna, Austria

E.l Human mast cells produce monocyte chemotactic protein-l M. BAGHESTANIAN, D. BEVEC, R. HOFBAUER, M. R. MOLLER, H . G. KRESS, M. WILHELM, H. AGIS, W. FOREDER, K. LECHNER, and P. V ALENT Chemokines are proinflammatory peptide mediators involved in the activation and chemotaxis of various leukocytes. In the present study expression of chemokines in isolated human lung mast cells (MC) (> 90 %) and the human MC line HMC-l was analyzed by Northern blotting using oligonucleotides. We found that lung MC and HMC-l constitutively express MCP-l (Monocyte Chemotactic Protein-I) mRNA, but not IL-8 (Interleukin-8), MIP-la (Macrophage Inflammatory Protein la), MIP-l~ or RANTES (Regulated on Activation, Normal T Expressed and Secreted) mRNA. Incubation of lung MC or HMC-l with rh (recombinant human) SCF (stem cell factor) for 2 to 4 hours resulted in an increased expression of MCP-l mRN A. As assessed by ELISA, HMC-l cells contained 100-300 pg MCP-l per 10' cells . A constant release of MCP-l over time from HMC-l cells into the supernatants (sups) was seen. Functional significance of MCP-l expression in MC could also be demonstrated: MC-derived sups as well as rhM CP-l-induced migration of purified (80-90 %) human blood monocytes (control: 3034 + 37 MC vs rhMCP-l, 10 ng/ml: 15.200 ± 49 vs HMC-l sups: 13.350 ± 480) and an antibody against MCP-l abrogated the effect of both stimuli. Together these data show that human MC are a source of MCP-I. MPC-l may be involved in the pathogenesis of mast cell-dependent immune processes associated with monocyte/ macrophage accumulation.

Joint annual meeting 1995 GGAI/GFI

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Biochemical Pharmacology, Faculty of Biology, University of Konstanz, Konstanz, Germany

E.2 Granulocyte-macrophage colony-stimulating factor (GMCSF) and interferon-y (IFN-y) counteract LPS-refractoriness in vitro in various macrophage populations and in vivo in mice

J. BARSIG, D. S. BUNDSCHUH, and A. WENDEL We have recently shown that the hematopoietic cytokine GM-CSF potentiates lipopolysaccharide (LPS)-induced Tumor Necrosis Factor (TNF) release and toxicity in mice (TIEGS et al. 1994. J. Clin. Invest. 93: 2616). IFN-y is also known to increase lethality in experimental endotoxemia and to prime for LPS-induced TNF release in mice. We were interested in investigating the immunomodulating capacity of GM-CSF and IFN-y in immunocompromised animals. Various macrophage populations from control mice were incubated with GM-CSF or IFN-y (10 ng/ml) 2 h before 1 [!g/ml LPS. These preincubations resulted in an enhanced release of TNF 6 h after LPS. Cells prepared from mice made tolerant by pretreatment with sublethal amounts of LPS (300 [!g/kg i.p., -24 h) did not respond to LPS stimulation. In contrast, cells exposed to GM-CSF or IFN-y partly regained their sensitivity for LPS. These in vitro findings were confirmed in vivo: GMCSF or IFN-y pretreatment increased plasma TNF release as well as mortality in mice challenged with 3 mg/kg LPS. Mice made tolerant by administration of 30 [!g/kg LPS (-24 h) failed to produce TNF after LPS (3 mg/kg) and survived LPS-induced shock. The impaired immunocompetence of such tolerized animals was restored by GM-CSF or IFN-y pretreatment (50 [!g/kg i.v., -45 min), i.e. the animals responded to LPS with increased TNF release and mortality compared to tolerant mice. These results demonstrate the GM-CSF and IFN-y are potent enhancers of LPS-induced TNF production in normal as well as in immunocompromised mice.

1 2

Biochemical Pharmacology, Faculty of Biology, University of Konstanz, Konstanz; Institute for Medical Immunology, Charite, Humboldt University, Berlin, Germany

E.3 Lipopolysaccharide-induced interleukin-10 in mice: role of endogenous tumor necrosis factor-a

Interleukin-l0 (IL-I0) is a potent anti-inflammatory cytokine, that attenuates lipopolysaccharide (LPS)-induced tumor necrosis factor-a (TNF-a)-release and protects animals from LPS-induced shock. We investigated interactions of LPS-induced circulating TNFa and IL-l 0 in mice. Injection of a lethal dose of LPS (5 mg/kg i.p.) caused a rapid rise in plasma levels of TNF-a as well as IL-I0 (peak concentrations 1-2 h and drop between 2 and 6 h after challenge). In contrast to TNF-a, IL-I0 showed a biphasic time course with a second rise between 8 and 12 h after LPS-injection. Neutralization of TNF-bioactivity in vivo, using a polyclonal TNF-a anti-serum, resulted in augmentation of early IL-I0 levels and reduction of late ones. Analysis of LPS-induced IL-I0 mRNA expression in different tissues 1 hand 8 h after LPS or LPS plus anti-TNF-a revealed that the amount of transcripts especially in livers correlated with circulating early and late IL-IO levels.

70 . Joint annual meeting 1995 0GAI/GFI These results demonstrate a regulation by LPS-stimulated circulating TNF-a of IL-I0 release in mice, predominantly originated from the liver. Furthermore, our findings have implication for anti-TNF strategies: Neutralization of TNF might not only be antiinflammatory by reducing detrimental effects of TNF itself but also by augmenting concentrations of IL-I0, that could further counteract inflammatory processes.

Department of Dermatology , University of Gottingen, Gottingen; I Department of Clinical Pharmacology, Hannover Medical School, Hannover, Germany

E.4 Nitric oxide inhibits cytokine production by activated human T cells H. BAUER, T. JUNG, B. MERZ, D. TSIKAS 1, D. STICHTENOTH 1, J. C. FROLICH 1, and C. NEUMANN At present the mechanisms which lead to the preferential development of Th2 cells in the skin of patients with atopy are still unknown. Nitric oxide (NO) has recently been shown to selectively inhibit the proliferation and secretion of cytokines of murine Thl cells. Furthermore, it has become clear that NO can be produced by cells of human skin. Therefore we investigated whether the secretion of Th2-(IL-4, IL-5, IL-I0) and Thlcytokines (IFN-y) of CD3/CD28-activated human peripheral blood-derived T cells were differently affected by NO. Cytokine secretion (ELISA) and T cell proliferation were measured in the presence of SIN-l and SNAP, which are well established as NO donating agents. SIN-l and SNAP markedly inhibited the production of IL-4, IL-5, IL10 and IFN-y as well. In contrast, SIN-l and SNAP did not affect T cells activated with PMA and Ionomycin. The NO-donors were equally effective when different T cell subpopulations were tested (CD4+, CD8+, CD45RA +, CD45RO+). As shown by a single cell assay NO-donors reduced the frequency of IFNy producing cells. We also showed that SOD and Catalase did not restore the effects of SIN-l and that 8-Br-cGMP reduced cytokine production to a similar extend as the NO-donors excluding oxygen radicals as a cause for the observed inhibition of T cell cytokines. Furthermore, high concentrations of SIN-l and SNAP strongly inhibited T cell proliferation. We also showed that the inhibiting effects of No were accompanied by a reduced calcium mobilization. Our results point to general suppressive effect of NO on various human peripheral blood T cell populations. It remains to be established whether human Thl- and Th2-cell clones will react differently as it was described in the mouse.

Joint annual meeting 1995 OGAI/GFI . 71 1Max-Planck-Institute

for Psychiatry, Department of N euroimmunology, Martinsried, Germany; 2Newcastle General Hospital, Muscular Dystrophy Group Research Laboratories, Newcastle Upon Tyne, Great Britain

E.S Localization of Fas-Receptor on inflammatory cells and muscle fibers in human inflammatory myopathies L. BEHRENS l , A. BENDER l , G. DIRKESl, M. A.JOHNSON 2, and R. HOHLFELD l The mechanism of muscle-fibre-destruction in the autoimmune deseases Polymyositis (PM) and Juvenile Dermatomyositis ODM) is not known. In order to investigate if the apoptosis-pathway via the Fas-Receptor (Fas-R) is involved in the pathogenesis of these diseases, cryopreserved muscle biopsy sections from 3 patients with PM and 3 patients withJDM and 3 healthy control specimens were stained immunohistochemically for FasR-expression. All patients' specimens bound anti-Fas-R monoclonal antibody (AntiApo-1) whereas the normal tissue was negative. Fas-R-expression was detected both on muscle fibres and on infiltrating cells. The expression in muscle was independent of inflammatory infiltration. To elucidate whether Fas-R-expression is developmentally regulated, the differentiation- and regeneration-marker N-CAM was colocalized. Because N-CAM is highly expressed on regenerating fibres in Duchenne-MuscularDystrophy (DMD), cryosections of three patients with DMD served as a positive control. The expression of N-CAM and Fas-R did not correlate. We conclude that: (i) Fas-R is expressed both in inflammatory and non-inflammatory myopathies but not in healthy muscle, (ii) in the inflammatory myopathies Fas-R is expressed both in inflammatory lesions and in areas without inflammation, (iii) Fas-R is expressed on a subpopu!ation of inflammatory cells and muscle fibres, (iv) apoptosis mediated via Fas-R may be involved in different muscle diseases. Supported by Wilhelm-Sander-Stiftung and DFG (SFB 217, C 13).

1 Institute of Pathology, Friedrich Schiller University J ena, J ena; 2 Clinic for Orthopedics, Friedrich Schiller University Jena at the Rudolf Elle Hospital, Eisenberg, Germany

E.6 Demonstration of matrix metalloproteinase 3 (stromelysin) mRNA in the synovial membrane of patients with rheumatoid arthritis by in situ transcription K. BELEITES\]. KRIEGSMANN\ G. SCHRODER\ A. ROTH 2 , D. KATENKAMpl, and R. BRAUER 1 Metalloproteinases may play an important role in the destruction of joints in rheumatoid arthritis. Matrix metalloproteinase 3 was localized in cells of the hyperplastic synovial lining layer of patients with rheumatoid arthritis. Conflicting results exist concerning the production of stromelysin by the fibroblast-like or macrophage-like synoviocytes. We have demonstrated stromelysin mRNA by in situ transcription, where the reverse transcription of the mRNA is initiated by a specific primer. The newly synthesized

72 . Joint annual meeting 1995 0GAI/GFI digoxigenin-Iabeled cDNA was detected by colloidal-gold conjugated anti-digoxigenin antibodies and a silver amplification reaction. Macrophage-like type A synovicytes were identified by simultaneous demonstration of CD68 by the APAAP-technique using a red substrate. The synovial lining layer contains focally numerous cells expressing stromelysin mRNA. Additionally, stromelysin mRNA containing cells could be detected in sublining areas. Most of the cells contained either stromelysin mRNA or were positive for CD68. These results suggest that the matrix metalloproteinase 3 is expressed in the fibroblastlike type B synoviocytes which are prone to attach and subsequently invade the articular cartilage.

Institute of Immunology, University of Heidelberg, Heidelberg, Germany

E.7 Modulation of C5a-induced neutrophil activation by tumor necrosis factor alpha (TNFa) and granulocyte/macrophage colony stimulating factor (GM-CSF) R.

BINDER,

A.

KRESS,

and M.

KIRSCH FINK

The polymorphonuclear neutrophilic granulocyte (PMN) is considered the most important cell type of acute inflammation. After activation by the complement-derived peptide C5a the binding of other inflammatory mediators like cytokines may modulate the response to the anaphylatoxin. In the present study we investigated in more detail the impact of TNFa and GM-CSF, which in previous experiments comparing various inflammatory mediators proved to be most efficient in modulating C5a-mediated PMN responses. After preincubation with TNFa as well as with GM-CSF (1-10 nM, 15 min, 4 DC) C5a-mediated chemotaxis and chemokinesis of isolated human PMN were significantly blocked, whereas the release of lysosomal enzymes (elastase) and the production of reactive oxygene radicals were markedly enhanced. On a molar base TNFa was more effective than GM-CSF. With regard to possible underlying mechanisms, we could not observe an alteration of actin polymerization or an upregulation of adhesion molecules (~2-integrins). However, in the presence of cytokines we found a dramatic downregulation of C5a receptor expression on PMN, as revealed by Scatchard analysis. Since treatment of the cells with pertussis toxin only partially antagonized the effect of TNFa and GM-CSF, we conclude that PMN migration is regulated independently from other PMN responses and that PMN activation may comprise at least two signal transduction pathways, one of them depending on G-proteins.

Joint annual meeting 1995 OGAIIGFI . 73 Institute for Immunology, University of Munich, Munich, Germany

E.8 IL-1 0 drives human monocytes to CD 16 positive macrophages ]. C. CALZADA-WACK, M. FRANKENBERGER, and H. W. L. ZIEGLER-HEITBROCK

Cells of the monocyte/macrophage lineage are heterogenous in that some types express the CD16 molecule (Fc receptor for IgG, type III), while others are negative. We now show that a culture of human blood monocytes with Interleukin-10 (IL-10) will induce high levels of cell surface CD16 within 18 h, and this goes along with increased transcript levels. After prolonged culture for 36 h, control cultures also become CD16 positive and this induction can be prevented not by anti IL-10, but not anti TNF antibody. The IL-10-induced CD16 is functional since Fc receptor mediated phagocytosis of antibody coated bacteria is increased from 9.6% in untreated to 20% positive cells in IL10 treated cultures. An in vitro culture of blood monocytes results in a spontaneous decrease on the myelomonocytic stem cell antigen CD33, suggesting that the cells undergo maturation. IL-10 treatment will accelerate this process and will result in a further reduction of cell surface CD33. These data indicate that IL-1 0 promotes monocyte maturation and directs these cells to develop into CD16 positive macrophages.

Department of Pharmacology, Hafslund Nycomed Pharma AG, Linz, Austria

E.9 Regulation of prostaglandin formation in cells of the human monocytic cell line MonoMac-6 T. CHRISTOPH, M. WIDERNA, A. BC)J)ENTEICH, and]. BERG To induce and maintain inflammation macrophages have been shown to secrete proinflammatory cytokines e.g. TNF-a, IL-1~, IL-6 and IL-8 as well as small molecular weight mediators of inflammation such as leukotrienes and prostaglandins. The formation of prostaglandins follows the induction of expression of £rosta§hndin H ~ynthase­ :? (PGHS-2) which has been shown to be inducible by pro-inflammatory stimuli. MonoMac-6 cells have been demonstrated to exhibit many structural and functional similarities to human peripheral blood monocytes. Upon stimulation with bacterial lipopolysaccharide (LPS) or IFN-y MonoMac-6 cells differentiate further and secrete pro-inflammatory cytokines e.g. TNF-a, IL-1~, IL-6, and IL-S. Now, we show that MonoMac-6 cells can be differentiated to secrete high amounts of prostaglandins (PGEz, PGl2 measured as PGF 1u and TXA2 measured as TXB 2 ) upon stimulation with LPS. The secretion of prostaglandins follows mRNA expression, protein synthesis and increase of enzymatic activity of PGHS-2. Prostaglandin formation was inhibited after administration of inhibitors of the enzymatic activity of PGHS e.g. indomethacin. These findings should pave the way to the study of structural and functional requirements of the expression of PGHS-2 in human monocytic cells in vitro.

74 . Joint annual meeting 1995 OGAI/GFI Institute of Clinical Immunology and Transfusion Medicine, University of Leipzig, Leipzig, Germany

E.10 Xenobiotics influence the IL-l- and IL-6-secretion of the human lung tumor cell line NCI-H 358 S. DEHMEL, F. RAABE, G. METZNER, and G. WICHMANN The relationships between environment and immune system are of special interest in epidemiology, environmental medicine and immunology as well. We have established an in vitro assay using the macrophage-like human lung cancer cell line NCI-H 358 to estimate the immunomodulating potency of different xenobiotics, e.g. polycyclic aromatic hydrocarbons (P AH), phenols, methylating agents and other possible products from incineration processes, in view of monokine production. The NCI-H 358 cells were cultured in microwell plates. On day 5, reaching a monolayer, they were challenged for 3 h with the test compounds solved and diluted in dimethylsulphoxide (DMSO). Then, after another incubation period of 14 h in fresh media, supernatants were harvested and the concentrations of IL-1 and IL-6 were determined in in vitro bioassays by using the murine Th 2 clone D10.G4.1 and the murine hybridoma 7TD1, respectively. Cytotoxicity data were obtained simultaneously by performance of MTT-Assay on the treated NCI-H 358. Interestingly, there are different categories of xenobiotics influencing mono kine production/secretion in NCI-H 358: - inhibitors of interleukine production/secretion, e.g. Benzo[a]pyrene, - agents that inhibit interleukine secretion at low concentrations, but stimulate at high concentrations, e.g. Methylmethanesulphonate, - stimulators, e.g. Dithranol and - compounds that do not influence IL-lIIL-6-production/secretion. The results suggest that some xenobiotics are able to modulate cytokine release of macrophages. This might be important in allergy, inflammation and immunosuppression.

Medizinische Mikrobiologie und Immunologie, AG Infektabwehr, Ruhr-Universitat Bochum, Bochum, Germany; 1 Sandoz Forschungsinstitut, Vienna, Austria

E.ll ERects of persulfates/sulfates on inflammatory mediator release from human eRector cells A. DRYNDA, B. KONIG, M. CESKA 1, R. HILGER, M. KOLLER, and W. KONIG A variety of chemicals lead to airway sensitization. Novel and reliable test assays are required to elucidate their allergy and inflammatory potential. Previously, we have shown that persulfates lead to histamine release from human basophils but inhibit the fMLP as well as calcium-ionophore A23187-induced leukotriene B4 release from human neutrophilic granulocytes and monocytes under non-toxic conditions. Now we show that persulfates modulate cytokine release from human peripheral blood mononuclear cells (PBMC). The persulfates versus sulfates differ with regard to their activity for interleukin-8 release in the absence as well as the presence of additional stimuli (fMLP, staphylococcal enterotoxin B[SEB)). This is also apparent when cells are sensitized with

Joint annual meeting 1995 0GAIIGFI . 75 persulfates or sulfates, primed with cytokines and subsequently stimulated with fMLP or SEB. The compounds (persulfates/sulfates) interact with defined components of the signal transduction cascade (ERK, RAF1) as was assessed with epithelial cells A549 which may explain the modulatory effects on the cellular target, e.g. bronchial epithelial cell. Thus, depending on the cellular and mediator environment a pro- or antiinflammatory reaction is induced by the substances which may account for their role in pseudoallergic intervention and airway sensitization.

Institute for Immunology, University of Munich, Munich, Germany

E.12 Interleukin-l 0 is upregulated in LPS tolerance M. FRANKENBERGER, H. PECHUMER, and H. W. L. ZIEGLER-HEITBROCK Lipopolysaccharide (LPS) stimulation of the human monocytic cell line MonoMac 6 leads to rapid expression of both the pro-inflammatory cytokine tumor necrosis factor (TNF) and the anti-inflammatory cytokine interleukin-10 (IL-10). Preculture of these cells with a low dose of LPS for 2 days rendered the cells tolerant to subsequent stimulation, in that TNF gene expression is only minimal, both at the mRNA and at the protein level. IL-10 shows a reciprocal pattern, however, as expression of this gene is upregulated in precultured cells, and it will further increase upon subsequent stimulation. Although TNF has been shown to induce IL-10 to downregulate TNF, this reciprocal regulation does not explain the pattern observed in LPS tolerance in MonoMac 6, since neutralizing antibodies against TNF and IL-10 could not prevent upregulation of IL-10 and downregulation of TNF, respectively. Treatment of MonoMac 6 cells during LPS preculture with interferon-y (IFN-y) could, however, reverse tolerance: LPS/IFN-y precultured cells produced high levels of TNF transcripts upon subsequent stimulation, while the response of the IL-10 gene was attenuated. The data show that LPS tolerance does not involve a passive downregulation of all types of monocyte functions, but it is an orchestrated response with downregulation of pro- and up regulation of anti-inflammatory cytokines.

Department of Dermatology, University of Wiirzburg, Wiirzburg; 1 Max Planck Institute for Biochemistry, Martinsried, Germany

E.13 Chemokine expression in human dermal microvascular endothelial cells: selective induction of chemokines MCP-l, RANTES, IL-8 and gro-alpha after stimulation with TNFalpha and IL-l M. GOEBELER, U. RITTER, S. WERNER\ A. TOKSOY, E.-B. BROCKER, and R. GILLITZER Recruitment of leukocytes to sites of inflammation is critically depending on the production of chemotactically active cytokines (chemokines) released by the endothelium. Earlier studies primarily focussed on the analysis of (macrovascular) human umbilical vein endothelial cells neglecting the fact that most pathophysiological events take place at

76 . Joint annual meeting 1995 OGAI/GFI

the level of the microvasculature. We therefore initiated a study to analyze the expression of chemokines by human dermal microvascular endothelial cells (HDMEC) after stimulation with IL-I, TNF-alpha and IFN-gamma. Employing in situ-hybridisation and/or RNAse protection assays we found a dose-dependent induction of the C-X-C chemokines gro-alpha and IL-8 as well as of the C-C chemokines MCP-I and, to a weaker extent, RANTES in response to stimulation with TNF-alpha and IL-I in HDMEC. ELISA assays revealed that these chemokines were actually translated into protein and release into the supernatant secondary to cytokine treatment. IFN -gamma dose-dependently induced expression of the C-X-C chemokine IP-IO in microvascular endothelium whereas it did not significantly influence the expression of other chemokines. The C-C chemokines MIP-I alpha and beta and 1309 as well as the C-X-C chemokine ENA 78 were induced by none of the cytokines tested. In summary, our data indicate that HDMEC selectively produce high levels of MCP1, IL-8, gro-alpha, IP-IO and moderate levels of RANTES upon appropriate cytokine stimulation. The selective expression of specific chemokines by microvascular endothelium may critically influence the recruitment of leukocytes from the vascular space into the dermal compartment and significantly contribute to the local cytokine network of the skin.

Department of Dermatology, University of Vienna Medical School, Vienna; 1 Laboratory of Immunoregulation, Internal Research Center at SFI, Vienna, Austria

E.14 Dermal microvascular endothelial cells express CD32 receptors (FcyRlla) in vivo and in vitro M. GROGER, G. SARMAYI, E. FIEBIGER, K. WOLFF, and P. PETZEL BAUER Immune complexes are thought to be the major cause of cutaneous necrotizing vasculitis but the mechanism of the immune complex targeting to specific vessels is largely unknown. Fcy receptors (FcyR) mediate immune complex-binding and receptor-mediated endocytosis in myelomonocytic cells. We asked, whether dermal microvascular endothelial cells (DMEC) express FcyRs. In cryostat sections of normal human skin mAb IV.3 or ATlO, both recognizing FcyRII (CD32), localize to the luminal surface of DMEC of the superficial but not of the deep vascular plexus. all DMEC are negative for FcyRI (CD64) or FcyRIII (CDI6). Adult skin-derived DMEC in culture express FcyRII molecules as measured by FA CS but are negative for Fcy RI or III, human umbilical vein endothelial cells (HUVEC), tested for comparison, do not express Fcy RI, II, or III proteins. By RTPCR and subsequent Southern blot analysis the isoform of the CD32 molecule expressed on DMEC is determined as FcyRIIa. HUVEC do not contain mRNA for FcyRIIa or b isoforms. In myelomonocytic cells IgG aggregates are most effectively internalized via FcyRIIa. In DMEC and monocytes, tested as a positive control, crosslinking of FcyRIIa with mAb IV.3-biotin and avidin rhodamin, results in fluorescence-staining of the cell surface only, when cells are incubated at 4°C. The fluorescence rapidly shifts to the cytoplasm when monocytes or DMEC are incubated at 3rc. We conclude that FcyRIIa molecules expressed on superficial vascular plexus DMEC are potentially involved in the pathogenesis of necrotizing vasculitis.

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Biochemical Pharmacology, Faculty of Biology, University of Konstanz, Konstanz, Germany

E.1S Granulocyte-mediated hepatocytotoxicity after endotoxin stimulation depends on adhesion and elastase release T. HARTUNG, A. SAUER, and A. WENDEL We used a coculture of neutrophils (PMN) and liver cells (LCC) to study endotoxin (LPS)-stimulated cell killing. The presence of PMN aggravated this process by 3 orders of magnitude. We found a release of human elastase from the PMN upon LPSstimulation in the LCC+PMN. The kinetics of LPS-induced elastase release correlated with that of LPS-induced toxicity. Serine protease inhibitors protected in our system and a polyclonal antibody against TNF prevented toxicity as well as elastase release. This suggests that TNF mediates elastase release and toxicity. When liver cells and PMN were separated by permeable membranes neither toxicity nor elastase release was found. This indicates that cell to cell contact between the PMN and the liver cells is needed for elastase-dependent toxicity. Scanning electron microscopy showed that PMN adhered to the hepatocytes in LPS-stimulated cultures. We conclude that adhesion of PMN to hepatocytes is necessary, but not sufficient for induction of LPS-mediated toxicity.

Biochemical Pharmacology, University of Konstanz, Konstanz, Germany

E.16 Blood from G-CSF treated volunteers shows an altered eicosanoid response T. HARTUNG and A. WENDEL We assessed human whole blood eicosanoid release, i.e. the formation of thromboxane metabolite B2 (TxB 2), prostaglandin E2 (PGE 2) and leukotriene B4 (L TB4). Blood was stimulated with a variety of immunostimulators each resulting in an individual eicosanoid response. In blood taken from volunteers pretreated with 500 mg aspirin p.o., TxB2 and PGE 2 was attenuated while LTB4 formation was not affected. Thus this model appears to be suitable to assess the ex vivo effect of immunomodulatory drug treatment. In 21 healthy volunteers treated s.c. with 480 [tg G-CSF (Neupogen®), the ex vivo stimulated blood showed reduced net LTB4 release per neutrophilic granulocyte while total PGE 2 formation was transiently 10-fold increased 8 h after G-CSF injection. We conclude that G-CSF treatment induced a shift towards an anti-inflammatory blood eicosanoid response.

78 . Joint annual meeting 1995 OGAIIGFI Department of Internal Medicine III, Division of Rheumatology, Department of Orthopedics, and Department of Internal Medicine I, Division of Hematology, Vienna, Austria

E.17 Phenotypic characterization of human synovial mast cells H. P. KIENER, M. BAGHESTANIAN, W. R. SPERR, A. WANIVENHAUS, W. B. GRANINGER, W. FUREDER, K. LECHNER, and P. VALENT Mast cells (MC) are multifunctional perivascular cells and supposedly play an active role in various inflammatory processes. MC functions (differentiation, recognition, mediator release) are associated with expression of distinct cell surface receptors (R). The aim of the present study was to analyze the R profile of human synovial MC (SyMC). Synovial tissue was obtained from four pts with rheumatoid arthritis (RA) by synovectomy and dispersed with collagenase and DNAse. Isolated SyMC were analyzed for R expression by mABs using a combined toluidine blue/immunofluorescence staining technique. Isolated SyMC were recognized by mABs against p24 (CD9) and gp67 (CD33), the leukocyte recognition molecules leukosialin (CD43), Pgp-1 (CD44), ICAM-1 (CDS4), the ~ chain of ~1 (CD29) and ~3 integrins (CD61) and the receptor for the mast cell agonist SCF (= c-kit R, CDl17). SyMC did not express TcR (Cm), T4 (CD4), C3biR (CDllb), LPSR related Ag (CD14), 3-FAL (CD1S), FcyRIII (CD16), lactosylceramid (CDw17), the B cell antigen CD19, IL-2Ra (CD122), IL-7R (CD127) or CR1 (CmS). SyMC were also found to lack the skin MC-associated marker CD88 (CSaR) as well as cytokine receptors expressed on human blood basophils, including IL-2R~ (CD2S) and IL-3Ra (CDw123). In summary, we have established the immunological surface marker profile of human synovial Me. This phenotype corresponds well with the phenotype of lung MC and includes a number of recognition molecules and receptors for immunomodulating compounds. The pathophysiologic role of SyMC and/or of cell surface R expressed on SyMC in RA remains to be elucidated.

Institute of Medical Microbiology, Hannover, Germany

E.1B Generation and modification of C5a antagonistic scFv-fragments in E. coli

J. KOHL, A. KOLA, M. BAENSCH, M. HENNECKE, A. KLOS, W. BAUTSCH, and P. REIN The anaphylatoxins CSa and CSadesArg are generally believed to be causally involved in a variety of acute and chronic inflammatory diseases, e.g. ARDS, sepsis syndrome or the rheumatoid arthritis. A suitable approach to attenuate the deleterious AT-effects in such clinical situations could be to generate mAb's directed against an AT-neoepitope, which is not expressed on the respective mother molecule. Immunization using the C-terminal nonapeptide hCSa-(6S-73) resulted in two mAb's (292S and 2906), which recognized hCSa/hCSadesArg but not CS. In competitive binding studies with Bt2cAMP differentiated U937 cells both mAb 292S and mAb 2906 were found to displace 12S1 rhCSa with an IC so of 3 x 10-7 M, and 4.2 x 10-7 M. Recently the phage display technology was shown to be a powerful tool to generate recombinant antibody fragments and to increase the affinity of such fragments. We have

Joint annual meeting 1995 OGAI/GFI . 79 used this technology (i) to express the neoepitope-specific mAb's described above as scFv fragments fused to the N-terminus of the gene III protein of the filamentous M13 phage or as soluble svFv in E. coli and (ii) to modify scFv 2925, i.e. to perform affinity maturation. The heavy chain was shuffled with a light chain library from an unimmunized mouse and tested for both binding to C5a/C5adesArg and C5. After one round of biopanning 4 new scFv fragments specific for hl5a/hC5adcsAr~ could be isolated.

Medizinische Mikrobiologie und Immunologie, AG Infektabwehrmechanismen, RuhrUniversitat Bochum, Bochum, Germany

E.19 Inhibition of leukotriene formation and 11-8 release by a platelet-activating factor receptor antagonist M. KOLLER, R. HILGER, and W. KONIG Inflammation, allergy, anaphylaxis and shock are induced and maintained by the interaction of various cell populations and soluble proinflammatory mediators such as lipid mediators or cytokines. Among them the stereospecific 5-lipoxygenase (5-LOX) metabolite leukotriene B4 (L TB 4) and the platelet-activating factor (P AF) are potent activators of various phagocyte responses. Recent reports provide evidence that an autocrine enhancement of LT synthesis by endogenous P AF and LTB4 represents an important amplification mechanism of cellular mediator release. Thus, it was the purpose of this study to investigate the role of the new PAF receptor antagonist (+)-cis-3.5dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-12502) on the generation of lipid mediators (leukotrienes, monohydroxylated eicosatetraenoic acids) and IL-S formation of peripheral leukocytes with the Ca ionophore A231S7, the bacterial-derived peptide fMLP, or with an activator of heterotrimeric G-proteins, the sodium fluoride (NaF, in the presence of AP+). The PAF receptor antagonist led to a concentration and time-dependent inhibition of LTB4 formation and IL-S release from PMN and LMB. Our data clearly indicate an inhibitory effect of the PAF receptor antagonist SM-12502 on the formation of mediators of the lipoxygenase pathway and on the release of IL-S.

'Department of Ophthalmology, 2 Department of Internal Medicine, 4 Department of Biostatistics,S Computer Center, University of Innsbruck, and 3 Blood Bank, Innsbruck, Austria

E.20 Genetic predisposition and risk factors of uveitis anterior A. KOLLMANN" W. GOTTfNGER" K. HOCK" S. MUR2 , D. SCHONITZER3 , W. NUSSBAUMER 3 , H. ULMER\ A. LOCHS S, and E. U. IRSCHICK' Genetic risk factors are described for various diseases e.g. Mb. Bechterew and HLA-B27. 143 patients were examined in order to investigate the relationship between uveitis anterior and several HLA class I loci. We further investigated associations with age, sex and blood group antigens like ABO, Rhesus and Kell. Patients with order types of uveitis or with associated systemic diseases such as Mb. Bechterew or Mb. Reiter were excluded

80 . Joint annual meeting 1995 OGAI/GFI from the study as well as patients with a history of eye infection or ophthalmological surgery. Our patients were compared with a control group of 1500 healthy volunteers. For statistical analysis Chi-Square-Test and Logistic Regression was used. The results showed a high risk of uveitis anterior accompanied with HLA-B27 for both men and women. HLA-Bw51 and HLA-Cw1 were of high risk for male patients only. The highest relative risk was determined for men with the haplotype HLA-B27-Cw1. Blood group 0 appeared in 70% of the male patients with uveitis anterior. Rhesus and Kell antigens showed no association with the disease. The age at onset of the disease showed two peaks in female and one in male patients. Our results suggest, that there are strong associations between several of our investigated antigens and uveitis anterior and that there is a genetic predisposition for the development of this possible autoimmune disease.

Biochemical Pharmacology, University of Konstanz, Konstanz, Germany

E.21 TNF-induced hepatocyte apoptosis as a mechanism of endotoxic liver injury in mice M. LEIST, F. GANTNER, 1. BOHLINGER, G. TIEGS, and A. WENDEL Tumor necrosis factor (TNF) is a central mediator of systemic inflammatory response syndromes such as endotoxic shock. It has been shown to cause lethality and tissue injury, including hepatic failure, but the mechanism eventually leading to hepatocyte death has not been elucidated. We addressed the question whether and under which conditions lipopolysaccharide (LPS)-injection into mice may induce programmed cell death in murine livers. In GaINsensitized mice endotoxin injection caused hepatocyte apoptosis, associated with internucleosomal DNA-fragmentation at time points well before cell lysis was detected. Hepatic apoptosis as well as subsequent liver failure in this model were completely prevented by passive immunization of mice against TNF. The causative role of TNF was confirmed since firstly, direct injection of this cytokine into sensitized mice also caused hepatocyte apoptosis and secondly gene targeted mice lacking the 55 kDa TNF receptor were completely resistant towards this TNF-induced apoptosis. The direct interaction of TNF with the target cell in vivo, i.e. the hepatocyte, and the resulting induction of programmed cell death was confirmed by in vitro experiments using primary murine hepatocyte cultures. In this system TNF-induced apoptosis was observed in analogy to in vivo under the selective biochemical condition of transcriptional block.

Joint annual meeting 1995 OGAI/GFI . 81 Biochemical Pharmacology, University of Konstanz, Konstanz, Germany

E.22 TNF-induced apoptosis as an early marker of T celldependent liver injury in mice M. LEIST, F. GANTNER, 1. BOHLINGER, G. TIEGS, and A. WENDEL D-galactosamine (GaIN)-sensitized mice injected with either anti-CD3 mAb or with staphylococcal enterotoxin B (SEB) developed severe apoptotic and secondary necrotic liver injury associated with oligonucleosomal DNA-fragmentation. Injection of concanavalin A (Con A) to unsensitized mice resulted also in hepatic apoptosis which preceded necrosis. The results obtained in the Con A model demonstrate that TNFinduced apoptosis is a general pathophysiological phenomenon not only observed under the condition of transcriptional arrest, since neither GaIN-sensitization was required nor hepatic transcriptional arrest was observed. Anti-CD3 mAb as well as SEB or Con A induced the release of systemic TNF and various other cytokines. Passive immunization against TNF-a or pretreatment with immunosuppressive drugs protected mice from liver injury. T lymphocytes were identified as effector cells of ConA in vivo by antibody dependent T cell depletion prior to Con A injection, by proof of resistance of athymic nude mice against Con A-induced liver failure and by restoration of susceptibility in nude mice by lymphocyte transfer from immunocompetent control mice. Our data demonstrate that systemic TNF-release is the common distal pathway leading to hepatic apoptosis as a consequence of polyclonal T cell activation.

Biochemical Pharmacology, Faculty of Biology, University of Konstanz, Konstanz, Germany

E.23 Endotoxin-induced DNA-fragmentation in different organs of the mouse M. LEIST, 1. BOHLINGER, F. GANTNER, and A. WENDFJ Bacterial endotoxins (LPS) exert their proinflammatory effects by inducing the production of a variety of endogenous mediators in the host. Tumor necrosis factor a (TNFa) has been recognized as one of the key inflammatory mediators in various experimental settings and has been shown to mediate lethal shock and multi-organ failure. Treatment of mice with a lethal dose of endotoxin led to DNA-fragmentation in liver, lung and caecum. The kidney was shown to be uneffected. 24 h after administration of LPS, necrotic liver cell death was indicated by elevated plasma levels of liver enzymes. Liver cell necrosis as well as DNA-fragmentation in liver and lung were prevented by pretreatment with anti-TNFa antibody. The localization of DNA-fragmentation in the liver was characterized by labeling DNA-double strand breaks in paraffin-embedded liver sections using the terminal transferase (TUNEL) method. Since morphological features of apoptosis such as chromatin-condensation or the formation of apoptotic bodies could not be detected, it remains open, whether the observed DNA-fragmentation results from apoptotic or necrotic cell death.

82 . Joint annual meeting 1995 OGAI/GFI Institut fur Pathologie, Universitats-Klinikum Dresden, Dresden; IMedizinische Klinik I, Universitat Heidelberg, Heidelberg, Germany

E.24 Increased tissue factor expression on monocytes in response to endotoxin in vivo T. LUTHER, A. BIERHAUS, M. KASPER, M. KOTZSCH, P. P. NAWROTHi, and M. MOLLER Bacterial endotoxin (LPS) is known to stimulate monocytes to rapidly and transiently express a defined set of gene products, including TNFa and tissue factor (TF) in vitro. In vivo, however, the cascade of events leading from bacterial infection to activation of monocytes followed by septic shock and multiple organe failure is still poorly understood. This study examined the role of in vivo activation of monocytes in mice following administration of endotoxin. Adult female Balb/c mice, weighing 20-25 g were used for all experiments. 50 I!g LPS was diluted in 200 I!l sterile saline and injected intraperitoneally into mice. Control mice were injected with an equivalent volume of saline alone. For blood and tissue sampling mice were sacrificed at various time points after LPS intoxication. LPS administration resulted in reproducible elevation of serum TNF, peaking after 90-120 min. Using immunohistochemistry on paraffin embedded tissue we could demonstrate an enhanced accumulation of monocytes in the vasculature of lung and kidney in response to LPS. Adherence of monocytes to the endothelium started after 2 h and reached its maximum after 6-8 h. At the same time an increase in TF expression in the accumulated monocytes was obvious. 24 h after LPS treatment binding of monocytes and enhanced TF expression was not longer detectable. It is known, that stimulation of monocytes by LPS results in activation of NF-xB in vitro. In the present study, the Electrophoretic Mobility Shift Assays with nuclear extracts of peripheral blood cells demonstrate a time-dependent activation of proteins that bound to the binding site for NF-xB (p65/c-rel), a nucleotid sequences within the LPS response element of the TF promoter. The observed NF-xB activation preceeded TF expression. Therefore we speculate that the LPS-induced increase in TF gene expression in the lung, is due to enhanced transcriptional activity and TF gene expression in the activated adherent monocytes, mediated at least in part by activation of NF-xB. This might contribute to organ failure of these organs during Gram-negative sepsis.

Institut fur Hygiene, Leopold-Franzens-Universitat Innsbruck, Innsbruck, Austria

E.2S Generation of multinucleated giant cells in vitro depends on the stage of monocyte/macrophage maturation J. MOST, L. SPOTL, G. MAYR, A. GASSER, and M. P. DIERICH Multinucleated giant cells (MGC) are a common feature of granulomas that develop in the course of certain infections or foreign body reactions. MGC originate from fusion of monocytes and can be induced in vitro by the addition of cytokine-containing media, but the exact mechanism of their generation is still unclear. To obtain more knowledge about the formation of MGC we investigated the influence of monocyte to macrophage maturation on the ability of monocytes/macrophages to fuse with each other.

Joint annual meeting 1995 OGAIIGFI . 83 Maturation of monocytes was induced by the addition of 10 % human serum. Monocytes cultured with or without serum were stimulated with conditioned medium on the day of isolation (day 0), and on days 1, 3, 5, and 8. Three days after stimulation cells were stained with Giemsa and the fusion rate (number of nuclei within MGC per total number of nuclei) was determined. With freshly isolated monocytes fusion rates of about 60 % were obtained. When monocytes that had been cultured in human serum were stimulated, fusion rates gradually declined with advancing time of the preceding culture (corresponding to the stage of differentiation) and almost no MGC formation could be obtained with 8-days old macrophages. In contrast, fusion rates did not decrease when monocytes had been cultured for the same time under serum-free conditions. When freshly isolated monocytes were added to 1-week cultured macrophages, which had been membrane labeled with a fluorochrome, fusion between the two populations could be induced, indicating that the diminished fusion capacity of macrophages is relative rather than absolute. These results demonstrate that the monocytes' ability to fuse is lost during maturation in vitro. Clarifying the mechanism(s) responsible for this difference between monocytes and macrophages could be helpful to define the relevance of MGC but may also lead to a better understanding of membrane fusion in general.

lMax-Planck-Institut fur Psychiatrie, Abteilung Neuroimmunologie, Planegg-Martinsried; 2Deutsches Krebsforschungszentrum, Institut fur angewandte Tumorvirologie, Abteilung Organisation komplexer Genome, Heidelberg; 3Medizinische Universitatsklinik, Abteilung Klinische Biochemie, Wurzburg, Germany

E.26 Murine granzyme M: structure of the gene and serine protease and physical linkage to proteinase 3 (Wegener's autoantigen) on mouse chromosome 10

Cosmid clones containing the genes for the granule-associated killer (NK) cell-specific serine protease granzyme M (GzmM) and the neutrophil-derived proteinase 3 (Prtn3) (Wegener's auto antigen) were isolated by screening a murine cosmid library with human cDNA probes that we had cloned previously. Although the human GzmM and Prtn3 genes were shown to be closely clustered within less than 400 kb on 19p 13.3, we failed to find overlapping cosmids for both loci in the mouse genome. Nevertheless, interphase studies with differentially labelled cos mid probes for the murine GzmM and Prtn3 loci revealed a close physical association on mouse chromosome 10. Restriction site, hybridization, and sequence analyses indicate that the granzyme M gene consists of five functional exons with the expected homology to the human gene. The open reading frame predicted from the genomic sequence is highly related to the rat and human granzyme M sequences and shares very similar amino acid residues in those regions which determine substrate specificity for methionine residues. The knowledge of the murine cDNA and genomic structure will permit us to analyze the functions of GzmM in target cell killing by generating sufficient quantities of the recombinant protein and by constructing GzmM deficient mice.

84 . Joint annual meeting 1995 GGAI/GFI Institute for Medical Microbiology and Virology, Heinrich Heine University, Dusseldorf, Germany

E.27 Establishment of a quantitative ZO-l assay and correlation between ZO-l expression and electrical resistance of cell monolayers with special reference to the blood brain barrier S. PROLLER, R. PLOGMANN, G. TILLMANN, and H. P. HEINZ The blood brain barrier (BBB) is of great importance concerning the immuno- and pathophysiology of meningoencephalitis. So tight junctions (TJ) are an attribute of high electrical resistance (ER) endothelial and epithelial cell monolayers. One of the most important TJ -associated molecules is the membrane protein ZO-1. Although TJ are always associated with ZO-1 this protein is also found in many cell types developing only low ER. Therefore it is often supposed that not the functional state of ZO-1 may be important for development of high ER. However no quantitative estimation of ZO-1 expression is done so far. For comparison of ZO-1 expression with ER we used low resistance human umbilical vein endothelial cells (HUVEC), high resistance Madin Darby canine kidney (MDCK) cells and brain capillary endothelial cells (BCEC), which are the correlate of the BBB. Moreover we examined the influence of rat astrocyte coculture. Under this conditions BCEC monolayers are known to increase ER. ZO-1 quantitation was made by a newly established ZO-1 ELISA. ER was measured using an assembly containing currentpassing and voltage measuring electrodes. Low ER (20 Ohm x cm 2) of HUVEC correlated with low ZO-1 expression (optical density (OD) 0.06). High ER (380 Ohm x cm 2 ) of MDCK cells correlated with high ZO-1 expression (OD 0.92). Primary cultures of bovine BCEC-monolayers with ER about So Ohm x cm 2 achieved OD-values of 0.75 in ZO-1 ELISA. Astrocyte coc'~lture 'had ·no effect on ER and ZO-1 expression of HUVEC and MDCK « 3 %) but increased ZO-1 expression of BCEC about 30 % and ER about 700 %. This effect was not due to cell proliferation as controlled by estimation of protein content. In summary we could show a correlation between ZO-1 expression and ER in different cell types as well as in the same cell type by stimulation of ER development of bovine BCEC under astrocyte coculture conditions. We conclude, that besides the functional state of ZO-I, its quantity plays a fundamental role for TJ assembly and high ER development.

Clinic of Internal Medicine IV, Department Gastroenterology and Hepatology, and Institute for General and Experimental Pathology , University of Vienna, Vienna, Austria

E.28 In vivo induction of HLA-DR on human neutrophils in patients treated with interferon-gamma W. REINISCH, M. WILLHElM, C. LICHTENBERGER, W. TILLINGER, K. BAIER, C. GASCHE, C. DEJACO, G. STEGER, O. SCHEINER, and A. GANGL Polymorphonuclear neutrophils (PMN) have long been regarded as terminally differentiated cells with their primary function as effectors in non-specific immune reactions. Recently, it has been demonstrated that PMN could be induced to express MHC class II

Joint annual meeting 1995 OGAIIGFI . 85 antigens by GM-CSF, IFN-y and IL-3, in vitro. The aim of the present study was to investigate the induction of MHC class II on PMN under in vivo conditions. The expression of HLA-DR, -DP, -DQ, CD32 (FcyRII) and CD64 (FcyRI) was studied in 4 patients with metastatic hypernephroma before and after two weeks of treatment with IFN-y (Imukin®), 100 [Ag subcutaneously 3x/week. Nine nontreated healthy donors (HD) served as control. Peripheral blood PMN were analyzed by their characteristic forward/sideward scatter in whole blood lysis flow cytometry. Data were evaluated as percentage of positive neutrophils.

HD before IFN-y after IFN-y

HLA-DR

HLA-DP

HLA-DQ

<1 <1 25 (22-38)"

<1 <1 <1

<1 <1 <1

CD64

CD32

7 (0-23) 29 (7-59) 99 (90-100)"

>99 >99 >99

Median (range), "'p < 0.01 vs HD and vs before IFN-y. The upregulation of HLA-DR was accompanied by an increased expression of CD64, whereas CD32 was expressed on > 99 % of PMN without any shift in the median channel fluorescence, indicating a specific reaction. To confirm MHC class II induction on PMN, CD16/HLA-DR double staining was performed. Neutrophils were discriminated as CD16bright and CDI6bright/HLA-DR+ cells were sorted and cytocentrifuged onto slides. Histochemical staining confirmed that> 98 % of sorted cells were PMN. This is the first report on the induction of HLA-DR on PMN in vivo. The concomitant up regulation of CD64 and HLA-DR is in accordance with in vitro findings, suggesting a direct effect of IFN-y on PMN in our patients.

IDepartment of Anatomy I, University of Erlangen, Erlangen; 2IBM Deutschland GmbH, Sindelfingen; 3Department of Medicine III, University Hospital Charite, Berlin, Germany

E.29 Nitric oxide production by human peripheral blood monocytes in vitro as determined by an improved colorimetric assay M. G. RrrTIG 1, D. FAIlIANl, H. JANTSCHKE 2 , A. KRAUSE 3, B. STUHLMDLLER 3, and G. R. BURMESTER3 There is still some debate whether human monocytes/macrophages may use nitric oxide (NO) for antimicrobial defense. One major reason for this controversy is the difficulty to demonstrate NO biosynthesis in human. Therefore, we have tried to adapt the colorimetric method routinely used in the murine system to the use with human phagocytes. Freshly isolated peripheral blood monocytes from healthy volunteers were allowed to adhere on plastic dishes. Subsequently, they were exposed to Borrelia burgdorferi, IFNy, LPS, and NMMA, either alone or in combination. NO production was measured indirectly by the determination of N0 2 with Griess and Lunges reaction-based colorimetric methods. Using this system, N0 2 was detected in the supernatant of unstimulated monocytes (8-61 [AM/ml; n = 8). The addition of B. burgdorferi, IFN-y, or LPS to

86 . Joint annual meeting 1995 OGAIIGFI the monocytes increased the measurable amount of NO z up to 4-fold. However, NO z could only be detected using more than 103 monocytes per well, an incubation period longer than 36 h, a supernatant volume below 1 ml, and if the samples had been deproteinized before determination. Using the Lunges reagent the concentration of N0 2 alone was above threshold, whereas the Griess reagent required the catalytic reduction of N0 3 to NO z . It is concluded that freshly isolated human monocytes have a basic NO synthesis in culture which can be increased by certain stimuli. The amount of NO produced differs significantly among cells from different donors. The indirect colorimetric method routinely used to determine NO production in the murine system has to be optimized considerably to allow determination in the human system.

Institut fur Klinische Immunologie und Transfusionsmedizin, Universitat Leipzig, Leipzig, Germany

E.30 Induction of hu/mu SCID-arthritis: a crucial role for fibroblasts U. SACK,J, LEHMANN, and F. EMMRICH Recently we have demonstrated the successful induction of an arthritis in mice with severe combined immunodeficiency (SCID) by transfer of human synovial membrane tissue pieces into the knee joint of mice Rheumatol. 21: 10-16, 1994). As shown in Tc99m-scintigraphy, the arthritis became polyarticular within 6 weeks following implantation. The inflammation shows typical histological aspects of a rheumatoid arthritis. In contrast, transfer of mononuclear cell suspensions from RA synovial membrane or peripheral blood did not cause an arthritis, probably due to migration of injected cells out of the joint within 24 h. These findings indicate the presence of an arthritogenic cell type, agent, or substance in complete RA-SM. Human synovial membrane fibroblasts were isolated by long-term culture from rheumatoid arthritis patients. The injection of 2 x 106 synovial membrane fibroblasts induced an arthritis in mice. The additional application of 4.2 x 106 prestimulated (l00 IU rhIL-2/ml) peripheral blood mononuclear cells beside intraarticular injection of fibroblasts from the same patient induced an amplification and acceleration of the arthritic process. This indicates, that fibroblasts are required for successful induction of hu/mu SCID-arthritis. Nevertheless, human mononuclear cells modulate and regulate the inflammatory process and are able to increase articular damage. We conclude, that complex cell cooperation occurs in arthritis induction and that synergism can be detected following application of fibroblasts and additional cell types.

a.

Joint annual meeting 1995 0GAI/GFI . 87 Department of Immunology and Cell Biology, Research Institute Borstel, Borstel, Germany

E.31 Functional role of multinucleated giant cells in an in vitro granuloma model U. SElTZER, G. HEINEMANN, M. HAHN, H. HAAS, H.-D. FLAD, and]. GERDES Granulomatous inflammations are chronic, predominantly mononuclear host reactions to persisting, poorly degradable tissue irritants. Multinucleated giant cells (MGCs) are a characteristic feature of several granulomatous inflammations and are formed by fusion of mononuclear cells of the myelomonocytic cell lineage. The mechanism of MGC formation are not fully understood, however fusion is promoted by stimulatory agents and modulated by cytokines. Little is known concerning the specific role of MGCs in the inflammatory response to infection. Possibly these cells represent an adaptation for enhanced microbicidal activity and serve a useful purpose in the host response to intracellular organisms that are resistant to killing by macrophages. In a new in vitro model for granuloma formation MGCs develop by coincubation of the non-human pathogenic nematode Nippostrongylus brasiliensis and human PBMNCs. To elucidate the functional role of MGCs in this model, single MGCs were prepared from the culture and analyzed with an RT-PCR method for single cells with respect to their cytokine profile. Our results show the presence of TNFa and IL-1 ~ transcripts in these cells, suggesting that MGCs are involved in granuloma formation and thus may contribute to immune defense mechanisms in chronic persistant inflammation. This research was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG SFB 361/C1).

Institute of Immunology and Transfusion Medicine, Ernst Moritz Arndt University, Greifswald, Germany

E.32 Human recombinant soluble C014 protects mice from lethality in LPS-induced septic shock but not from development of shock symptoms F. STELTER, S. WITT, B. FORLL, R. S. JACK, and C. SCHOTT As much as 50 % of all cases of the clinical syndrom septic shock are attributed to Gramnegative bacteria or their cell wall compound lipopolysaccharide (LPS, endotoxin). LPS stimulates monocytes/macrophages to release a variety of mediators including TNFa, Ill, ILS, lipid mediators and reactive oxygen species. Eventually inflammatory responses triggered by these substances may progress into a septic shock. To interfere at an early stage with this process therapeutic approaches either target the binding of LPS to its cellular receptors or the LPS-induced mediator release and action. Membrane bound CD14 serves as the major endotoxin-receptor on the surface of monocytes/macrophages and neutrophil granulocytes. A soluble form (sCD14) exists in serum which also recognizes LPS. We have previously shown that sCD14 prevents binding of LPS to membrane bound CD14 and inhibits the LPS-induced oxygen radical

88 . Joint annual meeting 1995 OGAIIGFI burst of human mononuclear cells (1). In this study we have tested the endotoxinneutralizing capacity of human recombinant sCD14 in vivo. Septic shock was induced in BALB/c-mice by administration of 8 [!g/g LPS, Salmonella abortus equi, intraperitoneally. We demonstrate that simultaneous injection of 250 [!g sCD14 reduces the mortality from 71 % (32/45) in control animals to 28 % (15/53) in CD14-treated animals (p < 0.001). Injection of sCD14 30 min prior to LPS is also protective whereas treatment 30 min after LPS-challenge has no effect on lethality. However sCD14-treatment does not prevent development of shock symptoms. Thus the results indicate that sCD14 has beneficial effects in LPS-induced septic shock though the mode of action has not yet been understood.

c., T. SCHILLING, U. GRUNWALD, W. SCHONfELD, and C. KROGER. 1992. Endotoxin neutralizing capacity of soluble CD14. Res. Immunol. 143: 71-78.

l.~SCHOTI,

Departments of lImmunology and 2Dermatology, Otto von Guericke University, Magdeburg, Germany

E.33 Antioxidant effect of 13-cis-~-carotene on the neutrophilgenerated oxygen-derived free radicals H. STRUy 1, M. BOHNE 2 , A. GERBERl, H. GOLLNICK 2, and]. MORENZ 1 Human neutrophils generate reactive oxygen species as a consequence of membrane interaction with a number of stimuli. The phagocyte-derived free radicals are involved in microbicidal and tumoricidal activity as well as in tissue damage at sites of inflammation. Carotenoids have an important function in protecting cells from oxidant damage. We investigated the inhibitory effect of 13-cis-~-carotene on superoxide and hydroxyl radicals from human neutrophils stimulated by opsonized zymosan. Hydroxyl radicals were determined by electron spin resonance using spin trapping system a-4-pyridyl-loxide-N-tert-butylnitrone and ethanol and superoxide by the spin trap 5,5-dimethyl-lpyrroline-N-oxide and by superoxide dismutase inhibitable cytochrome c reduction. The 13-cis-~-carotene concentrations (0.25 [!moIlL-l [!mollL) were similar to serum concentrations of healthy subjects before and after 8 days of ~-carotene supplementation. Our result demonstrated a concentration-dependent inhibition of oxygen-derived radicals by stimulated neutrophils. Maximum inhibition by about 50 % was found at 1 [!mollL 13cis-~-carotene. The ability of 13-cis-~-carotene to scavenge reactive oxygen species suggests its potential use in medicine.

Joint annual meeting 1995 0GAI/GFI . 89 IDepartment of Dermatology and 2Institute of Experimental Dermatology, University of Munster, Munster; 3Department of Dermatology, Virchow Klinikum, Humboldt University Berlin, Berlin, Germany

E.34 Effects of dexamethasone and pentoxifylline on experimental (murine) leukocytoklastic vasculitis C. SUNDER KOTTER 1, K. STEINBRINK2, and G. KOLDE 3 Pronounced vascular damage during inflammation such as leukocytoklastic vasculitis is the final stage of different pathophysiological pathways. We have previously established 3 mouse models which all show leukocyte-dependent damage of small blood vessels, but differ with regard to pathomechanisms (e.g. deposition of complement or reaction of leukocytes). They are: 1) Arthus reaction (local immunecomplexes) (Art-r), 2) local Shwartzman reaction (Shw-r), 3) local reaction after injection of venom from Loxosceles deserta. Vascular deposition of C3 was constant in the Art-r, but did not precede vascular damage in the other two reactions. Depletion of complement by cobra venom factor (3 x 1.8 U) markedly suppressed endothelial necrosis in the Art-r. We then studied if dexamethasone (dexa) or pentoxifylline (PTX), two agents used in human vasculitis, would have different effects in the 3 models: PTX (1 mg all 6 h) and dexa (2.5 mg) did not suppress the inflammatory damage caused during the Arthus or the spider venom reaction. However, dexa or dexa + PTX significantly suppressed the Shw-r. Thus, i) therapeutic effects of steroids in human immunecomplex vasculitis may be based on inhibition of immunecomplex formation rather than on suppression of the inflammatory infiltration; ii) complement is mandatory for the immunecomplex vasculitis, whereas processes inhibitable by steroids and PTX (e.g. release of TNF-a) are more important for endothelial damage in the Shw-r.

Institut fur Immunologie, Universitat Heidelberg, Heidelberg, Germany

E.35 Transforming growth factor ~l (TGF~l) preferentially upregulates synthesis of the EDA-splice variant of fibronectin in human tubular epithelial cells in culture C. VIEDT, A. BORGER, and G. M. HANSCH Cocultivation of renal tubular epithelial cells in culture (TEC) with TGF~l increased the steady-state level of fibronectin (FN) mRNA up to 4-fold within 24-48h. By RT-PCR and Northern blotting it was found that preferentially the EDA-splice variant of FN was upregulated. To quantitate FN protein synthesis, TEC were cultivated in the presence of eSS]-methionine; and FN in cell supernatants, the celllysates or the extracellular matrix was identified and quantitated. In TGF~ treated cells a small increase (1.7-fold) of FN was seen in the cell supernatants, a more prominent increase in the celllysates (4.5-fold) and in the matrix (4-fold). The de novo synthesized FN of the cell lysates and the extracellular matrix reacted with an EDA-specific monoclonal antibody. By comparing its reactivity with that of an antiserum to FN it was seen that in TGF0 treated cells preferentially EDA-FN protein was synthesized. As a further stimulus dexamethasone was used. Again an increase in FN-specific mRNA was seen; the ratio between FN and

90 . Joint annual meeting 1995 OGAI/GFI EDA-FN, however, was not altered when compared to untreated cells. Thus, depending on the stimulus, not only the quantity of FN is increased, but also a particular FNisoform. The latter changes not only structural, but also functional properties of the protein.

Medizinische Mikrobiologie und Immunologie, AG Infektabwehrmechanismen, Ruhr-Universitat Bochum, Bochum, Germany

E.36 Arachidonic acid induces apoptosis in human polymorphonuclear neutrophil granulocytes P. WACHTLER, M. KOLLER, and W. KONIG Cell death in distinct parts of the immune system is clearly programmed. We analyzed apoptosis in human polymorphonuclear neutrophil granulocytes from healthy normal donors using flow cytometry (FACS-Analysis) after DNA-staining with propidium iodide (50 fAg/ml). The cells were incubated 18 h at 37°C in RPMI 1640. The addition of arachidonic acid (AA, UJ-6 fatty acid, 40 fAM) led to a significant increase of apoptosis up to 80 % compared to the RPMI-control. In contrast, incubation with eicosapentaenoic acid (EPA, UJ-3 fatty acid, 40 fAM) did not induce apoptosis. Preincubation with MK 886 (1 fAM, inhibitor of leukotriene biosynthesis) led to an enhanced apoptosis (up to 88 %) suggesting that a lipoxygenase product is involved in the prevention of apoptosis. Furthermore preincubation with pentoxifylline (1 mM) and cyclosporine A (1 fAM) reduce AA-stimulated DNA fragmentation. Our data provide evidence for the involvement of lip oxygenase products and cytokines (e.g. TNF) in the regulation of AA-induced apoptosis in neutrophils.

Dermatological Clinic, Medical School Hannover, Hannover; lDepartment of Immunology, Georg-August-Universitat Gottingen, Gottingen, Germany

E.37 The C5a receptor (C5aR) on human skin mast cells (MC) and on the human mast cell line HMC-l T. WERFEL, M. OPPERMANN l , G. BEGEMANN,]. ELSNER, A. KAPP, O. GOTZE l , and]. ZWIRNER l The expression of the C5aR (CD88) on MC was studied with four novel anti-C5aR monoclonal antibodies (mAb) directed to the N-terminal domain of the receptor. All mAb bound to the human mast cell line HMC-l. The binding could be blocked by rC5a and by peptide EX-1 representing amino residues 1-31 on the N-terminal domain of the C5aR. In addition, FITC-labeled C5a bound to HMC-1, and this binding could be blocked by unlabeled C5a or anti-C5aR mAb. C5aR-specific mRNA was detected in HMC-l cells by RT-PCR. Lymphocyte conditioned medium, interferon y or phorbol esters which induce to a downregulation of C5aR on myeloid cells did not influence the expression of C5aR on HMC-l. C5a but not C5a desArg led to a transient mobilization

Joint annual meeting 1995 GGAIIGFI . 91 of intracellular calcium in HMC-1 which could be inhibited by preincubation of C5a with a neoepitope-specific anti-C5a mAb. Normal human skin was investigated by immunohistology applied to sequential 2 [,1m sections. Anti-C5aR antibodies selectively stained c-kitR+ cells which were metachromic after toluidine blue staining. The binding was inhibitable by peptide EX-l. A similar expression of C5aR epitopes was found on MC in psoriatic plaques while C5aR were not detectable in wheal and flare reactions.

lDepartment of Neurology, Neuroimmunology Branch and Clinical Research Group for Multiple Sclerosis, Julius-Maximilians-Universitat Wiirzburg, Wiirzburg; 2Department of Neurology, Universitat Rostock, Rostock; 3Institute of Clinical Chemistry and Laboratory Medicine, Universitat Regensburg, Regensburg, Germany

E.38 Detection of reactive oxygen intermediates in cells of the cerebrospinal fluid (CSF) U. K. ZETTL l ,2, E. MIX2, G. ROTHE 3, H. MEYER-RIENECKER2, and H.-P. HARTUNG l Reactive oxygen intermediates (RO) are generated by phagocytes during the oxidative burst. They exert several effects, e.g. (I) intracellular bactericidal activity, (II) vasoconstriction, (III) tissue damage (e.g. degradation of myelin), (IV) inactivation of endogeneous mediators (e.g. proteases, chemotaxins, leukotriens), and (V) reduction of transplantation antigen expression. Such processes take place in the central nervous system during the course of infectious and autoimmune diseases. Because the target sites of these diseases are difficult directly in investigate, conclusions concerning the course of disease are drawn from the functional state of cerebrospinal fluid (CSF) cells. In this context two parameters of CSF phagocytes are of special interest, i.e. (I) spontaneous oxidative burst activity, (II) capacity for production of ROI in response to stimulants. Flow cytometric measurements of production of the prototype ROI H 20 2 by its ability to oxidize the nonfluorescent dihydrorhodamine 123 to fluorescent rhodamine 123 is suitable for assessment of the oxidative burst activity of cells. We have adapted this method from blood cells to CSF cells. Results of one CSF sample with low phagocyte content are presented. The advantages and the validity of the method are demonstrated. Submaximum ROI stimulation was brought about by the chemotactic peptide N-formyl-Imethionyl-leucyl-phenylalanin (FMLP) and maximum stimulation by the activator of protein kinase C 12-0-Tetradecanoylphorbol-13-acetate (TPA). The spontaneous activity was highest in monocytes. In response to TP A stimulation the activity of the whole monocyte population increased 20-fold. Neutrophil granulocytes showed no spontaneous activity. TPA stimulation led to the occurrence of three sub populations with low, intermediate and high levels of burst activity (maximum 300-fold). This heterogeneity was not found in the peripheral blood. The procedure allows parallel testing of CSF and blood cells as well as the investigation of the influence of CSF and serum factors. It is appropriate for comparative examination in the course of diseases and between different patient groups.

92 . Joint annual meeting 1995 OGAIIGFI IDepartment of Neurology, Neuroimmunology Branch and Clinical Research Group for Multiple Sclerosis, Julius Maximilians Universitat Wiirzburg, Wiirzburg; 2Department of Neurology, Universitat Rostock, Rostock; 3Department of Neurology, Klinikum Benjamin Franklin, Freie Universitat Berlin, Berlin, Germany

E.39 Reactive oxygen species (ROS) and nitric oxide (NO) induce apoptosis in skeletal myoblasts U. K. ZETTL1,2, E. MIX 2 , M. STANGEL 1,3,]. ZIELASEK 1, H. MEYER-RIENECKER2 , K. V. TOYKA 1 , H.-P. HARTUNG,I and R. GOLDI Estimation of production of ROS and NO by inflammatory cells is of interest due to the role of ROS and NO in tissue damage, tissue regeneration or defense against infections. Inflammatory mediators like ROS and NO are potent inducers of apoptosis in lymphoid cells. Only little information is available whether apoptosis plays a role in muscle disorders. It was the aim of our study to investigate, whether exposure to ROS and NO causes apoptosis of myogenic cells. Skeletal myoblasts obtained from adult Lewis rats were cultured and allowed to fuse to myotubes. Both myoblasts and myotubes were exposed to the ROS hydrogen peroxide (H 20 2 ), the ROS generating system hypoxanthine/xanthine oxidase (HX/XO), and the NO liberator S-nitroso-N-acetylpenicillamine (SNAP). Apoptosis was assessed by morphological criteria and DNA gel electrophoresis, and estimated quantitatively by molecule labeling, i.e. by tailing and FACS analysis (for myoblasts) and in situ nick translation (for myotubes). After 16-hours exposure to H 2 0 2 (10-5-3 X 10-4 M), HX (10-4-5 x 10--4 M) and SNAP (10-3-5 x 10-3 M) a dose-dependent increase in the number of apoptotic myoblasts was observed with a maximum of 40 % for the higher concentrations of H 2 0 2 and SNAP. Apoptosis was specifically inhibited by catalase and hemoglobin, scavengers of H 20 2 and NO, respectively. Apoptosis of myotubes was observed at 3 x 10-4 M H 2 0 2. It is concluded that exposure to ROS and NO causes apoptosis of myogenic cells. This may have pathogenetic relevance for muscle cell damage and regeneration. Also, inhibitions of apoptosis may have therapeutic value in inflammatory and degenerative myopathies.

Sandoz Forschungsinstitut, Vienna, Austria; lVacsyn, Paris, France

E.40 MOP (Lysyl)GOP, a synthetic derivative of muramyl dipeptide, specifically inhibits synthesis of pro-inflammatory cytokines and iNOS in vitro M. ZUNIC, G. M. BAHRt, P. DUKOR, and C. LAM High levels of pro-inflammatory cytokines and nitric oxide (NO) are thought to mediate pathophysiological mechanism(s) associated with allergic disorders, such as immune complex diseases and asthma. In this study, we have compared anti-inflammatory activity of a non-pyrogenic, non-toxic MDP(Lys)GDP derivative with that of the parent molecule MDP, on the production of TNF-a, IL-1~, IFN-y and the expression of inducible NO synthase (iN OS) in murine thioglycollate-elicited peritoneal macrophages

Joint annual meeting 1995 OGAIIGFI . 93 (McI>s) stimulated by endotoxin. RNA transcripts for iNOS, TNF-a, IFN-y, and IL-1~ were not detected in unstimulated McI>s or cells incubated with MDP(Lys)GDP alone. Similarly, no cytokines and nitrites were detected in the culture supernatants. Macrophages incubated with MDP expressed mRNA encoding TNF-a and IL-1f:l and released the cytokines in cultures. Incubation of the cells with LPS for 4 or 6 h resulted in significant synthesis of iNOS, IL-1~, TNF-a mRNAs or iNOS and TNF-a after incubation with IFN-y, and accumulation of high levels of nitrites in cultures at 24 h. Coincubation of the McI>s with LPS (10 ng/ml) and MDP resulted in a dose-dependent up regulation in the production of nitrites, whereas MDP(Lys)GDP (1-100 [lg/ml) dosedependently suppressed the levels of the steady-state transcripts for iNOS, TNF-a and IL-1~ but not those induced by INF-y. The suppressive effect of MDP(Lys) GDP was reversed by cycloheximide, a protein synthesis inhibitor and was only specific for LPSinduced effects. On the contrary, MDP(Lys)GDP synergized with IFN-y in the production of the pro-inflammatory mediators in these macrophages. Together, these data show that MDP(Lys)GDP has a anti-inflammatory activity which can be exploited for prophylactic treatment of diseases in which endotoxin is thought to cause overproduction of pro-inflammatory mediators.