Workshop K: Cytokines and Cytokine Receptors: K.1–K.21

Workshop K Cytokines and Cytokine Receptors

1

Universit‰t Erlangen, Institut f¸r Klinische Mikrobiologie, Immunologie und Hygiene, Erlangen, Germany, 2Trudeau Insitute, and 3Trudeau Institute, Saranac Lake, USA

1

K. 1 Regulation of IL-4 expression in the mast cell lineage

K. 2 IL-2 is necessary for the efficient development of allergen- and helminth-induced Th2 responses in vivo

A. Gessner1, K. Mohrs2, C. Gie˚ler1, M. Rˆllinghoff1, and M. Mohrs3

Mast cells are critical effector cells of type 2 immunity and hypersensitivity. In addition they play a pivotal role in innate immune responses by rapid release of soluble mediators including multiple cytokines, such as IL-4. We used bicistronic IL4-gfp reporter mice (4get) to visualize IL-4 expression in mast cells. Unexpectedly, gene expression, as assessed by gfp-fluorescence and RT-PCR, was spontaneously initiated in cultures of developing bone marrow derived mast cells (BMMC). The development of IL-4 expressing BMMC was independent of IL-4 receptor (IL-4R)-mediated signals, as there was no impairment in cultures obtained from IL-4R or STAT6-deficient 4get mice. In contrast to IL-4 mRNA expression cytokine production was strictly dependent on stimulation by calcium-flux. Intracellular IL-4 could not be detected prior to stimulation while we could stain for pre-formed TNF-a. IL-4 production was restricted to gfp-positive cells. Thus the presence of the transcript is necessary but not sufficient to result in cytokine synthesis, suggesting a mechanism of post-transcriptional regulation. In vivo we detected gfp fluorescence in mature connective tissue mast cells (CTMC) but not in committed mast cell precursors. We propose a model whereby IL-4 expression is initiated as part of the lineage development of mast cells. Pre-formed transcripts correlate with rapid cytokine responses elaborated by these cells in the periphery.

Research Center for Infectious Diseases and 2 Institute of Virology and Immunobiology, University of W¸rzburg, W¸rzburg, Germany

G. Wohlleben1, B. Sandner-Nanan2, A. Schimpl2, and K.J. Erb1

IL-2 a cytokine usually associated with Th1 responses has also been suggested to be necessary for the efficient development of Th2 cells in vitro and in vivo. For this reason we analysed whether IL2 deficient mice could mount allergen- and helminth-induced Th2 type responses in vivo. Our data indicate that IL-2 is not necessary to induce the production of IgE and IgG1 by B cells. However, IL2 is absolutely necessary for the induction of IL-5 producing Th2 cells and airway eosinophilia. Furthermore, although IL-4 secreting Th2 cells are present in IL-2 deficient mice they are reduced in numbers in comparison to control mice. This suggests that IL-2 is also necessary for the efficient induction of Th2 cell development. However, the reduction of Th2 cells present in the lymphnodes (indicated by less IL-4 and IL-5 production) does not explain the almost total lack of airway eosinophilia in the IL-2 deficient mice. For this reason we investigated whether Th2 cells secreting IL-4 were present in the airways of IL-2 deficient mice by intracellular FACS-staining. The Th2 cells secreting IL-4 are strongly reduced in the airways of IL-2 deficient mice after infection with the helminth Nippostrongylus brasiliensis, thus explaining the lack of airway eosinophilia. Similar results were obtained when subjecting the IL-2 deficient mice to an ovalbumin based allergen-challenge model. Taken together our data clearly establish a role for IL-2 in the development of allergic- and helminth-induced Th2 responses. The strong inhibition of airway eosinophilia observed in both allergen- and helminth-treated IL-2 deficient mice suggests, that neutralizing IL-2 may also be useful in inhibiting the development of asthma in humans.

106 ¥ 34th Annual Meeting of the German Society of Immunology 1

Division of Viral Infections, Robert Koch-Institut, Berlin, Department of Virology, Max von Pettenkofer-Institut, M¸nchen, Germany, and 3 Department of Histology and Embryology, University of Rijeka, Rijeka, Croatia 2

K. 3 A viral protein reveals a biological role for STAT2 in IFN-g signaling H. Hengel1, M. Wilborn1, M. Wagner2, T. Ziade1, I. Bubic3, M. Trilling1, S. Jonjic3, U. Koszinowski2, and A. Zimmermann1

Cytomegaloviruses (CMVs) inhibit type I (a/b) and type II (g) interferon (IFN) receptor signaling through the JAK/STAT pathway and IFN-dependent expression of antiviral target genes. Based on a forward genetic procedure involving screening of a library of CMV mutants derived from random transposon mutagenesis by cis-acting reporter genes we identified the gene M27 of mouse CMV (MCMV) to mediate this effect. Expression of M27 prevented nuclear accumulation of STAT2 in infected cells and resulted in a rapid proteasomedependent degradation of STAT2 but not STAT1. Isolated expression of M27 was sufficient to mediate these effects. Deletion of M27 conferred susceptibility of MCMV replication to IFN-a/b. Unexpectedly, M27 had a much more drastic effect on IFN-g mediated antiviral activities which was strictly dependent on STAT2 and was also contingent on IFN-a/bR1 expression. Replication of the DM27MCMV mutant was dramatically attenuated in wildtype mice but partly restored in IFNa/bR-deficient as well as IFN-gR-deficient mice. The results establish a new mechanism for viral immune escape and document an unforeseen biological significance of STAT2 in IFN-gR signaling. The data support a model of molecular cross-talk between IFN-g and IFN-a/b signaling components which requires STAT2.

Institut f¸r Biochemie, RWTH Aachen, Aachen, Germany

K. 4 the regulation of the OSMR surface expression by Jak1 S. Radtke, A. Jˆrissen, C. Haan, P.C. Heinrich, and I. Behrmann

The oncostatin M receptor (OSMR) is part of a heterodimeric receptor complex which mediates signal transduction of the pleiotropic cytokine OSM via a signaling pathway involving Janus kinases (Jaks) and transcription factors of the signal transducers and activators of transcription (STAT) family. We have demonstrated in earlier studies that the surface expression of the OSMR is significantly enhanced by coexpression of Janus kinases. Here, we have further analysed the underlying mechanisms by using chimeric receptors as

well as GFP-tagged OSMRs. We have identified three dileucine-like signals within the OSMR box1/ 2-region that mediate the intracellular retention of the OSMR in the absence of associated Jaks. Even though one of these motifs fits the consensus sequence for lysosomal targeting, our results are not consistent with a lysosomal targeting mechanism. Rather we propose that the three dileucinelike motifs mediate the ER-retention of the OSMR. The presence of such a signal in the Jak1-binding region might ensure that only functional receptor/ Jak-complexes are expressed at the cell surface. 1 Department of Cell Biology, Medical Academy of Gdan¬sk, Postgraduate School of Molecular Medicine Warsaw, 2 Department of Cell Biology, Intercollegiate Faculty of Biotechnology, and 3 Department of Cell Biology, Intercollegiate Faculty of Biotechnology, Mediacal University of Gdan¬sk, Gdan¬sk, Poland

K. 5 Different response to soluble (sTNF) and transmembrane TNF (tmTNF) in U937 cell variants indicates requirement for cooperation between the two TNF receptors to induce cytocidal effect of tmTNF A. Pierzchalski1, K. Banach2, and J. Bigda3

Tumor necrosis factor is a pleiotropic cytokine that can induce apoptosis. Molecular mechanisms that mediate TNF?induced cellular responses, role of its two receptors: TNF?R55 and TNF?R75, as well as the engagement of two forms of TNF: secreted (sTNF) and transmembrane (tmTNF) in the process of apoptosis remain still not fully understood. The aim of this work was to analyse the cytocidal effect mediated by human tmTNF and sTNF and investigation of co-operation between two TNF receptors in mediating cytotoxicity. The experiments were carried out on three related U937 sublines: U937M, U937ATCC, U937G. Among three studied U937 cell variants only U937M cells revealed sensitivity to tmTNF and sTNF in the absence of the translation inhibitorcycloheximide. However, CHX did not increase sensitivity to tmTNF in U937M, as it did in U937ATCC and U937G cells in the case of tmTNF and sTNF. It has been also demonstated that selective blocking of TNF receptors (TNF?R55, TNF?R75 or TNF?R55 and TNF?R75 simultaneously) decreased the cytocidal effect mediated by cytokine in U937ATCC and U937G cells, but no synergistic activity could have been observed when blocking of both receptors. In the case of U937M cell line blocking of TNF?R55 required much higher doses of anti-receptor antibodies to inhibit the cytocidal effect of both soluble and transmembrane TNF. These results indicate

34th Annual Meeting of the German Society of Immunology ¥ 107

damage of protective mechanisms against TNF cytotoxic activity in U937M cells. This also suggests disturbed signaling mechanism of tmTNF in U937M cells, which may arise from abnormal cooperation between TNF?R55 and TNF?R75 necessary for tmTNF cytotoxicity. Thus our studies presenting different response to sTNF and tmTNF in U937 cell variants can indicate requirement for cooperation between the two TNF receptors in inducing apoptosis by tmTNF. Therefore we believe that our comparative model can be very useful for identification of a molecular mechanism governing cooperation between the two receptors. The cooperation is crucial for the transmembrane form of TNF in order to exert its effects on cells. As this form of TNF can be of prime importance to the local inflammation, identifying the molecular mechanism of TNF receptor cooperation may help in proposing new targets for antiinflammatory therapy. 1

Clinic of Rheumatology, University Magdeburg, Magdeburg and 2 Institute for Immunology, University Rostock, Rostock, Germany

K. 6 Biological treatment in rheumatoid arthritis – modulation of gene expression S. Drynda1, D. Koczan2, H.-J. Thiesen2, and J. Kekow1

Treatment with biologicals (TNF neutralizing substances, interleukin-1 receptor antagonist) induces a rapid and sustained decline in disease activity in patients with inflammatory rheumatic diseases. Biologicals have been shown to control disease activity in patients with refractory rheumatoid arthritis (RA). However, about 30% of patients receiving this expensive therapies fail to respond. Today neither clinical nor routine laboratory parameters exist, which enable the prediction of therapy outcome. It was the aim of this study to determine candidate genes with prognostic importance for the therapeutic responsiveness of patients to biologicals. DNA microarrays have been used to study the effects of anti-TNF (etanercept) and IL-1ra (anakinra) treatment on gene expression in mononuclear cells from peripheral blood (PBMC) in the course of treatment. Blood samples were taken from 14 RA patients before and 72 h after initiation of etanercept (n=10) or anakinra (n=4) therapy. Total RNA from PBMC was prepared with the RNeasy kit (Qiagen). Affymetrix chip technology (HG-U133A Set) was used to analyse the expression levels. Selected chip data were confirmed by real-time PCR. The therapy response was determined by changes in the disease

activity score (DAS28) 3 months after the start of treatment. Under anti-TNF therapy, the expression of genes for cytokines such as TNFa IL-1b IL-1ra and PBEF, for chemokines like IL-8, MIP-1a MIP-1b as well as other disease-associated proteins like COX-2, ICAM-1, tristetraprolin and manganese superoxide dismutase changed differentially. Interestingly, changes in the expression profile detected 3 days after the start of treatment were found to be closely associated with the outcome of therapy as reflected 3 months later by the DAS. Using RT-PCR, affymetrix data were confirmed and expression of low abundant transcripts, which were not detected on microarrays such as IL-6, were observed. The application of anakinra influenced the expression levels of L-1b, IL-8, and COX-2 in a similar fashion. In contrast to anti-TNF treatment expression levels of TNFa, MIP-1a, MIP-1b remained unchanged. Our data give new insights into the effects of biologicals used in RA treatment on the transcriptional level, and contribute to the understanding of the complex network of cytokines and their regulation. Marker genes have been identified which seem to be associated with the outcome of therapy. The data indicate the presence of genetic heterogeneities within the group of RA patients, suggesting the presence of genetic polymorphisms in the identified genes. Supported by BMBF grant (FZK 01GG0201)

Institut f¸r Biochemie, RWTH Aachen, Aachen, Germany

K. 7 Upregulation of gp130 surface expression by Janus kinases S. Diefenbach, S. Radtke, C. Haan, P.C. Heinrich, and I. Behrmann

Gp130 is the common signal transducing subunit of Interleukin-6-type cytokines. Tyrosine kinases of the Janus family associate non-covalently with the cytoplasmic region of gp130. They are necessary for activation of signaling pathways involving e.g. STAT transcription factors and MAP kinases. Here we show that Jaks may additionally be important for the regulation of the gp130 surface expression. Coexpression of the Janus kinases Jak1, Jak2 and Tyk2, but not Jak3 with gp130 results in an increased surface expression of gp130 in transiently transfected Cos7 cells, whereas the overall amount of gp130 was not changed. Mutations of Jak1 and gp130 revealed that Jak binding to the receptor but not the kinase activity is required for the effect. If the dileucine motif within the cytoplasmic region of gp130 is mutated to alanine, the effect is abolished. All of these results were confirmed with gp130-eGFP constructs whichal-

108 ¥ 34th Annual Meeting of the German Society of Immunology

lowed the simultaneous detection of surfaceexpressed receptors and overall expressed receptors by flow-cytometric analysis. Additionally we found that the surface expression of endogenous gp130 is decreased in Jak1-deficient fibrosarcoma cells compared to their parental cells, indicating that the observed effect is relevant for cells expressing physiological receptor levels. Our results indicate that Jaks may upregulate gp130 surface expression probably by negatively influencing endocytosis.

Department of Histology and Immunology, Medical University of Gdan¬sk, Gdan¬sk, Poland

K. 8 Serum interleukin-6 (IL-6) and its soluble receptor (sIL-6r) levels during oral contraceptive use L. Hak, K. Kotewicz, D. Rachon, and J. Mysliwska

Oral contraceptives (OC) pills beside its contraceptive role provide many short and long-term benefits. Long-term OC use confers protections against benign breast disease and colorectal cancer, help prevent rheumatoid arthritis, decrease hospitalisation for pelvic inflammatory disease, and helps preserve bone mineral density to reduce risk of fractures. Interleukin-6 (IL-6) is a multifunctional cytokine produced by immune and nonimmune cells. Inappropriate expression and production of IL-6 is though to be involved in the pathogenesis of numerous diseases, including osteoporosis, atherosclerosis and Alzheimer's disease. Various hormones can influence IL-6 productions. Our recent studies showed that 17b-estradiol decrease IL-6 productions by peripheral blood mononuclear cells. Ethynylestradiol (EE) is a potent synthetic estrogen, which is the main constituent of OC pills, and its impact on IL-6 production and activity is not known. Thus, we decided to study influence of OC use among young healthy women on serum IL6 levels and its soluble receptor (sIL-6r). Sixty women were included into the study and were divided into two groups. Group one consisted of 30 regular menstruating healthy women who did not used OC (age range: 21-31yrs). Group two consisted of 30 healthy women who used OC pills (age range: 22-30yrs). Serum IL-6 and sIL-6r levels were measured by immunoenzymatic assays (ELISA). There was no differences into IL-6 serum levels between those two groups, however serum sIL-6r levels among women who used OC, which contained 35mg of EE were significantly lower compared to other women. We can speculate that favorable effects of OC use in part be mediated by the inhibition of IL-6 activity.

St. Vincents Hospital and Univeristy of New South Wales, Centre for Immunology, Sydney, Australia

K. 9 The in vivo biology of the TGF-b superfamily cytokine MIC-1 and its role in inflammatory diseases H. Johnen, M.R. Qiu, D.A. Brown, T. Kuffner, W.D. Fairlie, P. Russel, and S.N. Breit

MIC-1 (macrophage inhibitory cytokine) is a divergent member of the TGF-b superfamily, first cloned by us on the basis of increased expression during macrophage activation. The functions of MIC-1 are poorly defined, however serum MIC-1 levels are increased in patients with chronic inflammatory processes. For example, patients with serum MIC-1 levels in the top decile have an almost 3 fold increased risk of developing a vascular event such as heart attack or stroke. Serum MIC-1 levels are also elevated in patients with prostate, breast and colon cancer, perhaps because of induction of MIC-1 by activation of the p53 pathway. MIC-1 is also highly expressed in the trophoblast and during pregnancy serum MIC-1 levels rise dramatically. These findings suggest an important role of MIC-1 in the regulation of immune, inflammatory and malignant processes but provide little information about the biological mechanisms regulating MIC1 production and its biological effects. Activated macrophages are a major source of MIC-1 in vivo. To help define the in vivo biology of MIC-1 we have generated transgenic mice that overexpress MIC-1 under the control of the monocyte/macrophage specific c-fms (CSF-1 receptor) promotor. These mice constitutively produce large amounts of MIC-1, which is detectable in macrophages and serum. These mice grow and breed normally and have no obvious phenotype. We predict they will exhibit aberrant behaviours in animal models of inflammation and cancer. We are performing experiments to validate these hypotheses.

Chair of Clinical Immunology, University of Medical Sciences, Poznan, Poland

K. 10 Cytokine expression in malignant cells from human tumor cell lines and malignant pleural effusions M. Kaczmarek, E. Mizera-Nyczak, J. Sikora, and J. Zeromski

The tumor cells may influence directly on the immunological system. The aim of study was assessment of some cytokines synthesis and secretion in tumor cells from human cell lines and isolated from malignant pleural effusions. In the cells from cell lines genes expression for IL-2, IL-4,

34th Annual Meeting of the German Society of Immunology ¥ 109

IL-10 and IL-18 was evaluated by RT-PCR. In the supernantants from these cultures were investigated TNFb, TGFb and IL-10 content by ELISA. Intracellural and surface expression of TNFa, TNFb, TGFb, IL-2, IL-4, IL-10 and IL-2Ra was evaluated by flow cytometry, both in cell lines and malignant pleural effusions. Molecular analysis confirmed the presence of mRNA for IL-2 in the one case and for IL-10 in the all studied cell lines. It wasn't demonstrated mRNA for IL-2, IL-4 and IL-18 in none of established cell lines. In supernatants was observed elevated presence of TGFb, however TNFb and IL-10 were not demonstrated in spite of early confirmed expression these cytokines in studied cells. Flow cytometry analysis performed in the cell lines had showed expression of TNFb, IL10, and TGFb. It wasn't observed the TNFa, IL-2, IL-4 and IL-2Ra (CD25) expression. The cytometry assessment of the tumor cell isolated from malignant pleural effusion showed the TNFb, IL-10 expression and weak presence of the IL-2, IL-4 and IL-2Ra. However, it wasn't showed TNFa in none of case. The performed preliminary studies confirmed the possibility of synthesis immunosupressive cytokines by tumor cells.

Deutsches Krebsforschungszentrum, Heidelberg, Germany

K. 11 Molecular cloning of the mouse IFRG28 gene and functional characterization of its promoter M. Klehr and R. Zawatzky

The Interferon-regulated-Gene28 (IFRG28) is activated on transcriptional level starting from 1-2h of IFN-a/b treatment. The gene spans 3.8 kb from the transcription start site to the polyadenylation signal, and is made up of 2 exons. The protein sequence encoded by the mouse IFRG28 gene is 43% identical to that of the human gene. Rapid amplification of cDNA 5©-ends (5©-RACE) identified one major transcription start site (+1) and several minor transcription start sites upstream and downstream. Analysis of the 5©-flanking sequence of the gene showed the presence of three interferonstimulated response elements (ISRE) at positions 22 to -33, -45 to ±56 and -71 to -82 as well as one GAS (gamma-interferon activation site) element at position ±219 to 228, relative to the transcription start site. Genomic fragments encompassing a portion of the IFRG28 5© flanking region were inserted into vectors containing a luciferase reporter gene. 290 bp of upstream sequence were found to be sufficient for IFN-a mediated induction of luciferase activity in the LMTK- cell line. Point mutations in the ISRE sites were performed in order to analyse their indiviudual influence on the

promoter strength. Mutation of all three ISRE sites resulted in complete unresponsiveness of the reporter gene to IFN-a treatment. Inactivation of only one out of three ISRE elements lead to an unexpected augmentation of the stimulation index, whereas mutation of two ISRE sites resulted in similar reporter gene activity after IFN treatment as observed with the wild-type promoter. 1

Fachbereich Humanmedizin Zentrum fuer Innere Medizin Haematologie/Onkologie, Georg-August-Universitaet Goettingen, Goettingen, 2 Institut fuer Vorlogie, Universitaet Koeln, Koeln, and 3 Fachbereich Humanmedizin Zentrum fuer Kinderheilkunde, Georg-August-Universitaet Goettingen, Goettingen, Germany

K. 12 Comparative analysis of LMP1 associated cytokine regulation in lymphoma cell lines D. Kube1, S. Smola2, and M. Vockerodt3

IL-10 can modulate the immune response at certain levels, playing a crucial role in balancing humoral and cellular responses. Moreover, it can function as a growth and differentiation factor for B cells. In addition, the expression of other cytokines, their balanced expression profile, regulated by EBV, may play a role in the patho-physiology of lymphoma development. Here we demonstrate that IL-6, IL-10 and TNF-alpha as well as IP-10 were specifically induced by LMP1 in certain lymphoma cell lines. The activation of NF-kappaB by LMP1 plays a crucial role for IP-10 and TNF-alpha and in part for IL-6, but not for IL-10 expression. LMP1 derivatives of the CTARs, by deletion or mutation, underline the differences in signaling associated with expression of TNF-alpha, IP-10, IL-6 or IL-10. A protein kinase cascade involving p38 MAP kinase regulates IL-10 an IP-10 mRNA stability, whereas the NF-kappaB binding site within the IP-10 promoter is crucial for transcriptional regulation. The overexpression of MEKK1 or MEK1 in comparison to LMP1 reveals additional differences between the analysed cytokines as well as differences in their regulation comparing Burkitt and Hodgkin lymphoma cell lines. Our results provide a striking example of usage of several signal transduction pathways for regulating the expression level of endogenous genes by LMP1. Furthermore, we have characterized more than 9kb of the 5' flanking region of the IL-10-gene identifying new DNA-sequence variations and their frequencies in different geographic regions. Their functional role in in vitro EBV infected B cells compared to in vitro stimulated PBMc by LPS or cAMP reveals significant differences (supported by SFB502, DFG KU 954/4-1, DFG KU 954/5-1 & Wilhelm-Sander Stiftung).

110 ¥ 34th Annual Meeting of the German Society of Immunology Medical Microbiology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany

K. 13 Toll-like receptor 2 mRNA expression in human neutrophils differs in response to CXC chemokines B. Kˆnig, R. Arnold, and W. Kˆnig

Neutrophil activating chemotactic factors including the CXC chemokines belong to the main proinflammatory mediators in innate immunity. They are induced by bacteria, bacterial products, as well as by a variety of viruses. The Toll-like receptors (TLRs) function as sensors of infection and are another main player in innate immunity. TLR ligands present molecular products derived from all of the main classes of pathogens. We hypothesized that the profile of the chemotactic response directs the recognition of microorganisms via TLRs. First we report that the CXC chemokines IL-8, GCP-2, GRO-alpha, NAP-2 and ENA-78 differ in their chemotactic potential to attract human neutrophils. With regard to the TLRs we found that neutrophils expressed high levels of TLR2 mRNA and cell surface TLR2, while TLR4, TLR7 and TLR9 were only detected at lower level. Transmigration of neutrophils towards a chemotactic gradient of IL-8 is combined with a dose-dependent downregulation of TLR2 mRNA expression, most prominent at the highest concentration. Similar results were obtained for GCP-2, GRO-alpha, NAP-2 as well as for ENA-78. However, the chemotactic factors differ markedly in their doseresponses. The exposure to the respective chemotactic factors also led to a modulation of TLR2 mRNA expression in the non-transmigrated neutrophils in a dose-dependent manner. Our results clearly show that neutrophil activating chemotactic factors differ in their potency to modulate TLR2 mRNA expression in neutrophils. One may suggest that both the basal levels of TLR2 and its inducible regulation may influence and direct fast responses to microbial infection. 1

Department of Immunology and Serology and 2 Department of Immunology of Serology, Silesian Medical Academy, Katowice, Poland

K. 14 Concentrations of NPT and IL-10 in peritoneal fluid and serum of women with and without endometriosis Z. Kondera-Anasz1, J. Sikora2, A. Mielczarek-Palacz1, and A. Mertas1

Introduction: Endometriosis is defined as the presence and growth of viable glands and stromal

endometrial tissue outside the uterine cavity. Many studies have demonstrated that disorders of immune response and immunological components in peritoneal fluid (PF) of women with endometriosis play an important role in the maintenance and development of this disease. Increased number, activation of peritoneal macrophages, release of macrophage-derived cytokines and growth factors are characteristic for endometriosis. The aim of our work was to study level of neopterin (NPT) and interleukin (IL)-10 in peritoneal fluid (PF) and serum of women with endometriosis in relation to stage of disease. Materials and methods: The levels of NPT and IL10 in PF and serum were determined by cytokinespecific enzyme-linked immunosorbent assay (ELISA) in PF and serum of 47 women; 32 with endometriosis and 15 with no endometriosis (control group). Conclusion: Increased levels of NPT and IL-10 in PF and serum of women with endometriosis indicate enhanced macrophage activity in this disease. The higher amounts of NPT and IL-10 in the PF than in serum suggest local immune activation. 1

Department of Immunology and Serology and 2 Department of Immunology of Serology, Silesian Medical Academy, Katowice, Poland

K. 15 Significantly increased serum levels of IL-1 a, and IL-12 in women Z. Kondera-Anasz1, A. Mielczarek-Palacz2, and B. Popczyk1

Introduction: Cytokines play a key role in the regulation of cells of the immune system and also have been implicated in the pathogenesis of malignant diseases. Some cytokines have been shown to have potential in diagnosis of cancer. Materials and methods: The aim of this study was to examine the levels of intrleukin-1a (IL-1 a), interleukin-6 (IL-6) and interleukin-12 (IL-12) in sera of women with ovarian tumours. The study was perfomed on 17 women aged between 21 and 77 years (mean age: 51,213,8 years) with benign or malignant ovarian tumours. This group involved 10 women with ovarian cyst and 7 women with ovarian carcinoma. Blood samples were taken from each patient before surgery. The control group consisted of 23 healthy women, aged 24 to 74 years (mean age: 50,214,6 years). Quantitative determination of IL-1a and IL-12 serum concentrations was perfomed using enzyme immunoassay kits Quantikine (R&D Systems Inc., Minneapolis, USA). The serum level of IL-6 was measured using enzyme immunoassay kit Milenia (DPC, Los Angeles, USA). The assays were per-

34th Annual Meeting of the German Society of Immunology ¥ 111

formed in duplicate, according to the manufacture's instructions. Results: In our study, high concentrations of IL-1 a (mean: 77,1 pg/ml), IL-6 (mean: 105,8 pg/ml) and IL-12 (mean: 6,3 pg/ml) were observed in patients with benign and malignant ovarian tumours. These mean levels were significantly higher than in the control group (p<0,0001). In women with ovarian carcinoma the mean serum levels of IL-1 a (mean: 179,8 pg/ml) and IL-12 (mean: 10,8 pg/ml) were significantly higher than in women with ovarian cyst (p<0,0001). Conclusion: With the excepion of IL-6 these results suggest that cytokine produktion in studied patients is a result of nonspecific inflammation. We assume, that interleukins IL-1 alpha and IL-12 may be regarded as a markers of ovarian carcinoma. 1

Abteilung H‰matologie und Onkologie, Universit‰tsklinik Regensburg, 2 Institut f¸r Pharmazie, Abteilung Pharmazeutische Chemie II, Universit‰t Regensburg, Regensburg, 3 Institut f¸r Pharmakologie, Medizinische Hochschule Hannover, Hannover, and 4 Klinik und Poliklinik f¸r Innere Medizin I, Universit‰tsklinik Regensburg, Regensburg, Germany

K. 16 Recruitment of MyD88 to the IL-1 receptor complex requires interactions between the conserved motifs box 1/2 and box 3 within the TIR domain of IL-1RAcP J. Radons1, S. Dove2, D. Neumann3, and W. Falk4

The Toll/IL-1 receptor family plays an important role in both innate and adaptive immunity. These receptors are characterized by a C-terminal homology motif called the TIR domain. A principal function of the TIR domain is mediating homotypic protein-protein interactions in the signal transduction pathway. To suggest interaction sites of TIR domains in the IL-1 receptor complex, we modelled the putative 3D structure of the TIR domain within the co-receptor chain, IL-1 receptor accessory protein (IL-1RAcP). The model was calculated on the basis of the crystal structures of human TLR1 and TLR2. The final structure of the IL1-RAcP TIR domain suggests the conserved regions box 1 and 2, including Pro446, as well as box 3 within the Cterminal a-helix as possible protein-protein interaction sites due to their exposure and their electrostatic potential. Pro446, analogous to the Pro/His mutation in dominant negative TLR4, is located in the third loop from the N-terminus at the outmost edge of the TIR domain and does not play any structural role. Inhibition of IL-1 responsiveness seen after substitution of Pro446 by charged amino acids is due to the loss of an interaction site for other TIR domains. Amino acids 527-534 as part of the loop close to the conserved box 3 are critical for

recruitment of MyD88 and to a lesser extent for IL1 responsiveness. Modeling suggests that native folding of the TIR domain may be approached by the responsive deletion mutants D528-534 and D527-533, whereas the C-terminal b-strand and/ or a-helix is displaced in the nonresponsive mutant D527-534.

Dept of Pathophysiology and Immunology, Institute of Rheumatology, Warsaw, Poland

K. 17 Interleukin 15 gene transfer into mouse muscle cells using electroporation in vivo method W. Rudnicka, M. Kurowska, M. Chorazy, M. Ziolkowska, and W. Maslinski

Background: Interleukin 15 plays a key role in synovial inflammation in rheumatoid arthritis (RA). Thus, the blockade of IL-15 bioactivity is a potent target for the therapeutic intervention in RA. Relatively simple and inexpensive technique of gene delivery using plasmids is limited by low protein expression levels in transfected tissues. However, the additional application of electropulses in the gene injection site rises protein expression significantly. The aim: To test the applicability of in vivo electroporation for gene transfer into mouse muscle, using plasmid DNA expressing human fusion proteins, respectively: IL-15 or IL-15R antagonist conjugated with Fc fragment of mouse IgG2a, as the vectors. Results: The simultaneous delivery of plasmids carrying cDNA for human IL-15 related genes injected into mice muscles, and electric pulses into the DNA injection site, resulted in effective, transient gene delivery of both: mRNA (human IL-15/ Fcg2a and human IL-15 mutant/Fcg2a) (evaluated by RT-PCR) and circulating protein (human IL-15/ Fcg2a) (measured by specific ELISA using set of antibodies recognizing human, but not mouse, IL15). The serum IL-15 levels peaked 3 day after electroporation (20 ng/ml) and maintained high until the 6th day after transfection (15 ng/ml). Then, gradually decreased reaching lowest decectable level (50 pg/ml) by day 15th. Conclusion: Electroporation in vivo may be used to test the effects of IL-15 or IL-15 antagonist transient overexpression in mice.

112 ¥ 34th Annual Meeting of the German Society of Immunology University of Regensburg, Institute of Immunology, Regensburg, Germany

K. 18 Characterization of a novel splice form of TNFReceptor 2 C. Scher¸bl, T. Hehlgans, and D.N. M‰nnel

Tumor necrosis factor (TNF) plays an important role in inflammation and immune responses. The effects of TNF are mediated through two distinct membrane bound receptor molecules with apparent molecular masses of 55 kDa (p55TNFR, TNFR type 1) and 75 kDa (p75TNFR, TNFR type 2), respectively. Whereas activation of p55TNFR induces the classical apoptotic pathways, the precise cellular effects of interaction of TNF with p75TNFR have not been completely identified. Activation of NFkB via TNF- p75TNFR interaction in a TRAF2 (TNF receptor associated factor 2)dependent manner has been demonstrated. While analyzing regulation of the p75TNFR expression we identified a novel p75TNFR isoform, termed icp75TNFR, which is generated by the use of an alternative transcriptional start site within the p75TNFR gene and differs only in the expression of the first exon coding for the presequence. Studies with YFP-tagged icp75TNFR constructs showed no membrane staining, but distinct intracellular localization, and colocalization with golgi complex and endosomes. The extracellular domain was cleaved by MMP's and released into the supernatant as soluble TNF inhibitor. Overexpression of icp75TNFR lead to higher resistance of L929 cells toTNF- mediated cytotoxicity. This effect was TNF- and TRAF2- dependent, and not due to the soluble TNF inhibitor.

Klinische Chemie und Molekulare Diagnostik, Klinikum der Philipps Universit‰t Marburg, Marburg, Germany

K. 19 Impaired lymphocyte proliferation in the BDNF knock-out mouse B. Schuhmann, B. Rost, S. Sel, H. Renz, and W.A. Nockher

The brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin (NT) family such as the well known nerve growth factor (NGF). These highly conserved and structurally related proteins are primarily known for their influence on growth, survival, and differentiation of neurons. BDNF exerts its biological effect by interacting with two kinds of cell-surface receptors, the low affinity p75NTR receptor, and the high affinity tyrosine kinase receptor TrkB. In addition to their neurotrophic functions, neurotrophins have recently been shown to be produced

by immune cells and to influence the immune system. While the effect of NGF on lymphocyte proliferation, survival and antibody synthesis have been specified, the immune modulatory role of BDNF has not yet been characterised. To elaborate the influence of BDNF on lymphocytes, we analysed the proliferation of isolated spleenocytes from mice with a homozygous deletion of the BDNF gene. We could detect a significant reduced in vitro proliferation either after unspecific stimulation or after lineage specific stimulation of B- lymphocytes (LPS, anti-IgM) or T-lymphocytes (ConA) compared to spleenocytes from wildtyp mice. We also analysed the expression of p75-NTR and TrkB by RT-PCR, because a direct action of BNDF on lymphocytes depends on the existence of these NT receptors. Here we detect the mRNAs for the p75-NTR receptor in both B- and T-cells, whereas TrkB was only present in B-cells. Our preliminary results indicate that BDNF is strongly involved in the modulation of the immune response. 1

Institut f¸r Immunologie, 2 Klinik f¸r Neurochirurgie, Universit‰tsklinikum Schleswig-Holstein, Campus Kiel, Kiel, and 3 Institut f¸r Medizinische Mikrobiologie und Hygiene, Universit‰t Regensburg, Regensburg, Germany

K. 20 Application of a novel preparative magnetic immunoaffinity system for the isolation of intracellular membrane compartments like TNF receptosomes S. Sch¸tze1, V. Tchikov1, J. Held-Feindt2, M. Heinrich1, S. Winoto-Morbach1, M. Jakob1, J. Neumeyer1, W. Schneider-Brachert3, and D. Kabelitz1

The isolation of defined cell subpopulations from mixtures of different cell populations is successfully being performed by magnetic cell sorting (e.g., MACS or Dynabead systems). Based on very low magnetic labelling properties and lack of appropriate magnetic sorting devices, the isolation of subcellular components like organelles, bacteria, viruses, parasites or soluble macromolecules for proteom analysis has not been possible to date. We developed a magnetic system based on targetspecific immuno-magnetic labelling and a freeflow magnetic chamber with specific properties allowing selective purification of biological materials from cellular lysates (German patent). With this system, we investigate the mechanism and functional significance of internalization and vesicular trafficking of activated TNF receptors as a novel signalling mechanism. We demonstrate the maturation of internalized TNF receptosomes from clathrin-coated vesicles, to form endosomal, multivesicular and lysosomal vesicles. The recruitment of death domain associated (DISC) proteins during

34th Annual Meeting of the German Society of Immunology ¥ 113

TNF receptor endocytosis and the fusion of TNF receptosomes with trans-golgi vesicles, recruiting acid sphingomyelinase and pro-cathepsin D is shown. The application of our magnetic sorting system for the analysis of other receptor systems and for investigations of intracellular trafficking of biologically active vesicles will be discussed. Our patented device will be commercially available soon. 1

Dept of Pathophysiology and Immunology and 2 Dept of Biochemistry, Institute of Rheumatology, Warsaw, Poland

K. 21 Reduced oxygen and proinflammatory cytokines enhance mesenchymal stem cell proliferation in vitro E. Warnawin1, T. Burakowski1, E. Kaminska1, M. Gajewski2, and W. Maslinski1

Background: Mesenchymal stem cells (MSC) are present in adult bone marrow and other tissues. Upon specific stimulation/culture conditions these cells differentiate into specialized cells capable to form tissues like bone, cartilage, muscle or fat. Due to these properties, there is increasing interest in MSC as a potential source of cells/tissues that could be used for tissue regeneration. Although some progress has been made, the optimal conditions for MSC propagation have to be elucidated. The aim: To test the hypotheses that (i) lower oxygen concentration, and (ii) the presence of select growth factors, facilitates MSC proliferation in vitro. Materials and methods: MSC were isolated from bone marrow of rheumatoid arthritis (RA) and osteoarthritis patients undergoing joint replacement. Cells were cultured in normal (21%) or reduced (5%) oxygen concentration in the presence of media or FGF-2, PDGF-BB, TNF-a, IL-1b, IL15. Cell proliferation was judged based on [3H]TdR incorporation. Results: MSC cultured in reduced oxygen (5%) proliferated more rapidly than in normal oxygen concentration (21%). Although all tested cytokines stimulated MSC proliferation, the best effect was observed in the presence of PDGF-BB. Neither oxygen concentration nor used cytokines exerted effect on cell morphology. There were no differences between proliferation of MSCs isolated from patients with OA and RA. Conclusion: MSC can be propagated in vitro in reduced oxygen tension in medium supplemented with PDGF-BB.