Workshop D: Chemokines, Cell Traffic and Adhesion: D.1–D.24

Workshop D Chemokines, Cell Traffic and Adhesion

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Experimentelle Rheumatologie, Charitÿ-Universtit‰tsmedizin Berlin, Berlin and 2 Mukosale Immunit‰t, GBF, Braunschweig, Germany

Institut of Immunology, Otto-von-Guericke-University, Magdeburg, Germany

D. 1 The integrin aE b7 identifies inflammationseeking regulatory T cell subsets

D. 2 The cytosolic adapter protein SKAP55 is involved in CXCR4-mediated b1-integrin adhesion and migration of T cells

K. Siegmund1, U. Haubold1, J. Buer2, A. Hamann1, and J. H¸hn1

S. Kliche, J. Hoppe, A. Nehring, and B. Schraven

CD25+CD4+ regulatory T cells (Treg) fulfil an important role in the regulation of immune reactions and maintenance of self-tolerance. Recently, we have reported that expression of the integrin aE allows the subdivision of the CD25+ Treg compartment and furthermore identifies CD25- murine Tregs. These subsets were shown to be heterogenetic in their suppressive capacity and cytokine expression profile. To identify the molecular differences between aECD25+, aE+CD25+ and aE+CD25- Tregs we performed gene expression profiling. Strikingly, many molecules associated with migration turned out to be differentially expressed. Confirmation by flow cytometry and chemotaxis assays revealed that CD25 single positive cells express to a large extent L-selectin and CCR7. In contrast, aE positive cells express high levels of E- and P-selectin-binding ligands and other adhesion molecules as well as receptors for inflammatory chemokines. We performed homing experiments to investigate the migratory behaviour of isolated Treg subsets in vivo. In naive mice, CD25 single positive cells recirculated significantly through lymph nodes, whereas aE expressing cells showed preferential accumulation in the liver. Striking differences between the Treg subsets were observed in a model of acute skin inflammation, where only aE single positive cells and not CD25 bearing Tregs showed an efficient migration into the inflamed site. Our data on the localization of Treg subsets suggest the existence of two functionally distinct Treg types, one mainly suppressing priming of naÔve T cells at lymphoid sites, and the other preferentially acting at sites of ongoing inflammatory reactions.

The Src Kinase-Associated Phosphoprotein of 55 kDa (SKAP55) is a T-cell specific cytosolic adapter protein that represents a substrate of Src-kinases. It contains a PH-domain, several tyrosine phosphorylation sites and a C-terminal SH3-domain the latter mediating a stable interaction with the cytosolic adapter proteins SLAP-130/ADAP. Previously, it has been shown that SKAP55 is capable of regulating T-cell-APC conjugate formation and b1and b2- integrin-mediated adhesion to fibronectin and ICAM-1 after stimulation of the TCR. Besides the TCR, the G-protein coupled chemokine receptor CXCR4 mediates b1-integrin activation and migration of T cells through an inside-outsignaling pathway whose molecular basis is so far not well understood. To assess a potential role of SKAP55 in CXCR4 mediated signaling, Jurkat-Tcells were generated which overexpress EGFPSKAP55. After treatment with SDF-1b (the natural ligand of CXCR4) these transfectants show a 2.5fold increase in adherence to the??1-ligand fibronectin in comparison to cells expressing GFP alone. Similarly, GFP-SKAP55 expressing T-cells display enhanced migration in a transwell system upon stimulation with SDF-1b. Finally, pull down assays revealed enhanced activity of the small GTPase Rac1, a downstream effector of CXCR4, in cells overexpressing SKAP55. Collectively, these data suggest that SKAP55 is involved in regulating T-cell adhesion and migration by connecting the CXCR4 chemokine receptor with b1-integrins via a mechanism that is located upstream of Rac1. Further studies are aimed at identifying the domains of SKAP55 which are responsible for its function and to further elucidate the role of SKAP55 in CXCR4mediated inside-out signaling.

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Department of Tumor Genetics and Immunogenetics, Department of Hematology, Oncology and Tumorimmunology, Max-Delbr¸ck-Center for Molecular Medicine, Berlin, Germany, 3 Transplantation and Immunology Research, Novartis Institutes for Biomedical Research-Basel, 4 Transplantation and Immunology Research, Novartis Institutes for Biomedical R, and 5 Department of Tumor Genetics and Immunogenetics, Max-Delbr¸ck-Center for Molecular Medicine, Basel, Switzerland

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D. 3 The chemokine receptor CCR7 controls allogeneic cytotoxic T cell priming in alloimmune responses

A. Soruri1, U. Forssmann2, E. Kl¸ver2, N. Spodsberg2, B.O. Gerber3, and J. Zwirner1

2

U.E. Hˆpken1, J. Droese2, J.-P. Li3, J. Joergensen4, D. Breitfeld5, H.-G. Zerwes4, and M. Lipp1

The chemokine receptor CCR7 and its ligands regulate migration and co-localization of T cells and mature dendritic cells to and within T cell-rich zones of lymph nodes, Peyer©s patches and spleen. We used two different in vivo models to demonstrate a role for CCR7 in the initiation of an alloimmune response and in the development of transplant rejection. First, in a model of acute allogeneic tumor rejection, CCR7-/- mice completely failed to reject subcutaneously injected MHC class I mismatched tumor cells. Cytotoxic activity of allo-specific T cells was severely compromised in CCR7-/- mice. Administration of allogeneic dendritic cells or reconstitution with in vitro primed effector T lymphocytes restored rejection of tumor cells in CCR7-/- mice. Next, we transplanted solid tumors bearing donor-type passenger leukocytes. CCR7-/- mice were capable of rejecting solid tumor allografts when transplants were derived from wild type mice, whereas allografts transplanted from CCR7-/- donors onto CCR7-/- recipients showed allograft survival up to 28 days. Second, in a heterotopic heart transplantation model CCR7 deficiency significantly prolonged allograft survival. Additional prolongation of graft survival was observed when hearts from CCR7-/- mice were used as donor organs. Our results define a key role for CCR7 in allogeneic T cells priming within the context of secondary lymphoid organs. However, CCR7 is not essential for direct priming of alloreactive immune responses in the allograft itself.

Immunology, University of Goettingen, Goettingen, 2IPF PharmaCeuticals, Hannover, Germany, and 3Institut for Research in Biomedicine, Bellinzona, Switzerland

D. 4 Human b-defensins-2 and -3 recruit macrophages and immature monocyte-derived dendritic cells in a SCID mouse model independent of CCR6

b-Defensins are antimicrobial peptides involved in host defense at epithelial surfaces. Some b-defensins recruit dendritic cells (DC) and lymphocytes via binding to the chemokine receptor CCR6. In the present study we investigated whether human bdefensins 2 and 3 (HBD-2 and -3) are chemotactic for immunocompetent cells not expressing CCR6. For this purpose, b-defensins were injected into the peritoneal cavity of SCID mice which at the same time received human monocytes or derivates (macrophages or monocyte-derived DC) i.v.. HBD-2 and -3 were unable to mobilize freshly isolated monocytes. However, monocyte-derived macrophages were recruited as early as 4 h after injection in a pertussis toxin-sensitive manner. IL-4 treatment down-regulated the responsiveness of macrophages to HBD-2 and HBD-3. Immature DC which were generated from monocytes in the presence of GMCSF and IL-4 migrated with delay as they were detectable 24 h but not 4 h after the i.p. injection of HBD-2 and HBD-3. Maturation left DC completely unresponsive to b-defensins. Monocyte-derived DC and macrophages were unresponsive to the CCR6 ligand CC chemokine ligand (CCL)20 but could be mobilized by the CCR4 ligand CCL22 similar to beta-defensins. However, in vitro chemotaxis and calcium mobilization experiments demonstrated that CCR4 did not function as a receptor for HBD-2 and -3. Our results identify macrophages as part of the innate leg of immunity to be in the first line of immune cells mobilized by b-defensins to the sites of infection and inflammation via specific Gai protein-coupled receptor(s) different from CCR4 and CCR6.

Experimental Pathology, Lund university, Lund, Sweden

D. 5 The reticular network of the lymph node as a delivery system for soluble antigens M. Sixt and L. Sorokin

The lymph node is the central organ of the immune system, where soluble mediators, antigens and immune cells that are carried by the lymph fluid meet with lymphocytes that are emigrating from the

42 ¥ 34th Annual Meeting of the German Society of Immunology

blood stream. The complex anatomy of the lymph node guarantees a highly organized and efficient interaction between these components, resulting in a controlled adaptive immune response. The non-hematopoietic backbone of the lymph node that maintains it's anatomy is a scaffold of fibroblastic reticular cells that are closely associated with the reticular fibres, a specialized extracellular matrix. Based on tracer studies, it has been hypothesized that the reticular fibres provide a transport system for lymphatic fluid that permits delivery of soluble molecules to their destination within the lymph node, without having to cross the lymphocyte-containing parenchyma. Using high resolution light microscopy and three dimensional reconstruction in combination with tracer studies, we have investigated the microanatomy and molecular basis of this ™conduit∫ system, which consists of a collagen core and a surrounding basement membrane which is again ensheeted by a layer of fibroblastic reticular cells. We found that occasional gaps in the fibroblast layer are occupied by resident dendritic cells which are able to take up soluble tracer within minutes after subcutaneous injection. The reticular fiber network is therefore a separated compartment of the lymph node that acts as a delivery system for small molecular weight substances like chemokines or soluble antigens. Via this infrastructure resident dendritic cells that are attached to the reticular fibres can be either activated or provided with soluble antigens immediately after a substance enters the afferent lymph. 1

Department of Immunology and 2 Department of Pathology, University of Gˆttingen, Gˆttingen, Germany

D. 6 A nonpeptide CCR5 antagonist inhibits colitis in SCID mice reconstituted with CD4+CD62L cells A. Bˆer1, P. Middel2, and M. Oppermann1

The migration of leukocytes into the intestinal mucosa is a multistep process mediated, in part, by chemokines and their receptors. The chemokine receptor CCR5 is preferentially expressed on monocytes and activated TH1 lymphocytes, which are likely to play key roles in the pathogenesis of murine colitis models and human Crohn's disease. Here, we investigated the effects of a synthetic CCR5 antagonist (TAK-779) in a CD4+CD62L T cell transfer model of colitis in SCID mice. As shown by the inhibition of RANTES-induced calcium mobilization and ERK1/2 activation in CCR5-expressing cells, TAK-779 is a functional antagonist for both human and mouse CCR5. Whereas untreated recipients reproducibly developed clinical signs of colitis within 3 wks after T cell

transfer and lost 17% of their initial body weight within the 8 wk observation period, weight loss (4% compared to initial values) and diarrhea scores (1.2  1.3 vs. 2.4  0.8 in untreated mice) were significantly (p<0.001) retarded and reduced in mice treated with daily injections of TAK-779. Histopathological and immunohistological examination revealed that TAK-779 treated recipients were partially protected from mucosal ulcerations, goblet cell depletion and mononuclear cell infiltration. Histological and clinical signs of colitis were also improved with delayed TAK-779 treatment which started 3 wks after T cell transfer. We conclude that CCR5 plays an important role in T cell recruitment to the intestine during the effector phase of colitis. 1

Department of Hematology,Oncology and Tumorimmunology, Department of Tumor Genetics and Immunogenetics, MaxDelbr¸ck-Center for Molecular Medicine, and 3 Forschungsinstitut for Molecular Pharmacology, Berlin, Germany 2

D. 7 Surface expression and endocytosis of the human cytomegalovirus-encoded chemokine receptor US28 is regulated by the GTPase dynamin J. Droese1, T. Mokros2, R. Hermosilla3, R. Sch¸lein3, M. Lipp2, B. Dˆrken1, U.E. Hˆpken2, and A. Rehm1

Infection with human cytomegalovirus causes a transient immunosuppression and additionally accelerates the risk for patients that already suffer from malignant or infectious diseases. It has been shown that immunsurveillance can be eluded by a number of HCMV gene functions that are targeted at both, the innate and adaptive immune response. Among those immunosubversive gene functions several were identified that interfere with chemokine receptors and their ligands. The HCMV encoded G-protein coupled receptor US28 binds inflammatory chemokines and sequesters them from the environment of infected cells. Here we analyze the mechanisms of rapid receptor endocytosis and ligand uptake. US28 internalizes independently of arrestin, but endocytosis is inhibited by a dominant negative mutant of the small GTPase dynamin. In general, dynamin controls two pathways, the clathrin coated pit pathway and the caveolae pathway. Although US28 partitions into a detergent resistant fraction, colocalization with the marker molecule caveolin was not found. Instead, internalization is mediated predominantly by a clathrin coated pit dependent pathway. To resolve the discrepancy between arrestin independent and clathrin dependent internalization, we currently explore structural motifs for the US28 endocytosis.

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c/o DRFZ, Charite, Experimental Rheumatology, AG Prof. Hamann and 2 c/o DRFZ, Charite, Experimental Rheumatology, Berlin, Germany

D. 8 Effect of antigen on the localization of murine CD4+ effector cells in inflamed skin S. Ghani1 and A. Hamann2

Adaptive immune responses lead to the generation of antigen-(Ag) specific effector cells. Accumulation of these Ag- specific T lymphocytes at the site of immune reaction is a well known phenomenon. However, the impact of the Ag for the recruitment of effector cells is poorly defined. Therefore we investigated the effect of localized antigen on the accumulation of CD4+ Th1 effector cells at the site of inflammation using a TCR- transgenic model. Skin inflammation was elicited by adoptive transfer of OVA- specific DO11.10 Th1 cells followed by subcutaneous injection of OVA peptide or protein in the presence of adjuvans. After transfer of radioactive or CFSE- labeled Th1 effector cells generated in vitro from Balb/c or DO11.10 mice, the accumulation of Ag- specific vs. non- specific cells into the site of inflammation was monitored. To our surprise the results show that Th1 effector cells accumulate within the inflammatory tissue independently of their antigen specificity, a process which is greatly enhanced by the presence of Agreactive ™pioneer∫ T cells. However, at late time points a strong enrichment of Ag- specific Th1 effector cells was observed which might be due to local proliferation or subsequent immigration of recently activated cells. In conclusion, in the initial phase recruitment of T cells is not antigen specific whereas in the late phase Ag- specific T cells dominate in the site of inflammation.

Department of Medicine, University of California, San Francisco, San Francisco, USA

D. 9 A novel mechanism for downregulation of sphingosine 1-phosphate G protein-coupled receptors mediates immunosuppression by FTY720 M.H. Gr‰ler and E.J. Goetzl

FTY720 is an immunosuppressant that reduces circulating levels of naÔve lymphocytes by both increasing homing to and decreasing exitation from secondary lymphoid organs (SLOs). Although originally proposed to be an agonist for sphingosine 1phosphate (S1P) G protein-coupled receptors (Rs) after phosphorylation, FTY720 now is shown to be a potent non-competitive inhibitor for types 1, 2,

and 5 S1P-Rs, without agonist activity. In rat hepatoma HTC4 transductants expressing single S1P-Rs, FTY720 blocks [32P]S1P binding, chemotaxis to S1P, and S1P-elicited increases in [Ca2+]i through S1P1,2,5-Rs but not through S1P3,4-Rs. It also blocks primary mouse splenocyte chemotaxis to S1P. Only 5 seconds of incubation with FTY720 at 37 8C but not at 4 8C is sufficient to induce FTY720 inhibitory effects on S1P1-Rs which peak earlier and last for up to 10 days at higher FTY720 concentrations. Confocal microscopy and flow cytometry reveal that dose-dependent FTY720inhibition of S1P-R signaling is induced by prolonged internalization and partial degradation of S1P1- but not S1P4-Rs. FTY720 thus is a potent noncompetitive inhibitor, which is selective for S1P1,2,5Rs. FTY720 induction of internalization and degradation of these S1P-Rs, without functional activation, reduces S1P suppression of lymphocyte chemotaxis, augments lymphocyte sequestration and diminishes T cell-dependent immunity. 1

Humorale Immunologie, Deutsches RheumaForschungszentrum, 2 Charite, Experimentelle Rheumatologie, and 3 Experimentelle Rheumatologie, Deutsches RheumaForschungszentrum, Berlin, Germany

D. 10 Chemotaxis of antibody-secreting cells A.E. Hauser1, G.F. Debes2, S. Arce1, G. Cassese1, A. Hamann3, A. Radbruch3, and R.A. Manz1

Plasma cell homing to the bone marrow is a prerequisite to develop persitent memory antibody titers. We analyzed the chemokine responsiveness of IgG-secreting plasma blasts formed in the spleen after secondary immunization with OVA. Starting from day 4 and within approximately 48 hours, OVA-specific plasma blasts emigrate from the spleen and appear in the bone marrow. These cells migrate toward CXCL12 (SDF-1a), and toward the inflammatory chemokines CXCL9 (MIG), CXCL10 (IP-10) and CXCL11 (I-TAC). The responsiveness of plasma blasts to these chemokines is restricted to a few days after their emigration from the spleen, indicating a role for these molecules and their cognate receptors, i.e. CXCR3 and CXCR4, in the regulation of plasma blast migration into the bone marrow and/or inflamed tissues. Mature bone marrow plasma cells do not migrate towards CXCL12 anymore, however, CXCL12 can promote the survivalof these cells. It is currently under investigation if the differences in the localisation of plasma cells generated in primary and secondary immune responses are due to an altered chemokine response of the IgG-ASC.

44 ¥ 34th Annual Meeting of the German Society of Immunology Institut f¸r Biochemie, Christian-Albrechts-Universit‰t, Kiel, Germany

D. 11 The transmembrane CXC-chemokine CXCL16 is expressed by cytokine-stimulated endothelial cells and fibroblasts and released by secretases of the disintegrin-like metalloproteinase family S. Abel, C. Hundhausen, S. Rose-John, and L. Andreas

The CXC-Chemokine CXCL16 is expressed as a transmembrane molecule on the surface of antigen presenting cells but is also released from the cells as a soluble chemoattractant for activated T cells. Here we demonstrate the expression of CXCL16 mRNA and the release of soluble protein by IFNg/ TNFa-stimulated endothelial cells and fibroblasts. By transfection of CXCL16 in COS-7 cells and ECV-304 cells we show that soluble CXCL16 is constitutively generated by proteolytic cleavage and that this cleavage results in reduced surface expression of the transmembrane molecule. Inhibition experiments with hydroxamate inhibitors suggest a role of the disintegrin-like metalloproteinase ADAM10 in the cleavage. In cytokine-stimulated embryonic fibroblasts generated from ADAM10 deficient mice constitutive cleavage of endogenous CXCL16 was markedly reduced and could be reconstituted by retransfection of ADAM10. Cleavage of transfected CXCL16 in COS-7 cells and murine fibroblasts was enhanced by cell stimulation with PMA. However, the PMA-induced cleavage was neither suppressed by inhibitors of ADAM10 nor modulated by the deletion or overexpression of ADAM10. Instead, PMA-inducible cleavage was completely blocked by inhibitors of TACE. These data demonstrate that ADAM10 is relevant for the majority of constitutive CXCL16 release from cytokine-stimulated cells and that the PMA-inducible cleavage could be mediated by TACE. Therefore strategies to block ADAM10 or TACE are likely to have an impact on the generation of soluble CXCL16 and its role as T cell chemoattractant.

Experimentelle Rheumatologie, Charitÿ, Berlin, Germany

D. 12 Differential regulation of P-selectin-ligand expression on naive and effector lymphocytes S. Jennrich, U. Syrbe, and A. Hamann

Expression of P-selectin-ligands (P-lig) is restricted to memory/effector lymphocytes displaying a specific homing capability to peripheral sites of inflammation, notably within the skin. Our previous data show that initial induction of Pselectin ligands on naÔve T cells depends on cell

cycle passage. This suggests that chromatin remodeling is involved in the regulation and might fix a particular homing phenotype directing effector cells back to the compartment of initial priming (topographical memory). If imprinting occurs primary induction should result in either constitutive expression on memory cells or cell cycle independent recall of expression. Indeed, activation of memory/effector cells in the presence of the cell cycle inhibitor L-Mimosine led to upregulation of P-lig on a significant fraction (30%) in addition to those cells expressing the ligand already after isolation ex vivo (10%). This indicates that most of the memory cells do not permanently express P-selectin ligands but show facilitated re-expression. We confirmed this in a further study monitoring purified P-lig positive cells (98%+) after adoptive transfer. The percentage of P-lig expressing cells rapidly declined over the first 7 days reaching about 30% of cells that remained positive for the observation period of 60 days. Moreover, preliminary data show that in vivo reinduction on transferred cells by Ag-specific challenge is more efficient than primary induction on naÔve T cells. How chromatin remodelling contributes to this peculiar type of regulation on memory/effector cells will be a major focus of our future work.

department of pharmakology, Clinic of the Johann-WolfgangGoethe University, Pharmazentrum Frankfurt, Frankfurt, Germany

D. 13 Specific binding of a vector derived RNA oligonucleotide to a GC-rich double-stranded MCP-1 promoter sequence and inhibition of chemokine gene expression K. Kautz, M. Schwarz, and H.H. Radeke

The potential of small-interfering RNA, antisense and triple-helix forming oligonucleotides (TFOs) to selectively inhibit expression of a target gene has been of great interest for gene therapy strategies. RNA oligonucleotides that bind homopurine/ homopyrimidine DNA sequences by forming triple-helical complexes (TFOs) can be rationally designed, but intracellular delivery of TFOs needs improvement. Extending our previous work here we describe an optimized method for TFO delivery. We constructed expression vectors generating RNA transcripts potentially capable of binding to the 19 bp GC-rich AP-1/SP1 site of the human MCP-1 promoter and thereby blocking MCP-1 transcription. A re-designed eukaryotic expression vector directed the transcription of either a 19 nt triplexforming CU-RNA sequence or two different 19 nt

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GU or CA-control sequences. These sequences are transcribed together with the RNA of a hygromycin resistance gene as one fusion transcript. HEK293 cells were stably transfected with the different vectors The presence of CU-TFO or control sequences inside the nucleus was proven by RTPCR analysing RNA from isolated nuclei. Specific binding of the 19 nt CU-RNA to the MCP-1 promoter duplex was verified in vitro by triplex blotting at pH 5.5 and 6.7. ELISA data showed a 58  13,1% inhibition of basal MCP-1 protein secretion in CU-TFO releasing cell lines in comparison to control cell lines and 76  10,2% inhibition of MCP-1 protein secretion in TNF-a stimulated CU-TFO releasing cell lines in comparison to control cell lines. Expression of IL-8 as a TNF-a inducible control gene was not affected by CUTFO. Thus, we demonstrated both highly specific and effective chemokine gene repression by triplex interference. Charite, HU-Berlin, Experimetal Rheumatology, 2 DRFZ, MPI for Infection Biology, 4 UKBF, FU-Berlin, Institut f¸r Infektionsmedizin, Berlin, 5 University W¸rzburg, Zentrum f¸r Infektionsforschung, W¸rzburg, Germany, 6 University of Rochester, Medical Centre, Centre for vaccine Biology and Immumology, Rochester, USA, and 7 Charite, HU-Berlin, Experimental Rheumatology, Berlin, Germany

homeostatic conditions, the percentage of selectin binding cells was dependent on the tissue harbouring the effector/memory cells. During infection, increased frequencies of P-selectin binding cells were found only among the dominating effector cell type, i.e. either Th1 or Th2 and only within the inflamed tissue, but not systemically. This suggests either a selective accumulation of these cells at the site of inflammation or a link between expression and recent activation. 1

Dept. of Neuroimmunology, Max-Planck-Institute of Neurobiology, Martinsried, 2 Dpt. of Neurology, LudwigMaximilians University, Munich, 3 Dpt. of Neurology, Philipps University, Marburg, 4 Institute for Clinical Neuroimmunology, Ludwig-Maximilians University, Munich, Germany, and 5 Dpt. of Neuroinflammation, Insitute of Neurology, London, UK

D. 15 Homeostatic B cell attracting chemokines in the normal brain and in multiple sclerosis (MS) lesions

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D. 14 Selectin ligands on in vivo generated murine CD4+ effector cells U. Kretschmer1, K. Bonhagen2, H.-W. Mittr¸cker3, G. Debes1, U. Syrbe1, O. Liesemfeld4, K. Erb5, D. Zaiss6, K. Thomas2, and A. Hamann7

Functional selectin ligands become induced upon activation and differentiation into effector/memory cells on a fraction of T cells. Binding of these ligands to endothelial selectins is an initial step for the recruitment of the cells into sites of inflammation. In vitro studies suggested an rather exclusive selectin ligand expression on Th1 effector/memory cells. The relevance for in vivo generated subsets, however, remained controversial. Therefore, we analysed the capacity to bind P-selectin among IFNg, IL-4 and IL-10 producing CD4+ T cells from noninfected mice and mice infected with influenza virus, Listeria monocytogene, Toxoplasma gondii (all Th1-models) or Nippostrongylus brasiliensis (Th2 model) to determine the relation between cytokine phenotype and expression of selectin ligands on T effector/memory cells in vivo. Our results demonstrate that in vivo functional selectin ligand expression is not restricted to Th1 effector cells (IFN-g +). Also IL-4-, and especially IL-10 ± producing cells showed a high frequency of selectin binding cells under appropriate conditions. Under

M. Krumbholz1, D. Theil2, S. Cepok3, B. Hemmer3, M. Hofbauer4, C. Farina1, T. Derfuss4, J. Newcombe5, R. Hohlfeld4, and E. Meinl1

Macrophages and T cells are by number the predominant cell types in the MS-lesions. However, two prominent immunological abnormalities regularly occurring in MS, an intrathecal Ig production and persistent oligoclonal bands, are based on B cells. This study examined B cell attracting chemokines in the brain in MS combining quantitative PCR and immunohistochemistry of frozen blocks of MSlesions with quantification of chemokines and immune cell subsets in the spinal fluid (CSF). Thereby we have identified four B cell attracting homeostatic chemokines, namely SDF-1a, MIP-3b, BCA-1, and MIP-3a that are overexpressed in MSlesions as compared to control brains. We then assessed the amount of B cells by the transcript levels of Iga and CD19. Considering all brain specimens the presence of B cells correlated significantly with BCA-1 and MIP-3b, but not MIP-3a. Histological analysis showed that MIP-3b and SDF1 were constitutively expressed in blood vessels in the parenchyma of normal noninflamed adult human brain. In MS-lesions, the overexpressed chemokines were localized at different positions suggesting a distinct role in extravasation and retention of the immune cells. CSF analysis revealed that BCA-1 correlated with the presence of B cells and plasma cells in this compartment in MSpatients. Since receptors for the analyzed chemokines are not only expressed on B-cells, this work has also implications for our understanding of the recruitment of T cells and myeloid cells to MSlesions.

46 ¥ 34th Annual Meeting of the German Society of Immunology Institut f¸r Biochemie, Christian-Albrechts-Universit‰t, Kiel, Germany

D. 16 The disintegrin-like metalloproteinase ADAM 10 is involved in constitutive fractalkine cleavage and regulates fractalkine-mediated cell-cell adhesion

1

Humoral Immunology, DRFZ, Berlin, 2 Research and Development, Miltenyi Biotec, Cologne, Germany, 3 Microbiology, University of Buffalo, Buffalo, USA, and 4 Cell Biology, DRFZ, Berlin, Germany

D. 17 Regulation of chemokine receptor expression during plasma cell differentiation

C. Hundhausen, S. Rose-John, and A. Ludwig

G. Muehlinghaus1, H. Leyendeckers2, S. Arce3, H. Mei1, A. Radbruch4, and R.A. Manz1

The CX3C-chemokine fractalkine (FKN) exists as a membrane-expressed protein promoting cell-cell adhesion and as a soluble molecule inducing chemotaxis. Transmembrane FKN is converted into its soluble form by defined proteolytic cleavage (shedding) which can be enhanced by stimulation with phorbol-12-N-myristate-13-acetate (PMA). PMA-induced FKN shedding has been shown to involve the TNFa-converting enzyme (TACE) whereas the constitutive cleavage in unstimulated cells remains elusive. Here we demonstrate a role of the closely related disintegrin-like metalloproteinase 10 (ADAM10) in the constitutive FKN cleavage. The hydroxamate GW28X, capable of blocking TACE as well as ADAM10, proved to be an effective inhibitor of the constitutive and the PMAinducible FKN cleavage in FKN-expressing ECV304 cells (FKN-ECV-304), whereas GI25X, preferentially blocking ADAM10 but not TACE, reduced the constitutive cleavage only. Over-expression of ADAM10 in Cos-7 cells enhanced constitutive cleavage of FKN. More importantly, in murine fibroblasts deficient of ADAM10 constitutive FKN cleavage was markedly reduced and could be reconstituted by stable retransfection of ADAM10. Thus, ADAM10 contributes to the constitutive shedding of FKN in unstimulated cells. Addressing the functional role of FKN shedding for cell adhesion via membrane-expressed FKN, we found that THP-1 cells adhere to FKNECV-304 cells but detach in the course of vigorous washing. Inhibition of ADAM10-mediated FKN shedding not only increased adhesive properties of FKN-ECV-304 cells but also prevented de-adhesion of bound THP-1 cells. Our data demonstrate that ADAM10 is involved in the constitutive cleavage of FKN and thereby may regulate the recruitment of monocytic cells to FKN-expressing cell layers.

The duration of specific antibody titers ranges from a few weeks up to several years, e.g. protective antibody titers against antigens like tetanus are found in sera of humans for decades. While the formation of antibody secreting plasma cells takes place in secondary lymphoid tissues, long-lasting antibody responses are provided by bone marrow plasma cells. The great majority of plasma cells that remain in secondary lymphoid tissues die within a few days. In contrast, plasma cells migrating into the bone marrow and possibly into chronically inflamed tissues can survive for much longer periods of time (estimated half-life of long-lived murine bone marrow plasma cells: 6 months). The capacity of these cells to migrate into these tissues is regulated by chemokine receptors. Expression of CXCR4 is important for plasma cell migration into the bone marrow. CXCR3 ligands are expressed in large quantities in inflamed tissues and may lead to plasma cell accumulation at those sites. Here we analyzed the regulation of chemokine receptor expression during plasma cell differentiation in culture. Purified PBMC were activated in a two-step culture system, for three days by CD40 ligand together with IL2 and IL-10 and for another five days by IL2 and IL-10 only. Addition of LPS or monocyte/T-cell culture supernatant to the initial culture, but not at late stages induced the expression of CXCR3 on plasma cells suggesting that the regulation of this receptor is an early event during plasma cell differentiation. In contrast, CXCR4 was present on all plasma cells cultured with the minimal stimuli resulting in plasma cell formation, i.e. CD40L, IL-2 and IL-10. Whether expression of CXCR4 is generally associated with differentiation into plasma cells remains to be determined.

Immunology, Indian Institute of Chemical Biology, Calcutta, India

D. 18 Mammalian chemokine receptor homologue in Leishmania donovani S. Roy

The protozoan parasite Leishmania donovani (LD) when exposed to a chemokine MIP1a, showed

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alteration in overall shape and size indicating a probable docking site of the chemokine on the parasites. The LD parasites could be stained with antibodies to the chemokine receptor, CCR5. The infection of macrophage with Leishmania parasites in vitro could be reduced to a great extent in the presence of antibodies to CCR5. Thus we tend to believe the presence of chemokine receptor or receptor like molecule in the parasites that may act as yet another receptor for entry to the host cells. Appropriate primers were designed from the two conserved regions of the mammalian chemokine receptor CCR5 gene and by RT-PCR; a single product could be detected from the parasite. Southern blot analysis of genomic DNA of LD either with RT-PCR product or human CCR5 DNA probe revealed that the putative chemokine receptor is a single copy gene. The promastigote genomic DNA library was screened with RT-PCR product and numbers of positive clones were obtained. Sequence of one of the representative clones showed 40% homology with human CCR5 and CCR1 at the nucleotide level. The above observation indicated the presence of chemokine receptor or receptor like molecule in the Leishmania parasite. The receptor appeared to be functional because intracellular Ca++ could be mobilized in response to human MIP1a, one of the ligands of human CCR5 and CCR1. There are about 22500 and 22000 receptor molecules for MIP-1a on the Leishmania donovani and Leishmania major respectively with KD of almost 5.9pM and almost 3.9pM respectively. 1

Immunpharmacology, J.W. Goethe University Frankfurt/Main, Pharmazentrum, Frankfurt am Main, 2FH Hamburg, Hamburg, 3 Frankfurt, 4Pharmazentrum Frankfurt, Clinic of the Goethe University, and 5 Immunpharmacology, Pharmazentrum Frankfurt, JW. Goethe University, Frankfurt am Main, Germany

D. 19 Development of chemokine receptor antagonists for combined chemokine receptor blockade in inflammatory disease models S. Rubant1, E. Werner2, M. Schwarz3, J.M. Pfeilschifter4, and H.H. Radeke5

Therapeutic studies blocking one single chemokine receptor only led to partial improvement of inflammatory diseases. In order to analyse whether combined blockade of several pro-inflammatory chemokine receptors simultaneously might be more effective, we cloned a set of three chemokine antagonists. As a test system for inflammatory chemokine receptors (CCR2, CCR5 and CXCR3) we defined the receptor profile of an antigen-specific, central memory Th1 clone (CCR7+, p-selectin+) by cDNAArray, RT-PCR and FACS analysis. As expected,

cDNA-array analysis showed that e.g. CCR7 mRNA was upregulated following stimulation of Th1 cells, while CCR2 was down regulated. Data of FACS analysis confirmed that unstimulated Th1 express CCR2 and CCR5 at a frequency of 74% and 62%, respectively. In parallel, we generated three recombinant murine chemokine antagonists, MCP-1(1-8)del, I-TAC(1-3)del and the viral broad spectrum antagonist vMIP-II in the eukaryotic Pichia pastoris. Proper structure and immune reactivity of these antagonists was proven by SDSPAGE, ELISA and Western Blot. Biological activity was confirmed in chemotaxis assays. All three antagonists blocked specific agonist-induced Th1 migration. Whereas MCP-1(1-8)del and vMIP-II reduced MCP-1 migration up to 60%, I-TAC(13)del inhibited IP-10 induced Th1 chemotaxis at 5fold molar excess by 80%. Using murine glomerular mesangial cells as a model of inflamed renal tissue we confirmed there inflammatory chemokine profile, e.g. MCP-1, MIP-1a, and IP-10, which in vivo specifically attracts monocytes and T cells. Thus, with both at hand now the inflammatory chemokine mixture and specific chemokine antagonists we are in the position to confirm our hypothesis in vitro and in vivo. (* supported by DFG-grant RA 525/7-3) 1

Experimentelle Rheumatologie, Charitÿ ± Universit‰tsmedizin Berlin, Berlin, 2 Institut f¸r Molekulare Medizin und Experimentelle Immunologie, Rheinische Friedrich-WilhelmsUniversit‰t, Bonn, 3 Abteilung Vaskul‰re Zellbiologie, MaxPlanck-Institut f¸r Vaskul‰re Biologie, M¸nster, and 4 Medizinische Klinik I, Charitÿ ± Universit‰tsmedizin Berlin, Berlin, Germany

D. 20 Liver sinusoidal endothelial cells (LSECs) promote transmigration of CD4+ T cell subsets A. Schrage1, F. Blumenthal-Barby1, P. Knolle2, B. Engelhardt3, A. Hamann1, and K. Klugewitz4

Data from liver transplantation studies and animal models suggest that the liver is involved in the modulation of CD8+ and also CD4+ T cells. In the present study we aimed to clarify how immune cells are trapped by the liver and migrate into the parenchyma. LSECs forming the hepatic sinusoidal barrier are good candidates to be involved in the process of trapping and transmigration of immune cells. To investigate the biological functions of LSECs, especially their influence to CD4+ T cells, we developed a MACS-based ex vivo separation technique for endothelial cells. Our experiments focused on the chemotaxis of ex vivo isolated T cells in the presence of LSECs in response to the

48 ¥ 34th Annual Meeting of the German Society of Immunology

ubiquitously chemokine SDF-1? as well as the proinflammatory chemokine MIG. Ex vivo isolated LSECs were used in chemotaxis assays to clarify their function in the transendothelial migration of various CD4+ T cell populations. LSECs themselves seemed to stimulate the transmigration of naive and CD45RBlow memory T cells even in the absence of chemokines. Furthermore, our data showed that LSECs support a more rapid transmigration of CD4+ T cells subsets in response to SDF-1? and MIG but not to other tested chemokines. In contrast, other endothelial cells rather reduced the transmigration of CD4+ T cells. By use of ICAM-1 knock out mice we were able to demonstrate that the chemotactic response to SDF-1? is not dependent on ICAM-1. By use of LSECs from CXCR3 knock out mice we could show that the increased transmigration of CD4+ T cells do not depend on direct effects of MIG to LSECs. Taken together, our data support the view that LSECs possess distinct functional properties with respect to trapping, transmigration and modulation of T cells. 1

Institute for Medical Microbiology and Hygiene, University of Freiburg, Freiburg and 2 Department of Nephrology, University of Kiel, Kiel, Germany

D. 21 Constitutive CCR7-transgene expression on Tcells: Impact on T-cell migration and LCMV clearance H. Unsˆld1, D. Voehringer1, S. Krautwald2, and H. Pircher1

To investigate the role of the CC chemokine receptor 7 (CCR7) on migration of activated CD8 T cells, transgenic (tg) mice constitutively expressing CCR7 on CD8 T cells were generated. After infection with lymphocytic choriomeningitis virus (LCMV), CD62L but not CCR7 was downregulated on CCR7-tg effector CD8 T cells. Nevertheless, in vivo clonal expansion after LCMV infection was not impaired by constitutive expression of CCR7 on CD8 T cells. Although CCR7 and its ligands CCL21 and CCL19 are active in inducing integrin-mediated arrest of lymphocytes on HEVs, constitutive expression of CCR7 failed to promote migration of CCR7+ effector T cells to lymph nodes. However, constitutive expression of CCR7 led to accumulation of CD8 effector cells in T cell zones in the spleen whereas CCR7- effector T cells failed to enter white pulp areas and localized mainly in the red pulp. This different localization pattern was still observed in the memory phase when both CCR7-tg and wild type (wt) T cells were stained positive for CCR7. This may be due to a 2-fold increased CCR7 expression on CCR7-tg memory T cells compared to memory T cells from wt mice.

Finally, our data show that constitutive CCR7 expression on effector T cells results in the predominant localization of activated T cells in the splenic white pulp associated with an impaired early LCMV clearance when compared to CCR7- wt effector T cells that were present in marginal zone and red pulp. 1

Dept. of Tumor Genetics and Immunogenetics, Max-Delbr¸ckCenter, MDC, Berlin and 2 Institute of Pathology, FriedrichSchiller University, Jena, Jena, Germany

D. 22 Role of chemokine receptor CCR7 in the initiation and progression of antigen-induced arthritis (AIA) A. Wengner1, U. Hˆpken1, P.K. Petrow2, R. Br‰uer2, and M. Lipp1

Chronic inflammation is associated with the accumulation of large numbers of mononuclear cells at the site of inflammation. Leukocyte recruitment is mediated by inflammatory chemokines but there is now considerable evidence for the involvement of homeostatic chemokines such as CXCL13 and CCL21. The corresponding chemokine receptors CXCR5 and CCR7 are principal regulators for targeting T cells, B cells, and dendritic cells into secondary lymphoid organs and their compartmentalization into functionally separated T and B cell zones. In addition, CCR7 and CXCR5 have been identified as useful markers in the classification of functional distinct subsets of memory and effector T cells. In order to study the role of CCR7 on lymphocyte infiltration under chronic inflammatory conditions the initiation and progression of antigen-induced arthritis (AIA) was analyzed after intra-articular injection of mBSA (methylated bovine serum albumin) into the knee joints of preimmunized wild type and CCR7-deficient mice. The course of arthritis was evaluated by means of joint swelling and histological evaluation of cartilage degradation and infiltration of T and B cells and macrophages. Monitoring was carried out at day 3 during the acute inflammatory phase and at day 21 during the early chronic phase. Interestingly, joint swelling and inflammatory infiltrates of lymphocytes were significantly reduced in CCR7deficient mice suggesting a regulatory role of CCR7 in the acute and early chronic phase of AIA. Currently, CXCR5-deficient and CCR7/ CXCR5 double-deficient mice are used in the AIA model in order to prove our hypothesis, that the homeostatic chemokine receptors CCR7 and CXCR5 are critical in the maintenance of the late phase of chronic inflammatory diseases.

34th Annual Meeting of the German Society of Immunology ¥ 49 Abt. Pneumologie, Medizinische Klinik und Poliklinik Freiburg, Freiburg, BR Deutschland

D. 23 CXCR3-agonistic chemokines in interstitial lung diseases D. Vlachakis, J. M¸ller-Quernheim, and G. Zissel

The chemokines MIG (CXCL9), IP-10 (CXCL10), and ITAC (CXCL11) bind to the chemokine receptor CXCR3 mainly expressed on activated Th1 T cells. Therefore it is hypothesised that these chemokines may be important in the recruitment of activated T cells. A feature of interstitial lung diseases like pulmonary sarcoidosis (SAR) and exogen-allergic alveolitis (EAA) is the accumulation of activated T cells in the lung as reflected by an increase in T cells in bronchoalveolar lavage (BAL). We therefore analysed the release of CXCL9, CXCL10, and CXCL11 by BAL cells from patients with SAR, EAA, usual interstitial pneumonia (UIP), tuberculosis (TB) and controls and correlated the amount of the released chemokines with BAL cytology. IP-10 and MIG was released even by BAL cells from controls. IP-10 release was increased in SAR and EAA. MIG release was increased in EAA, SAR, and UIP. Although ITAC release was very low, it was significantly increased in all four patient groups. Simple regression analysis revealed different correlations of the chemokines analysed with various BAL parameters. However, employing multiple regression analysis a positive correlation of the lymphocyte percentage with IP-10 could be found in UIP. In SAR percentage of CD4+ lymphocytes correlated positively with IP-10 release but negatively with ITAC release. In EAA ITAC and the percentage of CD4+ cells disclosed a positive correlation. No synergistic activity of IP-10, MIG and ITAC in attracting T cells could be established. Our data demonstrate that the importance of CXCR3-agonistic chemokines varies in different interstitial lung diseases and may act independently. The negative correlations of ITAC with various BAL parameters imply the hypothesis that at least in the diseases investigated ITAC is may inhibit lymphocyte accumulation.

1

Immunology, Max-Planck-Institute for Infection Biology and Department for Molecular Tumor and Immunogenetics, MaxDelbr¸ck-Center for Molecular Medicine, Berlin, Germany

2

D. 24 Differential requirement for the chemokine receptor CCR7 for the induction of T cell responses during infection with intracellular bacteria M. Kursar1, U.E. Hˆpken2, A. Kˆhler1, M. Lipp2, S.H.E. Kaufmann1, and H.-W. Mittr¸cker1

For effective priming of T cell responses, naive T cells have to come into intimate contact with professional antigen presenting cells. This contact is the consequence of highly coordinated migration processes, in which the chemokine receptor CCR7 and its ligands CCL19 and CCL21 appear to play a central role. Here we analyze CCR7-deficient mice using the L. monocytogenes infection model with two major purposes. Firstly, we attempt to evaluate the concept of a crucial role of the CCR7/CCR7ligand system for T cell priming during infection. Secondly, we investigate differences in the activation requirements of listeria-specific T cell subsets. We demonstrate that naive MHC class Ia-restricted CD8+ T cells are highly dependent on CCR7 for efficient priming. Surprisingly, MHC class Ib and MHC class II-restricted T cells appear to be less dependent on CCR7 and memory T cell responses are completely independent. Transfer experiments indicate that CCR7 expression on MHC class Ia restricted CD8+ T cells alone is insufficient. CCR7 has to be present on additional cells to promote efficient priming. In this context, we currently analyze the role of CCR7 for the migration of dendritic cells during L. monocytogenes infection. In summary, our results demonstrate differential requirements of CCR7 for T cell activation depending on the T cell subpopulation and the state of the T cell response.