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Workshop T: Mucosal Immunity
Workshop T Mucosal Immunity
1
Mucosal Immunity Group, German Research Center for Biotechnology (GBF), Braunschweig and 2Department of Pathology, School of Veterinary Medicine, Hannover, Germany
T. 1 Mucosal antigen expression leads to lung-specific autoimmunity in a transgenic mouse model D. BRUDER1, A.M. WESTENDORF1, A.D. GRUBER2, and J. BUER1 Reactivity to a lung-specific antigen was studied by transgenic expression of hemagglutinin (HA) under the transcriptional control of the surfactant protein C (SP-C) promoter. HA was found to be expressed in type II alveolar epithelial cells, as well as in the thymus of all founder lines. Transfer of HA-specific CD4 T cells led to a strong perivascular infiltration and inflammation of the lung. Moreover, concomitant expression of HA and a class II restricted transgenic TCR specific for the HA in double transgenic TCR-HA¥SPC-HA mice led to the development of a severe immune-mediated lung disease. Characterization of thymocytes from these mice revealed that HA-specific T cells escaped from thymic deletion and migrate into the periphery. These mature peripheral CD4 T cells showed an activated phenotype and a normal proliferative response to HA. Histological examination of the lungs indicated progressive interstitial pneumonitis characterized initially by lymphocytic and plasmacytic infiltration of interalveolar septa, especially around bronchioles and venules, followed by an exuberant mononuclear cell infiltration that correlated with restrictive pulmonary function. Therefore we conclude that due to inefficient thymic deletion and the obvious lack of peripheral tolerance induction towards HA double transgenic animals developed severe lung-specific autoimmunity. The phenotype of inflammation strongly resembles clinical symptoms observed in virus induced pneumonitis and tissue rejection after lung transplantation and may have profound implications concerning the cellular and molecular characterization of mucosa– T cell interactions in immune-mediated lung diseases.
1
Medizinische Klinik I, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Berlin, Germany and 2National Institute of Allergy and Infectious Diseases, NIH, Bethesda MD, USA
T. 2 Epithelial IL-10 production is essential for mucosal tolerance F. HELLER1, W. STROBER2, and M. ZEITZ1 IL-10 is a pleiotropic cytokine with immuno-suppressive properties. IL-10 knockout mice develop a chronic enterocolitis that is driven by bacterial antigens in the gut flora and depends on CD4 T lymphocytes. These lymphocytes are tolerized in wildtype mice. However, IL-10 is produced by various cells: lymphocytes, antigen-presenting cells and epithelial cells. In the presented experiments IL-10 was selectively knocked-out in epithelial cells.
298 · 33rd Annual Meeting of the German Society of Immunology Epithelial-IL-10 knockout mice (IL-10EKO) were generated by transplanting bone marrow from IL-10+/+ wildtype mice into IL-10–/– mice after myeloablative irradiation. In IL-10EKO all hematopoietic cells were donor-derived IL-10+ cells while the epithelial cells remained IL-10–. Oral tolerance was induced by feeding ovalbumin. Mice were rechallenged with antigen in the ear lobe, and ear swelling was measured to demonstrate systemic unresponsiveness. Dendritic cells (DC) and lymphocytes were isolated from mesenteric lymph nodes (MLN) and spleen (SP) and stimulated with LPS or antigen. IL-10 production from total colon culture was measured by RT-PCR and ELISA. IL-10 production from wildtype colons was 50-fold higher than from IL-10EKO. IL-10 mRNA from IL-10EKO was barely detectable. DC from IL-10EKO produced 2–5 fold higher concentrations of IL-12, TNF-a and IL-10. While feeding antigen induced tolerance in WT, IL-10EKO reacted with increased DTH to antigen rechallenge. T cells from WT showed deletion and reacted anergic to antigen stimulation, but cells from IL-10EKO exhibited increased proliferation and expansion and produced increased amounts of inflammatory cytokines. Epithelial cells are the major source of IL-10 in the mucosal immune system. Without epithelial cell derived IL-10 the immune response to luminal antigens is skewed to an inflammatory response. Epithelial IL-10 has a down modulatory effect on local DCs. While luminal antigens induce anergy and deletion of lymphocytes specific for such antigens the lack of epithelial-IL-10 allows for activation and expansion of T cells. As a result systemic tolerance to gut antigens is not achieved.
Institut für Immunologie, Ruprecht-Karls-Universität, Heidelberg, Germany
T. 3 Physiological expression of heme oxygenase 1 in enterocytes and macrophages of the human colon J. KOCH, P.O. BERBERAT, T. GIESE, and B.M. SCHÄFER Introduction: The stress responsive gene HO-1 plays a cytoprotective role in a variety of inflammatory diseases. The anti inflammatory effect of HO-1 is presumably mediated by its enzymatic products biliverdin/bilirubin, iron/ferritin and carbon monoxide. In most of the tissues inflammatory stimuli lead to a rapid upregulation of HO-1. In the intestine however controversial data were reported: whereas pouchitis in humans did not show any induction of HO-1, HO-1 is rapidly and significantly upregulated in the colon of mice suffering from experimentally induced colitis. To clarify the physiological role of HO-1 in the human colon we performed immunohistology in combination with rtPCR on normal human colon tissue, Westernblot analysis of isolated lamina propria cells and enterocytes. Methods: Immunohistochemistry, rtPCR, Western blot. Results: Immunohistology revealed expression of HO-1 protein in the intestinal epithelium, i.e. mainly in the luminal enterocytes (enterocytes in crypts were found to be HO-1 negative), and in some lamina propria cells. Immunophenotyping of HO-1 positive lamina propria cells showed a predominance of macrophages. The expression of HO-1 was confirmed on mRNA(rtPCR) level and protein level via westernblotting of isolated lamina propria cells and enterocytes. Conclusions: In contrast to other tissues HO-1 is constitutively expressed at high level in the non-inflammed human colon predominantly in enterocytes and macrophages. Since HO-1 is
33rd Annual Meeting of the German Society of Immunology · 299 upregulated upon stress in other tissues these findings suggest its upregulation by local luminal factors in the gut such as e.g. endotoxins. The resultant HO-1 produced at this site might act as an endogeneous protection system to control the so called physiological inflammation in the human gut.
Department of Virology, Institute of Medical Microbiology and Hygiene, Homburg, Germany
a4b7+) Th1 T. 4 Inactivated poliovirus vaccination induces a gut homing (a memory response in orally poliovirus pre-vaccinated volunteers C. KRIEG, T. HEINTEL, R. MAIER, and A. MEYERHANS The mucous membrane is the main interface between the human body and the surrounding environment. Upon contact of this surface with a pathogen a pool of gut homing lymphocytes (a4b7+) is activated which is subsequently detectable in the blood. The effect of booster immunization either intramuscularly with inactivated poliovirus vaccine (IPV) or orally with attenuated poliovirus vaccine (OPV) in pre-immunised volunteers on this pool of gut derived cells was investigated. Cellular immune response was tested in the proliferation assay and with FACS analysis of whole blood. Furthermore poliovirus neutralising antibody response was determined. After oral poliovirus booster immunization of three volunteers only a marginal T cell response and a small increase of neutralising antibodies was detected. In contrast the immune response after intramuscular poliovirus booster immunization in 4 volunteers at day 7 shows: a) an increase of neutralising antibody titre, b) poliovirus-specific lymphocyte proliferation and c) an increase of a poliovirus-specific CD4+ T cell response. Surprisingly the further characterization of these cells revealed that they were Th1 lymphocytes (IL-4–, IFN-g+ and IL-2+). The cell differentiation type is: CCR7–, CD45R0+, CD45RA–, CD27+. The examination of homing receptors showed that they were positive for the gut homing receptor a4b7 and negative for the lymph node homing receptor CD62L (L-selectin). Our data indicate that the observed Th1 response after intramuscular poliovirus booster immunization of orally pre-vaccinated volunteers is recruited from a poliovirus-specific gut (a4b7+) memory pool. This could be essential for future mucous prime and intramuscular booster immunization strategies, e.g. future HIV vaccination strategies.
Abteilung Immunologie, Max-Planck-Institut für Infektionsbiologie, Berlin, Germany
T. 5 Expression of a non-pairing TCR a chain leads to elevated levels of a–b+ T cells and exaggerates chronically activated CD4+ TCRa inflammatory bowel disease I. PRINZ, U. KLEMM, S.H.E. KAUFMANN, and U. STEINHOFF The precise etiology of inflammatory bowel disease (IBD) is incompletely understood, nevertheless CD4+ T cells have been identified as pathogenic T cells in colitis. In TCR a–/– mice an unu-
300 · 33rd Annual Meeting of the German Society of Immunology sual CD4+TCRa–b+ T cell population is associated with the development of IBD resembling human ulcerative colitis (UC). However, the significance of such CD4+TCRa–b+ T cells in immunocompetent individuals remains unclear. Using a transgenic approach, an in-frame rearranged but non-pairing TCRa chain was expressed in the T cells of TCRa–/– mice. Although not detected on the cell surface, this TCRa chain stabilized newly synthesized TCRb chains, likely acting as a «private molecular chaperone». This lead to increased frequencies of CD4+TCRa–b+ T cells and exaggerated the course of IBD in TCRa–/– mice expressing the transgenic TCRa chain. Additional evidence that this unconventional T cell population is the causative agent of IBD is provided by the finding that adoptive transfer of these cells into TCRb–/– mice induced colitis in the recipients. Furthermore, we show that the CD4+TCRa–b+ T cells are chronically activated both in normal and transgenic TCRa–/– mice with regard to expression of the cell surface markers CD44, CD62L, and CD69. Thus, regular TCRa chain rearrangement can promote the formation of readily activated colitogenic CD4+TCRa–b+ T cells. We conclude that these T cells are present under physiological conditions and play a role in the etiology of UC.
Institute of Medical Microbiology, Immunology and Hygiene, Technical University, Munich, Germany
T. 6 Lymphocytes are essential for the development of chronic enterocolitis in mice with a myeloid cell-specific Stat3 deficiency W. REINDL and I. FÖRSTER Stat3 is the key signal transducer downstream of cytokine receptors signaling through gp130. Using the Cre loxP recombinase system, mice with a cell type specific disruption of the Stat3 gene in macrophages and neutrophils (lysozymeM (LysM)cre/Stat3flox/–) were generated (Takeda et al. Immunity 10:39–49). Analysis of these mice demonstrated that Stat3 is indispensable for IL-10 signaling in macrophages and neutrophils. This signaling defect causes the spontaneous, chronic enterocolitis and impaired weight gain observed in LysMcre/Stat3flox/– mice. The hyperreactivity of myeloid cells is the reason for the high susceptibility to endotoxin shock seen in these animals. To elucidate the significance of lymphocytes in the pathogenesis of colitis and endotoxin shock we crossed the conditional knockout mice to a RAG deficient background (LysMcre/ Stat3flox/–¥RAG–/–). Peritoneal macrophages isolated from LysMcre/Stat3flox/– and LysMcre/ Stat3flox/–¥RAG–/– showed identical levels of IFN-g and NO production after activation with LPS. Astonishingly, histology displayed no signs of colitis in LysMcre/Stat3flox/–¥RAG–/– mice and their increase in body weight was similar to littermate controls. Despite the similar potential of myeloid cells to produce proinflammatory mediators, RAG–/–¥LysMcre/Stat3flox/– mice showed enhanced resistance to LPS challenge compared to LysMcre/Stat3flox/– mice. Nevertheless LysMcre/Stat3flox/–¥RAG–/– mice were far more susceptible to endotoxin than wildtype controls. These results demonstrate, that despite the fact that myeloid cells predominate in fully developed lesions of LysMcre/Stat3flox/– mice, T and/or B lymphocytes play a key role in the initiation and perpetuation of colitis.
33rd Annual Meeting of the German Society of Immunology · 301 Institutes of 1Immunology and 2Medical Microbiology, Johannes Gutenberg-University, Mainz, Germany
T. 7 The streptococcal virulence factor streptolysin O activates mast cells to a by p38 MAP-kinase- and protein kinase C-dependent produce TNF-a pathways C. RICHTER1, S. BHAKDI2, T. BOPP1, C.H.M. MÜLLER1, C. NEUDÖRFL1, M. STASSEN1, I. WALEV2, and E. SCHMITT1 Streptolysin O (SLO), a major virulence factor of pyogenic streptococci, binds to cholesterol in membranes of eukaryotic cells and oligomerizes to form large transmembrane pores. While high toxin doses are rapidly cytocidal, low doses are tolerated because a limited number of lesions can be resealed. Here, we report that, at sublethal doses, SLO activates primary murine bone marrow-derived mast cells (BMMC) to degranulate and to rapidly induce or enhance the production of several cytokine mRNAs including TNF-a. Mast cell-derived TNF-a plays an important protective role in murine models of acute inflammation, and the production of this cytokine was analysed in more detail. Release of biologically active TNF-a peaked at about 4 h after stimulation with SLO. Production was blunted upon depletion of protein kinase C by pretreatment of the cells with PMA. Furthermore, transient permeabilization led to activation of stress-activated protein kinases p38 MAP-kinase and JNK, and inhibition of p38 MAP-kinase markedly reduced production of TNF-a. Transcriptional activation of mast cells following transient permeabilization might contribute to host defence against infections via the beneficial effects of TNF-a. However, SLO-mediated stimulation of mast cells might also promote the development of toxic streptococcal syndromes.
Medizinische Klinik I, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Berlin, Germany
T. 8 Effects of highly active antiretroviral therapy on the gut mucosal immune system in HIV-infected patients H. SCHULBIN, H. BODE, T. ZIPPEL, W. SCHMIDT, M. ZEITZ, and R. ULLRICH Introduction: The effect of highly active antiretroviral therapy (HAART) on intestinal immune function, which is the major site of virus replication and CD4+ T cells depletion in patients infected with the human immunodeficiency virus (HIV), is unknown. We therefore investigated cytokine mRNA expression in intestinal biopsies from HIV-infected patients receiving HAART. Methods: We established a quantitative real-time PCR using a LightCycler to measure mRNA-expression of: IL-2, IL-4, IL-6, IL-10, IL-16, IFN-g, TNF-a, RANTES, MIP-1-a, and the housekeeping-gene GAPDH. Rectal biopsies were obtained from 7 controls and 11 HIVinfected patients before and 4 months after initiation of HAART. HIV-RNA was measured using the Amplicor-Kit. Mucosal CD4+ T cells were determined by cytofluorometry after isolation of immune cells from biopsies.
302 · 33rd Annual Meeting of the German Society of Immunology Results: Mucosal HIV load decreased by about 2 logs and the CD4+ T cell count increased by about 1–2 logs after Cytokine mRNA-expression in HIV-infected patients was frequently higher than in controls independent of HAART.The mucosal IL-4 and IL-10 mRNA expression was higher in HIV-infected patients than in controls during the whole examination period. The mucosal RANTES mRNA expression was higher in HIV-infected patients before HAART than in controls. The mucosal mRNA expression of IL-6 and TNF-a was higher in HIV-infected patients after four months of HAART than in controls. HAART. Conclusions: The high levels of IL-4, IL-10, IFN-g, RANTES, IL-6 and TNF-a, mRNA in mucosal biopsies from HIV-infected patients receiving HAART for 4 months indicates ongoing mucosal immune activation in spite of effective inhibition of viral replication.
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Medizinische Klinik I, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Berlin, 2Department of Pathology, Johannes Gutenberg-University, Mainz, Germany, and 3 Department of Medicine, University of Colorado, Denver CO, USA
T. 9 A critical role for leptin in different experimental models of colitis B. SIEGMUND1, H.A. LEHR2, C.A. DINARELLO3, M. ZEITZ1, and G. FANTUZZI3 Leptin, the product of the ob gene, regulates the balance of Th1/Th2 cytokines and modifies T cell immunity. Leptin-deficient ob/ob as well as leptin receptor (Ob-Rb)-deficient db/db mice are resistant against acute and chronic DSS-induced colitis, as evaluated by body weight, diarrhea, bleeding, histology, induction of proinflammatory cytokines, STAT-3 activation in the colon and rate of apoptosis in lamina propria lymphocytes (LPL). Similar resistance could be observed in the model of TNBS-induced colitis, in which reduced cytokine production and T cell activation associated with an increase of apoptosis in LPL was observed in ob/ob mice. In vitro studies revealed that leptin can directly induce STAT-3 activation in LPL and intraepithelial lymphocytes. To evaluate the role of T cells, we compared the effect of transferring CD4+CD45RBhigh cells from WT and db/db mice into scid recipients. These experiments indicate that colitis induction by CD4+CD45RBhigh cells from db/db mice is significantly reduced when compared to the transfer of WT cells, suggesting that leptin receptor expression on T cells is critical for development of intestinal inflammation. These findings are indicating for the first time in vivo, that the direct peripheral effect of leptin is crucial for T cell activation and subsequent inflammation. In conclusion, these results demonstrate that leptin represents a link between the endocrine and the immune system, which requires further investigations in experimental models and human IBD. Supported by a grant from the Deutsche Forschungsgemeinschaft DFG SI 749/2–1 and the E. and E. L. Broad Foundation
33rd Annual Meeting of the German Society of Immunology · 303 Abteilungen 1Tumorimmunologie, Institut für Pathologie and 2Innere Medizin I, Universitätsklinikum Regensburg, Regensburg, Germany
b receptor activation is critical in colitis T. 10 Lymphotoxin-b P. STOPFER1, F. OBERMEIER2, W. FALK2, D.N. MÄNNEL1, and T. HEHLGANS1 The Lymphotoxin-b receptor (LTbR) is a member of the TNF receptor family. So far two functional ligands have been identified which activate the LTbR the cell surface-bound heterotrimeric LTa1b2 complex and LIGHT, a membrane-anchored homotrimeric complex. The LTbR signaling pathway is involved in both the development of secondary lymphoid organs and the maintenance of organized lymphoid tissues. In order to investigate the role of LTbR in intestinal inflammation, LTbR activation was inhibited in a mouse model for acute or chronic colitis induced by dextran sulfate (DSS). As functional inhibitor of LTbR activation a fusion protein consisting of the extracellular domain of the murine LTbR and the Fc part of human IgG1 (LTbR:Ig), was used. The inhibitory activity of LTbR:Ig in vivo was confirmed by the transient dedifferentiation of the splenic follicular dendritic cell network after a single injection of LTbR:Ig. In the acute form of DSS-induced colitis, treatment with LTbR:Ig significantly aggravated the disease indicating a protective role of LTbR activation in this experimental model. However, treatment of the chronic form of DSS-induced colitis with LTbR:Ig significantly ameliorated the development and histological manifestations of the disease. Semiquantitative PCR analysis demonstrated strong expression of the ligand LTb in mice with chronic colitis compared to mice with acute or without colitis. When LTa1b2/LIGHT expression on lymphocytes from mesenterial lymphnodes was investigated, lymphocytes from mice treated with LTbR:Ig showed decreased LTa1b2/LIGHT expression after PMA stimulation. LTbR:Ig treatment seems to interfere with T cell activation, thus disturbing T cell mediated functions in this experimental model. Taken together these results demonstrate that activation of the LTbR pathway is critically involved in both the acute and the chronic form of colitis.
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Institute of Immunology and Transfusion Medicine, Ernst-Moritz-Arndt-University, Greifswald and 2Institute of Bacteriology and Mycology, University of Leipzig, Leipzig, Germany
T. 11 A role for lipopolysaccharide-binding protein in local innate immunity of the intestine? M. VOGLER1, D. SCHRÖDER1, W. SCHRÖDL2, N. HABERLAND2, B. FÜRLL1, R.S. JACK1, B. AHRENS1, M. KRÜGER2, and C. SCHÜTT1 Lipopolysaccharide-binding protein (LBP) is a hepatocyte derived acute phase protein which catalyses the binding of LPS to CD14 and TLR4 on macrophages and thus leads to cellular activation. Extrahepatic expression of LBP has been described for lung, kidney and epithelial cell lines. In this study we asked whether or not LBP plays a role in the normal barrier function of the intestinal mucosa. As the gut histology of LBP-deficient mice looks regular and the animals
304 · 33rd Annual Meeting of the German Society of Immunology are healthy LBP does not seem to have an essential function in preventing translocation of endotoxin or bacteria. Using in situ hybridisation we find a constitutive expression of LBP-mRNA which is restricted to the bottom of the crypts in the small intestine, where the Paneth cells are located. We were not able to detect LBP protein in the lumen of the gut, but LBP was found in the mucus of the small intestine. Interestingly, mice deficient for LBP show a significantly reduced number of Gram-negative bacteria in the microflora as compared to wild type mice. These preliminary results illustrate a possible role for LBP in influencing mucus-resident commensal bacteria.
1
Mucosal Immunity Group, German Research Center for Biotechnology (GBF), Braunschweig and 2Department of Pathology, School of Veterinary Medicine, Hannover, Germany
T. 1 Mucosal antigen expression leads to lung-specific autoimmunity in a transgenic mouse model D. BRUDER1, A.M. WESTENDORF1, A.D. GRUBER2, and J. BUER1 Reactivity to a lung-specific antigen was studied by transgenic expression of hemagglutinin (HA) under the transcriptional control of the surfactant protein C (SP-C) promoter. HA was found to be expressed in type II alveolar epithelial cells, as well as in the thymus of all founder lines. Transfer of HA-specific CD4 T cells led to a strong perivascular infiltration and inflammation of the lung. Moreover, concomitant expression of HA and a class II restricted transgenic TCR specific for the HA in double transgenic TCR-HA¥SPC-HA mice led to the development of a severe immune-mediated lung disease. Characterization of thymocytes from these mice revealed that HA-specific T cells escaped from thymic deletion and migrate into the periphery. These mature peripheral CD4 T cells showed an activated phenotype and a normal proliferative response to HA. Histological examination of the lungs indicated progressive interstitial pneumonitis characterized initially by lymphocytic and plasmacytic infiltration of interalveolar septa, especially around bronchioles and venules, followed by an exuberant mononuclear cell infiltration that correlated with restrictive pulmonary function. Therefore we conclude that due to inefficient thymic deletion and the obvious lack of peripheral tolerance induction towards HA double transgenic animals developed severe lung-specific autoimmunity. The phenotype of inflammation strongly resembles clinical symptoms observed in virus induced pneumonitis and tissue rejection after lung transplantation and may have profound implications concerning the cellular and molecular characterization of mucosa– T cell interactions in immune-mediated lung diseases.
1
Medizinische Klinik I, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Berlin, Germany and 2National Institute of Allergy and Infectious Diseases, NIH, Bethesda MD, USA
T. 2 Epithelial IL-10 production is essential for mucosal tolerance F. HELLER1, W. STROBER2, and M. ZEITZ1 IL-10 is a pleiotropic cytokine with immuno-suppressive properties. IL-10 knockout mice develop a chronic enterocolitis that is driven by bacterial antigens in the gut flora and depends on CD4 T lymphocytes. These lymphocytes are tolerized in wildtype mice. However, IL-10 is produced by various cells: lymphocytes, antigen-presenting cells and epithelial cells. In the presented experiments IL-10 was selectively knocked-out in epithelial cells.
298 · 33rd Annual Meeting of the German Society of Immunology Epithelial-IL-10 knockout mice (IL-10EKO) were generated by transplanting bone marrow from IL-10+/+ wildtype mice into IL-10–/– mice after myeloablative irradiation. In IL-10EKO all hematopoietic cells were donor-derived IL-10+ cells while the epithelial cells remained IL-10–. Oral tolerance was induced by feeding ovalbumin. Mice were rechallenged with antigen in the ear lobe, and ear swelling was measured to demonstrate systemic unresponsiveness. Dendritic cells (DC) and lymphocytes were isolated from mesenteric lymph nodes (MLN) and spleen (SP) and stimulated with LPS or antigen. IL-10 production from total colon culture was measured by RT-PCR and ELISA. IL-10 production from wildtype colons was 50-fold higher than from IL-10EKO. IL-10 mRNA from IL-10EKO was barely detectable. DC from IL-10EKO produced 2–5 fold higher concentrations of IL-12, TNF-a and IL-10. While feeding antigen induced tolerance in WT, IL-10EKO reacted with increased DTH to antigen rechallenge. T cells from WT showed deletion and reacted anergic to antigen stimulation, but cells from IL-10EKO exhibited increased proliferation and expansion and produced increased amounts of inflammatory cytokines. Epithelial cells are the major source of IL-10 in the mucosal immune system. Without epithelial cell derived IL-10 the immune response to luminal antigens is skewed to an inflammatory response. Epithelial IL-10 has a down modulatory effect on local DCs. While luminal antigens induce anergy and deletion of lymphocytes specific for such antigens the lack of epithelial-IL-10 allows for activation and expansion of T cells. As a result systemic tolerance to gut antigens is not achieved.
Institut für Immunologie, Ruprecht-Karls-Universität, Heidelberg, Germany
T. 3 Physiological expression of heme oxygenase 1 in enterocytes and macrophages of the human colon J. KOCH, P.O. BERBERAT, T. GIESE, and B.M. SCHÄFER Introduction: The stress responsive gene HO-1 plays a cytoprotective role in a variety of inflammatory diseases. The anti inflammatory effect of HO-1 is presumably mediated by its enzymatic products biliverdin/bilirubin, iron/ferritin and carbon monoxide. In most of the tissues inflammatory stimuli lead to a rapid upregulation of HO-1. In the intestine however controversial data were reported: whereas pouchitis in humans did not show any induction of HO-1, HO-1 is rapidly and significantly upregulated in the colon of mice suffering from experimentally induced colitis. To clarify the physiological role of HO-1 in the human colon we performed immunohistology in combination with rtPCR on normal human colon tissue, Westernblot analysis of isolated lamina propria cells and enterocytes. Methods: Immunohistochemistry, rtPCR, Western blot. Results: Immunohistology revealed expression of HO-1 protein in the intestinal epithelium, i.e. mainly in the luminal enterocytes (enterocytes in crypts were found to be HO-1 negative), and in some lamina propria cells. Immunophenotyping of HO-1 positive lamina propria cells showed a predominance of macrophages. The expression of HO-1 was confirmed on mRNA(rtPCR) level and protein level via westernblotting of isolated lamina propria cells and enterocytes. Conclusions: In contrast to other tissues HO-1 is constitutively expressed at high level in the non-inflammed human colon predominantly in enterocytes and macrophages. Since HO-1 is
33rd Annual Meeting of the German Society of Immunology · 299 upregulated upon stress in other tissues these findings suggest its upregulation by local luminal factors in the gut such as e.g. endotoxins. The resultant HO-1 produced at this site might act as an endogeneous protection system to control the so called physiological inflammation in the human gut.
Department of Virology, Institute of Medical Microbiology and Hygiene, Homburg, Germany
a4b7+) Th1 T. 4 Inactivated poliovirus vaccination induces a gut homing (a memory response in orally poliovirus pre-vaccinated volunteers C. KRIEG, T. HEINTEL, R. MAIER, and A. MEYERHANS The mucous membrane is the main interface between the human body and the surrounding environment. Upon contact of this surface with a pathogen a pool of gut homing lymphocytes (a4b7+) is activated which is subsequently detectable in the blood. The effect of booster immunization either intramuscularly with inactivated poliovirus vaccine (IPV) or orally with attenuated poliovirus vaccine (OPV) in pre-immunised volunteers on this pool of gut derived cells was investigated. Cellular immune response was tested in the proliferation assay and with FACS analysis of whole blood. Furthermore poliovirus neutralising antibody response was determined. After oral poliovirus booster immunization of three volunteers only a marginal T cell response and a small increase of neutralising antibodies was detected. In contrast the immune response after intramuscular poliovirus booster immunization in 4 volunteers at day 7 shows: a) an increase of neutralising antibody titre, b) poliovirus-specific lymphocyte proliferation and c) an increase of a poliovirus-specific CD4+ T cell response. Surprisingly the further characterization of these cells revealed that they were Th1 lymphocytes (IL-4–, IFN-g+ and IL-2+). The cell differentiation type is: CCR7–, CD45R0+, CD45RA–, CD27+. The examination of homing receptors showed that they were positive for the gut homing receptor a4b7 and negative for the lymph node homing receptor CD62L (L-selectin). Our data indicate that the observed Th1 response after intramuscular poliovirus booster immunization of orally pre-vaccinated volunteers is recruited from a poliovirus-specific gut (a4b7+) memory pool. This could be essential for future mucous prime and intramuscular booster immunization strategies, e.g. future HIV vaccination strategies.
Abteilung Immunologie, Max-Planck-Institut für Infektionsbiologie, Berlin, Germany
T. 5 Expression of a non-pairing TCR a chain leads to elevated levels of a–b+ T cells and exaggerates chronically activated CD4+ TCRa inflammatory bowel disease I. PRINZ, U. KLEMM, S.H.E. KAUFMANN, and U. STEINHOFF The precise etiology of inflammatory bowel disease (IBD) is incompletely understood, nevertheless CD4+ T cells have been identified as pathogenic T cells in colitis. In TCR a–/– mice an unu-
300 · 33rd Annual Meeting of the German Society of Immunology sual CD4+TCRa–b+ T cell population is associated with the development of IBD resembling human ulcerative colitis (UC). However, the significance of such CD4+TCRa–b+ T cells in immunocompetent individuals remains unclear. Using a transgenic approach, an in-frame rearranged but non-pairing TCRa chain was expressed in the T cells of TCRa–/– mice. Although not detected on the cell surface, this TCRa chain stabilized newly synthesized TCRb chains, likely acting as a «private molecular chaperone». This lead to increased frequencies of CD4+TCRa–b+ T cells and exaggerated the course of IBD in TCRa–/– mice expressing the transgenic TCRa chain. Additional evidence that this unconventional T cell population is the causative agent of IBD is provided by the finding that adoptive transfer of these cells into TCRb–/– mice induced colitis in the recipients. Furthermore, we show that the CD4+TCRa–b+ T cells are chronically activated both in normal and transgenic TCRa–/– mice with regard to expression of the cell surface markers CD44, CD62L, and CD69. Thus, regular TCRa chain rearrangement can promote the formation of readily activated colitogenic CD4+TCRa–b+ T cells. We conclude that these T cells are present under physiological conditions and play a role in the etiology of UC.
Institute of Medical Microbiology, Immunology and Hygiene, Technical University, Munich, Germany
T. 6 Lymphocytes are essential for the development of chronic enterocolitis in mice with a myeloid cell-specific Stat3 deficiency W. REINDL and I. FÖRSTER Stat3 is the key signal transducer downstream of cytokine receptors signaling through gp130. Using the Cre loxP recombinase system, mice with a cell type specific disruption of the Stat3 gene in macrophages and neutrophils (lysozymeM (LysM)cre/Stat3flox/–) were generated (Takeda et al. Immunity 10:39–49). Analysis of these mice demonstrated that Stat3 is indispensable for IL-10 signaling in macrophages and neutrophils. This signaling defect causes the spontaneous, chronic enterocolitis and impaired weight gain observed in LysMcre/Stat3flox/– mice. The hyperreactivity of myeloid cells is the reason for the high susceptibility to endotoxin shock seen in these animals. To elucidate the significance of lymphocytes in the pathogenesis of colitis and endotoxin shock we crossed the conditional knockout mice to a RAG deficient background (LysMcre/ Stat3flox/–¥RAG–/–). Peritoneal macrophages isolated from LysMcre/Stat3flox/– and LysMcre/ Stat3flox/–¥RAG–/– showed identical levels of IFN-g and NO production after activation with LPS. Astonishingly, histology displayed no signs of colitis in LysMcre/Stat3flox/–¥RAG–/– mice and their increase in body weight was similar to littermate controls. Despite the similar potential of myeloid cells to produce proinflammatory mediators, RAG–/–¥LysMcre/Stat3flox/– mice showed enhanced resistance to LPS challenge compared to LysMcre/Stat3flox/– mice. Nevertheless LysMcre/Stat3flox/–¥RAG–/– mice were far more susceptible to endotoxin than wildtype controls. These results demonstrate, that despite the fact that myeloid cells predominate in fully developed lesions of LysMcre/Stat3flox/– mice, T and/or B lymphocytes play a key role in the initiation and perpetuation of colitis.
33rd Annual Meeting of the German Society of Immunology · 301 Institutes of 1Immunology and 2Medical Microbiology, Johannes Gutenberg-University, Mainz, Germany
T. 7 The streptococcal virulence factor streptolysin O activates mast cells to a by p38 MAP-kinase- and protein kinase C-dependent produce TNF-a pathways C. RICHTER1, S. BHAKDI2, T. BOPP1, C.H.M. MÜLLER1, C. NEUDÖRFL1, M. STASSEN1, I. WALEV2, and E. SCHMITT1 Streptolysin O (SLO), a major virulence factor of pyogenic streptococci, binds to cholesterol in membranes of eukaryotic cells and oligomerizes to form large transmembrane pores. While high toxin doses are rapidly cytocidal, low doses are tolerated because a limited number of lesions can be resealed. Here, we report that, at sublethal doses, SLO activates primary murine bone marrow-derived mast cells (BMMC) to degranulate and to rapidly induce or enhance the production of several cytokine mRNAs including TNF-a. Mast cell-derived TNF-a plays an important protective role in murine models of acute inflammation, and the production of this cytokine was analysed in more detail. Release of biologically active TNF-a peaked at about 4 h after stimulation with SLO. Production was blunted upon depletion of protein kinase C by pretreatment of the cells with PMA. Furthermore, transient permeabilization led to activation of stress-activated protein kinases p38 MAP-kinase and JNK, and inhibition of p38 MAP-kinase markedly reduced production of TNF-a. Transcriptional activation of mast cells following transient permeabilization might contribute to host defence against infections via the beneficial effects of TNF-a. However, SLO-mediated stimulation of mast cells might also promote the development of toxic streptococcal syndromes.
Medizinische Klinik I, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Berlin, Germany
T. 8 Effects of highly active antiretroviral therapy on the gut mucosal immune system in HIV-infected patients H. SCHULBIN, H. BODE, T. ZIPPEL, W. SCHMIDT, M. ZEITZ, and R. ULLRICH Introduction: The effect of highly active antiretroviral therapy (HAART) on intestinal immune function, which is the major site of virus replication and CD4+ T cells depletion in patients infected with the human immunodeficiency virus (HIV), is unknown. We therefore investigated cytokine mRNA expression in intestinal biopsies from HIV-infected patients receiving HAART. Methods: We established a quantitative real-time PCR using a LightCycler to measure mRNA-expression of: IL-2, IL-4, IL-6, IL-10, IL-16, IFN-g, TNF-a, RANTES, MIP-1-a, and the housekeeping-gene GAPDH. Rectal biopsies were obtained from 7 controls and 11 HIVinfected patients before and 4 months after initiation of HAART. HIV-RNA was measured using the Amplicor-Kit. Mucosal CD4+ T cells were determined by cytofluorometry after isolation of immune cells from biopsies.
302 · 33rd Annual Meeting of the German Society of Immunology Results: Mucosal HIV load decreased by about 2 logs and the CD4+ T cell count increased by about 1–2 logs after Cytokine mRNA-expression in HIV-infected patients was frequently higher than in controls independent of HAART.The mucosal IL-4 and IL-10 mRNA expression was higher in HIV-infected patients than in controls during the whole examination period. The mucosal RANTES mRNA expression was higher in HIV-infected patients before HAART than in controls. The mucosal mRNA expression of IL-6 and TNF-a was higher in HIV-infected patients after four months of HAART than in controls. HAART. Conclusions: The high levels of IL-4, IL-10, IFN-g, RANTES, IL-6 and TNF-a, mRNA in mucosal biopsies from HIV-infected patients receiving HAART for 4 months indicates ongoing mucosal immune activation in spite of effective inhibition of viral replication.
1
Medizinische Klinik I, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Berlin, 2Department of Pathology, Johannes Gutenberg-University, Mainz, Germany, and 3 Department of Medicine, University of Colorado, Denver CO, USA
T. 9 A critical role for leptin in different experimental models of colitis B. SIEGMUND1, H.A. LEHR2, C.A. DINARELLO3, M. ZEITZ1, and G. FANTUZZI3 Leptin, the product of the ob gene, regulates the balance of Th1/Th2 cytokines and modifies T cell immunity. Leptin-deficient ob/ob as well as leptin receptor (Ob-Rb)-deficient db/db mice are resistant against acute and chronic DSS-induced colitis, as evaluated by body weight, diarrhea, bleeding, histology, induction of proinflammatory cytokines, STAT-3 activation in the colon and rate of apoptosis in lamina propria lymphocytes (LPL). Similar resistance could be observed in the model of TNBS-induced colitis, in which reduced cytokine production and T cell activation associated with an increase of apoptosis in LPL was observed in ob/ob mice. In vitro studies revealed that leptin can directly induce STAT-3 activation in LPL and intraepithelial lymphocytes. To evaluate the role of T cells, we compared the effect of transferring CD4+CD45RBhigh cells from WT and db/db mice into scid recipients. These experiments indicate that colitis induction by CD4+CD45RBhigh cells from db/db mice is significantly reduced when compared to the transfer of WT cells, suggesting that leptin receptor expression on T cells is critical for development of intestinal inflammation. These findings are indicating for the first time in vivo, that the direct peripheral effect of leptin is crucial for T cell activation and subsequent inflammation. In conclusion, these results demonstrate that leptin represents a link between the endocrine and the immune system, which requires further investigations in experimental models and human IBD. Supported by a grant from the Deutsche Forschungsgemeinschaft DFG SI 749/2–1 and the E. and E. L. Broad Foundation
33rd Annual Meeting of the German Society of Immunology · 303 Abteilungen 1Tumorimmunologie, Institut für Pathologie and 2Innere Medizin I, Universitätsklinikum Regensburg, Regensburg, Germany
b receptor activation is critical in colitis T. 10 Lymphotoxin-b P. STOPFER1, F. OBERMEIER2, W. FALK2, D.N. MÄNNEL1, and T. HEHLGANS1 The Lymphotoxin-b receptor (LTbR) is a member of the TNF receptor family. So far two functional ligands have been identified which activate the LTbR the cell surface-bound heterotrimeric LTa1b2 complex and LIGHT, a membrane-anchored homotrimeric complex. The LTbR signaling pathway is involved in both the development of secondary lymphoid organs and the maintenance of organized lymphoid tissues. In order to investigate the role of LTbR in intestinal inflammation, LTbR activation was inhibited in a mouse model for acute or chronic colitis induced by dextran sulfate (DSS). As functional inhibitor of LTbR activation a fusion protein consisting of the extracellular domain of the murine LTbR and the Fc part of human IgG1 (LTbR:Ig), was used. The inhibitory activity of LTbR:Ig in vivo was confirmed by the transient dedifferentiation of the splenic follicular dendritic cell network after a single injection of LTbR:Ig. In the acute form of DSS-induced colitis, treatment with LTbR:Ig significantly aggravated the disease indicating a protective role of LTbR activation in this experimental model. However, treatment of the chronic form of DSS-induced colitis with LTbR:Ig significantly ameliorated the development and histological manifestations of the disease. Semiquantitative PCR analysis demonstrated strong expression of the ligand LTb in mice with chronic colitis compared to mice with acute or without colitis. When LTa1b2/LIGHT expression on lymphocytes from mesenterial lymphnodes was investigated, lymphocytes from mice treated with LTbR:Ig showed decreased LTa1b2/LIGHT expression after PMA stimulation. LTbR:Ig treatment seems to interfere with T cell activation, thus disturbing T cell mediated functions in this experimental model. Taken together these results demonstrate that activation of the LTbR pathway is critically involved in both the acute and the chronic form of colitis.
1
Institute of Immunology and Transfusion Medicine, Ernst-Moritz-Arndt-University, Greifswald and 2Institute of Bacteriology and Mycology, University of Leipzig, Leipzig, Germany
T. 11 A role for lipopolysaccharide-binding protein in local innate immunity of the intestine? M. VOGLER1, D. SCHRÖDER1, W. SCHRÖDL2, N. HABERLAND2, B. FÜRLL1, R.S. JACK1, B. AHRENS1, M. KRÜGER2, and C. SCHÜTT1 Lipopolysaccharide-binding protein (LBP) is a hepatocyte derived acute phase protein which catalyses the binding of LPS to CD14 and TLR4 on macrophages and thus leads to cellular activation. Extrahepatic expression of LBP has been described for lung, kidney and epithelial cell lines. In this study we asked whether or not LBP plays a role in the normal barrier function of the intestinal mucosa. As the gut histology of LBP-deficient mice looks regular and the animals
304 · 33rd Annual Meeting of the German Society of Immunology are healthy LBP does not seem to have an essential function in preventing translocation of endotoxin or bacteria. Using in situ hybridisation we find a constitutive expression of LBP-mRNA which is restricted to the bottom of the crypts in the small intestine, where the Paneth cells are located. We were not able to detect LBP protein in the lumen of the gut, but LBP was found in the mucus of the small intestine. Interestingly, mice deficient for LBP show a significantly reduced number of Gram-negative bacteria in the microflora as compared to wild type mice. These preliminary results illustrate a possible role for LBP in influencing mucus-resident commensal bacteria.