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TGF beta in autoimmune hepatitis: impairment of TGF beta signaling in T-cells increases susceptibility to experimental autoimmune hepatitis
Saturday, April 21,200l
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amounts of AFP thereby causing autoimmune liver disease. Materials and Methods: C57BL/6 mice were immunized with Mock-DNA or DNA encoding mouse AFP. For induction of liver regeneration partial hepatectomy was performed and hepatocellular injury was monitored by serially measuring serum ALT activities and histopathological examination. AFP specific in vivo T cell precursor frequency was determined by ELISPGT technique. Results and Diicussion: In AFP DNA immunized animals a significant hepatocyte damage characterized by necrosis and lymphocytic infiltrations was observed in regenerating liver that correlated well with the number of AFP specific T cells, the activity of liver regeneration, and the level of AFP synthesis. No autoimmune liver damage was observed in Mock-DNA immunized mice and in AFP-DNA immunized MHC class I and II knock-out mice suggesting the participation of both AFP specific CD4+ and CD8+ T cells for mediating the hepatocyte damage. Autoimmunity was generally self-limiting after completion of liver regeneration. In conclusion, the use of non-tumor restricted antigens as targets for tumor immunotherapy may carry the risk for severe autoimmune disease. Careful selection of HCC patients for AFP directed immunotherapy may reduce this risk.
I 290
TGF BETA IN AUTOIMMUNE HEPATITIS: IMPAIRMENT OF TGF BETA SIGNALING IN T-CELLS INCREASES SUSCEPTIBILITY TO EXPERIMENTAL AUTOIMMUNE HEPATITIS
that liver dendritic cells (DCs) play a role. Subsets of DCs capable of generating different immune responses have been described. Human liver DCs were isolated by migration from tissue overnight, and purified by density gradient centrifugation. In the same way DCs were isolated from normal skin. The phenotype of the cells was compared by immunocytochemistry and flow cytometry, and function compared by ELISA measurement of IL-10 and IL-12~70 production by pure DCs. Cells from normal liver expressed CD14 (50%), CD1 lb, CDllc, as well as HLA-DR and CD86. After culture cells lost expression of CDllb and CD14 but increased CD83 and CD86. Skin cells expressed similar myeloid DC markers, and expressed the same. markers of immaturity (CD1 lb and CD14)initially, but a majority of cells were CDla and SlOO +ve (Langerhan’s cell markers). Chemokine receptor expression was consistent with DCs migrating from tissue to lymph nodes. CCR7 and CXCR4 were present, they are up-regulated in mature DCs. There were low levels of CCR5 and no CCRl expression. Liver DCs produce IL-10 (414pg/ml), whilst skin DCs do not, DCs from diseased liver produce increased IL-lo, but not significantly (673). None of the DCs produce IL-12~70 in the conditions tested. DCs from human skin and liver have a similar phenotype, but liver DCs do not express CDla. Using this technique, DCs of the same stage of maturation, and therefore functional stage, can be obtained and compared. IL- 10 is associated with tolerance and generation of regulatory T cells, therefore IL-10 production by liver DCs may be involved in the generation and maintenance of allograft tolerance.
C. Schramm, M. Protschka, H.H. KBhler, P.R. Galle, A.W. Lohse, M. Blessing. I. Medizinische Klinik und Poliklinik, Johannes Gutenberg University, Mainz, Germany
In Autoimmune Hepatitis strong TGFbetal-expression can be found in the inflamed liver. TGFbeta might be produced in order to suppress autoreactive T-cells in the liver. To test this hypothesis we determined whether imairment of TGFbeta signaling in T-cells leads to increased susceptibility to Experimental Autoimmune Hepatitis (EAH). Methods: Transgenic FVB/N mice were generated expressing a dominant negative TGFbeta type II receptor in T-cells under the control of the human CD2 promoter/enhancer. For induction of EAH, mice were immunized with the 100.000 g supematant of syngeneic liver homogenate in complete Freund’s adjuvant. Results: Three transgenic lines were generated and characterized. RNAexpression in T-cells was determined by Northern blot analysis. In contrast to wildtype T-cells, IL-2 production of transgenic T-cells could not be inhibited by addition of 10 nglml TGFbetal to the culture (100% vs. 17% (wildtype) of baseline values), confirming the impairment of TGFbeta signaling in transgenic T-cells. In addition, transgenic splenocytes secreted more IFNgamma than wildtype splenocytes (52 vs. 34 ng/ml) upon CDS/CD28 crosslinking. Although transgenic mice did not show spontaneous inflammation of the liver, they were more susceptible to induction of EAH (ALT: 66 vs. 40 U/I, pt0.02). Liver histology showed portal and periportal lymphocytic infiltration with hepatocellular necroses (median histologic score: 1.8 vs 0.75, ~~0.01). Conclusions: Impairment of TGFbeta signaling in T-cells increases susceptibility to EAH in mice. The attempt to restore immune homeostasis might be the reason for the liver to produce TGFbeta upon T-cell mediated injury. Background:
I
946
A COMPARISON OF DENDRlTlC CELLS MIGRATED FROM HUMAN LIVER AND SKIN
S. Goddard, P.J. Lane, D.H. Adams. Liver Unit Laboratories, University of Birmingham, UK There is evidence of organ specific allograft acceptance in the absence
of immune suppression following liver transplantation, it is suggested
S19
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777
Working Party 4 - Viral hepatitis
POLYMORPHISMS IN THE IL-10 LIOAND AND THE IL-10 RECEPTOR /3 CHAIN INFLUENCE SUSCEPTIBILlTY TO PERSISTENT HBV INFECTION
M.R. Thursz, L. Zhang, A. Frodsham, U. Dumpis, M. Chiaramonte, C. Venturi-Pasini, S. Lobello, A.V. Hill, H.C. Thomas. Medicine, Imperial College School of Medicine, London, UK
Host genetic factors play an important role in determining the outcome of hepatitis B virus (HBV) infection. The aim of this study was to identify the gene(s) responsible for increased susceptibility to persistent HBV infection. We have previously reported a genome wide scan on affected sibling pairs which identified a susceptibility locus for persistent HBV infection on the long arm of chromosome 21 (maximum lod score 3.55, p = 0.00002). Transmission disequilibrium tests implicated the IL-10 receptor II and/or the interferon gamma receptor II genes. Disease association studies were performed by comparison of allele frequencies between cases (persistent HBV infection) and controls (self limiting HBV infection). Polymorphisms in the IL-10 promoter (A- 1082G), IL- 10 receptor II(K47E) and the interferon gamma receptor II (T2087C-3’UTR) were investigated in a population from the Gambia. The G allele at position -1082 in the IL-10 promoter was found more frequently in persistent infection (41% v 29%. OR = 1.73, P = 0.0003); this association was reproduced in a European population. In addition the KK genotype at codon 47 in the IL10 receptor II was more frequently in self limiting infection (83.4% v 92.4%. OR = 0.41, P = 0.014). There was no significant difference in allele frequencies in the IFN gamma receptor gene. Our data suggests that genetic variation in the IL-10 ligand-receptor axis influences susceptibility to HBV infection.
14
amounts of AFP thereby causing autoimmune liver disease. Materials and Methods: C57BL/6 mice were immunized with Mock-DNA or DNA encoding mouse AFP. For induction of liver regeneration partial hepatectomy was performed and hepatocellular injury was monitored by serially measuring serum ALT activities and histopathological examination. AFP specific in vivo T cell precursor frequency was determined by ELISPGT technique. Results and Diicussion: In AFP DNA immunized animals a significant hepatocyte damage characterized by necrosis and lymphocytic infiltrations was observed in regenerating liver that correlated well with the number of AFP specific T cells, the activity of liver regeneration, and the level of AFP synthesis. No autoimmune liver damage was observed in Mock-DNA immunized mice and in AFP-DNA immunized MHC class I and II knock-out mice suggesting the participation of both AFP specific CD4+ and CD8+ T cells for mediating the hepatocyte damage. Autoimmunity was generally self-limiting after completion of liver regeneration. In conclusion, the use of non-tumor restricted antigens as targets for tumor immunotherapy may carry the risk for severe autoimmune disease. Careful selection of HCC patients for AFP directed immunotherapy may reduce this risk.
I 290
TGF BETA IN AUTOIMMUNE HEPATITIS: IMPAIRMENT OF TGF BETA SIGNALING IN T-CELLS INCREASES SUSCEPTIBILITY TO EXPERIMENTAL AUTOIMMUNE HEPATITIS
that liver dendritic cells (DCs) play a role. Subsets of DCs capable of generating different immune responses have been described. Human liver DCs were isolated by migration from tissue overnight, and purified by density gradient centrifugation. In the same way DCs were isolated from normal skin. The phenotype of the cells was compared by immunocytochemistry and flow cytometry, and function compared by ELISA measurement of IL-10 and IL-12~70 production by pure DCs. Cells from normal liver expressed CD14 (50%), CD1 lb, CDllc, as well as HLA-DR and CD86. After culture cells lost expression of CDllb and CD14 but increased CD83 and CD86. Skin cells expressed similar myeloid DC markers, and expressed the same. markers of immaturity (CD1 lb and CD14)initially, but a majority of cells were CDla and SlOO +ve (Langerhan’s cell markers). Chemokine receptor expression was consistent with DCs migrating from tissue to lymph nodes. CCR7 and CXCR4 were present, they are up-regulated in mature DCs. There were low levels of CCR5 and no CCRl expression. Liver DCs produce IL-10 (414pg/ml), whilst skin DCs do not, DCs from diseased liver produce increased IL-lo, but not significantly (673). None of the DCs produce IL-12~70 in the conditions tested. DCs from human skin and liver have a similar phenotype, but liver DCs do not express CDla. Using this technique, DCs of the same stage of maturation, and therefore functional stage, can be obtained and compared. IL- 10 is associated with tolerance and generation of regulatory T cells, therefore IL-10 production by liver DCs may be involved in the generation and maintenance of allograft tolerance.
C. Schramm, M. Protschka, H.H. KBhler, P.R. Galle, A.W. Lohse, M. Blessing. I. Medizinische Klinik und Poliklinik, Johannes Gutenberg University, Mainz, Germany
In Autoimmune Hepatitis strong TGFbetal-expression can be found in the inflamed liver. TGFbeta might be produced in order to suppress autoreactive T-cells in the liver. To test this hypothesis we determined whether imairment of TGFbeta signaling in T-cells leads to increased susceptibility to Experimental Autoimmune Hepatitis (EAH). Methods: Transgenic FVB/N mice were generated expressing a dominant negative TGFbeta type II receptor in T-cells under the control of the human CD2 promoter/enhancer. For induction of EAH, mice were immunized with the 100.000 g supematant of syngeneic liver homogenate in complete Freund’s adjuvant. Results: Three transgenic lines were generated and characterized. RNAexpression in T-cells was determined by Northern blot analysis. In contrast to wildtype T-cells, IL-2 production of transgenic T-cells could not be inhibited by addition of 10 nglml TGFbetal to the culture (100% vs. 17% (wildtype) of baseline values), confirming the impairment of TGFbeta signaling in transgenic T-cells. In addition, transgenic splenocytes secreted more IFNgamma than wildtype splenocytes (52 vs. 34 ng/ml) upon CDS/CD28 crosslinking. Although transgenic mice did not show spontaneous inflammation of the liver, they were more susceptible to induction of EAH (ALT: 66 vs. 40 U/I, pt0.02). Liver histology showed portal and periportal lymphocytic infiltration with hepatocellular necroses (median histologic score: 1.8 vs 0.75, ~~0.01). Conclusions: Impairment of TGFbeta signaling in T-cells increases susceptibility to EAH in mice. The attempt to restore immune homeostasis might be the reason for the liver to produce TGFbeta upon T-cell mediated injury. Background:
I
946
A COMPARISON OF DENDRlTlC CELLS MIGRATED FROM HUMAN LIVER AND SKIN
S. Goddard, P.J. Lane, D.H. Adams. Liver Unit Laboratories, University of Birmingham, UK There is evidence of organ specific allograft acceptance in the absence
of immune suppression following liver transplantation, it is suggested
S19
u
777
Working Party 4 - Viral hepatitis
POLYMORPHISMS IN THE IL-10 LIOAND AND THE IL-10 RECEPTOR /3 CHAIN INFLUENCE SUSCEPTIBILlTY TO PERSISTENT HBV INFECTION
M.R. Thursz, L. Zhang, A. Frodsham, U. Dumpis, M. Chiaramonte, C. Venturi-Pasini, S. Lobello, A.V. Hill, H.C. Thomas. Medicine, Imperial College School of Medicine, London, UK
Host genetic factors play an important role in determining the outcome of hepatitis B virus (HBV) infection. The aim of this study was to identify the gene(s) responsible for increased susceptibility to persistent HBV infection. We have previously reported a genome wide scan on affected sibling pairs which identified a susceptibility locus for persistent HBV infection on the long arm of chromosome 21 (maximum lod score 3.55, p = 0.00002). Transmission disequilibrium tests implicated the IL-10 receptor II and/or the interferon gamma receptor II genes. Disease association studies were performed by comparison of allele frequencies between cases (persistent HBV infection) and controls (self limiting HBV infection). Polymorphisms in the IL-10 promoter (A- 1082G), IL- 10 receptor II(K47E) and the interferon gamma receptor II (T2087C-3’UTR) were investigated in a population from the Gambia. The G allele at position -1082 in the IL-10 promoter was found more frequently in persistent infection (41% v 29%. OR = 1.73, P = 0.0003); this association was reproduced in a European population. In addition the KK genotype at codon 47 in the IL10 receptor II was more frequently in self limiting infection (83.4% v 92.4%. OR = 0.41, P = 0.014). There was no significant difference in allele frequencies in the IFN gamma receptor gene. Our data suggests that genetic variation in the IL-10 ligand-receptor axis influences susceptibility to HBV infection.