Background: Non-suppurative destructive cholangitis (NSDS), T cell- mediated apoptotic destruction of biliary epithelial cells (BEC), occurs in autoimmune (primary biliary cirrhosis, PBC) and alloimmune (chronic graft-versus host disease, CGVHD or liver allograft rejection) diseases. The presence of NSDC in both PBC and CGVHD after engraftment of HLA- identical, MLR-negative marrow indicates that BEC-specific antigens presented by self-HLA can sensitize CTL. Our MHC-matched, MLR-negative B10.D2>BALB/c CGVHD model also causes NSDC when grafts contain CD4 T cells. Hypothesis: Class II, MHC-restricted donor CD4 T cell responses to recipient BEC antigens cause NSDC. To define the immunopathogenesis of NSDC at the cellular level, we developed methods to isolate: a) anatomic sties of NSDC from CGVHD livers; b) hepatic T cells and c) BEC from normal or CGVHD mice. We also created SV40 transformed, immortalized BEC (IBEC) lines that exhibit a BEC phenotype.
Specific Aim 1 : To define the phenotype and functions of T cells isolated from inflamed portal tracts during evolution of NSDC we will assess: a) Th1/Tc1 versus Th2/Tc2 cytokine phenotypes of CD4/CD8 T cells during evolution of NSDC by flow cytometry; b) histopathological consequences of polarizing intrahepatic inflammation to either a Th1/Tc1 or Th2/Tc2 response by antagonism of IL-12 or IL-4 using monoclonal antibodies, rIL-12 or immunization; c) effector functions and capacity of BEC- specific CD4/CD8 T cell clones to mediate NSDC in vivo after adoptive transfer; and d) Fas and perfornin/granzyme B mechanisms involved in CTL- and cytokine-mediated BEC cytotoxicity.
Specific Aim 2 : To characterize and compare fresh BEC and IBEC lines, in the presence or absence of cytokines, we will assess: a) mechanisms and regulation of apoptosis induced by anti-Fas antibodies, cell-bound FasL and TNFalpha in vitro and b) endogenous and stimulated cytokine gene and protein expression using flow cytometry, molecular techniques and ELISA. New insights into T cell- and cytokine-mediated mechanisms of NSDC immunopathogenesis are needed to develop more effective therapies for PBC and CGVHD.
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