The objective of this project proposal is to gain further insight into the pathophysiology of liver graft rejection. We will focus our efforts on the bile duct cell as a target for liver rejection. Utilizing murine strains with well-defined genetic barriers, we will explore in vitro and in vivo mechanisms of isografted and allografted liver rejections. Using our recently described technique for the purification of isolated bile duct cells from mice, we propose to analyze the interactions between the immune effector cells (lymphocytes) and liver target cells (isolated bile duct cells, endothelial cells). Using responder and stimulator strains which differ at MHC class I + class II, capacity of bile duct cells alone or in the context of antigen presenting cells (Kupffer cells or splenocytes) as stimulator cells for bile duct cell lysis. The effector cell populations involved in cytolysis in these contexts will be further investigated by cell population depletion of the responder cells, blockage of ligand receptor interactions involved in cytolysis (LFA-1:ICAM-1; CD2:LFA-3; CD8:Class I; CD4:Class II), and correlate these studies with phenotyping of bile duct cells from normal mice, bile duct cell ligated mice, and mice undergoing liver rejection. Alterations in immune response will be investigated by stimulating bile duct cells with recombinant proteins (interferon-gamma; IL-1; TNF-alpha; IL-6) to stimulate MHC class I and/or class II and/or ICAM-1 expression. An in vivo model system of liver rejection will be used to analyze the pathophysiology of liver graft rejection by determining which MHC antigens contribute to this process, phenotyping infiltrating cell populations at sequential time periods post- grafting, isolating interhepatic effector cell populations and comparing their cytolysis to bile duct cells versus extrahepatic lymphocyte populations in liver transplanted recipients, and to induce mice that tolerate allografts (example class I or class II only disparate) by in vivo priming with irradiated splenocytes and/or substituting soluble mediators from CD4 cells in class I only disparities and CD8 cells in class II only disparities. Cumulatively, this data will further our understanding of the process of liver graft rejection and will provide future experimental strategies to reverse liver rejection in preclinical and clinical model systems.
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