The human cholangiopathies including primary biliary cirrhosis and primary sclerosing cholangitis are chronic liver diseases in which the bile duct epithelial cell, or cholangiocyte, is the target of immune-mediated destruction. A model of experimental autoimmune cholangitis was recently described: after immunization with purified cholangiocytes, rodents developed an in vivo T cell-mediated response to bile duct cells. Serum antibodies to antigens uniquely present on bile duct cells also developed. Finally, cholangiography revealed distortion of the intrahepatic biliary tree following this immunization regimen. The current proposal will extend the initial validation of this model to determine the molecular basis of the recognition of bile duct cells by the immune system and provide novel therapeutic approaches for autoimmune cholangiopathies. The hypothesis of this proposal is that unique cholangiocyte autoantigens trigger both cellular (i.e., T cell) and humoral (i.e., B cell) arms of the immune system to target cholangiocytes for destruction.
The specific aims are: 1) determine the role of T cells and adhesion/costimulatory molecules. Liver will be analyzed with T cell subset-specific and adhesion marker monoclonal antibodies. Specific T cell subsets will be depleted with antibody and complement during adoptive transfer studies. Cholangiocyte-specific T cells will be cloned and characterized by their cytokine secretion profile. T cell antigen receptor expression will be assessed by the polymerase chain reaction (PCR) to confirm that unique bile duct cell antigens drive selective T cell expansion. 2) identify biliary antigens which incite experimental autoimmune cholangitis. Using serum from animals immunized with cholangiocytes, membrane and cytosolic proteins from freshly isolated and long term cultures of rodent cholangiocytes will be probed by immunoblotting. Biliary proteins will be fractionated by routine biochemical methods and the ability of biliary antigens to support T cell proliferation in vitro will be determined to confirm that specific biliary antigens have been identified after systemic cholangiocyte immunization. 3) induce tolerance to experimental autoimmune cholangitis. Oral immunization regimens have been successful in down regulating organ specific autoimmune disease. Prior to systemic immunization with cholangiocytes, oral immunization with bile duct cells and purified rat cholangiocyte autoantigens will be performed; the effects of tolerance induction on the development of cholangitis will be assessed by histology, cholangiography and T cell proliferative responses. The ability of oral tolerance to decrease the level of cholangitis induced by systemic immunization will provide a rational treatment regimen for autoimmune cholangiopathies. This study will define important molecular interactions between biliary epithelial cells and the immune system.
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