Lack of surface expression for the B-cell autoepitope of cytochrome P450 IID6 evidenced by flow cytometry

The major target antigen of LKM-1 autoantibodies in chronic active hepatitis is cytochrome P450 IID6. The role of LKM-1 autoantibodies in the pathogenesis of the disease is not clear. A crucial point is the expression of the target epitope on the hepatocyte membrane. Cultured rat hepatocytes were used in this study. Time dependent analysis of cytochrome P450 IID6 specific RNA-level revealed a constant decrease in culture. A high amount of the specific RNA-level was present after culturing the hepatocytes for 24 hours at the time of the membrane studies. To investigate membrane surface expression, we used recombinant human cytochrome P450 IID6 to affinity purify the specific autoantibody from patients sera. Indirect immunofluorescence studies and flow cytometry analysis were performed with viable, unfixed rat hepatocytes. As positive control, a monoclonal antibody against rat MHC class I antigens was used. More than 85% of viable hepatocytes showed a positive membrane staining after the incubation with the monoclonal antibody to MHC class I antigens. Affinity purified monospecific LKM-1 autoantibody, sera from control subjects and from patients with liver diseases showed a membrane staining of less than 10%. Thus, no significant expression of the B cell autoepitope of LKM-1 antigen was found on the plasma membrane of viable hepatocytes with the methods applied.