CYTOCHROME-P450IID6 RECOGNIZED BY LKM1 ANTIBODY IS NOT EXPOSED ON THESURFACE OF HEPATOCYTES

LKM1 autoantibody, directed against P450IID6, is accepted as a marker of a particular type of autoimmune hepatitis, but its role in the path ogenesis of the disease is controversial. Localization of P450IID6 on the cell surface of rat hepatocytes was previously reported, suggestin g that membrane-bound P450IID6 could be the target of LKM1 antibodies, thus allowing immune lysis of hepatocytes. The objective of the prese nt study was to determine, using various methods, the cell localizatio n of P450IID6 in human and rat hepatocytes. Incubation of rat and huma n hepatocytes with LKM1-positive serum showed slight, if any, cell mem brane staining using immunofluorescence, immunoperoxidase and immunoel ectron microscopic studies. No staining of the plasma membrane of huma n hepatocytes was observed when incubations were carried out with immu noaffinity-purified antibody directed against peptide 254-271, the mai n epitope of P450IID6 recognized by all LKM1 sera tested. Chinese hams ter ovary cells, transfected with the complete P450IID6 cDNA and incub ated with the supernatant from a B cell lymphoblastoid cell line prepa red with the lymphocytes of a LKM1-positive patient, did not show any staining of the cell surface by immunofluorescence. Incubation of rat microsomal fraction vesicles with LKM1-positive serum, followed by pro tein A-gold immunoelectron microscopy, displayed a staining of almost all vesicles, confirming that P450IID6 is present on the cytoplasmic s ide of the microsomal membrane, which makes it unable to be expressed on the cell surface even if it were transported from the endoplasmic r eticulum (ER). Sulpho NHS Biotin labelling of rat hepatocyte cell memb ranes did not show the presence of a 50-kD molecule that could have re acted with LKM1 antibody. DNA sequencing of exon 1 of the CYP2D6 gene of a patient positive for LKM1 antibody did not show any difference fr om that of the normal published sequence of the gene. This does not fa vour an alteration of the NH2 terminal sequence of the P450IID6 molecu le that could explain a translocation of the molecule to the luminal s ide of the ER, allowing its expression on the cell surface. These resu lts indicate that, in all likelihood, P450IID6 molecule is not present on the cell surface of normal rat and human hepatocytes. Other mechan isms than antibody-mediated cell lysis directed against membrane P450I ID6 antigenic determinants must be found to account for the destructio n of hepatocytes observed in this disease.