Pig aortic endothelial cell antigens recognized by human IgM natural antibodies

Human-to-pig xenoantibodies may constitute a major obstacle to the successful use of pigs as xenograft donors for human transplantation. Our studies demonstrate that normal human serum contains antibodies, primarily IgM, that are cytotoxic for pig aortic endothelial cells (PAECs). These antibodies bind to several antigens isolated from PAECs, lymphocytes, platelets, red blood cells, and the kidney. Absorption of human serum with pig lymphocytes removes the cytotoxic activity to PAECs and some, but not all, of the IgM antibodies capable of binding in an ELISA assay to the PAECs. The cytotoxic antibodies are inactivated by 2-mercaptoethanol, suggesting that they are primarily IgM. Whole cell extracts of PAEC, lymphocytes, platelets, red blood cells, and kidney were prepared and analyzed by Western blots to establish the cellular distribution of the xenoantigens that react with human IgM in pooled human serum. Results showed that several of the most intensely stained bands migrated between 24 and 66 kDa. High molecular weight bands (> 100 kDa) were observed only in kidney, platelet, and PAEC preparations. Human IgM xeniantibodies also reacted strongly in Western blots to endothelial cell membranes proteins with molecular weights of 62, 48, 42, 36, 34, 28, and 26 kDa. Absorption of human serum with pig lymphocytes removes IgM binding to all bands except for a 34-kDa Treatment of the PAEC membrane proteins with proteinase K disrupts the binding of the human IgM antibodies. Similar treatment with glycosidase F) resulted in a decrease in molecular weight of the 28- and 26-kDa bands, suggesting that these xenoantigens are glycoproteins and that antibody binding to some xenoantigens may not require glycosylation.