PIG AORTIC ENDOTHELIAL-CELL ANTIGENS RECOGNIZED BY HUMAN-IGM NATURAL ANTIBODIES

Human-to-pig xenoantibodies may constitute a major obstacle to the suc cessful use of pigs as xenograft donors for human transplantation. Our studies demonstrate that normal human serum contains antibodies, prim arily IgM, that are cytotoxic for pig aortic endothelial cells (PAECs) . These antibodies bind to several antigens isolated from PAECs, lymph ocytes, platelets, red blood cells, and the kidney. Absorption of huma n serum with pig lymphocytes removes the cytotoxic activity to PAECs a nd some, but not all, of the IgM antibodies capable of binding in an E LISA assay to the PAECs. The cytotoxic antibodies are inactivated by 2 -mercaptoethanol, suggesting that they are primarily IgM. Whole cell e xtracts of PAEC, lymphocytes, platelets, red blood cells, and kidney w ere prepared and analyzed by Western blots to establish the cellular d istribution of the xenoantigens that react with human IgM in pooled hu man serum. Results showed that several of the most intensely stained b ands migrated between 24 and 66 kDa. High molecular weight bands (>100 kDa) were observed only in kidney, platelet, and PAEC preparations. H uman IgM xeniantibodies also reacted strongly in Western blots to endo thelial 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 protein, suggest ing this protein is unique to PAECs. Treatment of the PAEC membrane pr oteins with proteinase K disrupts the binding of the human IgM antibod ies. Similar treatment with glycosidases (neuramidase, O-glycosidase, and N-glycosidase F) resulted in a decrease in molecular weight of the 28- and 26-kDa bands, suggesting that these xenoantigens are glycopro teins and that antibody binding to some xenoantigens may not require g lycosylation.