EVIDENCE THAT RAT XENOREACTIVE ANTIBODIES RECOGNIZE MULTIPLE PROTEIN ANTIGENS ON GUINEA-PIG ENDOTHELIAL-CELLS AND PLATELETS

Xenoreactive antibodies are an integral part of the natural immune bar rier to successful xenotransplantation between phylogenetically dispar ate species. Studies in primates suggest that the critical targets inv olved in hyperacute rejection of pig organs are glycoproteins expresse d on endothelial cells and platelets. However, there is little informa tion regarding the targets of xenoreactive antibodies and their cellul ar distribution in other experimental models of hyperacute xenograft r ejection, including the commonly used guinea pig-to-rat model. The aim of this study was to characterize the target antigens on guinea pig p latelets and endothelial cells that are recognized by rat anti-guinea pig antibodies. Using guinea pig platelet membrane proteins or intact guinea pig endothelial cells as antigens in an ELISA, we demonstrated that rat serum contains IgG and IgM anti-guinea pig antibodies; level s of antiplatelet antibodies correlated with those against endothelial cells. Serum from naive or complement-depleted rats transplanted with a guinea pig heart was investigated by immunoblotting against membran e proteins extracted from guinea pig platelets and endothelial cells. Normal rat serum revealed numerous bands on either platelet or endothe lial cell immunoblots, without obvious similarities in banding pattern under reduced or nonreduced conditions. Absorption of rat serum with intact guinea pig endothelial cells reduced reactivity against numerou s bands on endothelial cell membrane blots, but only partially reduced reactivity with three platelet bands (141, 155, and 210 kDa). Absorpt ion of rat serum with intact guinea pig platelets resulted in reductio n in reactivity against both platelet and endothelial cell immunoblots . Antibodies eluted from those intact guinea pig endothelial cells and platelets used to absorb rat sera were found to yield patterns of mem brane blot reactivity similar to those with unabsorbed sera, suggestin g that the proteins recognized are expressed on the cell surface. Lect in affinity blotting demonstrated many of the guinea pig endothelial c ell and platelet membrane proteins to be glycosylated. However, digest ion of endothelial cell and platelet immunoblots with a mixture of gly cosidases failed to change the xenoreactivity with naive rat serum. Im munoblot analysis of serum samples taken daily from complement-deplete d rats carrying a functioning guinea pig heart for 3-4 days showed an increase in intensity for specific protein bands on platelets (38, 48, 56, 141, and 155 kDa) and endothelial cells (30, 210, and 270 kDa); n o new protein bands were observed. Increased reactivity at several ban ds correlated with highly elevated rat serum anti-guinea pig antibody titers, as determined by ELISA. We conclude that (1) in contrast to th e pig-to-primate species combination, anti-guinea pig antibodies in ra ts recognize membrane proteins of different size on endothelial cells and platelets; (2) although the proteins recognized on endothelial cel ls by anti-guinea pig antibodies have a different size pattern than th ose on platelets, there is evidence of shared antigenic determinants o r crossreactivity; (3) carbohydrate substitutions on these xenoantigen s do not appear to be major targets of rat anti-guinea pig antibodies; and (4) immune xenoreactive antibodies and natural antibodies in rat serum react with similar glycoproteins on guinea pig endothelial cell or platelet membranes.