ALPHA-GALACTOSYL EPITOPE-MEDIATED ACTIVATION OF PORCINE AORTIC ENDOTHELIAL-CELLS - TYPE-II ACTIVATION

Background. Xenoreactive natural antibodies (XNAs) and complement medi ate hyperacute rejection of discordant xenografts. Inhibition of compl ement alone results in some prolongation of graft survival, but delaye d xenograft rejection still precludes longterm graft survival. In vitr o data provide evidence for the direct proinflammatory activation of e ndothelial cells (ECs) by XNAs. These antibodies are primarily directe d against galactose alpha(1-3)-galactose (alpha-gal), the major xenoan tigen in the pig to primate xenotransplant model. Previous studies hav e shown EC activation by XNAs but failed to address the question of wh ether alpha-gal-specific ligands can induce EC activation. The aim of this study was to investigate whether agonist binding to the alpha-gal epitope by alpha-gal-specific lectins as compared with XNAs or elicit ed xenoreactive antibodies can directly elicit type II porcine aortic EC (PAEC) activation (i.e., activation that requires protein synthesis ). Methods and Results. The tetravalent, alpha-gal-binding Bandeiraea simplicifolia lectin I (BS-I), the wholly alpha-gal-specific BS-I isol ectin B-4, and elicited primate anti-pig xenoreactive antibodies (deco mplemented cynomolgus monkey anti-porcine serum) induced E-selectin pr otein expression in PAECs. This induction was alpha-gal-specific, as p reincubation with synthetic alpha-gal carbohydrate or adsorption of le ctin or serum to rabbit, but not human, red blood cells removed the ac tivating component. E-selectin expression, induced by BS-I, was inhibi ted in the presence of genistein, a tyrosine kinase inhibitor, and by mepacrine, an inhibitor of phospholipase A2. Human and primate XNAs la cked this activity when tested at relevant concentrations; however, st imulation of PAECs with affinity-purified human XNA (IgM and IgG) resu lted in slightly increased interleukin-8 and P-selectin mRNA levels bu t had no apparent effects on E-selectin transcription, BS-I strongly i nduced E-selectin, P-selectin, intercellular adhesion molecule-1, and interleukin-8 mRNA in an NF-kappa B-dependent manner. Conclusions. Sev eral agonists that specifically bind to alpha-gal can evoke type II EC activation. Hence, anti-Gal antibodies may contribute directly to xen ograft rejection in the absence of complement activation.