XENOREACTIVE ANTIBODY RECOGNITION OF RELEVANT ANTIGENS IN SWINE KIDNEY AND LUNG

Abstract

535 Background: The role of antibody in swine-to-baboon lung xenograft injury is different from that in hyperacute rejection of heart and kidney xenografts, which involves the binding of xenoreactive antibody to Galα(1-3)Gal epitopes on the endothelium. Removal of xenoantibody by pretransplant ex-vivo column absorption or kidney perfusion is not beneficial to subsequent pulmonary graft survival; however, the use of pretransplant lung perfusion does have a beneficial effect on subsequent pulmonary graft outcome. One possible explanation for this finding is that pretransplant lung perfusion removes additional antibodies that are specific for unique lung antigens. The purpose of this study was to determine if these relevant unique antigens exist on the pulmonary microvasculature. Methods: (1)Xenoreactive Antibody Depletion: Human plasma was perfused through porcine lungs and kidneys harvested by standard technique in order to absorb out xenoantibody. (2)Antigen Extraction: Antigens from porcine lungs and kidneys were extracted by organ perfusion with a solution of Triton-X 100 and protease inhibitors. Antigens from porcine aortic endothelial cells (PAEC) were obtained from culture for comparison. (3)Western Blot Analysis: Membrane antigens were separated by SDS page, transferred to membranes, and stained with normal human serum, human serum perfused through porcine lung, or human serum perfused through porcine kidney, and labeled with anti-IgM. Results: Perfusion of serum through both the lung and through the kidney removed higher molecular weight (HMW) bands, between 97.4kD and 220 kD, which were recognized by normal IgM in human serum; this removal was consistent across all three cell extract types (PAEC, lung microvascular, and kidney microvascular). These HMW bands were previously found to correspond to antigens that possess Galα(1-3)Gal epitopes. Normal human serum also stained numerous lower molecular weight (LMW) antigens, corresponding to bands between 30kD and 46kD, in kidney and lung extracts; these were not present in PAEC extracts. However, antibodies recognizing these LMW antigens were not significantly depleted from human serum by lung or kidney perfusion. Conclusions: Porcine lung perfusion did not remove any additional xenoreactive antibody from human serum as compared to kidney perfusion. This suggests that there are no lung-specific antigens that are recognized by human xenoreactive antibodies, and therefore that antibody removal does not explain the beneficial effects of pretransplant porcine lung perfusion on pulmonary xenograft survival.