Carbohydrate antigens on porcine endothelial cells. Structural characterisation, cellular localisation and reactivity with human anti-pig antibodies

Abstract

As a result of the succesful development of clinical organ transplantation, more patients have become eligible for transplantation and there is now a severe shortage of human organs suitable for grafting. Xenotransplantation, i.e. transplantation of cells, tissue or organs between individuals of different species, may be a solution to this problem. For different reasons, the pig is now generally considered to be the most suitable donor species for xenografting to humans. However, all humans have naturally occuring antibodies against carbohydrate structures present on the cell surface of pig cells, e.g. the endothelial cells lining the blood vessels. These antibodies are one of the most essential of various factors that cause the immediate, hyperacute, rejection of transplanted porcine organs.The aim of this study was to identify and characterise the antigenic carbohydrate structures on porcine endothelial cells and to localise them subcellularly. Methods to remove the anti-pig antibodies from a presumptive recipient has also been studied.Glycolipids were prepared and isolated from pig aorta. Structural characterisation was achieved by mass spectrometry and 1H NMR spectroscopy on native and derivatised compounds. Monoclonal antibodies and carbohydrate specific lectins were used for immunostaining of glycolipids on thin layer chromatography plates.A cell-EIA assay with a primary cell culture was established for the study of antibody reactivity to pig aortic endothelial cells (PAEC). The binding of serum antibodies to PAEC and pig lymphocytes was also investigated by e.g. flow cytometry and lymphocytotoxicity tests.To localise a certain carbohydrate structure subcellularly we used lectin-gold silverstaining on cryosections from pig aorta. The sections were studied by both light and electron microscopy.Carbohydrate structures with a terminal galactose in an a linkage to a second galactose (Gala1,3Gal) has been shown to be the major target for human anti-pig antibodies. We have shown that pig aorta contain two glycolipid structures with that determinant - Gala1,3neolacto-tetraosylceramide and Gala1,3Lewisx. The presence of longer carbohydrate structures with the same terminal saccharides can not be excluded. These epitopes are mainly found on endothelial cells and are located to the apical side.Further, we have shown that the anti-pig antibodies in patients who have undergone repeated plasmapheresis treatments is reduced to very low levels, but return to the original level in a week or two. The binding of the anti-pig antibodies can also be inhibited by soluble Gala1,3Gal saccharide. A combination of these two methods might be a way to temporarily reduce the anti-pig antibodies to reach a state where the graft is tolerated despite the subsequent return of the antibodies. This phenomenon is called accommodation, but the mechanisms for its origin is not yet fully understood.