Endothelial cells derived from pigs lacking Gal alpha(1,3)Gal: no reduction of human leukocyte adhesion and natural killer cell cytotoxicity.

BACKGROUND: The expression of galactose-alpha(1,3)galactose (Gal) on porcine cells represents a major barrier to xenotransplantation. The generation of Gal-/- pigs to overcome this barrier redirected the focus of research to other rejection mechanisms, including cellular immunity. The present in vitro study investigated (1) the adhesive interactions between human leukocyte subsets and primary endothelial cells derived from inbred Gal-/- and Gal+/+ pigs, and (2) the susceptibility of such Gal-/- porcine endothelial cells to human natural killer (NK) cell cytotoxicity. METHODS: Primary porcine aortic endothelial cells (PAEC) were isolated from Gal-/- (PAEC-Gal-/-) and Gal (PAEC-Gal+/+) pigs. Human peripheral blood mononuclear cells (PBMC), polymorphonuclear neutrophils (PMN), and NK cells were isolated from healthy volunteers and tested in functional adhesion and cytotoxicity assays. RESULTS: Adhesion of human PBMC, PMN, or purified NK cells on PAEC-Gal-/- cells was not different from that on PAEC-Gal+/+ cells. Comparing the different leukocyte subsets of PBMC, a preferential adhesion of NK and B cells on both PAEC-Gal-/- and PAEC-Gal+/+ was detected. Tumor-necrosis factor-alpha stimulation of PAEC-Gal-/- and PAEC-Gal+/+ induced an increase of CD62E and CD106 expression and increased cellular adhesion, in particular, of PMN. The lack of Gal-/- expression on PAEC-Gal cells did not prevent xenogeneic human NK-cell cytotoxicity mediated by freshly isolated or interleukin-2-activated NK cells. CONCLUSIONS: Neither human leukocyte adhesion nor xenogeneic NK-cell cytotoxicity against PAEC are impaired by the lack of Gal, indicating that Gal is not a dominant target of cellular rejection.

Human leukocyte transmigration across Galalpha(1,3)Gal-negative porcine endothelium is regulated by human CD18 and CD99.

BACKGROUND: In pig-to-human xenotransplantation cross-species receptor interactions mediate cellular infiltration and rejection of porcine grafts. However, the mechanisms responsible for recruitment of human leukocyte subsets across porcine endothelial cells (EC) remain largely unknown. Here, we investigated the role of CD99, CD18, and Galalpha(1,3)Gal (Gal) in this process. METHODS: Adhesion and transmigration of human peripheral blood mononuclear cell (PBMC) subsets on Gal and Gal porcine EC (pEC) and on human EC was analyzed using a two-compartment system separated by a permeable membrane. The mechanisms of human PBMC recruitment to pEC were investigated by blocking cell surface receptors and by differentially measuring adhesion and transendothelial migration (TEM). RESULTS: Blocking of CD18, but not CD99, decreased human PBMC adhesion on pEC, whereas blocking of CD18 or CD99 strongly reduced the subsequent human PBMC TEM across pEC. The inhibitory effect of CD99 blockade was slightly stronger across pEC as compared with human EC. A critical role for Gal in TEM of human monocytes, B, natural killer (NK), NK/T, and T cells was excluded by evaluating TEM across pEC derived from Gal and Gal pigs. CONCLUSIONS: CD99 and CD18, but not Gal, play a critical role in human monocyte and lymphocyte TEM across pEC, and their respective porcine ligands may serve as targets to specifically inhibit human leukocyte recruitment in pig-to-human xenotransplantation.