Introduction: Cellular xenogeneic rejection (CXR) is one of the important immunological obstructions that need to be overcome, if xenogeneic organs are to be used in the clinic. In particular, innate immunity by NK cells, macrophages and neutrophils cause severe rejections in xenotransplantation. Therefore, the development of strategies designed to suppress innate immune cells have considerable potential in practical applications of xenotransplantation. Because the macrophage is one of main sources of proinflammatory cytokines and the fact that proinflammatory cytokines orchestrate a variety of inflammatory responses, the regulation of macrophages could result in solving the problem of xenogeneic rejection. We recently found that human CD31 on swine endothelial cells (SEC) suppresses neutrophil-mediated xenogeneic rejection through homophilic binding. Since a significant amount of CD31 is expressed, not only on neutrophils, but also on macrophages, we hypothesized that human CD31 on SEC may suppress the macrophage-mediated cytotoxicity.
Materials and Methods: To validate our hypothesis, SEC and hCD31-transfected SEC (SEC/hCD31) were cocultured with macrophages and macrophage-related cytotoxicity was evaluated using a WST-8 assay. Next, peripheral blood-derived macrophages were generated by culturing peripheral blood monocytes with 100ng/ml GM-CSF for 7 days and the cytotoxicity caused by peripheral blood-derived macrophages was assessed using a WST-8 assay. Furthermore, to confirm whether or not inhibitory signals are induced by hCD31 homophilic binding, the phosphorylation of SHP-1 was investigated by western blotting.
Results: While PMA-activated THP-1 cells (monocyte-like cells) induced a significant level of cytotoxicity against SEC (cytotoxicity: 44.0±5.8%), a significant reduction in cytotoxicity by THP-1 was observed in SEC/hCD31 (21.1±10.6%; p<0.001, n=6). The cytotoxicity of macrophages against SEC was also significantly suppressed by hCD31 on SEC (SEC: 43.5±14.3%, SEC/CD31: 17.2±5.7%). Western blotting analyses revealed that a significant phosphorylation of SHP-1 was induced in macrophages and THP-1 that had been cocultured with SEC/hCD31.
Conclusion: Taken together, we conclude that human CD31 on porcine cells might suppress, not only neutrophils, but also macrophage-mediated cytotoxicity in a CD31 homophilic ligation-dependent manner. Our findings suggest that the generation of hCD31 transgenic pigs for use in xenografts is very attractive in terms of preventing xenogeneic rejection.