Role and regulation of pig CD59 and membrane cofactor protein/CD46 expressed on pig aortic endothelial cells

Background. Hyperacute rejection in xenotransplantation is caused by activa tion of complement (C) on endothelium. We have previously shown that purifi ed C-regulators of the pig (CD59 and membrane cofactor protein [MCP]) are e fficient regulators of human C (HuC). The aim of this study was to clarify the role of endogenously expressed C-regulatory molecules on pig endotheliu m in the protection against hyperacute rejection. Methods. Porcine aortic endothelial cells (PAEC) were harvested and culture d for various passages. PAEC were examined for the expression of endogenous pig CD59 and MCP by flow cytometry. PAEC were assessed for their susceptib ility to lysis by HuC. The effect of phorbol la-myristate 13-acetate and va rious cytokines on the expression of MCP and CD59 and C-susceptibility was assessed. Results. Primary PAEC showed an initial high level of expression of pig CD5 9, however, upon culturing, CD59 levels decreased dramatically to about 20% after five passages. In contrast, levels of MCP doubled upon culturing of PAEC to confluency and remained stable during at least five passages. Prima ry cells and cells in the early passages were more resistant to HuC than ce lls that were cultured for longer. Blocking the function of CD59 but not of MCP using monoclonal antibody increased the susceptibility to HuC, Purifie d human CD59 incorporated to a level of expression similar to that of pig C D59 reversed the increased C-susceptibility, suggesting that pig and human CD59 are similarly protective against HuC. Increase of C-resistance and of expression of pig MCP, but not of CD59, was achieved upon incubation with p horbol 12-myristate 13-acetate, Tumor necrosis factor-alpha, interleukin-1 beta, interleukin-4, or interferon-gamma had no effect on C-regulator expre ssion or C-susceptibility. Conclusions. These data demonstrate the importance of using primary PAEC or cells in the first passages of culturing in in vitro models of xenotranspl antation and show that pig MCP and, in particular, pig CD59 play an importa nt role in protection of PAEC from HuC.