Introduction: Macrophage activation plays important roles in xenograft rejection.  Overexpression of human CD200 (hCD200) in porcine endothelial cells (PECs) has been reported to suppress xenogeneic immune responses of human macrophages against PECs. The current study aimed to address whether the above-mentioned beneficial effect of hCD200 is mediated by overcoming the molecular incompatibility between porcine CD200 (pCD200) and hCD200 receptor or simply by increasing the expression levels of CD200 without any molecular incompatibility across the two species.
Method: We overexpressed hCD200 or pCD200 using lentiviral vectors with V5 marker in porcine endothelial cells (PECs) and compared their suppressive activity against U937-derived human macrophage-like cells as well as human primary monocyte-derived  macrophages. After co-culture of hCD200- or pCD200-overexpressing PECs and human macrophages, assays of phagocytosis, cytotoxicity, and cytokine secretion were performed, and signal transduction downstream of CD200 receptor was analyzed using immunoblot analysis.
Results: In xenogeneic co-culture of PECs and human macrophage-like cells, hCD200-PECs suppressed phagocytosis and cytotoxicity of human macrophage-like cells to a greater extent than pCD200-PECs. Secretion of tumor necrosis factor-α, interleukin-1β, and monocyte chemoattractant protein-1 from human macrophage-like cells and expression of M1 phenotypes (inducible nitric oxide synthase, dectin-1, and CD86) were also suppressed by hCD200 to a greater extent than pCD200. Furthermore, in signal transduction downstream of CD200 receptor, hCD200 induced Dok2 phosphorylation and suppressed IκB phosphorylation to a greater extent than pCD200. As primary monocytes were differentiated into macrophages, expression of CD200 receptor increased. Xenogeneic co-culture of PECs and human monocyte-derived macrophages showed similar results as co-culture of PECs and human macrophage-like cells.
Conclusion: These data supported the possibility of a significant molecular incompatibility between pCD200 and human CD200 receptor, suggesting that the beneficial effects of hCD200 overexpression in PECs could be mediated by overcoming the interspecies molecular incompatibility rather than by simple overexpression effects of CD200. hCD200-transgenic pigs may be promising for the control of xenogeneic activation of human macrophages and subsequent xenograft rejection by overcoming interspecies molecular incompatibility.