Xenogeneic human serum promotes leukocyte adhesion to porcine endothelium under flow conditions, possibly through the activation of the transcription factor NF-κB

Endothelial cell activation and leukocyte infiltration are a consistent feature of discordant xenograft rejection. Here we evaluated whether xenogeneic serum, as a source of xenoreactive natural antibodies and complement, induced endothelial cell activation with consequent leukocyte adhesion under flow conditions. Porcine aortic endothelial cells (PAEC) were incubated for 1 hr 30 min or 5 hr with 10% homologous porcine serum (control) or 10% xenogeneic human serum and then perfused with total human leukocytes in a parallel plate flow chamber under laminar flow (1.5 dynes/cm2). Adherent cells were counted by digital image analysis. Xenogeneic human serum significantly (P < 0.01) increased the number of adherent leukocytes as compared with porcine serum. A similar adhesive response was elicited by TNFα (100 U/ml), one of the most potent inducers of endothelial cell adhesive properties, here used as positive control. In order to elucidate possible mechanisms underlying endothelial cell activation by xenogeneic serum, we focussed on transcription factor NFκB, a central regulator for the induction of different genes, including adhesive molecules and chemoattractants. By confocal fluorescence microscopy, we observed a positive staining for NF-κB (p65 subunit) in the nuclei of PAEC exposed for 1 hr 30 min to human serum, which indicated NF-κB activation in this setting. At variance, in PAEC incubated with the homologous serum, NF-κB was strictly localized in the cell cytoplasm. Treatment of PAEC exposed to xenogeneic serum with the NFκB inhibitors pyrrolidinedithiocarbamate (PDTC, 25 μM) and tosyl-phechloromethylketone (TPCK, 25 μM) significantly (P < 0.01) reduced leukocyte adhesion in respect to PAEC treated with human serum alone. Findings that xenogeneic serum promotes leukocyte-endothelium interaction possibly through NF-κB activation might be relevant for designing future therapeutic strategies aimed at prolonging xenograft survival.