XENOGENEIC HUMAN SERUM PROMOTES LEUKOCYTE ADHESION TO PORCINE ENDOTHELIUM UNDER FLOW CONDITIONS, POSSIBLY THROUGH THE ACTIVATION OF THE TRANSCRIPTION FACTOR NF-KAPPA-B

Endothelial cell activation and leukocyte infiltration are a consisten t 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 leukoc yte adhesion under flow conditions. Porcine aortic endothelial cells ( PAEC) were incubated for 1 hr 30 min or 5 hr with 10% homologous porci ne serum (control) or 10% xenogeneic human serum and then perfused wit h total human leukocytes in a parallel plate flow chamber under lamina r flow (1.5 dynes/cm(2)). 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 simil ar adhesive response was elicited by TNF alpha (100 U/ml), one of the most potent inducers of endothelial cell adhesive properties, here use d as positive control. In order to elucidate possible mechanisms under lying endothelial cell activation by xenogeneic serum, we focussed on transcription factor NF-kappa B, 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-kappa B (p65 subunit) in the nuclei of PAEC exposed for 1 hr 3 0 min to human serum, which indicated NF-kappa B activation in this se tting. At variance, in PAEC incubated with the homologous serum, NF-ka ppa B was strictly localized in the cell cytoplasm. Treatment of PAEC exposed to xenogeneic serum with the NF-kappa B inhibitors pyrrolidine dithiocarbamate (PDTC, 25 mu M) and tosyl-phechloromethylketone (TPCK, 25 mu M) significantly (P<0.01) reduced leukocyte adhesion in respect to PAEC treated with human serum alone. Findings that xenogeneic seru m promotes leukocyte-endothelium interaction possibly through NF-kappa B activation might be relevant for designing future therapeutic strat egies aimed at prolonging xenograft survival.