Xenogeneic serum promotes leukocyte-endothelium interaction under flow through two temporally distinct pathways: Role of complement and nuclear factor-kappa B

Endothelial cell activation and mononuclear cell infiltration are consisten t features of discordant xenograft rejection. This study evaluated whether xenogeneic serum-as a source of xenoreactive natural antibodies and complem ent-induced endothelial activation with consequent leukocyte adhesion and t ransmigration under flow conditions. Porcine aortic endothelial cells (PAEC ) were incubated for 30 min, 1 h 30 min, or 5 h with 10% human serum or 10% porcine serum and then perfused with human leukocytes in a parallel plate flow chamber under flow (1.5 dynes/cm(2)). Adherent and transmigrated cells were counted by digital image analysis. Results showed that human serum si gnificantly (P < 0.01) increased over time the number of adherent leukocyte s compared with porcine serum. Stimulation of PAEC with human serum also pr omoted a progressive increase in leukocyte transmigration that reached stat istical significance (P < 0.01) at 1 h 30 min and at 5 h compared with porc ine serum. Studying the role of complement in leukocyte-endothelium interac tion in xenogeneic conditions, a marked complement C3 deposition on PAEC ex posed to human serum was shown by immunofluorescence, whereas cells incubat ed with porcine serum were negative. Next, it was documented that human ser um decomplemented by heating and C3-deficient human serum failed to promote both leukocyte adhesion and transmigration, results that were comparable t o porcine serum. To elucidate the intracellular mediators involved in endot helial cell activation by xenogeneic serum, this study focused on transcrip tional factor nuclear factor-kappa B (NF-kappa B), a central regulator for the induction of different genes, including adhesive molecules and chemoatt ractants. Positive nuclear staining of NF-kappa B (p65 subunit) found by co nfocal fluorescence microscopy of PAEC exposed to human serum was taken to reflect NF-kappa B activation. NF-kappa B was instead strictly localized in the cell cytoplasm in PAEC incubated with the homologous serum. Heat-inact ivated human serum failed to activate NF-kappa B. Electrophoretic mobility shift assay of nuclear extracts from PAEC exposed to human serum revealed a n intense NF-kappa B activation that was inhibited by the NF-kappa B inhibi tor pyrrolidinedithiocarbamate. The NF-kappa B inhibitors pyrrolidinedithio carbamate and tosyl-phe-chloromethylketone did not affect the number of adh erent and transmigrated leukocytes in PAEC exposed to human serum for 30 mi n and 1 h 30 min. Both inhibitors instead significantly reduced leukocyte a dhesion and transmigration induced by human serum at 5 h. Confocal fluoresc ence microscopy studies showed that human serum induced an increase in the expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1. Functional blocking of these adhesive molecules with the corres ponding antibodies significantly inhibited xenogeneic serum-induced leukocy te adhesion. These data suggest that leukocyte adhesion and transmigration are directly dependent on complement deposited on PAEC in the early phase o f cell activation (30 min and 1 h 30 min) induced by xenogeneic serum, wher eas leukocyte adhesive events observed after 5 h of incubation of endotheli al cells with xenogeneic serum are possibly regulated by transcription of N F kappa B-dependent genes. The finding that xenogeneic serum promotes leuko cyte-endothelial interaction depending on NF-kappa B activation might be re levant for designing future therapeutic strategies intended to prolong xeno graft survival.