BACKGROUND: Rapid leukocyte adherence to donor organ vasculature is a hallmark of hyperacute xenograft rejection. However, the molecular interactions required for leukocyte binding to vascular endothelium have not been characterized. METHODS AND RESULTS: Binding assays performed between human neutrophils and porcine aortic endothelial cells (PAEC) after exposure to human complement demonstrated that adhesion was mediated by both surface-bound C3b and C5b-9 activity. C5b-9-dependent adhesion was blocked by neuraminidase treatment of the neutrophils, suggesting that this binding was mediated by porcine P-selectin. Porcine P-selectin was isolated from a PAEC cDNA library. The porcine P-selectin primary sequence contained an open reading frame encoding 646 amino acids with 82% identity to human P-selectin. Recombinant soluble porcine P-selectin specifically bound to human neutrophils and HL-60 cells. Transfection of COS cells with the full-length porcine P-selectin cDNA resulted in surface expression of the protein and markedly increased the binding of human neutrophils to these cells. The binding of both soluble and COS-expressed porcine P-selectin to human neutrophils was blocked by pretreatment of the neutrophils with neuraminidase or the addition of EDTA. Finally, treatment of PAEC with human thrombin or normal human serum but not purified human C5a- or C8-deficient human serum resulted in the rapid expression of porcine P-selectin on the cell surface. CONCLUSIONS: This report establishes that porcine P-selectin supports the binding of human neutrophils to PAEC in vitro. Further, these data suggest that sublytic deposition of C5b-9 during hyperacute rejection results in the expression of porcine P-selectin, which may contribute to the rapid adhesion of neutrophils to porcine xenografts.
BACKGROUND: Rapid leukocyte adherence to donor organ vasculature is a hallmark of hyperacute xenograft rejection. However, the molecular interactions required for leukocyte binding to vascular endothelium have not been characterized. METHODS AND RESULTS: Binding assays performed between human neutrophils and porcine aortic endothelial cells (PAEC) after exposure to human complement demonstrated that adhesion was mediated by both surface-bound C3b and C5b-9 activity. C5b-9-dependent adhesion was blocked by neuraminidase treatment of the neutrophils, suggesting that this binding was mediated by porcine P-selectin. Porcine P-selectin was isolated from a PAEC cDNA library. The porcine P-selectin primary sequence contained an open reading frame encoding 646 amino acids with 82% identity to humanP-selectin. Recombinant soluble porcine P-selectin specifically bound to human neutrophils and HL-60 cells. Transfection of COS cells with the full-length porcine P-selectin cDNA resulted in surface expression of the protein and markedly increased the binding of human neutrophils to these cells. The binding of both soluble and COS-expressed porcine P-selectin to human neutrophils was blocked by pretreatment of the neutrophils with neuraminidase or the addition of EDTA. Finally, treatment of PAEC with humanthrombin or normal human serum but not purified human C5a- or C8-deficienthuman serum resulted in the rapid expression of porcine P-selectin on the cell surface. CONCLUSIONS: This report establishes that porcine P-selectin supports the binding of human neutrophils to PAEC in vitro. Further, these data suggest that sublytic deposition of C5b-9 during hyperacute rejection results in the expression of porcine P-selectin, which may contribute to the rapid adhesion of neutrophils to porcine xenografts.
Authors: Sonia I Vlaicu; Alexandru Tatomir; Violeta Rus; Armugam P Mekala; Petru A Mircea; Florin Niculescu; Horea Rus Journal: Immunol Res Date: 2016-02 Impact factor: 2.829
Authors: I Kourtzelis; A Ferreira; I Mitroulis; D Ricklin; S R Bornstein; C Waskow; J D Lambris; T Chavakis Journal: Horm Metab Res Date: 2014-10-28 Impact factor: 2.936