Role of porcine P-selectin in complement-dependent adhesion of human leukocytes to porcine endothelial cells.
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.


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.
Rollins
S A
SA
Alexion Pharmaceuticals, Inc., New Haven, Connecticut 06511, USA.
Johnson
K K
KK
Li
L
L
Birks
C
C
Matis
L A
LA
Rother
R P
RP
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000502:physiology; D019556:COS Cells; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003179:Complement C3b / Q000502:physiology; D015938:Complement Membrane Attack Complex / Q000502:physiology; D003165:Complement System Proteins / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D006801:Humans; D007962:Leukocytes / Q000502:physiology*; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology; D019007:P-Selectin / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D013552:Swine / Q000276:immunology*; D014162:Transfection
D015938:Complement Membrane Attack Complex; D019007:P-Selectin; D011994:Recombinant Proteins; D003179:Complement C3b; D003165:Complement System Proteins