Alpha-galactosyl epitope-mediated activation of porcine aortic endothelial cells: type II activation.
BACKGROUND
Xenoreactive natural antibodies (XNAs) and complement mediate hyperacute rejection of discordant xenografts. Inhibition of complement alone results in some prolongation of graft survival, but delayed xenograft rejection still precludes long-term graft survival. In vitro data provide evidence for the direct proinflammatory activation of endothelial cells (ECs) by XNAs. These antibodies are primarily directed against galactose alpha(1-3)-galactose (alpha-gal), the major xenoantigen in the pig to primate xenotransplant model. Previous studies have shown EC activation by XNAs but failed to address the question of whether alpha-gal-specific ligands can induce EC activation. The aim of this study was to investigate whether agonist binding to the alpha-gal epitope by alpha-gal-specific lectins as compared with XNAs or elicited xenoreactive antibodies can directly elicit type II porcine aortic EC (PAEC) activation (i.e., activation that requires protein synthesis).


RESULTS
The tetravalent, alpha-gal-binding Bandeiraea simplicifolia lectin I (BS-I), the wholly alpha-gal-specific BS-I isolectin B4, and elicited primate anti-pig xenoreactive antibodies (decomplemented cynomolgus monkey anti-porcine serum) induced E-selectin protein expression in PAECs. This induction was alpha-gal-specific, as preincubation with synthetic alpha-gal carbohydrate or adsorption of lectin or serum to rabbit, but not human, red blood cells removed the activating component. E-selectin expression, induced by BS-I, was inhibited in the presence of genistein, a tyrosine kinase inhibitor, and by mepacrine, an inhibitor of phospholipase A2. Human and primate XNAs lacked this activity when tested at relevant concentrations; however, stimulation of PAECs with affinity-purified human XNA (IgM and IgG) resulted in slightly increased interleukin-8 and P-selectin mRNA levels but had no apparent effects on E-selectin transcription. BS-I strongly induced E-selectin, P-selectin, intercellular adhesion molecule-1, and interleukin-8 mRNA in an NF-kappaB-dependent manner.


CONCLUSIONS
Several agonists that specifically bind to alpha-gal can evoke type II EC activation. Hence, anti-Gal antibodies may contribute directly to xenograft rejection in the absence of complement activation.
Palmetshofer
A
A
Novartis Center for Immunobiology, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Galili
U
U
Dalmasso
A P
AP
Robson
S C
SC
Bach
F H
FH
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D000918:Antibody Specificity; D001011:Aorta / Q000166:cytology; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D003167:Complement Activation; D004187:Disaccharides / Q000276:immunology* / Q000378:metabolism; D019040:E-Selectin / Q000096:biosynthesis; D004730:Endothelium, Vascular / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D000939:Epitopes / Q000276:immunology* / Q000378:metabolism; D019833:Genistein / Q000494:pharmacology; D000882:Haplorhini; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D007075:Immunoglobulin M / Q000378:metabolism; D037102:Lectins / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D037121:Plant Lectins; D011796:Quinacrine / Q000494:pharmacology; D011817:Rabbits; D013552:Swine; D014183:Transplantation, Heterologous / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000906:Antibodies; D004187:Disaccharides; D019040:E-Selectin; D000939:Epitopes; C045475:Griffonia simplicifolia lectins; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D037102:Lectins; D037121:Plant Lectins; D014409:Tumor Necrosis Factor-alpha; C055075:galactosyl-(1-3)galactose; D019833:Genistein; D011796:Quinacrine