Characterization of human IgG-binding xenoantigens expressed by porcine aortic endothelial cells.
Xenoreactive antibodies (XAb) play a major role in the rejection of xenografts. In this study, human IgG XAb that bind to xenoantigens expressed by porcine aortic endothelial cells (PAEC) were characterized, together with their corresponding xenoantigens. Using an ELISA with both fixed and unfixed confluent monolayers of PAEC, XAb of both IgG and IgM classes in pooled and individual normal human serum were identified. The binding of these IgG XAb to the endothelium is mediated by F(ab')2 and the only detectable subclasses that bind to the endothelium are IgG1 and IgG2. On the basis of direct binding experiments, inhibition and antibody adsorption studies, and enzymatic digestions, it is shown that only a minor component of the XAb binding is directed against galactose in an alpha 1,3 linkage with galactose on PAEC surfaces. There is some cross-reactivity with antigens expressed on porcine lymphocytes, but not porcine red blood cells. Histological examination of sections of porcine aortae, snap-frozen and stained using immunoperoxidase techniques, confirmed interaction with the vascular endothelium. Labeling of the PAEC with 125I, followed by cell lysis and immunoprecipitation under reducing conditions, showed binding of IgG XAb to several components on the endothelial cell surface, the most prominent of which have apparent molecular masses of 75 kDa, 110 kDa, 180 kDa, and 210 kDa. The 110-kDa component and the 180-kDa component were sensitive to digestion with endoglycosidase F, which suggests the participation of N-linked carbohydrate structures. These studies demonstrate that human IgG XAb recognize multiple determinants expressed by PAEC, a minor population of which contain alpha 1,3-linked galactose residues. Cross-reactive determinants are expressed on porcine lymphocytes but not porcine red blood cells.
Vascular Biology Research Centre, Kings College, London, United Kingdom.
S P
SP
Cooke
R A
RA
Hederer
J D
JD
Pearson
C O
CO
Savage
D000818:Animals; D015478:Antigens, Heterophile / Q000737:chemistry / Q000276:immunology*; D001011:Aorta; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000276:immunology*; D000939:Epitopes; D005697:Galactosides / Q000276:immunology; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D008970:Molecular Weight; D013552:Swine
D015478:Antigens, Heterophile; D000939:Epitopes; D005697:Galactosides; D006023:Glycoproteins; D007074:Immunoglobulin G; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase