title: Cytokine secretion depends on Galalpha(1,3)Gal expression in a pig-to-human whole blood model.
abstract: Transplants from alpha1,3-galactosyltransferase (Gal) gene-knockout pigs to nonhuman primates are largely protected from hyperacute but not acute humoral xenograft rejection. The present study investigates the role of Gal in cytokine responses using a novel pig-to-human whole blood in vitro model, developed for species-specific analysis of porcine and human cytokines. Porcine (n = 7) and human (n = 27) cytokines were measured using ELISA or multiplex technology, respectively. Porcine aortic endothelial cells from control (Gal(+/+)) and Gal-deficient (Gal(-/-)) pigs were incubated with human lepirudin anticoagulated whole blood from healthy donors. E-selectin expression was measured by flow cytometry. The C3 inhibitor compstatin and a C5aR antagonist were used to study the role of complement. Cytokine species specificity was documented, enabling detection of 2 of 7 porcine cytokines and 13 of 27 human cytokines in one single sample. Gal(+/+) porcine aortic endothelial cells incubated with human whole blood showed a marked complement C5b-9 dependent up-regulation of E-selectin and secretion of porcine IL-6 and IL-8. In contrast, Gal(-/-) cells responded with E-selectin and cytokine expression which was so weak that the role of complement could not be determined. Human IL-6, IL-8, IFN-gamma, MIP-1alpha, MIP-1beta, eotaxin, and RANTES were detected in the Gal(+/+) system, but virtually no responses were seen in the Gal(-/-) system (p = 0.03). The increase in human cytokine release was largely complement dependent and, in contrast to the porcine response, mediated through C5a. Species-specific analysis of cytokine release revealed a marked, complement-dependent response when Gal(+/+) pig cells were incubated with human whole blood, compared with Gal(-/-) cells which induced virtually no cytokine release.
authors.affiliation: Institute of Immunology, Rikshospitalet University Hospital and Faculty of Medicine, University of Oslo, Oslo, Norway.
authors.forename: Marit
authors.initials: M
authors.lastname: Saethre
authors.forename: Mårten K J
authors.initials: MK
authors.lastname: Schneider
authors.forename: John D
authors.initials: JD
authors.lastname: Lambris
authors.forename: Paola
authors.initials: P
authors.lastname: Magotti
authors.forename: Guttorm
authors.initials: G
authors.lastname: Haraldsen
authors.forename: Jörg D
authors.initials: JD
authors.lastname: Seebach
authors.forename: Tom E
authors.initials: TE
authors.lastname: Mollnes
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000276:immunology; D001011:Aorta / Q000276:immunology; D001773:Blood Cells / Q000276:immunology*; D002478:Cells, Cultured; D018920:Coculture Techniques; D015936:Complement C5a / Q000276:immunology; D016207:Cytokines / Q000276:immunology*; D019040:E-Selectin / Q000276:immunology; D042783:Endothelial Cells / Q000276:immunology*; D005700:Galactosyltransferases / Q000235:genetics / Q000276:immunology*; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000276:immunology*; D006801:Humans; D008954:Models, Biological; D013045:Species Specificity; D013552:Swine; D014183:Transplantation, Heterologous
chemical_list: D016207:Cytokines; D019040:E-Selectin; D015936:Complement C5a; D005700:Galactosyltransferases