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(1,3)Gal Expression in a Pig-to-Human Whole Blood Model1
* Institute of Immunology, Rikshospitalet University Hospital and Faculty of Medicine, University of Oslo, Oslo, Norway;
Laboratory of Transplantation Immunology, Department of Internal Medicine, University Hospital, Zurich, Switzerland;
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104;
Laboratory for Immunohistochemistry and Immunopathology, Division of Pathology, Rikshospitalet University Hospital and Faculty of Medicine, University of Oslo, Oslo, Norway; and
¶ Service of Immunology and Allergology, Department of Internal Medicine, Geneva University Hospital and Faculty of Medicine, Geneva, Switzerland
Transplants from 
1,3-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-
, MIP-1, MIP-1β, 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.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by The Norwegian Research Council, The Research Council of Rikshospitalet, The Norwegian Council on Cardiovascular Disease, The Family Blix Foundation, The Odd Fellow Foundation, Swiss National Science Foundation Research Grant 3200B0-109921, and National Institutes of Health Grants GM-62134 and AI-068730.
2 Address correspondence and reprint requests to Dr. Tom Eirik Mollnes, Institute of Immunology, Rikshospitalet University Hospital, N-0027 Oslo, Norway. E-mail address: t.e.mollnes{at}medisin.uio.no
3 Abbreviations used in this paper: HAR, hyperacute rejection; Gal, Gal1–3Gal; AHXR, acute humoral xenograft rejection; PAEC, porcine aortic endothelial cell; KO, knockout; PMN, polymorphonuclear cell; EC, endothelial cell; MFI, median fluorescence intensity; FGF, fibroblast growth factor; PDGF, platelet-derived growth factor; IP-10, IFN--inducible protein 10; VEGF, vascular endothelial growth factor.
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