Expression of Complement Regulatory Proteins ... - Wiley Online Library

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mediated lysis in vitro. Collectively, these studies sug- gest that the up-regulation of complement regulatory proteins may abrogate complement-mediated rejec-.
American Journal of Transplantation 2008; 8: 32–40 Blackwell Munksgaard

 C 2008 The Authors C 2008 The American Society of Journal compilation  Transplantation and the American Society of Transplant Surgeons

doi: 10.1111/j.1600-6143.2007.02016.x

Expression of Complement Regulatory Proteins in Accommodated Xenografts Induced by Anti-a -Gal IgG1 in a Rat-to-Mouse Model J. Wen Dinga , T. Zhoua , L. Maa , D. Yina , J. Shena , C. P. Y. Dinga , I. Y. Tangb , G. W. Byrnec and A. S. Chonga, ∗ a

Section of Transplantation, Department of Surgery, Section of Nephrology, Department of Medicine, University of Chicago, Chicago, IL, c Division of Cardiovascular Surgery, Mayo Clinic, Rochester, MN ∗ Corresponding author: Anita Chong, [email protected] b

Anti-graft antibodies are often associated with graft rejection. Under special conditions, grafts continue to function normally even in the presence of anti-graft antibodies and complement. This condition is termed accommodation. We developed a xenograft accommodation model in which baby Lewis rat hearts are transplanted into Rag/GT-deficient mice, and accommodation is induced by repeated i.v. injections of lowdose anti-a -Gal IgG 1 . The accommodated grafts survived a bolus dose of anti-a -Gal IgG 1 , while freshly transplanted second grafts were rejected. To study the mechanism of anti-a -Gal IgG 1 -mediated accommodation, both real-time PCR and immunohistochemical staining revealed elevated expression of DAF, Crry and CD59 in the accommodated grafts. In vitro exposure of rat endothelial cells to anti-a -Gal IgG 1 also induced the up-regulation of DAF, Crry and CD59, as revealed by Western blot analyses, and was associated with an acquired resistance to antibody and complementmediated lysis in vitro. Collectively, these studies suggest that the up-regulation of complement regulatory proteins may abrogate complement-mediated rejection and permit the development of xenograft accommodation. Key words: Accommodation, a -Gal, complement regulatory proteins, endothelial cells, monoclonal antibodies, xenograft Received 30 March 2007, revised. 07 September 2007 and accepted for publication 10 September 2007

Introduction The pathogenic activities of graft-reactive antibodies are illustrated by their ability to induce hyperacute, acute and 32

chronic rejection of xenografts and allografts. Paradoxically, the presence of graft-reactive antibodies does not invariably lead to graft rejection, and under special circumstances, grafts have been observed to function normally despite the return of high titers of graft-reactive antibodies (1–3). Graft accommodation has been reported to occur in both xenografts and allografts (4–6). The mechanism underlying accommodation is unknown, however, changes in the function of antibodies, in the expression of target antigen, and in the graft, which imparts resistance to injury, have been implicated (7,8). Attempts have been made to understand the biochemical basis for accommodation. Early studies by Dalmasso et al. indicated that human xenoreactive IgM was able to induce porcine endothelial resistance to complementmediated injury (9). Williams et al. reported that xenograft accommodation in a pig-to-baboon model was associated with a premature interruption of the complement cascade and heightened expression of heparan sulfate and syndecan-4-phosphate (10). In the hamster-to-rat model, in which xenograft rejection is controlled by cobra venom factor (CVF) and cyclosporine A, accommodation to T-cellindependent antibodies was postulated to be mediated by an elevated expression of HO-1 and the anti-apoptotic genes, A-20, Bcl-x L and Bcl-2 (3,6). A role of Bcl-2 and Bclx L , nitric oxide and adenosine A2 receptors has also been implicated in the acquired resistance of endothelial cells to antibody-mediated lysis (11,12). More recently, specific upregulation of cytoprotective genes such as nitric oxide synthase, Bcl XL and indoleamine 2,3 dioxygenase, and a poor in situ expression of immunoglobulin chain gene has been reported for long-term tolerated allografts induced by a 20-day treatment with a deoxyspergualin (DSG) analogue, LF15–0195 (13). Up-regulation of CD59 in pig endothelial cells after exposure to a-Gal binding lectins has also been reported to confer partial protection against complementmediated endothelial lysis (14,15). Microarray studies of accommodated ABO-incompatible renal grafts reported an up-regulation of a different set of genes, including SMADs, protein tyrosine kinases, TNF-alpha and mucin 1 (7). It is unclear whether these different gene associations with graft accommodation reflect fundamentally different accommodated states or differences resulting from different analytical approaches. Furthermore, in some of the studies involving human or nonhuman primate tissues, it is unclear whether differences in expression levels were

Mechanism of Anti-a -Gal IgG 1 -Mediated Accommodation

functional or simply served as markers of the accommodated state. To prevent by-stander cells from the deleterious effects of autologous complement activation, endothelial cells express a number of complement regulatory proteins, including the decay accelerating factor (DAF), membrane cofactor protein (MCP) and CD59. In rodent endothelial cells, MCP is replaced by complement receptor-related protein y (Crry), which has overlapping functional features with DAF and MCP (16). Thus, DAF and Crry inhibit complement activation at the C3 and C5 convertase level of both classical and alternative pathways, while CD59 prevents the formation of the membrane attack complex (MAC) (17–21). In a porcine-to-primate xenotransplantation model, hyperacute rejection (HAR) is mediated by the binding of xenoreactive natural anti-a-Gal antibodies to galactose a-1,3 galactose (a-Gal) epitopes expressed on endothelial cells (22). The binding results in the activation of the complement cascade, the destruction of the endothelial cells and thrombus formation in the donor organ vasculature. The transgenic expression of complement regulatory proteins, DAF, MCP and CD59, has been shown to effectively inhibit HAR (23– 27). Thus, we have hypothesized that graft accommodation to pathogenic titers of anti-a-Gal antibodies may be due to the enhanced expression of complement regulatory proteins. We have developed a xenograft accommodation model by transplanting baby Lewis rat hearts into Rag/ Galactosyltransferase-deficient mice (Rag/GT dKO mice), and inducing graft accommodation with repeated administrations of low doses of anti-a-Gal IgG 1 . Graft accommodation induced by anti-a-Gal IgG 1 was associated with an increased expression of the protective complement regulatory proteins, DAF, Crry and CD59 on the xenograft endothelium. The protective properties of these regulatory proteins were confirmed with in vitro assays.

Carlsbad, CA) and purified by saturated ammonium sulfate precipitation, as previously described (30). Both saturated ammonium sulfate and phosphate buffered saline (PBS) for dialysis were made in lipopolysaccharide (endotoxin)-free water. After precipitation of hybridoma culture supernatant with saturated ammonia sulfate overnight at 4◦ C, the monoclonal antibodies (mAbs) were collected by centrifugation at 20 000 × g for 60 min at 4◦ C. The purity and the isotype of the anti-Gal mAbs were examined by SDS-PAGE gel and by IsoStrip (Roche, Indianapolis, IN). The protein concentration was determined by a GeneSys spectrophotometer at 280 nm (Thermo Electron Corporation, Milford, MA). Endotoxin level in anti-a-Gal IgG 1 was