深見 直彦

Using a murine model, we demonstrated that endobronchial administration of antibodies (Abs) to major histocompatibility complex (MHC) class I results in cellular infiltration, epithelial metaplasia, fibrosis and obstruction of the small airways (obliterative airway disease [OAD]) mediated predominantly by Th17 responses to self-antigens. This resembles bronchiolitis obliterans syndrome developed following human lung transplantation. Since B cells play a crucial role in induction of autoimmune responses, we defined the role of B cells and its antigen presenting properties in induction of OAD in this study. Anti-MHC class I was administered endobronchially in B-/- and wild-type mice. In contrast to wild type, B-/- animals did not demonstrate cellular infiltration, epithelial metaplasia and obstruction of airways following anti-MHC. Frequency of K-a1 tubulin and CollagenV-specific IL-17 cells was significantly decreased in B-/- mice. As expected, Abs against self-antigens and germinal center formation were not developed in B-/- mice. Thus, we conclude that B cells and its antigen presenting capacity play an important role in induction of immune responses to self-antigens and immunopathogenesis of OAD following the administration of anti-MHC. Therefore, strategies to block B-cell and its antigen presenting functions should be considered for preventing the development of chronic rejection.

Introduction: Mizoribine (MZR) is an inosine monophosphate dehydrogenase inhibitor. It has been widely used in Japan in the treatment of autoimmune diseases and is known to inhibit T and B cell proliferation. The aim of this study was to evaluate the efficacy of MZR as an immunosuppressive agent and determine its ability to synergize with a commonly used calcineurin inhibitor Cyclosporine A (CsA) in prolonging survival of murine islet cells and heart transplanted across major histocompatibility barrier. Methods: Murine allogeneic islet cell transplantation between Balb/c donor mice and C57BL/6 recipient mice and heterotopic heart transplantation was done between C3H/Fle donor mice and Balb/c recipient mice. Recipients were divided into groups based on immunosuppression: Group 1-No immunosuppression, Group 2-MZR alone (20 mg/kg/day), Group 3-CsA alone (20 mg/kg/day), Group 4-MZR + CsA (20 mg/kg/day). Donor specific IFN-gamma, IL-10, IL-2, IL-4 secreting cells were enumerated by ELISpot. Serum cytokine and chemokine concentration was measured by Luminex. Results: Islet cell allograft recipients treated with CsA and MZR had prolonged islet function compared to other groups [normoglycemia (blood glucose <200 mg/dL) up to 32 +/- 4 days, p<0.05]. Similarly, heart allograft survival was significantly improved in mice treated with CsA and MZR compared to other groups (50% 30-day survival, p = 0.04). Donor specific IFN-gamma, IL-4, IL-2 secreting cells were significantly decreased in recipients treated with CsA and MZR with marked increase in IL-10 secreting cells (p<0.05). There was also an increase in serum IL-10 with decrease in IFN-gamma, IL-4, IL-2, MCP-1, and IL-6 in mice treated with CsA and MZR Conclusion: MZR and CsA when used in combination are potent immunosuppressive agents in murine islet cell and heart transplantation models. These agents lead to a decrease in donor specific IFN-gamma with increase in IL-10 secreting cells leading to improved allograft survival and function. (C) 2011 Elsevier B.V. All rights reserved.

Background. Presence of donor-specific antibodies (Abs) is detrimental to posttransplant allograft function. Some sensitized recipients have successfully undergone transplantation after pretransplant conditioning regimen using plasmapheresis and/or intravenous immunoglobulin therapy, but underlying mechanisms that confer such allograft protection are undefined. Methods. We developed a single human leukocyte antigen (HLA)-mismatched heterotopic murine heart transplant model (HLA-A2 into HLA-A2-sensitized-C57BL/6) to determine whether pretreatment of donors with low concentration of HLA class I (W6/32) or control Ab (C1.18.4) will confer protection. Expression levels of survival genes, Bcl-2 and heme oxygenase-1, were analyzed by gene array analysis and quantitative real-time polymerase chain reaction. Expression levels of cytokine panel were analyzed by Luminex. Role of Bcl-2 in the induction of allograft protection was analyzed by silencing the Bcl-2 expression in the donor hearts using a small hairpin (shRNA) specific for Bcl-2. Results. Control Ab-pretreated hearts were rejected in less than 5 days demonstrating hemorrhage, Ab, and C4 deposition. In contrast, W6/32-pretreated hearts were rejected at 15 days (P < 0.05) that was prolonged to 25 days with antilymphocyte serum treatment. W6/32-pretreated hearts on day 5 exhibited increased expression of Bcl-2 (5.5-folds), Bcl-xl (5.5-folds), and heme oxygenase-1 (4.4-folds); decreased expression of ICAM-1, VCAM-1 (3.2-fold), along with reduced levels of cytokines interleukin (IL)-1 beta (4.4-folds), tumor necrosis factor alpha (3.7-folds), IL-6 (7.5-folds), IL-12 (2.3-folds) and chemokines monocyte chemotactic protein 1 (4.5-folds), MIG (4.4-folds), MIP-1 alpha (3.4-folds), and IL-8 (3.1-folds). Silencing of Bcl-2 in accommodated hearts before transplant resulted in loss of protection with rejection (9 +/- 3 vs. 15 +/- 2days, P < 0.05). Conclusion. Pretreatment of hearts with low levels of anti-HLA Abs increases expression of antiapoptotic genes that inhibits caspases, leading to decreased inflammatory cytokines and chemokines, which promote allograft survival.

Inflammatory insults following islet transplantation (ITx) hinders engraftment and long-term function of the transplanted (Tx) islets. Using a murine model of ITx, we determined the role of LMP-420, a novel TNF-alpha inhibitor, both individually and in combination with the immunosuppressant cyclosporinc A (CSA) in islet engraftment and survival. Diabetic C57BL/6 mice were Tx with 500 BALB/c islets under the kidney capsule. Four cohorts were used: LMP-420 only, CSA only, combination of LMP-420 and CSA (LMP+CSA), and control (n = 12 per cohort). Serial monitoring of blood glucose levels revealed that LMP+CSA (35 +/- 5 days) prolonged stable blood insulin levels compared to control (6 +/- 4 days). Immunohistology demonstrated that coadministration (LMP+CSA) results in a significant decrease in CD8(+) T-cell infiltration (LMP+CSA: 31 18 vs. control: 224 +/- 51 cells, p < 0.001). Scrum cytolcine analysis revealed that LMP-420 administration resulted in an increase in the anti-inflammatory cytokine IL-10 (2.5-fold), and a decrease in TNF-alpha (three-fold) with no change in IL-2. However, coadministration resulted in a marked decrease in both IL-2 and TNF-alpha (threefold) along with increase in IL-10 (threefold). Coadministration also demonstrated increase of antiapoptotic SOCS-1 and Mn-SOD expression and significant reduction of donor-specific antibodies (p < 0.005). In conclusion. LMP-420 administration with CSA results in the upregulation of anti-inflammatory and antiapoptotic mechanisms which facilitate islet allograft engraftment and survival.