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The Journal of Heart and Lung Transplantation Volume 25, Number 2S

treated B6 mice (MST⫽60d, n⫽5) and in both groups lacking class II with demonstrable chimerism [CI⫹CII⫺ (MST⫽60d, n⫽8) and CI⫺CII⫺ (MST⫽60d, n⫽4) mice]. BALB grafts were prolonged but not indefinitely in CI⫺II⫹ mice (MST⫽30d, n⫽4). Untreated B6 and CI⫺CII⫺ mice rejected grafts promptly. Preliminary data show prolonged BALB skin graft survival in FLC-treated class II deficient recipients with persistent chimerism. Conclusions: Intact recipient MHC class II expression prevents persistent microchimerism after neonatal allogeneic FLC injection, however lack of chimerism does not impair cardiac allograft acceptance. Skin graft survival is prolonged under conditions in which chimerism can persist, suggesting different mechanisms of acceptance of different organ allografts after neonatal treatment.*Tao and Mai contributed equally. Supported by Heart/Stroke Fdn Canada and PSI Fdn. 328 MACROPHAGE DEPLETION SUPPRESSES CARDIAC ALLOGRAFT VASCULOPATHY IN MICE W.H. Kitchens,1 C.M. Chase,1 S. Uehara,1 R.B. Colvin,2 P.S. Russell,1 J.C. Madsen,3 1Department of Transplant Surgery, Massachusetts General Hospital, Boston, MA; 2Department of Pathology, Massachusetts General Hospital, Boston, MA; 3 Department of Cardiac Surgery, Massachusetts General Hospital, Boston, MA Background: Chronic rejection is the primary long-term complication in cardiac allografts, where it manifests as diffuse arteriosclerosis known as cardiac allograft vasculopathy (CAV). Although T, B and NK cells are implicated in its pathogenesis, the identity of the endeffectors that fuel CAV development is ill-defined. Because of their abundant presence in CAV lesions and their capacity to produce growth factors implicated in neointimal cell proliferation, macrophages are leading candidates to serve as these end-effectors. Methods: Macrophages were depleted in a murine heterotopic cardiac transplant system known to develop fulminant CAV lesions. C57BL/6 hearts were transplanted into (C57BL/6 x BALB/c)F1 recipients, which then received an anti-macrophage therapy for 30 days before euthanasia. Results: Attempts to deplete macrophages with clodronate-encapsulated liposomes were abandoned because they provoked allograft thrombosis. However, intraperitoneal carrageenan and aspirin anticoagulation enabled the successful depletion of macrophages with minimal adventitious effects upon T, B, or NK cells as confirmed by flow cytometry and NK cytotoxicity assays. CAV development was prevented in 70% of carrageenan-treated recipients, a significant contrast with the universal CAV occurring in controls treated with aspirin alone. The suppression of CAV in carrageenan-treated recipients correlated with the degree of macrophage depletion achieved. Inhibition of macrophage phagocytosis alone with gadolinium chloride failed to prevent CAV in transplant recipients, suggesting that macrophages primarily contribute to CAV through cytokine and growth factor production. Finally, treatment with carrageenan starting 30 days post-transplant failed to reverse established CAV, revealing that macrophages are not required for the maintenance of CAV lesions. Conclusions: These findings offer a new target for immunosuppression and raise the possibility that anti-macrophage strategies in humans may improve the long-term outcome of transplanted organs. 329 TLR2 SIGNALING BLOCKS REGULATORY T CELL SUPPRESSION LEADING TO IMMUNE ACTIVATION FOLLOWING CARIDAC ALLOTRANSPLANTATION

Abstracts

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S. Ramachandran,1 T.A. Goers,1 W. Liu,1 G.A. Patterson,1 T. Mohanakumar,1,2 1Surgery, Washigton University School of Medicine, St. Louis, MO; 2Pathology & Immunology, Washigton University School of Medicine, St. Louis, MO Intracellular pathways mediated by toll-like receptors (TLRs) have been shown to play a crucial role in linking innate immunity to adaptive immune responses. Mechanisms by which TLR signaling contributes to rejection of allografts is still not defined. Our objective was to define the mechansim by which TLRs activate the innate immune system and contributes to acute rejection. BALB/c cardiac allografts were transplanted heterotopically in the abdomen of C57BL/6 mice and harvested on day 3. TLR expression profile was analyzed by quantitative real-time PCR. Activated NF-kB levels were determined by TransAM NF-kB family kit. BALB/c cardiac grafts were also transplanted heterotopically into TLR2⫺/⫺ and TLR4⫺/⫺ mice to define the roles of TLRs in acute rejection. Further, TLR2⫺/⫺ mice were depleted of regulatory T cells (Tregs) pretransplant to define the role of TLR2⫺/⫺ in immune regulation. Serum cytokine and chemokine levels were analyzed by mouse 25-plex cytokine and chemokine luminex assay. Significant increase in the levels of TLR2 (3.6 fold), TLR4 (2.3 fold) and activated NF-kB (3.7 fold) was observed by post-transplant day 3. The TLR2⫺/⫺ recipients had significantly prolonged allograft survival (11 days in TLR2⫺/⫺ vs 7 days in C57BL/6, p0.05) whereas no difference was observed in TLR4⫺/⫺ recipients. Treg depletion in TLR2⫺/⫺ mice resulted in rejection of the cardiac allografts within 7 days as observed in wildtype recipients. The levels of IL-6, IL-1␤ and MCP-1 (7.2, 2.6, 9.9 folds respectively, p0.05) were significantly lower in the TLR2⫺/⫺ recipients. Treg depletion in TLR2⫺/⫺ mice resulted in a similar cytokine profile as observed in wild type recipients. In summary, we have shown that early post-transplant TLR2mediated innate immune activation blocks the regulation mediated by Tregs resulting in a pro-inflammatory cytokine mileu leading to acute rejection of cardiac allografts. 330 SIRNA-MEDIATED SILENCING OF THE IL-1 AND ITS IMPACT ON THE PATHOGENESIS OF OBLITERATIVE AIWAY DISEASE FOLLOWING TRACHEAL TRANSPLANTATION T.A. Goers,1 S. Ramachandran,1 N. Benshoff,1 T. Mohanakumar,1,2 1Surgery, Washington University School of Medicine, Saint Louis, MO; 2Immunology and Pathology, Washington University School of Medicine, Saint Louis, MO Inflammatory cytokines play an important role in the development of experimental obliterative airway disease (OAD) after transplantation. Neutralization of these cytokines would aid both in delaying OAD development and prolonging allograft function. Short interfering RNAs (siRNAs) that have been shown to function as key intermediaries in triggering sequence-specific RNA degradation during posttrasciptional gene silencing. This project’s aim was to develop a 27 nucleotide IL-1 siRNA that resulted in reduced levels of IL-1 and maintenance of allograft patency in a murine tracheal transplant model. In vitro studies using MSHS-1, a monocyte cell line constitutively expressing IL-1␤ was used to screen and identify the siRNA that could inhibit IL-1 secretion in this cell line with and without LPS stimulus. C57BL/6 mice were then injected with varying concentrations of the identified IL-1 siRNA for dose optimization. A second group of mice who received the siRNA injections received tracheal transplants after 72 hours. They were sacrificed on post-transplant days 7 and 24. In vitro studies demonstrated that the levels of IL-1␤ were 2-fold lower in cells treated with IL-1␤2 siRNA compared to controls. More significantly, subsequent to LPS stimulation, cells