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BACKGROUND.: Recruitment of recipient mononuclear cells to the donor heart is a central event in the development of cardiac allograft vasculopathy (CAV). The role of individual chemokine receptors in this process is incompletely defined. TAK-779 (N,N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbonyl]amino]benzyl]tetra-hydro-2H-pyran-4-aminium chloride) is a synthetic nonpeptide antagonist of CCR5 and CXCR3. The purpose of this study is to determine if combined CCR5 and CXCR3 blockade by TAK-779 would attenuate CAV in an experimental model. METHODS.: We used a previously characterized murine model of CAV. Recipient mice were treated with TAK-779 or vehicle control. Donor hearts were harvested on day 24 posttransplantation and analyzed for intimal thickening and cellular infiltration. Recipient splenocytes were analyzed for proliferative response and interferon (IFN)-gamma production by enzyme-linked immunosorbent spot assay. The donor hearts were also examined for heme oxygenase-1 (HO-1) gene induction. RESULTS.: CCR5 and CXCR3 blockade by TAK-779 reduced the severity of intimal lesions (53+/-10% vs. 16+/-2%; P<0.05) and decreased the number of graft infiltrating CCR5 and CXCR3 CD4 and CD8 lymphocytes. Moreover, treatment with TAK-779 (a) decreased alloantigen-specific T-lymphocyte proliferation and number of IFN-gamma producing cells and (b) increased HO-1 gene transcript level in the allografts. CONCLUSIONS.: Antagonism of CCR5 and CXCR3 by TAK-779 is effective in controlling CAV. The beneficial effects of TAK-779 may be because of (a) reduction in CCR5 and CXCR3 T-lymphocyte subset infiltration into the graft, (b) attenuation of alloantigen-specific T-lymphocyte proliferative response and IFN-gamma production, and (c) induction of HO-1 gene. This compound may offer a novel approach in clinical management of CAV.

Chemokine-chemokine receptor interactions orchestrate mononuclear cells recruitment to the allograft, leading to acute and chronic rejection. Despite biologic redundancy, several experimental studies have demonstrated the importance of CXCR3 and CCR5 in acute rejection of allografts. In these studies, deficiency or blockade of CXCR3 or CCR5 led to prolongation of allograft survival, yet allografts were ultimately lost to acute rejection. Given the above findings and the specificity of mononuclear cells bearing CXCR3 and CCR5, we hypothesized that combined blockade of CXCR3 and CCR5 will lead to indefinite (>100 days) graft survival in a full MHC-mismatched murine cardiac allograft model. The donor hearts in the control group were rejected in 6 +/- 1 days after transplantation. Combined blockade of CXCR3 and CCR5 prolonged allograft survival >15-fold vs the control group; all allografts survived for >100 days. More importantly, the donor hearts did not display any intimal lesions characteristic of chronic rejection. Further analysis of the donor hearts in the CXCR3/CCR5 blockade group demonstrated graft infiltration with CD4(+)CD25(+) T cells expressing the Foxp3 gene. Depletion of CD25(+) cells in the combined CXCR3 and CCR5 blockade group resulted in acute rejection of the allografts in 22 +/- 2 days. Combined CXCR3 and CCR5 blockade also reduced alloantigen-specific T lymphocyte proliferation. Combined CXCR3 and CCR5 blockade is effective in preventing acute and chronic rejection in a robust murine model. This effect is mediated, in part, by CD25(+) regulatory T cell recruitment and control of T lymphocyte proliferation.

Background: Chronic rejection in transplanted hearts or cardiac allograft vasculopathy (CAV) is the leading cause of late death among heart transplant recipients. Strategies to control CAV traditionally have focused on lymphocyte functions. We hypothesized that D-4F, an apoA-I mimetic peptide with potent anti-inflammatory/antioxidant properties, will attenuate CAV., Methods: We used a previously characterized murine model of CAV. B6.C-H2 hearts were heterotopically transplanted into C57BL/6 mice. Recipient mice were treated with either 20 microg of D-4F or carrier daily. Donor hearts were harvested on day 24 after transplantation., Results: Treatment of recipients with D-4F reduced the severity of intimal lesions (62.5+/-3.4% vs. 31.1+/-8.7%, P<0.009). Treatment also resulted in a decrease in the number of graft-infiltrating CD4 and CD8 lymphocytes and CXCR3+ T-lymphocyte subsets. Heme oxygenase-1 (HO-1) gene transcript in the donor hearts was up-regulated with D-4F treatment, and HO-1 blockade partially reversed the beneficial effects of D-4F. In vitro studies showed that D-4F reduced allogeneic T-lymphocyte proliferation and effector cytokine production. These processes were HO-1 independent., Conclusion: This study suggests that D-4F, a prototypical apoA-I mimetic peptide, is effective in controlling CAV via induction of HO-1 in the graft and a direct effect on T-lymphocyte function. This class of peptides with anti-inflammatory/antioxidant properties provides a novel strategy in the treatment of CAV.

Chronic rejection in transplanted hearts or cardiac allograft vasculopathy (CAV) is the leading cause of late death among heart transplant recipients. We hypothesized that induction of HO-1 by D4-F, an apoA-I mimetic peptide with potent antiinflammatory/antioxidant properties, attenuated CAV. We utilized a previously characterized murine model of CAV. B6.C-H2(bml2) hearts were heterotopically transplanted into C57BL/6 mice. In the control group, recipient mice were treated with 20 microg of saline daily. In experimental group I, mice were treated daily with 20 microg of D4-F. In experimental group II, mice were treated daily with 20 microg of D4-F daily, plus CuPP, which does not have any effect on HO-1 activity. In experimental group III, recipient mice were treated with 20 mug of D4-F daily, plus SnPP, which is a competitive inhibitor of HO-1. Donor hearts were harvested on day 24 after transplantation. The donor hearts in the control group developed severe intimal lesions. In experimental group I, treatment with D4-F was associated with upregulation of HO-1 and a marked reduction in intimal lesions, which was consistent in experimental group II. In experimental group III, inhibition of HO-1 was associated with partial restoration of intimal lesions. Induction of HO-1 by an apoA-1 mimetic peptide was effective to control CAV. This class of antiinflammatory peptides, which show an ability to induce HO-1, provides a novel strategy for the treatment of CAV.

Unlabelled: Chemokines are known to participate in allograft rejection by mediating leukocyte trafficking. Despite redundancy in chemokine family, several chemokine-chemokine receptor interactions have proven critical in alloimmune responses. We sought to determine the effect of combined blockade of CXCR3 and CCR5, two critical chemokine receptors, in acute rejection., Methods: Heterotopic heart transplantation was performed using BALB/c to B6/129 mice deficient in CCR5. Following transplantation these mice were treated with goat anti-CXCR3 serum every other day. In the control group, BALB/c hearts were transplanted in wild type B6/129 recipients and treated with goat serum alone. No immunosuppression was given to either group. Recipient mice were then assessed daily for allograft function by abdominal palpation, and graft survival was confirmed by laparotomy., Results: The donor hearts in the control group were rejected at 6 +/- 1 days posttransplantation. Combined blockade of CXCR3 and CCR5 prolonged allograft survival versus control; all allografts survived to 24 days. In addition, there was a decrease in graft infiltrating CD4 and CD8 lymphocytes in the experimental group at 24 days., Conclusion: Combined CXCR3 and CCR5 blockade is effective in prolonging allograft survival in a fully MHC mismatched murine model. Combined chemokine blockade holds promise in control of acute rejection in organ transplantation.

Background: The role of CD8 lymphocytes, in chronic rejection or cardiac allograft vasculopathy (CAV), is incompletely understood. The purposes of this study were to determine whether CD8 lymphocytes, in the absence of CD4 lymphocytes, are capable of causing the intimal lesions of CAV; and if so, to define the effector mechanism(s) of CD8 lymphocytes., Methods: We modified a previously characterized major histocompatibility complex class II mismatched murine model of CAV. Wild-type CD8 lymphocytes were transferred to nude mice followed by heterotopic heart transplantation. Recipient mice were then treated with a CD40 activating antibody, which is known to provide help for CD8 lymphocyte activation, in the absence of CD4 lymphocytes. Donor hearts were harvested on day 40 posttransplantation and analyzed for cellular infiltrates and intimal thickening. In separate experiments, isolated perforin -/-, Fas ligand (FasL) -/-, and interferon (IFN)-gamma -/- CD8 lymphocytes were transferred to nude mice followed by identical experimented protocol., Results: With adaptive transfer of wild-type CD8 lymphocytes, the donor hearts were infiltrated with activated CD8 lymphocytes and displayed significant intimal lesions. Adoptive transfer of perforin -/- and FasL -/- CD8 lymphocytes to nude mice resulted in similar patterns of CD8 lymphocyte infiltration and similar severity of intimal lesions. The donor hearts from IFN-gamma -/- CD8 lymphocyte reconstituted recipients displayed minimal intimal lesions, although CD8 lymphocytes were present in the allografts., Conclusions: Unprimed CD8 lymphocytes in the absence of CD4 lymphocytes can cause intimal lesions of CAV. CD8 lymphocytes production of IFN-gamma, but not the perforin or the FasL-mediated cytotoxicity, is the critical step in the development of intimal lesions.