G.R. Hsieh

Chemokine monokine induced by IFN-γ/CXC chemokine ligand 9 stimulates T lymphocyte proliferation and effector cytokine production

Monokine induced by IFN-γ (MIG; CXC chemokine ligand (CXCL)9) is important in T lymphocyte recruitment in organ transplantation. However, it is not known whether this chemokine, in addition to its chemotactic properties, exerts any effect on T lymphocyte effector functions. For in vivo studies, we used a previously characterized murine model of chronic rejection. The recipient mice were treated with anti-MIG/CXCL9 Ab; graft-infiltrating cells were analyzed for IFN-γ production. For in vitro studies, exogenous CXCR3 ligands were added to CD4 lymphocytes in MLRs, and the proliferative responses were measured. Separate experiments quantitated the number of IFN-γ-producing cells in MLRs by ELISPOT. Neutralization of MIG/CXCL9, in the in vivo model, resulted in significant reduction in the percentage of IFN-γ-producing graft-infiltrating T lymphocytes. In vitro experiments demonstrated that 1) exogenous MIG/CXCL9 stimulated CD4 lymphocyte proliferation in a MHC class II-mismatched MLR, 2) MIG/CXCL9 also increased the number of IFN-γ-producing CD4 lymphocytes in ELISPOT, 3) neutralization of MIG/CXCL9 in MLR reduced T lymphocyte proliferation, 4) IFN-γ-inducible protein 10/CXCL10 and IFN-inducible T cell α chemoattractant/CXCL11 had similar effects on T lymphocyte proliferation, 5) MIG/CXCL9 stimulated T lymphocyte proliferation in MHC class I- and total MHC-mismatched MLRs, 6) neutralization of CXCR3 reduced MIG/CXCL9-induced T lymphocyte proliferation and the number of IFN-γ-positive spots on ELISPOT, and 7) the proliferative effects of MIG/CXCL9 were mediated via an IL-2-independent pathway and were controlled by IFN-γ. This study demonstrates that MIG/CXCL9 stimulates T lymphocyte proliferation and effector cytokine production, in addition to its chemotactic effects. This novel observation expands our current understanding of MIG/CXCL9 biology beyond that of mediating T cell trafficking.

Combined CXCR3/CCR5 blockade attenuates acute and chronic rejection.

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.

Inflammation/oxidation in chronic rejection: apolipoprotein a-i mimetic peptide reduces chronic rejection of transplanted hearts.

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-H2bm12 hearts were heterotopically transplanted into C57BL/6 mice. Recipient mice were treated with either 20 &mgr;g 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.

CD8 lymphocytes are sufficient for the development of chronic rejection

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)-&ggr; −/− 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-&ggr; −/− 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-&ggr;, but not the perforin or the FasL-mediated cytotoxicity, is the critical step in the development of intimal lesions.

Chemokine Receptor Blockade With a Synthetic Nonpeptide Compound Attenuates Cardiac Allograft Vasculopathy

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)-γ 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-γ 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-γ production, and (c) induction of HO-1 gene. This compound may offer a novel approach in clinical management of CAV.