Amelioration of human allograft arterial injury by atorvastatin or simvastatin correlates with reduction of interferon-gamma production by infiltrating T cells

Abstract

Background: Graft arteriosclerosis (GA) is an important factor limiting long-term outcomes after organ transplantation. We have used a chimeric humanized mouse system to model this arteriopathy in human vessels, and found that the morphologic and functional changes of experimental GA are interferon (IFN)-gamma dependent. This study evaluated whether 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, described as inhibitors of IFN-gamma production, affect GA in our model.

Methods: C.B.-17 severe combined immunodeficiency-beige mice were transplanted with human artery segments as aortic interposition grafts and inoculated with allogeneic human peripheral blood mononuclear cells (PBMCs) or replication-deficient adenovirus encoding human IFN-gamma. Transplant arteries were analyzed from recipients treated with vehicle vs. atorvastatin or simvastatin at different doses. The effects of statins on T-cell alloresponses to vascular endothelial cells were also investigated in vitro.

Results: Graft arteriosclerosis-like arteriopathy induced by PBMCs was reduced by atorvastatin at 30 mg/kg/day or simvastatin at 100 mg/kg/day that correlated with decreased graft-infiltrating CD3+ T cells. Circulating IFN-gamma was also reduced, as were graft IFN-gamma and IFN-gamma-inducible chemokine transcripts and graft human leukocyte antigen-DR expression. Graft arteriosclerosis directly induced by human IFN-gamma in the absence of human PBMCs was also reduced by atorvastatin, but only at the highest dose of 100 mg/kg/day. Finally, atorvastatin decreased the clonal expansion and production of interleukin-2, but not IFN-gamma, by human CD4+ T cells in response to allogeneic endothelial cells in coculture.

Conclusions: Our results suggest that a benefit of statin administration in transplantation may include amelioration of GA primarily by inhibiting alloreactive T-cell accumulation and consequent IFN-gamma production and secondarily through suppression of the arterial response to IFN-gamma.

Figure 1. Atorvastatin and simvastatin effects on intimal expansion

Panels a and c show representative EVG stains (100X magnification) of human artery grafts from animals that received allogeneic PBMCs plus vehicle control demonstrating the characteristic myointimal proliferative lesions with associated lymphocytic infiltration. Panel b shows a representative result when atorvastatin (30 mg/kg/day) was administered simultaneously with PBMC introduction. Panel d shows similar findings with a higher dose of simvastatin (100 mg/kg/day). The intima area measurements from 6 individual experiments were used to calculate the mean ± SEM. Results from control animals (left columns, Panels e and f) were normalized to “1”, and each treatment group was described as a fraction of that value. Atorvastatin at 30 mg/kg/day (Panel e) and simvastatin at 100 mg/kg/day (Panel f) were associated with a significant reduction in mean intimal area (P <0.001; t-test).

Figure 2. Effects of atorvastatin and simvastatin on circulating cholesterol levels and human CD3⁺ lymphocytic infiltration of artery grafts

Plasma total cholesterol was determined for animals receiving PBMC plus vehicle control vs. atorvastatin at 30 mg/kg/day (Panel a) or simvastatin at 100 mg/kg/day (Panel b). Aggregate analysis has been expressed as the mean ± SEM. At the statin doses used in these normocholesterolemic recipients, there were no differences in plasma cholesterol values comparing control animals with those receiving atorvastatin or simvastatin (P = N.S.; t-test). CD3⁺ lymphocyte infiltration was also evaluated using immunohistochemistry in sections of artery grafts obtained from control animals receiving PBMC plus vehicle for comparison to those receiving atorvastatin at 30 mg/kg/day (Panel c) or simvastatin at 100 mg/kg/day (Panel d). Aggregate analysis (expressed as the mean ± SEM) of 6 individual experiments found a significant reduction in the number of infiltrating CD3⁺ cells per high power field (HPF) of grafts from statin-treated animals (P <0.05; t-test).

Figure 3. Effects of atorvastatin and simvastatin on IFN-γ production and responses

Artery-bearing animals received PBMC plus vehicle vs. atorvastatin at 30 mg/kg/day (Panel a) or simvastatin at 100 mg/kg/day (Panel b), and representative sections of the grafts were stained with mouse anti-human HLA-DR antibody and evaluated microscopically. Aggregate analysis (mean ± SEM) of statin-treated animals exhibited a significant reduction in HLA- DR⁺ graft cells per high power field (HPF) compared to the respective controls (P <0.05; t-test). Panel c describes the concentration of circulating IFN-γ (mean ± SEM) in artery-bearing animals receiving PBMC plus vehicle vs. PBMC plus atorvastatin at 30 mg/kg/day, showing a modest, but significant reduction of IFN-γ plasma levels among atorvastatin-treated animals (P <0.05; t-test). Panel d demonstrates that the amount of IFN-γ mRNA (mean ± SEM) was reduced to an even greater extent within the artery grafts from animals receiving PBMC plus atorvastatin at 30 mg/kg/day (P <0.05; t-test). Panel e demonstrates that the transcript expression (mean ± SEM) of the IFN-γ-inducible chemokine, Mig was decreased to a lesser extent than intra-graft IFN-γ mRNA in the same recipients (P <0.05; t-test). Panel f demonstrates that the expression of IFN-γ mRNA (mean ± SEM) in these artery grafts was not significantly reduced when normalized to the T cell marker, CD3ε (P = 0.10; t-test), suggesting that the effects of atorvastatin on accumulation of allogeneic T cells was greater than that on IFN-γ production.

Figure 4. Effects of atorvastatin on directly introducing IFN-γ

A human IFN-γ-encoding, replication-defective adenovirus was used to directly stimulate an inflammatory response in the absence of leukocytes. The resulting, robust intimal proliferative lesion (Panel a) was similar to that observed with allogeneic PBMC and was prevented only at the highest dose of atorvastatin of 100 mg/kg/day (Panel b). Aggregate analysis of 6 experiments (mean ± SEM) with control grafts from vehicle-treated animals indexed as a value of “1” demonstrates in Panel c essentially no response when atorvastatin at 30 mg/kg/day was given (P = N.S.; t-test), in contrast to Panel d where a modest, but significant reduction of intimal expansion was observed if 100 mg/kg/day was given (P <0.05; t-test).

Figure 5. Effects of atorvastatin on allogeneic T cell-EC co-cultures

CD4⁺ T cells proliferated in response to allogeneic, IFN-γ-pretreated, HLA-DR-expressing ECs as indicated by CFSE dilution in a subset of cells by 7 days of co-culture (Panel a). Addition of atorvastatin at 1 µM (Panel b) and 3 µM (Panel c) did not prevent T cell alloresponses, but at 10 µM (Panel d) abrogated T cell proliferation and formation of CFSE^low cells. Assessment of cytokine levels (n = 6) from the supernatants after 1 day of allogeneic T cell-EC co-culture demonstrated that the higher doses of atorvastatin inhibited the secretion of IL-2 (Panel e), but not of IFN-γ (Panel f) compared to vehicle alone (P <0.01; One-way ANOVA). The data shown are from a single pair of EC and T cell donors and are representative of three independent experiments.