Treatment of relapsing paralysis in experimental encephalomyelitis by targeting Th1 cells through atorvastatin

Abstract

Statins, known as inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, exhibit numerous functions related to inflammation, such as MHC class II down-regulation, interference with T cell adhesion, and induction of apoptosis. Here we demonstrate that both subcutaneous and oral administration of atorvastatin inhibit the development of actively induced chronic experimental autoimmune encephalomyelitis in SJL/J mice and significantly reduce the inflammatory infiltration into the central nervous system (CNS). When treatment was started after disease onset, atorvastatin reduced the incidence of relapses and protected from the development of further disability. Both the reduced autoreactive T cell response measured by proliferation toward the encephalitogenic peptide PLP139-151 and the cytokine profile indicate a potent blockade of T helper cell type 1 immune response. In in vitro assays atorvastatin not only inhibited antigen-specific responses, but also decreased T cell proliferation mediated by direct TCR engagement independently of MHC class II and LFA-1. Inhibition of proliferation was not due to apoptosis induction, but linked to a negative regulation on cell cycle progression. However, early T cell activation was unaffected, as reflected by unaltered calcium fluxes. Thus, our results provide evidence for a beneficial role of statins in the treatment of autoimmune attack on the CNS.

Figure 1.

Atorvastatin suppresses the clinical severity of EAE in a preventive and therapeutic manner. (A) For the preventive treatment paradigm, 200 μg atorvastatin (n = 12; shaded circles) or vehicle alone (n = 12; open circles) were injected subcutaneously daily from the day of immunization onward. Both the maximum disease scores and the mean clinical disease scores were significantly reduced in the atorvastatin group. (B) For the nonpreventive therapy, mice were randomized into two groups (n = 8 for both groups) after the establishment of disease at day 10 and subsequently treated. Significant differences were noted for the maximum disease scores, the relapse incidence, and the mean disease scores for the time of the relapse. (C) Mice were treated subcutaneously with 20 μg atorvastatin (n = 10 for atorvastatin group, n = 7 for vehicle group). (D) For oral therapy, mice were treated with 200 μg (n = 7 for both groups). For all trials, treatment periods are indicated by horizontal hatched bars. The treatment effect was statistically significant as outlined in the marked range for all presented EAE courses (*, P < 0.05, analysis of variance).

Figure 2.

Decreased inflammation in spinal cords of atorvastatin-treated mice. Representative histology of spinal cord longitudinal sections was obtained from the EAE course presented in Fig. 1 A. Inflammatory infiltration was visualized by hematoxylin and eosin staining. (A) Vehicle-treated animals showed typical perivascular infiltrates (arrowheads) comprised of lymphocytes and macrophages. (B) In contrast, atorvastatin-treated animals showed a markedly reduced inflammatory reaction. Average number of inflammatory foci/ocular field ± SEM in spinal cords of atorvastatin- and vehicle-treated mice as detected in hematoxylin and eosin–stained sections (P < 0.01, Student's t test).

Figure 3.

Atorvastatin treatment in vivo suppresses the PLP-specific priming response. Three mice per group were treated with either atorvastatin (200 µg per mouse) or vehicle from day 1 after immunization with PLP as described in Materials and Methods. (A) Proliferation of splenocytes in response to PLP (at the indicated concentrations) was assessed in quadruplicate cultures and expressed as stimulation index (mean ± SEM; atorvastatin, shaded bars; vehicle, open bars). (B) Accordingly, IL-2 was determined in the supernatants. Whereas IL-12 (C) as well as IFN-γ (D) were found at lower concentrations in the atorvastatin-treated mice than in those vehicle-treated, levels of IL-4 (E) and IL-10 (F) were higher in supernatants from splenocytes after treatment with atorvastatin. Data are presented as mean ± SEM; differences between atorvastatin and vehicle treatment are significant (P < 0.05; Student's t test).

Figure 4.

Atorvastatin inhibits antigen-dependent proliferation of murine and human TCLs. The murine CD4⁺ PLP-specific TCL Je1 (A) and the human BP-specific TCL MB8 (B) were stimulated with (+) or without (−) antigen presented by irradiated autologous APC in the absence or presence of different concentrations of atorvastatin (shaded bars) or vehicle alone (open bars), and proliferation was assessed. Striped bars correspond to controls without atorvastin or vehicle.

Figure 5.

Pathways used by atorvastatin to interfere with T cell cycle. Hatched bars (A and B) correspond to controls without atorvastatin, vehicle, or l-mevalonate. (A) The representative human BP-specific TCL MB7 was stimulated with (+) or without (−) antigen presented by irradiated autologous APC. Atorvastatin (shaded bars) or vehicle (open bars) was added immediately (0 h) or at later stages after 24 or 48 h, respectively. (B) MB7 was stimulated with (+) or without (−) anti-CD3/CD28 in the presence or absence of atorvastatin or vehicle, respectively. Gray bars indicate the addition of l-mevalonate (200 μM). (C) Accordingly, IFN-γ was measured in the supernatant. The cytokine levels, derived from cells treated with (gray bars) or without (black bars) l-mevalonate, are given in relation to vehicle control (dotted line). (D) The representative MBP-specific TCL EG8 is demonstrated as an example for the measurement of Ca²⁺ influx by flow cytometry. Cells were incubated for 24 h in the absence (control) or presence of atorvastatin and Ca²⁺ influx was monitored by a Tg model of CRAC activation (Materials and Methods). Gd³⁺, a Ca²⁺ entry blocker, was used as a positive control. (E) Expression of CDK4 was assessed 24 h after anti-CD3/CD28 stimulation of the representative MBP-specific TCL EG7 by immunoblotting. (F) MB7 was used to demonstrate the CDK inhibitor p27^kip1 regulation by atorvastatin and mevalonate blockade. (G) Jurkat T cells were incubated with (+) or without (−) 10 U/ml murine CD95 ligand as controls (hatched bars) or with the indicated concentrations of atorvastatin or vehicle, respectively. After 24 h, specific DNA fragmentation was measured by flow cytometry.