Immunoregulatory effects of atorvastatin on experimental autoimmune myocarditis in Lewis rats

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1 Immunology and Cell Biology (2006) 84, doi: /j x Research Article Immunoregulatory effects of atorvastatin on experimental autoimmune myocarditis in Lewis rats WEI-MIN LI, 1 WEI LIU, 1 CHENG GAO 2 and BAO-GUO ZHOU 3 Departments of 1 Cardiology, 2 Neurosurgery and 3 Surgery, The First Affiliated Hospital, Harbin Medical University, Harbin, Heilongjiang, China Summary Experimental autoimmune myocarditis (EAM) in rats is a T-cell-mediated disorder and has been shown to involve immune imbalance. The aim of this study was to examine the immunomodulatory effects of 3-hydroxy-3- methyl-glutaryl coenzyme A reductase inhibitor, atorvastatin, on the expression of MHC class II molecules in the myocardium of rats with EAM, and to examine its therapeutic potential for EAM. EAM was induced in Lewis rats by injection of porcine cardiac myosin. High-dosage (10 mg/kg per day) or low-dosage (1 mg/kg per day) atorvastatin or vehicle was given orally for 3 weeks. On day 21 after immunization, echocardiography was carried out and the severity of myocarditis was evaluated by histopathological investigations. Immunohistochemistry techniques were used to examine the expression of MHC class II molecules in the myocardium. Type I, III and IV class II transactivator (CIITA) promoter transcription was evaluated by reverse transcription PCR. Cardiomyocytes were isolated and the expression of MHC class II molecules by them was detected using cytometry. Serum Th1/Th2 cytokines were examined on day 21 by ELISA. Cardiac function was improved in the two atorvastatin-treated groups compared with the untreated one. In atorvastatin groups, the histopathological severity of myocarditis was attenuated and the expression of MHC class II molecules on the nonprofessional APC, the cardiomyocytes, was reduced. mrna level of type IV CIITA promoter was downregulated in the statin-treated groups in a dosage-dependent manner, but levels of type I and III CIITA mrna did not differ between the groups statistically. Levels of IFN-g and IL-2 increased, whereas levels of IL-4 and IL-10 decreased, in immunized rats from day three through day 21. Atorvastatin reversed these trends in the treated groups. Atorvastatin improves cardiac function and histopathology of the myocardium in EAM by inducing Th2-biased immune responses, and thus 3-hydroxy-3-methyl-glutaryl coenzyme A reductase blockade may be a promising new strategy for the treatment of cardiac autoimmune impairments. The underlying mechanisms may be related to downregulation of MHC class II Ag expression due to silencing of the CIITA mrna transcription. Key words: autoimmunity, CIITA, HMG-CoA reductase inhibitor, MHC class II antigen, Th1/Th2 cytokine. Introduction Myocarditis is the major cause of sudden unexpected death in patients less than 40 years of age. In 30% of cases, myocarditis can give rise to dilated cardiomyopathy (DCM) with progression to heart failure, a major cause of morbidity and mortality among young adults, whose prognosis is poor with a 10 year survival rate of less than 40%. 1 It has been reported to be a CD4 1 T-cellmediated disease and is thought to be related to autoimmune pathogenesis. 2 Experimental autoimmune myocarditis (EAM) induced in rats is an animal model of human myocarditis and post-myocarditis DCM. In autoimmune situations, MHC class II molecules present autoantigens to Th cells and direct the subsequent cardiac injury. 3 Statins are widely used oral cholesterol-lowering drugs that competitively inhibit 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase, the enzyme that catalyses conversion of HMG-CoA to L-mevalonate, a key intermediate in cholesterol Correspondence: Wei Liu, Department of Cardiology, The First Affiliated Hospital, Harbin Medical University, Harbin, Heilongjiang , China. doctorliuwei@yahoo.com.cn Received 14 November 2005; accepted 29 December synthesis. Certain metabolites of mevalonate are also involved in post-translational modification of specific proteins involved in cell proliferation and differentiation. Thus, statins have important biologic effects that may be independent of their cholesterol-reducing properties. Recent studies indicate that statins have anti-inflammatory and immunoregulatory properties that may be beneficial in the treatment of multiple sclerosis as well as other autoimmune diseases. 4 This study was designed to examine the immunoregulatory effects of atorvastatin on MHC class II molecule expression in EAM and to elucidate their probable mechanisms. Materials and methods Induction and treatment of EAM model Male Lewis rats, aged 6 8 weeks, purchased from Beijing Vital River Laboratory Animal Technology, Beijing, China (certificate number SCXK ), were maintained in our animal facilities. Purified porcine cardiac myosin (Sigma Chemical, Shanghai, China) was dissolved in 0.01 mol/l PBS and emulsified with an equal volume of CFA supplemented with Mycobacterium tuberculosis H37RA at a concentration of 10 mg/ml. On days zero and Ó 2006 The Authors Journal compilation Ó 2006 Australasian Society for Immunology Inc.

2 Immunoregulatory effects of atorvastatin 275 Table 1 Results of echocardiography Group FS (%) LVEDd (mm) LVEDs (mm) IVSTd (mm) PWTd (mm) Group C (n = 6) 29.7 ± ± ± ± ± 0.89 Group H (n = 9) 41.3 ± 3.2* 4.93 ± 0.40* 3.05 ± ± 0.47* 1.36 ± 0.27* Group L (n = 8) 35.2 ± 2.9** 4.87 ± ± ± 0.33* 1.40 ± 0.69* Normal control (n = 3) 40.9 ± 6.7* 4.90 ± 0.53* 3.08 ± ± 0.61* 1.29 ± 0.33* *P < 0.05 compared with group C; **P < 0.05 compared with group H and normal control group. FS, fractional shortening; IVST, diastolic interventricular septum thickness; LVEDd, left ventricular end-diastolic diameter; LVEDs, left ventricular end-systolic diameter; PWTd, diastolic posterior wall septum thickness. seven, 26 rats were injected s.c. in the footpad with 0.2 ml emulsion. Three of them were used for adoptive transfer study. Three other rats that were never immunized were used as normal control (normal control group). Twenty-three rats induced as EAM were divided into three groups. High-dosage atorvastatin (10 mg/kg per day, group H, n = 9), low-dosage atorvastatin (1 mg/kg per day, group L, n = 8) or vehicle (group C, n = 6) was given orally by gastric gavage for 3 weeks from day 0 to day 21. Induction of EAM by adoptive transfer of purified porcine cardiac myosin-primed lymphocytes Adoptive transfer was carried out as follows. Briefly, Lewis donors were immunized twice with cardiac myosin on days zero and seven. Spleen cells were obtained on day 10 for in vitro stimulation. Splenocytes were cultured ( cells/ml) in RPMI-1640, 10% fetal bovine serum and 2.5 mg Con A for 48 h at 37 C. Con A-stimulated cells ( cells/ml) were injected i.v. into naive recipients. Recipient rats received 200 ng of pertussis toxin in 200 ml of PBS i.p. on days zero and two. All recipients were killed 10 days after transfer. Prevalence and disease severity were determined histologically on haematoxylin eosin (HE)-stained heart sections, and disease severity was scored as follows. 0, No inflammation; 1, presence of a few small lesions, not exceeding 5% cross-section area; 2, inflammatory lesions account for 5 10% cross-section area; 3, inflammation infiltrated 10 25% cross-section area; 4, inflammatory infiltration accounts for ³25% cross-section area. Where indicated, cultures also contained myosin (10 pg/ml) and recombinant murine IL-4 (R&D Systems, Minneapolis, MN, USA; 20 pg/ml) for Th2 deviation. Some of these cells were injected i.v. into naive Lewis recipients ( to viable cells per recipient) to test their disease-induction potential (adoptive transfer group, and myosin plus IL-4 adoptive transfer group). 1, presence of a few small lesions, not exceeding 0.25 mm 2 ; 2, presence of multiple small lesions or a few moderately sized lesions, not exceeding 6.25 mm 2 ; and 3, presence of multiple moderately sized lesions or more, larger lesions. Areas of the entire heart and region with infiltration by inflammatory cells and myocardial necrosis were determined with the NIH Image 1.63 program (National Institutes of Health, Bethesda, MD, USA), and the area ratio (affected area/entire area, as a percentage) was calculated. All histological evaluations were carried out in a double-blind manner by two professional staff. Expression of MHC class II molecules was examined with immunohistochemistry techniques, using clone OX6 mab as previously described. 5 Echocardiography A transthoracic echocardiogram was obtained on day 21 by using HP Sonos 2500 (Hewlett Packard, New Orleans, LA, USA) with a 10 MHz imaging linear scan probe transducer. Left ventricular (LV) end-diastolic diameter, LV end-systolic diameter and fractional shortening (FS) were determined in the M-mode recordings. Histopathological examination Twenty-one days after the immunization or 10 days after adoptive transfer, hearts were removed and processed for HE staining. After heartweight was measured, the ratio of heartweight to bodyweight was evaluated. Then, the heart was fixed with perfusion of 3.8% formaldehyde, embedded in paraffin, sectioned into 4 mm slices and stained with HE for histological examination. Microscopic scores of the severity of inflammation were classified into four grades: 0, no inflammation; Figure 1 Echocardiography results. (A) Typical 2-D results and M-mode results of the untreated Lewis rats with experimental autoimmune myocarditis (EAM). (B) Magnified M-mode results of the untreated EAM Lewis rats. (C) Magnified M-mode results of the EAM Lewis rats treated with high-dosage atorvastatin (10 mg/kg per day).

3 276 W-M Li et al. Table 2 rats Donor of DC Splenocytes from tolerized rats transferred tolerance to naive Recipient with EAM after challenge Isolation of cardiac APC and flow cytometry for cardiomyocyte MHC class II expression Mean score (0 4) ± SD Adoptive transfer 9/9 3.2 ± 2.1* group Myosin plus IL-4 adoptive 0/ D transfer group Naive 7/8 2.1 ± 1.1 Dendritic cells (DC) (10 6 ) were concomitantly challenged by active immunization with myosin emulsified with CFA after transfer, and rats were killed 10 days after challenge. Myocarditis was identified in the fixed heart sections by histopathological examination. *P < 0.05 for the adoptive transfer group versus the myosin plus IL-4 adoptive transfer group; DP < 0.05 versus the other two groups. EAM, experimental autoimmune myocarditis. Hearts were harvested on day 21. Isolation of EAM Lewis rat cardiomyocytes has been described previously. 6 Briefly, hearts were obtained from rats, minced finely, and subjected to sequential digestion with 0.25% pancreatin (GIBCO, Carlsbad, CA, USA) and 0.4% collagenase (Worthington Biochemical, Freehold, NJ, USA). The single-cell suspension was washed and depleted of endothelial cells and fibroblasts by two sequential 1 h adsorptions to plastic. The nonadherent population was retrieved for MHC class II flow cytometry. Rat cardiomyocytes were incubated with FITC-conjugated specific MHC class II antibody (60 min, 4 C) and analysed in a Becton Dickinson FACScan flow cytometer as described previously. 7 At least viable cells were analysed. Data were analysed using Cell- Quest software (Becton Dickinson, San Jose, CA, USA). Reverse transcription PCR mrna preparation and reverse transcription (RT) were carried out according to Ryan et al The following PCR primers were used: promoter (p)i class II transactivator (CIITA) sense: 5 -CCA GTT CAG CAA GCT GTT GCA G-3 ( ), antisense: 5 -CCA TGG CTA CCA GAG GAC CAC TT-3 ( ); piii CIITA sense: 5 -CAG AAG ACT GTG TGT CC-3 (4 20), antisense: 5 -CCT CAC ATG CCT CCT ATA CC-3 ( ); piv CIITA sense: 5 -TTG GGC TG A GCG TGA A-3 (3 18), antisense: 5 -GCA GTC TCC TGG CAG CTA CC-3 ( ); and internal control (elongation factor [EF]-1) sense: 5 -TGG ACA AAC TGA AAG CTG AGC G-3, antisense: 5 -TCT GCC CGT TCT TGG AGA TAC C-3. Thereafter, PCR was carried out as follows using TaKaRa LA Taq (TaKaRa, Otsu, Japan): cdna products of the RT reaction were denatured for 5 min at 94 C, and the amplification steps involved denaturation at 94 C for 60 s, annealing at 58 C for 90 s and extension at 72 C for 90 s. Thirty-four amplification cycles were carried out. ELISA Serum concentrations of IFN-g, IL-2, IL-4 and IL-10 were determined on days 3, 9, 15 and 21 with an ELISA kit (BioSource International London, UK) according to the manufacturer s instructions. The ratio of IFN-g to IL-4 was used as an indicator for Th1/Th2 polarization. Figure 2 Results of haematoxylin eosin (HE) staining of the myocardium ( 400). In the untreated experimental autoimmune myocarditis group (group C), severe myocardium destruction was found, accompanied by a large amount of inflammatory cell infiltration. Atorvastatin treatment lessened the inflammatory damage of cardiac tissue, with fewer inflammatory lesions witnessed (groups L and H). The HE staining of myocardium in the normal control group showed the appearance of a few mononuclear cells.

4 Immunoregulatory effects of atorvastatin 277 Figure 3 Immunohistochemistry results of MHC class II molecules in the myocardium ( 400). Expression of MHC class II molecules in the myocardium of the two atorvastatin-treated experimental autoimmune myocarditis (EAM) groups, groups L and H, was significantly lower than that in the untreated group C (P < 0.05). In the normal control group, which did not have induced EAM, the expression of MHC class II protein did not differ statistically; that is, atorvastatin has no significant effects on MHC class II molecule expression in normal situations. In the normal myocardium, expression of MHC class II is minimal, but has been found to be highly upregulated in EAM Lewis rats in our research. Statistical analysis All data are expressed as mean ± SEM. Mean difference among the groups was tested by one-way ANOVA. Analysis of the EAM grades (non-parametric) was carried out by Mann Whitney U-test. SPSS 11.0 was used for the statistical analysis (SPSS, Chicago, IL, USA). Values of P < 0.05 were considered statistically significant. Results Improvement of cardiac function by atorvastatin Echocardiographic results showed that FS was greatly impaired in group C, and that treatment with atorvastatin prevented the LV dysfunction and inhibited an increase in LV wall thickness (Fig. 1; Table 1). These results suggest that treatment with atorvastatin prevents development of LV remodelling and progression to heart failure following myocarditis. Cardiac function of myosin plus IL-4 adoptive transfer rats was markedly improved compared with the myosin adoptive transfer group (data not shown). Adoptive transfer results To directly address the question of whether the Th2 response regulates target organ susceptibility, adoptive transfer studies were carried out. Th2 deviation protected recipient rats from EAM induction in the adoptive transfer test (Table 2). Improvement of the myocarditis-affected areas and inhibition of MHC class II expression by atorvastatin Severe inflammatory lesions were observed in the hearts of group C and myosin-stimulated lymphocyte adoptive transfer rats. These lesions were composed of extensive myocardial necrosis and showed infiltration by mononuclear cells, polymorphonuclear neutrophils and multinucleated giant cells. In contrast, the hearts of group H and myosin plus IL-4-treated adoptive transfer rats showed little infiltration by inflammatory cells. These results suggest that treatment with atorvastatin Group C Group L Group H * * MHC class II-positive cardiomyocytes (%) Figure 4 Ratio of MHC class II-positive cardiomyocytes to total cardiomyocytes in the myocardium of groups C, L and H. *P < 0.05 versus group C;.P < 0.05 versus group L. Atorvastatin inhibited the expression of MHC class II molecules in a dosagedependent manner.

5 278 W-M Li et al. Table 3 Grey scale results of promoter (p) type I, III and IV class II transactivator (CIITA) reverse transcription PCR in the four groups Group C (n = 6) Group L (n = 8) Group H (n = 9) Normal control group (n =3) pi CIITA 0.96 ± ± ± ± 0.55 piii CIITA 0.89 ± ± ± ± 0.61 piv CIITA 2.36 ± ± 0.16* 0.15 ± 0.10* ± 0.13 *P < 0.05 versus group C; P < 0.05 versus group L. reduces the infiltration of inflammatory cells into the myocardium. Also, a Th2 bias was associated with EAM protection (Fig. 2; Table 2). Expression of MHC class II molecules in the myocardium of the two atorvastatin-treated groups was significantly lower than that in group C (P < 0.05), and this reduction is dosage related (Figs 3,4). Results of flow cytometry analysis of MHC class II expression in isolated cardiomyocytes The percentage of MHC class II-positive cardiomyocytes in the two atorvastatin-treated groups was 10.2 ± 3.3 (group H) and 24.9 ± 6.2 (group L), significantly lower than that in the untreated EAM rats (60.3 ± 9.5; P < 0.01). A marked difference was seen between group H and group L (P < 0.05), indicating a dosage-dependent effect of atorvastatin. Downregulation of CIITA-IV promoter expression Semiquantitative RT PCR showed that atorvastatin downregulated type IV CIITA promoter expression in the myocardium of EAM rats in a dosage-dependent manner, whereas the expression of neither type I nor type III CIITA promoter mrna differed statistically ( Fig. 5; Table 3). Modulation of Th1/Th2 cytokines by atorvastatin Production of Th1-type cytokines, including IFN-g and IL-2, significantly increased in the untreated group, whereas Th2 cytokines, IL-4 and IL-10, decreased markedly from day 3 through day 21. Atorvastatin treatment reversed this trend in groups H and L. The ratios of Th1 to Th2 subsets in EAM on day 21 were 3.69 ± 0.89 in group C, 0.62 ± 0.09 in group H, and 0.96 ± 0.12 in group L (P < 0.05). These results suggest that treatment with atorvastatin changes the Th-cell balance from Th1 to Th2 and inhibits pro-inflammatory cytokine production (Table 4). macrophages. However, to our knowledge, no study has systematically examined the effects of atorvastatin on the nonprofessional APC, the cardiomyocytes. The class II MHC molecules are transmembrane glycoproteins that consist of a and b chains that associate through noncovalent interactions. Class II MHC can be expressed by both professional and nonprofessional APC. Professional APC (e.g. dendritic cells, macrophages and B cells) constitutively express low levels of class II MHC proteins, and when they are stimulated with IFN-g, class II expression is increased. Nonprofessional APC do not normally express class II MHC unless they are treated with IFN-g. Class II MHC proteins play a major role in the induction of specific immune responses by presenting fragments of exogenous Ag to CD4 1 Th cells, which results in their activation and differentiation. Expression of class II MHC is primarily regulated at the transcriptional level. 9 CIITA is considered to be the master regulator of class II expression, and in general, the level of CIITA expression in a cell directly correlates to the level of class II MHC expression. Several compounds have been shown to inhibit class II MHC expression, and most of these attenuate expression by inhibiting CIITA transcription. TGF-b, IL-4 and IL-10 are cytokines with immunosuppressive properties that inhibit class II MHC at the level of CIITA transcription. In addition, many human pathogens downregulate class II MHC as a mechanism of evading the immune system. Unlike professional APC, including dendritic cells, macrophages and B cells, which express cell surface class II molecules constitutively, cardiomyocytes and other nonprofessional APC require stimulation by IFN-g for class II expression. Whether endothelial cells or cardiomyocytes participate in the activation and regulation of CD4 1 T cells under cardiac inflammatory conditions in vivo is not clear. In vitro, cardiomyocytes stimulated by IFN-g upregulate class II molecules and can present Ag to CD4 1 T cells, indicating that they are also capable of processing native myocardial autoantigens. In this study, Discussion Myocarditis is a principal cause of heart disease among young adults and is often a precursor of heart failure due to DCM. Activation and differentiation of T cells play a critical role in the pathogenesis of myocarditis and DCM. They are mediated primarily by CD4 1 Th1 cells that recognize myocardial self- Ag in association with MHC class II molecules expressed on the surface of APC. In the present study, we used the HMG- CoA reductase inhibitor atorvastatin for in vivo studies in an attempt to decrease the clinical and pathological symptoms of the EAM model. Earlier studies have described the downregulation of MHC class II molecule expression and secretion of pro-inflammatory cytokines on the professional APC, the EF-1 (internal control) piv CIIT A pi CIIT A piii CIIT A Figure 5 Typical photograph of promoter (p) type IV, I and III class II transactivator (CIITA) reverse transcription PCR. 1, 2: group C; 3, 4: group L; 5: group H.

6 Immunoregulatory effects of atorvastatin 279 Table 4 Levels of Th1/Th2 cytokines evaluated by ELISA Group C (n = 6) Group L (n = 8) Group H (n = 9) Normal control (n =3) IFN-g (pg/ml) 58.6 ± ± 5.1* 15.3 ± 3.6* 14.3 ± 4.3* IL-4 (pg/ml) 16.6 ± ± 2.4* 28.3 ± 2.2* 30.4 ± 5.6* IL-10 (pg/ml) ± ± 34.8* ± 54.7* ± 43.9* IFN-g/IL-4 ratio * 0.62* 0.49* *P < 0.05 versus group C. upregulated MHC class II protein in the myocardium of EAM was reduced in groups H and L, and so was type IV CIITA promoter mrna, indicating that atorvastatin inhibited MHC class II expression by downregulation of CIITA. Aberrant MHC class II expression on non-immune cells has been associated with multiple autoimmune diseases. The assumption emerged that aberrant class II expression allowed cells to become APC, interact with T cells and initiate an immune response. 10 Our study supports this hypothesis and suggests that the expression pattern of CIITA dictates most qualitative and quantitative aspects of MHC class II gene expression. Th lymphocytes mediate critical effector and regulatory functions in autoimmune diseases. Th cells possess clonal receptors that recognize antigenic peptides that are complexed with self-molecules of the MHC on the surface of APC. Our study shows that production of Th1-type cytokines, including IFN-g and IL-2, was inhibited by atorvastatin, accompanied by an increase in IL-4 and IL-10, which are Th2-type cytokines. This result is consistent with reports that atorvastatin increased Th2 response and suppressed Th1 response in an experimental encephalomyelitis model. 4 IL-10 is a pleiotropic cytokine produced by Th2-type T cells, B cells, monocytes and macrophages that inhibits a broad array of immune parameters, including Th1-lymphocyte cytokine production, Ag presentation and Ag-specific T-cell proliferation. Its effector functions include induction of a shift of T-cell cytokine expression from a Th1 to a Th2 profile, and attenuation of the production of proinflammatory cytokines by macrophages and polymorphonuclear neutrophils. With regard to the underlying mechanisms, statins inhibit IFN-g-inducible MHC class II expression on macrophages and block stimulation of T cells, both of which might result in suppression of activation of pro-inflammatory Th1 cells. 11 Also, atorvastatin promoted differentiation of Th0 cells into Th2 cells. In adoptive transfer, these Th2 cells protected recipient mice from experimental autoimmune encephalomyelitis (EAE) induction, which is supported by our results. 12 Furthermore, a subset of CD4 1 cells that possess T-regulatory cell properties (CD4 1 CD25 1 phenotype) rely heavily on IL-10 for their development. IL-10 can also impair processes of allograft rejection by downregulation of class II MHC expression on monocytes and B7 costimulatory ligands on macrophages. Treatment with atorvastatin inhibited inducible transcription at the type IV CIITA promoter and suppressed class II upregulation. In the normal myocardium, expression of MHC class II is minimal, but class II has been found to be highly upregulated in EAM Lewis rats in our research. The MHC class II molecules present normal as well as disease-related and pathogenderived peptides to T cells to alert the immune system of the health status of a cell. Th1 cytokines play critical roles in the differentiation of cardiac myosin-specific T cells and pathogenesis of EAM. Our report shows that the tight regulation imposed in vivo on the cell-type specificity and level of CIITA and MHC class II expression is a key parameter in determining the balance between Th1 and Th2 responses. Otten et al. reported that proper expression of the CIITA gene seems to be important in maintaining proper Th cytokine profiles. 13 Both transgenic overexpression and a complete absence of CIITA at the wrong time would perturb the programming of Th-cell differentiation. 14 Statins are reported to lessen inflammatory diseases in murine autoimmune models by inhibiting Th1 responses and to influence T-cell activation. 15 Therefore, statins could be useful for modulating T-cell immunity through inhibition of Th1 cytokine production in EAM. Recent evidences have suggested that atorvastatin is a negative regulator of the immune system. 15,16 Our results show that downregulation of CIITA in T cells inhibits Th1 differentiation and function, suggesting that the expression of CIITA in T cells might play a role in the regulation of the Th1/Th2 balance during the T-cell lineage commitment. As statins have mechanisms of action different from those of currently approved myocarditis or DCM treatments, they may be useful in combination therapy. Our results also provide a rationale for testing atorvastatin in other organ-specific Th1-mediated autoimmune diseases. Acknowledgement This study was funded by the Harbin Medical University Postgraduate Creation Foundation (2005). References 1 Liu PP, Mason JW. Advances in the understanding of myocarditis. Circulation 2001; 104: Bergelson JM, Cunningham JA, Droguett G et al. Isolation of common receptor for Coxsackie B viruses and adenoviruses 2 and 5. 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