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Cardiovascular Research 2003 59(3):685-694; doi:10.1016/S0008-6363(03)00457-7
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Copyright © 2003, European Society of Cardiology

Peroxisome proliferation-activated receptor-{gamma} ligands ameliorate experimental autoimmune myocarditis

Zuyi Yuana,*, Yan Liua, Yu Liua, Jijun Zhanga, Chiharu Kishimotob, Yanni Wanga, Aiqun Maa and Zhiquan Liua

aDepartment of Cardiovascular Medicine, First Hospital of Xi’an Jiaotong University, No. 1 Jiankang Road, Xi’an, Shaanxi 710061, China
bDepartment of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan

zuyiyuan{at}mail.xjtu.edu.cn

* Corresponding author. Tel.: +86-29-532-4021; fax: +86-29-526-3190.

Received 11 December 2002; accepted 14 May 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) ligands have been shown to ameliorate a variety of inflammatory conditions. The present study tested the hypothesis that PPAR-{gamma} ligands reduce experimental autoimmune myocarditis (EAM) associated with inhibition of the expansion and activation of T cells, as well as suppression of the expression of proinflammatory cytokines. Methods and results: EAM was induced in Lewis rats by immunization with porcine cardiac myosin. PPAR-{gamma} ligands, 15-deoxy-{Delta}12,14-PGJ2 (15d-PGJ2) 200 µg/kg/day i.p. and pioglitazone (PIO) 10 mg/kg/day orally, were administered for 3 weeks to rats with EAM. The results showed that enhanced PPAR-{gamma} expression was prominently stained in the nuclear and perinuclear regions of infiltrating inflammatory cells. Administration of PPAR-{gamma} ligands markedly reduced the severity of myocarditis, as shown by comparing the heart weight/body weight ratio, pericardial effusion scores, macroscopic scores and microscopic scores. PPAR-{gamma} ligands suppressed myocardial mRNA expression of inflammatory cytokines and the expression of interleukin (IL)-1β protein in rats with EAM. In addition, 15d-PGJ2 and PIO treatment suppressed the proliferative response and interferon-{gamma} production of T cell-enriched splenocytes from rats with EAM. Furthermore, the cytotoxic activity and myocardiogenic potential of these T cells were inhibited by 15d-PGJ2 treatment. Conclusions: PPAR-{gamma} may play a role in the pathophysiology of EAM. PPAR-{gamma} ligands ameliorate the EAM associated with suppression of the expansion and activation of myocardiogenic T cells, as well as inhibition of the expression of proinflammatory cytokines. These results suggest that PPAR-{gamma} ligands such as 15d-PGJ2 and PIO may have the potential to modulate human inflammatory heart diseases such as myocarditis.

KEYWORDS Myocarditis; Immunity; T cells; PPAR-{gamma}; Cytokines


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Two mechanisms have been proposed to explain how myocarditis develops into dilated cardiomyopathy (DCM); one is a persistent viral infection, and the other is a progressive autoimmune myocardial injury [1]. Strong evidence supports a role of cellular immune mechanisms in the pathogenesis of myocarditis and subsequent DCM [2,3]. Experimental autoimmune myocarditis (EAM) in rodents has been reported to be T cell-mediated and the autoimmunity is transferable by active T cells [3]. EAM in rats mimics human fulminant myocarditis in the acute phase [4].

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcriptional factors that heterodimerize with the retinoid X receptor and bind to specific elements, termed PPAR-responsive elements, in target gene promoters [5]. PPARs have three isoforms, {alpha}, β (or {delta}), and {gamma}. PPAR-{alpha} regulates the genes involved in the β-oxidative degradation of fatty acids, whereas PPAR-{gamma} is expressed at high levels in adipose tissue and promotes adipocyte differentiation and glucose homeostasis. In addition, the expression of PPAR-{gamma} has been detected on macrophages, T cells, endothelial cells, vascular smooth muscle cells, and cardiac myocytes [6–9]. Recent data have shown that the natural prostaglandin (PG), 15-deoxy-{Delta}12,14-PGJ2 (15d-PGJ2), and synthetic antidiabetic thiazolidinediones (TZDs), which are PPAR-{gamma} ligands, suppress the T cell proliferative response in vitro [7] and inhibit inflammatory cytokine production by cells of monocyte/macrophage lineage [6,10]. These ligands inhibit gene expression in part by antagonizing the activities of transcription factors such as activator protein-1 (AP-1) and nuclear factor-{kappa}B (NF-{kappa}B) [6,10]. Importantly, PPAR-{gamma} ligands have been shown to ameliorate a variety of inflammatory conditions, including atherosclerosis [11], arthritis [12], inflammatory bowel disease [13], and experimental autoimmune encephalomyelitis [14,15].

To explore the role of PPAR-{gamma} ligands during the pathogenesis of EAM, we examined myocardial PPAR-{gamma} expression in rats with EAM. We also examined the effect of the PPAR-{gamma} ligands 15d-PGJ2 and pioglitazone on T cell proliferation and cytokine production in cultured splenocytes and lymph node (LN) cells, as well as on the cytotoxic activity of LN cells from rats with EAM. Our results demonstrate that the administration of PPAR-{gamma} ligands reduce myocardial inflammation, inhibit the activation and proliferation of T cell-enriched splenocytes, and suppress the myocardial proinflammatory cytokine expression in rats with EAM. These findings suggest that the anti-inflammatory and immunomodulating properties of PPAR-{gamma} ligands could provide therapeutic benefit in the treatment of myocarditis.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Induction of EAM in rats
Acute EAM was induced by immunization of 6-week-old Lewis rats with porcine cardiac myosin as previously described [16]. Control rats were immunized with Freund’s complete adjuvant (FCA) alone.

2.2 Treatment protocol
Rats with EAM were divided into three groups: 15d-PGJ2 (n=8), PIO (n=8), and Vehicles (n=8). 15d-PGJ2 was purchased from Cayman Chemicals and was administered daily by the i.p. route at 200 µg/kg/day [14] in 0.4 ml of sterile PBS for 3 weeks from day 1 to day 21 after immunization. Vehicles were injected with PBS. PIO tablets were purchased from a pharmacy, pulverized, and added to chow at 0.01% (w/w). Rats were provided free access to the diets and ingested approximately 20 g of chow per rat per day, providing approximately 10 mg/kg/day of PIO [15]. Normal controls were also prepared (n=7). Blood pressure and heart rate (HR) were determined by the tail-cuff method using a photoelectric tail-cuff detection system (Softron BP-98A, Tokyo, Japan). All rats were sacrificed on day 22.

The protocol was approved by the Institutional Animal Research Committee of Xi’an Jiaotong University and the care of animals was in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996).

2.3 Histopathology
At sacrifice, macroscopic findings were graded according to the following criteria: 0 (normal appearance), 1 (a focal discolored area), 2 (multiple or diffuse discolored areas not exceeding one-third of the heart), 3 (diffuse discolored areas not exceeding two-thirds of the heart), and 4 (diffuse discolored areas exceeding two-thirds of the heart). Pericardial effusion was graded: 0 (none), 1 (mild), and 2 (massive). After macroscopic examination, the heart was sliced transversely; a part of the ventricles was fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin to examine the degree of myocardial damage and infiltration of inflammatory cells. Microscopic findings were graded: 0 (normal), 1 (lesion extent not exceeding 1% of a transverse section), 2 (not exceeding 10%), 3 (not exceeding 50%), 4 (exceeding 50%). Three sections were obtained from each heart, and the mean score of the three sections was recorded as the microscopic score. We measured the lesion area using a square lattice scale in the eye lens of the microscope. Two observers scored the histopathological scores blindly. After pathologic examination, a part of the ventricles was embedded in OCT for immunohistochemistry; remanent ventricle tissues were kept at –80 °C for Western blotting and RNA extraction.

2.4 Ribonuclease protection assay
Total mRNA was extracted from the myocardium, and the cytokine mRNA levels were measured by ribonuclease protection assay (RPA) as described previously [17].

2.5 Western blotting
The myocardial lysates (10 µg protein/lane) were electrophoresed on 12% SDS–PAGE, and sequentially electrophoretically transferred to a membrane (Millipore). The membrane was incubated with anti-rat interleukin-1β (IL-1β) antibody (Secrotec), and then with a peroxidase-linked secondary antibody (Amersham). Chemiluminescence was detected and semiquantitatively analyzed using the NIH Image system.

2.6 Immunohistochemistry
We used an immunoperoxidase technique to perform immunohistochemistry for PPAR-{gamma}, IL-1β, and surface markers as described previously [16,17]. Primary antibodies: anti-PPAR-{gamma} (1:400, Santa Cruz, sc-7196), anti-IL-1β (1:200, Secrotec), ED1 (macrophages, Secrotec), W3/25 (CD4, Secrotec) and OX8 (CD8, Secrotec). IL-1β, macrophages, CD4 and CD8 positive-staining cells of heart tissue were counted blindly by two observers in six random fields at x400 magnification (within a 1 mm2 grid), and the total positive-staining cells of the six fields was recorded as the number of infiltrating cells in the lesions.

2.7 Cell culture
To culture splenocytes and LN cells, spleens and LNs were harvested from Lewis rats 12 days after immunization with cardiac myosin during the phase of the autologous regulatory process, and single-cell suspensions were obtained by passing through a stainless steel mesh screen. Cells were suspended in RPMI-1640 supplemented with fetal calf serum (FCS), 1% sodium pyruvate, 1% nonessential amino acids, 5x10–5 M 2-mercaptoethanol and penicillin–streptomycin mixture.

To culture neonatal rat cardiomyocytes, cardiac ventricles from 1- to 4-day-old Lewis rats were minced, and dissociated with 0.125% trypsin. Cardiomyocytes (2x104/well) were incubated in 96-well plates in 10% FCS-supplemented DMEM at 37 °C. Bromodeoxyuridine (100 µmol/l) was added during the first 48 h to prevent the proliferation of non-myocytes.

2.8 Proliferation assay and interferon-{gamma} measurement
Proliferative responses of T cell-enriched splenocytes and LN cells (2x105/well) were determined using porcine cardiac myosin in the presence of various concentrations of 15d-PGJ2, as indicated. The cells were incubated for 72 h. The wells were pulsed with [3H]thymidine at 0.5 µCi/well for the final 16 h of culture. Cells were harvested on glass-fiber filters, and incorporated [3H]thymidine was measured with a Betaplate counter. Results were determined as means from quadruplicate cultures.

For analysis of interferon-{gamma} (INF-{gamma}) production, supernatants were assayed using an antibody-sandwich enzyme-linked immunosorbent assay (ELISA) at the end of the culture period (R&D System).

2.9 Cytotoxicity assays
Lewis rats were immunized with cardiac myosin, treated with 15d-PGJ2 (200 µg/kg/day, i.p.), PIO (10 mg/kg/day, orally) or PBS, and killed on day 12. LN cells from rats treated with 15d-PGJ2, PIO or PBS were used as effector cells. Cardiomyocytes (2x104/well) plated in 96-micro-well plates were labeled with sodium chromate at 1 µCi/well (51Cr, Amersham International) for 1 h. After labeled target cells had been washed with PBS three times, LN cells were incubated at an effector–target ratio of 50:1, 100:1, and 200:1 for 4 h. The supernatant was collected and the radioactivity of 51Cr released into the supernatant was measured by a gamma counter. The percentage of cytotoxicity was calculated using the formula

Formula
where E is the counts per minute (cpm) released in the presence of effector cells, S is the spontaneous cpm released from target cells incubated in the medium, and M is the maximal cpm released from target cells incubated with 2% Triton X-100.

2.10 Adoptive transfer
Spleen and LN cells from normal rats, from rats with EAM and treated with 15d-PGJ2, or from rats with EAM and treated with PBS, respectively, were prepared as above and injected intraperitoneally into Lewis rats at a dose of 3x107 viable cells per animal. Three weeks later, rats were sacrificed and the histopathology was examined.

2.11 Statistics
All values are expressed as the mean±standard deviation (S.D.). Student’s t-test or one-way analysis of variance (ANOVA), followed by the Fisher protected least significant difference test, were performed. A value of P<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Myocardial PPAR-{gamma} expression in rats with EAM
To examine the expression pattern of PPAR-{gamma} in myocardium during the course of the acute stage of EAM, we performed an immunohistochemistry assay for PPAR-{gamma} expression. No immunoreactivity or only trivial immunoreactivity for PPAR-{gamma} was detected in myocardium of control rats (Fig. 1A). In rats with EAM, PPAR-{gamma} was strongly stained in infiltrating inflammatory cells, and the expression of PPAR-{gamma} was prominently located in the nuclear and perinuclear regions of inflammatory cells (Fig. 1B). Immunostaining with normal rabbit serum was completely negative in all animals (data not shown). Administration of PPAR-{gamma} ligands 15d-PGJ2 and PIO markedly suppressed the PPAR-{gamma} expression (Fig. 1C and D). These results suggest that PPAR-{gamma} might play a role in the pathophysiology of EAM.


Figure 1
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  		Fig. 1 Immunohistochemical staining for PPAR-{gamma} and histopathology in autoimmune myocarditis. (A–D) Immunohistochemical staining for PPAR-{gamma} in myocardium. (A) In normal control, no immunoreactivity or only trivial immunoreactivity for PPAR-{gamma} was detected. (B) In rats with EAM, PPAR-{gamma} was strongly stained in infiltrating inflammatory cells, and the expression of PPAR-{gamma} was prominently located in the nuclear and perinuclear regions of inflammatory cells (arrows). (C, D) Administration of PPAR-{gamma} ligands 15d-PGJ2 and PIO markedly suppressed the PPAR-{gamma} expression. (E–H) Histopathology. (E) Histopathology in a control (grade 0). (F) Representative histopathology in a rat with myocarditis treated with PBS, the myocardial inflammation is transmural (grade 4, arrowheads). Marked diffuse myocardial necrosis and cellular infiltration with multinuclear giant cells (inset, arrow) are shown in the inflammatory regions. (G) Representative histopathology in a rat with myocarditis treated with 15d-PGJ2. A small focus of cellular infiltration in the inflammatory region (arrowhead) is shown (grade 1). (H) Representative histopathology in a rat with myocarditis treated with pioglitazone. A small focus of cellular infiltration in the inflammatory regions (arrowhead) is shown (grade 1). (A–D) Original magnification, x200. (E–H) Hematoxylin and eosin, original magnification, x10; inset, x100.

 
3.2 PPAR-{gamma} ligands attenuate myocardial inflammation in rats with EAM
No rats died during the entire study period. On day 22 at sacrifice, the hearts showed severe and diffuse discolored myocarditis with massive pericardial effusion. Extensive injuries to myocytes with inflammatory changes, such as fragments of necrotic myocardial fibers, mononuclear cells, polymorphonuclear neutrophils, eosinophils, and multinucleated giant cells, were observed (Fig. 1F). Treatment with 15d-PGJ2 and PIO markedly reduced the severity of the disease, as assessed by measuring HW/BW, pericardial effusion, macroscopic and microscopic scores (Table 1 and Fig. 1G and H). The surface markers of infiltrating inflammatory cells in myocardium were detected by immunohistochemistry. The numbers of macrophages, CD4+, and CD8+ T cells recruited into the lesions were significantly reduced by 15d-PGJ2 and PIO treatment (Fig. 2). Taking these results together, PPAR-{gamma} ligand treatment suppressed the inflammatory infiltration of myocardium and ameliorated acute autoimmune myocarditis.


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  		Table 1 Hemodynamics, histopathology and heart weight/body weight ratio by PPAR-{gamma} ligands treatment in rats with EAM

 

Figure 2
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  		Fig. 2 Numbers of infiltrating cells in the lesions. Infiltrating macrophages, CD4+, and CD8+ T cells in the lesions were detected by immunostaining. The positive-staining cells were counted in six random fields at x400 magnification. The total positive-staining cells of the six fields was recorded as the number of infiltrating cells in the lesions. 15d-PGJ2 and PIO treatment significantly reduced the numbers of macrophages, CD4+, and CD8+ T cells recruited into the lesions. *P<0.05, **P<0.01 vs. Vehicle.

 
3.3 PPAR-{gamma} ligands suppress mRNA expression of inflammatory cytokines
In controls, the myocardial mRNA expression of cytokines was detected only for macrophage inhibitory factor (MIF) and interferon-{gamma}. In rats with EAM treated with PBS, mRNAs of Th1 cytokines (such as IL-18), Th2 cytokines (such as IL-6), and proinflammatory cytokines (such as MIF, interferon-{gamma}, IL-1β, and IL-1Ra) were markedly upregulated, and mRNA expressions of IL-1{alpha}, IL-12p35, IL-12p40 and IL-10 were slightly upregulated. Administration of 15d-PGJ2 and PIO markedly reduced the expression of cytokine mRNAs (Fig. 3), suggesting that PPAR-{gamma} ligand ameliorated EAM may be associate with inhibition of the expression of inflammatory cytokines.


Figure 3
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  		Fig. 3 Ribonuclease protection assay for mRNAs of Th1, Th2, and proinflammatory cytokines. In controls, the myocardial mRNA expression of cytokines was detected only for MIF and interferon-{gamma}. In myocarditis rats treated with vehicle, mRNAs of Th1 cytokines (such as IL-18), Th2 cytokines (such as IL-6), and proinflammatory cytokines (such as MIF, interferon-{gamma}, IL-1β, and IL-1Ra) were markedly upregulated, and mRNA expression of IL-1{alpha}, IL-12p35, IL-12p40, and IL-10 were slightly upregulated compared with controls. Treatment with 15d-PGJ2 and PIO markedly reduced the expression of cytokine mRNAs. INF-{gamma} indicates interferon-{gamma}. L32 and GAPDH are housekeeping genes. A representative finding of three distinct experiments is shown.

 
3.4 PPAR-{gamma} ligands suppress myocardial IL-1β protein expression in rats with EAM
Western blotting showed that myocardial IL-1β expression was upregulated 3.3-fold in rats with EAM treated with PBS compared with those in controls (Fig. 4A and B). Treatment with 15d-PGJ2 and PIO decreased the upregulated IL-1β expression. Immunohistochemistry showed that IL-1β immunoreactivity was strongly stained in infiltrating inflammatory cells (data not shown), and 15d-PGJ2 and PIO administration reduced the number of IL-1β-positive cells in the inflammatory lesions (Fig. 4). These results suggest that PPAR-{gamma} ligand treatment suppresses myocardial IL-1β protein expression in myocarditis.


Figure 4
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  		Fig. 4 Myocardial IL-1β protein expression by Western blot and immunostaining. (A) Western blot analysis. Ten micrograms of each protein sample were loaded. (B) Densitometric analysis of relative protein levels. In rats with myocarditis treated with PBS, the IL-1β protein expression was markedly increased; 15d-PGJ2 and PIO treatment decreased IL-1β expression. Values are derived from four animals and represented as percentage of controls. (C) The positive-staining cells of IL-1β were counted in six fields at x400 magnification. The numbers of IL-1β-positive cells in the lesions were significantly reduced by 15d-PGJ2 and PIO treatment. *P<0.05, **P<0.01 vs. Control; {dagger}P<0.01 vs. Vehicle.

 
3.5 PPAR-{gamma} ligands suppress splenocyte and LN cell proliferative response and INF-{gamma} production
Because EAM is caused by cardiac myosin-specific CD4+ T cells, we sought to determine whether 15d-PGJ2 and PIO had an effect on T cell proliferation. T cell-enriched splenocytes and LN cells from rats with EAM treated with 15d-PGJ2 and PIO exhibited a lower basal proliferation rate than those from rats with EAM treated with PBS (Fig. 5A). T cells from rats with EAM treated with 15d-PGJ2 and PIO were able to respond to cardiac myosin-specific stimulus by proliferation (Fig. 5B), indicating that they were energized rather than eliminated. Addition of 15d-PGJ2 to cells reduced the T cell proliferative response in the absence of stimulation and with cardiac myosin stimulation. Also, both basal and myosin-induced INF-{gamma} production were reduced in 15d-PGJ2-treated and PIO-treated rats compared with PBS-treated rats; addition of 15d-PGJ2 to cultures inhibited production in the absence of stimulus and with myosin-induced INF-{gamma} (Fig. 5C and D). These results suggest that 15d-PGJ2 and PIO inhibit cardiac myosin-specific T cell proliferation and Th1 responses in EAM.


Figure 5
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  		Fig. 5 15d-PGJ2 and pioglitazone suppress splenocyte and LN cell proliferative response and INF-{gamma} production. (A, B) T cell-enriched splenocytes and LN cells from rats with EAM treated with 15d-PGJ2 and pioglitazone exhibited a lower basal proliferation rate than those from rats with EAM treated with PBS. T cells from rats with EAM treated with 15d-PGJ2 and pioglitazone were able to respond to a cardiac myosin-specific stimulus by proliferation. Addition of 15d-PGJ2 to cells reduced the T cell proliferative response in the absence of stimulation and with cardiac myosin stimulation. (C, D) Both basal and myosin-induced INF-{gamma} production were reduced in 15d-PGJ2-treated and pioglitazone-treated rats compared with PBS-treated rats, and addition of 15d-PGJ2 to cultures inhibited production in the absence of stimulation and with myosin-induced INF-{gamma}. *P<0.01 vs. PBS-treated rats.

 
3.6 PPAR-{gamma} ligands suppress the cytotoxic activity of LN cells from rats with myocarditis
The cytotoxic activities of LN cells against cardiomyocytes, the specific antigen, were examined (Fig. 6). At an E/T ratio of 200:1, the cytotoxic activities of LN cells from rats immunized with cardiac myosin and treated with 15d-PGJ2 or PIO were significantly suppressed compared with those from rats immunized with cardiac myosin and treated with PBS (9.8±2.4% for 15d-PGJ2 treated, 10.9±5.8% for PIO treated vs. 36.9±6.9% for PBS treated, n=4 each, P<0.01). These findings suggest that PPAR-{gamma} ligands protect rats from myocarditis associated with suppression of the cytotoxic activity of immune effector cells.


Figure 6
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  		Fig. 6 15d-PGJ2 and pioglitazone suppress the cytotoxic activity of LN cells from rats with myocarditis. The cytotoxic activities of LN cells against cardiomyocytes, the specific antigen, were examined. At an E/T ratio of 200:1, the cytotoxic activities of LN cells from rats immunized with cardiac myosin and treated with 15d-PGJ2 and pioglitazone were significantly suppressed compared with those from rats immunized with cardiac myosin and treated with PBS. *P<0.01 vs. PBS-treated rats.

 
3.7 15d-PGJ2 inhibits the myocardiogenic potential of splenocytes and LN cells
Because 15d-PGJ2 inhibited the proliferation and INF-{gamma} production of cardiac myosin-specific T cells in vitro and in vivo, we then addressed whether this also inhibited the ability of these T cells to adoptively transfer EAM. When splenocytes and LN cells from rats with EAM treated with 15d-PGJ2 or PBS were transferred into Lewis rat recipients, cells from 15d-PGJ2-treated rats presented a lower myocardiogenicity which resulted in a decrease in the severity of disease compared with those from PBS-treated rats (Table 2). No evidence of myocarditis was observed in rats by adoptive transfer from control rats. These results suggest that 15d-PGJ2 can reduce the myocardiogenic potential of cardiac myosin-specific T cells


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  		Table 2 Incidence and histopathology of adoptively transferred EAM in rats

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present findings clearly demonstrate that PPAR-{gamma} ligands markedly ameliorate the severity of EAM in rats, which was associated with suppression of the proliferative response, cytokine secretion, and cytotoxic activities of T cells, and suppression of myocardial proinflammatory cytokine expression.

In the present study, immunohistochemical results showed that PPAR-{gamma} expression was strongly stained in infiltrating inflammatory cells, and the expression of PPAR-{gamma} was prominently located in the nuclear and perinuclear regions of inflammatory cells. Although Takano et al. reported that PPAR-{gamma} was expressed in neonatal normal cardiac myocytes [7], no immunoreactivity or only trivial immunoreactivity for PPAR-{gamma} was detected in myocardium of normal rats in the present study. Administration of PPAR-{gamma} ligands ameliorated EAM in rats and the PPAR-{gamma} expression of inflammatory cells in myocardial tissue was reduced. These results suggest that PPAR-{gamma} may play a role in the pathophysiology of EAM, and PPAR-{gamma} ligands have the potential to suppress myocardial inflammation in autoimmune myocarditis.

PPAR-{gamma} ligands have previously been shown to inhibit the antigen-stimulated responses of T cells [7]. A more recent study has shown that 15d-PGJ2 can inhibit T cell proliferation and the ability of those T cells to transfer experimental autoimmune encephalomyelitis when T cells were cultured with 15d-PGJ2 in vitro [14]. These results are consistent with our data, which showed that PPAR-{gamma} ligands, 15d-PGJ2 and PIO, suppressed the T cell proliferation and cytokine secretion in response to cardiac-specific myosin in myocarditis in vitro and in vivo. In addition, 15d-PGJ2 administration inhibited the ability of those T cells to transfer EAM in vivo.

Extensive cardiomyocyte injury with macrophages CD4+ and CD8+ were shown in rats with EAM. T cells play an important role in EAM [2,3], and the transfer of T cell-enriched splenocytes from rats with EAM induced myocarditis [3]. The cytotoxicity assay indicated that PPAR-{gamma} ligands reduced the cytotoxic responses of LN cells to specific antigens (cardiomyocytes), suggesting that PPAR-{gamma} ligand treatment ameliorated EAM, due, in part, to the suppression of the cytotoxic activities of immune effector cells.

It has been demonstrated that inflammatory cytokines play an important role in the development of inflammatory myocardial diseases [18,19]; IL-1β promotes coxsackie B3-induced myocarditis in resistant B10.A mice [20]. Suppression of inflammatory cytokines has beneficial effects on ameliorating acute myocarditis [17,19]. In the present study, administration of 15d-PGJ2 and PIO suppressed the myocardial mRNA expression of inflammatory cytokines and the expression of IL-1β protein in myocarditis. These results are consistent with reports that PPAR-{gamma} ligands inhibit proinflammatory cytokine INF-{gamma}, IL-1β, and TNF-{alpha} expression in isolated human CD4+ T cells [6,21]. Accordingly, in the present study, 15d-PGJ2 and PIO attenuated myocardial inflammation in EAM may, at least partly, be due to the suppression of inflammatory cytokines.

PPAR-{gamma} activators have previously been shown to effectively inhibit inflammatory disorders [11–15]. More recent studies have shown that PPAR-{gamma} ligands can reduce experimental myocardial infarction size [22] and attenuate left ventricular remodeling and failure after myocardial infarction [23]. Various mechanisms have been proposed to explain these therapeutic efficacies. Mechanistically, PPAR-{gamma} ligands act, at least in part, by inhibiting the activity of transcription factor AP-1, signal transducers and activators of transcription-1 (STAT-1), and NF-{kappa}B [10,24–26]. In the case of NF-{kappa}B, PPAR-{gamma} ligands inhibit the activity of I{kappa}B kinase, which normally phosphorylates the NF-{kappa}B inhibitor, I{kappa}B, resulting in trans-activation of NF-{kappa}B target genes [25,26]. Inhibition of NF-{kappa}B can attenuate EAM in rats [27].

There are a number of other possibilities to explain how PPAR-{gamma} ligands mediate its therapeutic effect in vivo. For example, it has recently been shown that 15d-PGJ2 inhibited the production of TNF-{alpha} and NO, and also inhibited the production of IL-12, which is important for the differentiation of T cells to a Th1 phenotype [28], and Th1 dominant is believed to play a pivotal role in EAM pathogenesis. 15d-PGJ2 also inhibited the secretion of chemokines, e.g. INF-inducible protein-10, monokine induced by INF-{gamma}, and INF-inducible T cell {alpha}-chemoattractant by endothelial cells [29]. These chemokines have been shown to play an important role in T cell recruitment to the sites of inflammation. Thus, another possibility for the suppression of EAM by PPAR-{gamma} ligands is their ability to inhibit the molecules that contribute to the development of inflammatory cell infiltration.

Overall, our results demonstrate that PPAR-{gamma} ligands suppress EAM. It probably accomplishes this through inhibition of the expansion and activation of myocardiogenic T cells as well as through inhibition of the expression of proinflammatory cytokines. Although PPAR-{gamma} ligands were administered simultaneously with myosin immunization and therefore were only preventive in this study, considering the fact that PPAR-{gamma} ligands have anti-inflammatory and immunomodulating effects on myocarditis, PPAR-{gamma} ligands such as 15d-PGJ2 and PIO may have the potential to modulate human inflammatory heart diseases such as myocarditis.

Time for primary review 27 days.


   Acknowledgements
 
Supported by the Natural Science Foundation of China (30170371).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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