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Cardiovascular Research 2003 59(1):95-104; doi:10.1016/S0008-6363(03)00334-1
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Copyright © 2003, European Society of Cardiology

Attenuation of experimental autoimmune myocarditis by blocking activated T cells through inducible costimulatory molecule pathway

Hideki Futamatsua, Jun-ichi Suzukia, Hisanori Kosugea, Osamu Yokosekib, Masafumi Kamadac, Hiroshi Itoa, Manabu Inobed, Mitsuaki Isobea,* and Toshimitsu Ueded

aDepartment of Cardiovascular Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
bThe First Department of Internal Medicine, Shinshu University School of Medicine, Nagano, Japan
cPharmaceutical Frontier Research Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco Inc., Kanagawa, Japan
dDivision of Molecular Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan

isobemi.cvm{at}tmd.ac.jp

* Corresponding author. Tel.: +81-03-5803-5951; fax: +81-03-5803-0238.

Received 12 December 2002; accepted 24 February 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Inducible costimulator (ICOS) is a member of the CD28 family. Although inflammation is an essential pathological feature of myocarditis, the role of ICOS in myocarditis remains unclear. Methods and Results: Lewis rats were immunized on day 0 with purified porcine cardiac myosin to establish experimental autoimmune myocarditis (EAM). Flow cytometry was used to examine expression of ICOS on myocardial infiltrating cells. Anti-ICOS antibody or ICOS-immunoglobulin (ICOSIg) was administered intravenously, and rats were killed on day 14 or 21 to study effects of ICOS/ICOS-ligand (ICOSL) pathway blockade during the antigen priming phase (days 0–14) or immune response phase (days 14–21), respectively. The heart weight to body weight ratio was determined, and histological examination and echocardiogram were performed to evaluate the severity of the disease. Cytokine expression in the heart and T cell proliferation against cardiac myosin were analyzed. Flow cytometry revealed that the majority of infiltrating cells, especially CD4-positive cells, expressed ICOS. Blockade of the ICOS/ICOSL pathway during the immune response phase attenuated EAM development. However, blockade of the ICOS/ICOSL pathway during the antigen priming phase did not attenuate and exacerbate EAM. Blockade of T cell activation through ICOS suppressed expression of cytokines including INF-{gamma}, IL-4, IL-6, IL-10, IL-1β, and TNF-{alpha} and inhibited T cell proliferation in vitro. Conclusions: Blockade of T cell activation through ICOS during the immune response phase regulates development of EAM, and therefore, ICOS may be an effective target for treating myocarditis.

KEYWORDS Heart failure; Immunology; Leukocytes; Lymphatic circulation; Myocarditis


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Acute myocarditis is a fatal disease and a major cause of dilated cardiomyopathy [1,2], however, the etiology of myocarditis is unclear, and an effective treatment does not yet exist. Autoimmunity plays an important role in myocarditis, in particular a reaction to cardiac myosin following viral infection may contribute to development of myocarditis [3]. Experimental autoimmune myocarditis (EAM) in rats is characterized by severe myocardial damage and the appearance of multinucleated giant cells and is used as an animal model of human giant cell myocarditis [4,5]. Previous study reported that this model had two stages. The first stage detected a focal inflammation mainly consisting of macrophages and lasted up to day 14. The next stage detects strong inflammation consisting of macrophages and CD4-positive T cells and the maximal inflammation lasted up to around day 21 [6]. We defined days 0–14 as the antigen priming (Ap) phase and days 14–21 as the immune response (Ir) phase in EAM. EAM is thought to be induced by T cell activation [7]. T cell activation requires distinct signals from an antigen-presenting cell (APC) [8]. The first signal is antigen-specific and is mediated by the T cell receptor (TCR), and the second signal occurs through costimulatory molecules such as CD28. The interactions of CD28 and CD40L with their respective ligands, B7-1/B7-2 and CD40, play a critical role in T cell activation; therapy with cytotoxic T lymphocyte antigen 4 (CTLA4)-Ig prevents development of EAM [9]. Inducible costimulator (ICOS) was identified as the third member of the CD28 family [10]. ICOS is expressed on the surface of T cells activated through the CD28 pathway or by phorbol 12-myristate 13-acetate (PMA)-inomycin or anti-CD3 [11–13]. Studies of ICOS-deficient mice have shown that ICOS costimulation is necessary for activation and function of effector T cells [14–16]. The ICOS ligand was identified as the third member of the B7 family and was termed B7 homolog (B7-H) [17].

It was previously shown that blockade of the ICOS/ICOSL pathway plays a significant role in graft-versus-host disease (GVHD), cardiac allograft survival, and experimental allergic encephalomyelitis (EAE) [18–20]. However, there have been no reports regarding the role of this molecule in myocarditis. In the present study, we investigated the effect of ICOS/ICOSL pathway blockade in EAM. We used anti-ICOS antibody and ICOSIg to examine the effect of ICOS blockade or B7-H blockade, respectively. We found that ICOS plays an important role in myocarditis and that blockade of the ICOS/ICOSL pathway inhibits development of myocarditis.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animals
Male Lewis rats (7-weeks-old; body weights 200 to 250 g) were purchased from Sankyo Laboratories. They were fed a standard diet and water and were maintained in compliance with the animal welfare guidelines of the Institute of Experimental Animals, Tokyo Medical and Dental University.

2.2 Antigen and immunization
Purified porcine cardiac myosin (Sigma Chemical Co.) was dissolved in 0.01 M phosphate-buffered saline (PBS) and emulsified with an equal volume of complete Freund's adjuvant (Difco) supplemented with Mycobacterium tuberculosis H37RA (Difco) at a concentration of 10 mg/ml. On day 0, rats were injected subcutaneously in the footpads with 0.2 ml of emulsion, yielding an immunizing dose of 1.0 mg/body of cardiac myosin per rat [21,22].

2.3 Reagents
Anti-rat ICOS monoclonal antibody (JMAb50, generated at JT Frontier Research Laboratory) and isotype-matched control IgG1{kappa} monoclonal antibody against keyhole limpet hemocyanin (JMAb211) were prepared as described previously [23]. Biotinylated isotype-matched control IgG, FITC-conjugated anti-CD4 antibody, FITC-conjugated anti-CD8 antibody, and streptavidin–PE were purchased from PharMingen. Human IgG was obtained from Jackson ImmunoResearch Laboratories.

2.4 Preparation of ICOSIg
Soluble ICOS (ICOSIg) was prepared by constructing an adenovirus vector containing cDNA encoding an extracellular domain of human ICOS and the Fc portion of human IgG. The ICOS cDNA was amplified by reverse transcription-polymerase chain reaction (RT–PCR) of mRNA isolated from human peripheral blood leukocytes stimulated with Con-A. PCR primers were forward, 5'-GGACTGAATTCTGTTTCTGGCAAACATG-3' and reverse, 5'-CATGGATCCGGTAACCAGAACTTCAGCTG-3'. The cDNA was inserted into the EcoRI/BamHI site of a plasmid carrying IgG1-Fc DNA [24]. The ICOSIg DNA was then removed by EcoRI/XbaI digestion, and a blunt-ended fragment was ligated into the SwaI site of the pAxCAwt cosmid vector to prepare recombinant adenovirus AxICOSIg. Preparation of recombinant adenovirus was done with the Adenovirus Expression Vector Kit (Takara) according to the manufacturer's protocol. ICOSIg protein was purified from lysate of AxICOSIg-infected COS7 as described [25]. In brief, concentrated COS7 cells were washed with PBS and cultured with DMEM for 4 days in the presence of AxICOSIg. The ICOSIg protein was purified from the supernatant with protein A-sepharose 4FF (Pharmacia) affinity chromatography.

2.5 Treatment
Because human disease is diagnosed on the basic of clinical symptoms, it is important to determine if a treatment is effective against progressing or established disease. It is reported that cellular infiltration of myocardium in EAM occurs around day 14 [7], so we administered anti-ICOS antibody either from day 0 or from day 14.

In the first set of experiments, rats in group Ab (n = 6) were injected intravenously with anti-ICOS antibody (3 mg/kg) on days 0, 4, 7, 11, 14, and 18. Rats in group Ir (n = 7) were given 3 mg/kg anti-ICOS antibody and those in group Ig (n = 6) were given 1 mg/kg ICOSIg intravenously on days 14 and 18. As controls, groups C1 and C2 were treated with isotype-matched IgG monoclonal antibody, and group C3 was treated with human IgG (n = 9 in each group). Rats were killed on day 21 to examine the effect of ICOS/ICOSL pathway blockade.

In the second set of experiments, rats in group Ap (n = 6) were given 3 mg/kg anti-ICOS antibody intravenously on days 0, 4, 7, and 11 and killed on day 14. Group C4 (n = 6) received isotype-matched IgG monoclonal antibody on days 0, 4, 7, and 11. These protocols are outlined in Fig. 1 [26,27].


Figure 1
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  		Fig. 1 Experimental protocols. Group Ab was treated with anti-ICOS antibody (Ab) from the antigen priming phase. Group Ir and Group Ig were treated with anti-ICOS Ab or ICOS Ig during the immune response phase. As controls, groups C1, C2, and C4 were injected with isotype-matched IgG monoclonal Ab and C3 was injected with human IgG. {blacktriangleup}, anti-ICOS Ab or control Ab; {triangleup}, ICOS Ig or control Ig; {dagger}, histological examination.

 
2.6 FACS analysis
Myocardial inflammatory cells and spleen cells were isolated from rats with myocarditis on day 21 after immunization with myosin emulsion as described previously [28]. Cells were incubated with biotinylated anti-ICOS antibody or isotype-matched control IgG on ice for 20 min. Cells were then incubated with FITC-conjugated anti-CD4 or anti-CD8 antibody and streptavidin–PE on ice for 20 min. Cells were washed with PBS and incubated with propidium iodide. Cells were then analyzed by flow cytometry on a FACScalibur (Becton Dickinson) with CellQuest software.

2.7 Histologic examination
Hearts were harvested immediately after rats were killed. We obtained five transverse sections per heart for histologic examination. Apex, midventricular, and basal level slices were stained with hematoxylin and eosin (HE). The area of myocardium and surrounding tissue affected by myocarditis (consisting of inflammatory cell and myocardial necrosis) was determined with a computer-assisted analyzer (Scion Image beta 4.0.2). The area ratio (affected/entire area as a percentage) was calculated as described previously [29]. Values for three ventricular regions were averaged for each heart, and the mean percentage of affected area for each group was calculated. All data were analyzed in a blind fashion by two independent investigators and averaged.

2.8 Echocardiography
Trans-thoracic echocardiography was performed on animals anesthetized by intraperitoneal administration of pentobarbital sodium on day 21. An echocardiographic machine with a 7.5 MHz transducer (Nemio, Toshiba) was used for M-mode left ventricular echocardiographic recordings. A 2D targeted M-mode echocardiogram was obtained along the short-axis view of the left ventricle at the papillary muscles. Left ventricular fractional shortening (LVFS) was calculated from M-mode echocardiograms over three consecutive cardiac cycles according to the American Society for Echocardiography leading edge method [30,31]. Measurements were made offline by two independent investigators.

2.9 Ribonuclease protection assay (RPA)
Trizole (Life Technologies) was used to isolate mRNA according to the manufacturer'sprotocol. Probe was synthesized by the in vitro transcription method with a Multi-Probe Template Set (Pharmingen), T7 polymerase, and [{alpha}-32P]UTP. Ten micrograms of total RNA was hybridized with probe at 56°C for 16 h. All samples were then treated with RNase before treatment with proteinase K. Samples were separated by electrophoresis on a 5% acrylamide denaturing gel. mRNA bands were detected with an image analyzer (BAS2000, Fujifilm). Messenger RNA levels were quantified and normalized against levels of GAPDH. The normalized level of mRNA in each control group was expressed as 1.0 [32].

2.10 T cell proliferation assay
Spleen cells were isolated from rats with myocarditis on day 18. Cells (5x105/well) were cultured in 96-well culture plates with 50 µg/ml purified porcine heart myosin. Anti-ICOS antibody or ICOSIg was added to each well at various concentrations. Cultures were incubated at 37°C under 5% CO2 for 3 days. Similarly, spleen cells isolated from rats in the control group or in the group with ICOS/ICOSL pathway blockade were cultured with purified porcine heart myosin at various concentrations. T cell proliferation was assessed with the Cell Counting Kit-8 (Dojindo). Cell proliferation was expressed as the optical density [33].

2.11 Enzyme-linked immunosorbent assay (ELISA)
Supernatant was collected from cultures used for T cell proliferation assays. Concentrations of INF-{gamma} and IL-2 were determined with an ELISA kit (BioSource International) according to the manufacturer's instructions.

2.12 Statistical analysis
Values are given as mean±S.D. Groups were compared with Scheffé's ANOVA (Stat View, SAS Institute, Inc.). We used Student's t-test for comparisons between two groups in the second experiment. Differences were considered statistically significant at a value of P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Expression of ICOS by inflammatory cells
ICOS was detected on myocardial inflammatory cells. CD4-positive cells showed stronger expression of ICOS than did CD8-positive cells (Fig. 2A and B). However, ICOS was not detected on either CD4- or CD8-positive spleen cells (Fig. 2C and D).


Figure 2
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  		Fig. 2 Expression of ICOS on T cells. T cells stained with anti-ICOS antibody are shown as a solid line, and T cells stained with isotype control IgG are shown in black. Panels A and C show CD4-positive cells. Panels B and D show CD8-positive cells.

 
3.2 ICOS/ICOSL pathway blockade reduced heart weight/body weight ratios
In the first set of experiments, the heart weight to body weight ratios in groups, Ab (n = 6), Ir (n = 7), and Ig (n = 6) were less than those of the control groups (n = 9 each) (Fig. 3A). In the second set of experiments, the heart weight to body weight ratio of group Ap (n = 6) was not remarkably different from that of group C4 (n = 6) (Fig. 3B). The heart weight to body weight ratios in the treatment groups were decreased in the first set of experiments, but that was not changed in the second set of experiments.


Figure 3
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  		Fig. 3 Heart weight/body weight ratio. The heart weight/body weight ratios in groups with ICOS/ICOSL pathway blockade were significantly lower than those in control groups in the first set of experiments (A). In the second set of experiments, the heart weight/body weight ratio in the group with ICOS/ICOSL pathway blockade was similar to that in the control group (B). *P<0.05. N.S., not significant.

 
3.3 Reduction of inflammatory cells in the heart
In the first set of experiments, myocardial lesions were rarely observed in hearts of rats treated with ICOS pathway blockade (Fig. 4A). In the groups treated with ICOS/ICOSL pathway blockade, there was little infiltration of inflammatory cells or myocardial necrosis (Fig. 4B). However, severe myocardial lesions were observed in hearts of all control rats on day 21 (Fig. 4C). These lesions were composed of extensive necrosis and infiltration by mononuclear cells and polymorphonuclear neutrophils (Fig. 4D). In the second set of experiments, myocardial lesions were observed in hearts of rats treated with ICOS/ICOSL pathway blockade as well as those of control rats on day 14 (Fig. 4E and F).


Figure 4
Figure 4
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  		Fig. 4 Representative cross-sections of heart. Panel A shows a representative cross-section of heart from rats with ICOS/ICOSL pathway blockade (group Ir). Panel B shows little infiltration by inflammatory cells in group Ir. Panel C shows a representative cross-section of heart from control rats (group C2) on day 21. Panel D shows severe myocarditis lesions, including giant cells, in group C2. A representative heart from rats with ICOS/ICOSL pathway blockade (group Ap) on day 14 is shown (E). Panel F shows a representative cross-section of heart from control rats (group C4) on day 14. Panels G and H show the myocarditis-affected area ratios in the respective groups. Original magnification in A, C, E, and F is x10. Original magnification in B and D is x400.

 
In the first set of experiments, the ratios of myocarditis-affected areas in groups Ab (n = 6), Ir (n = 7), and Ig (n = 6) were less than those of the control groups (n = 9 each) (Fig. 4G). In the second set of experiments, the ratio of group Ap (n = 6) was not significantly different from that of group C4 (n = 6) (Fig. 4H). Myocardial lesions were clearly reduced with ICOS/ICOSL pathway blockade in the first set of experiments, but were not in the second set of experiments.

3.4 ICOS/ICOSL pathway blockade improved cardiac function
On day 21, ICOS/ICOSL pathway blockade with anti-ICOS antibody improved LVFS in group Ir (Fig. 5A) in comparison with that in group C2 (Fig. 5B).


Figure 5
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  		Fig. 5 Echocardiograms. Panel A shows an M-mode echocardiogram from a rat in group Ir. A representative M-mode echocardiogram from a rat in group C2 on day 21 is shown in panel B. Effects of ICOS/ICOSL blockade on LV fractional shortening on day 21 are presented (C and D). *P<0.05. N.S., not significant.

 
In the first set of experiments, LVFS in groups Ab (n = 6), Ir (n = 7), and Ig (n = 6) showed greater improvement than those of each control groups (n = 9 each) (Fig. 5C). In the second set of experiments, LVFS in group Ap (n = 6) showed no significant change in comparison to that of group C4 (n = 6) (Fig. 5D). LVFS was improved with ICOS/ICOSL pathway blockade in the first set of experiments, but was not in the second set of experiments.

3.5 Cytokine mRNA expression during ICOS/ICOSL pathway blockade
RPA was used to examine expression of cytokine mRNAs in hearts. Levels of cytokine mRNAs for IL-4, IL-6, IL-10, IL-1β, and TNF-{alpha} in groups Ab (n = 4), Ir (n = 4), and Ig (n = 4) were markedly decreased comparison with that of each control group (n = 4 each) (Fig. 6A and B). Expression of cytokine mRNAs in group Ap (n = 4) and group C4 (n = 4) was enhanced (Fig. 6C and D). In the first set of experiments, expression of cytokine mRNA was reduced with ICOS/ICOSL pathway blockade, but was not reduced in the second set of experiments. RPA revealed that ICOS/ICOSL pathway blockade during only the inflammatory phase (days 14–21) reduced expression of IL-4, IL-6, IL-10, IL-1β, and TNF-{alpha} mRNAs as well as ICOS/ICOSL pathway blockade during the antigen priming phase and inflammatory phase. However, ICOS/ICOSL pathway blockade during only the antigen priming phase did not affect expression.


Figure 6
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  		Fig. 6 Expression of cytokine mRNAs in the heart. Panel A shows representative findings on day 21 in groups Ir and C2. Panel C shows representative findings on day 14 in group Ap and C4. Levels of mRNAs were normalized to those of GAPDH as control group (B and D). Data are the mean of four independent experiments. *P<0.05. N.S., not significant.

 
3.6 Suppression of cell proliferation
We performed cell proliferation assays to examine the effect of ICOS/ICOSL pathway blockade on antigen-induced T cell proliferation. Antigen-induced T cell proliferation was suppressed by both anti-ICOS antibody (Fig. 7A) and ICOSIg (Fig. 7B). Antigen-specific cell proliferation in rats treated with ICOS/ICOSL pathway blockade was reduced in comparison to that in rats in the control group (Fig. 7C).


Figure 7
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  		Fig. 7 Effects of ICOS/ICOSL blockade on antigen-induced proliferation of splenocytes. Splenocytes were isolated from group C1 on day 18. Both anti-ICOS antibody (anti-ICOSAb) and ICOSIg suppressed T cell proliferation (A and B). Rats in group C1 show greater myosin-specific splenocyte proliferation than those in group Ab (C). *P<0.05.

 
3.7 Th1-type cytokine change by ICOS/ICOSL pathway blockade
ELISA analysis of supernatants after incubation of spleen cells with cardiac myosin revealed that production of INF-{gamma} by group Ab (n = 4) cells was suppressed in comparison to that in group C1 (n = 4) (Fig. 8A). Production of IL-2 was not affected by cardiac myosin (Fig. 8B).


Figure 8
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  		Fig. 8 Th1-type cytokine production. Splenocytes from rats in group Ab produced significantly less INF-{gamma} after restimulation with cardiac myosin than did those in group C1 (A). Production of IL-2 in group Ab was similar to that of group C1 (B). *P<0.05.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Treatment of acute myocarditis in humans remains a major clinical problem [34]. Here, we clearly show that the ICOS/ICOSL pathway plays an important role in EAM, and blockade of the ICOS/ICOSL pathway with anti-ICOS antibody or ICOSIg suppresses the development of EAM.

It was previously reported that the CD28/CTLA4-B7 and CD40/CD40L pathways play crucial roles in EAM [8]. However, the role of ICOS, which is a novel costimulatory molecule, in EAM is not known. T cell proliferation is enhanced by stimulation of the ICOS/ICOSL pathway [10], and this response is inhibited by blockade of the ICOS/ICOSL pathway [35]. Development of EAM involves myosin-autoreactive T cell proliferation.

We confirmed that ICOS is expressed on myocardial infiltrating T cells by FACS analysis; however we could barely detect ICOS on spleen cells. This observation is consistent with that of a previous study indicating that ICOS is expressed strongly on activated T cells at the site of inflammation [36]. Thus, we hypothesized that development of EAM was inhibited by ICOS/ICOSL pathway blockade.

The pathogenic mechanism of this model involves three sequential processes. As the first step, autoreactive T cells are activated and expanded by a fragment of cardiac myosin. The second step is recruitment of activated T cells to the target organ, and the third step appears to be effector–target interaction [37]. Inflammation consists mainly of macrophages and CD4-positive T cells. During the inflammatory phase, Th1-type cytokines and proinflammatory cytokines, such as IL-2, INF-{gamma}, IL-1β, and TNF-{alpha}, are produced. The peak of inflammation in EAM is expected to occur around day 21 [6], thus, we killed rats on day 21. Our in vivo studies revealed that ICOS/ICOSL pathway blockade inhibits development of EAM by day 21. The heart weight to body weight and myocarditis-affected area ratios were similar between ICOS/ICOSL pathway blocked from day 0 and from day 14. Cardiac function was improved to the same degree with both treatments. A previous study showed that rats with EAM treated with CTLA4-Ig from day 0 showed less development of EAM in comparison with those treated from day 14 [9]. Additionally, to examine the effect of ICOS/ICOSL blockade during the antigen priming phase, rats were killed on day 14. We did not observe a clinical effect with ICOS blockade during the antigen priming phase. Thus, ICOS/ICOSL pathway blockade during the antigen priming phase is not useful. However, ICOS/ICOSL pathway blockade is effective against EAM only during the immune response phase. A previous study showed that ICOS/ICOSL pathway blockade during the antigen priming phase exacerbates disease in another autoimmune model, EAE, and in ICOS-deficient mice, clinical symptoms of EAE were exacerbated [14]. However, in the present study, ICOS/ICOSL pathway blockade during the antigen priming phase did not exacerbate the development of EAM. This may be due to preferential induction of ICOS after T cell activation at the site of inflammation. Because the inflammatory phase is important for clinical treatment, we examined the effect of ICOS/ICOSL pathway blockade with ICOSIg. Treatment with ICOSIg also had a beneficial clinical effect, indicating that blockade of the B7-H ligand has therapeutic value.

Additionally, ICOS is involved in modulation of secondary responses [38] and is expressed on resting memory cells [12]. Therefore, we used splenocytes harvested after myosin immunization for cell proliferation assays. Cell proliferation assays revealed that ICOS/ICOSL pathway blockade reduces myosin-specific T cell proliferation in EAM. These findings suggest that ICOS/ICOSL pathway blockade was an effective treatment in vivo.

Th1 and Th2 cytokines are produced by T cells and modulate inflammation in this model. ICOS/ICOSL pathway blockade during the immune response phase decreased expression of proinflammatory cytokines including IL-6, IL-1β, and TNF-{alpha}. Suppression of proinflammatory cytokines that produce Th1-type cytokines prevented development of EAM [9]. However, ICOS/ICOSL pathway blockade during the antigen priming phase did not. These results were consistent with those of previous studies that showed that ICOS is expressed on activated T cells [11–13] and that ICOS is not expressed on activated T cells at the site of inflammation during the antigen priming phase.

Changes in expression of Th1-type cytokines, such as INF-{gamma} and IL-2, caused by ICOS/ICOSL pathway blockade in response to cardiac myosin was analyzed in splenocytes, because EAM in Lewis rats is mediated by the Th1 response [6]. We found that production of INF-{gamma} was reduced, whereas that of IL-2 was not changed by ICOS/ICOSL pathway blockade. This result is supported by previous findings that the ICOS/ICOSL pathway promotes production of IFN-{gamma} but not IL-2 in ICOS-deficient mice [10]. Our results suggest that the role of INF-{gamma} is more important than that of IL-2 in the development of EAM. However, some studies in GVHD and lung mucosal inflammation models indicated that ICOS/ICOSL pathway blockade do not reduce expression of Th1-type cytokines and reduce Th2-type cytokines, such as IL-4 and IL-10 [18,36]. Our results in EAM are consistent with data from studies of allograft rejection, EAE, and collagen-induced arthritis models that ICOS/ICOSL pathway blockade suppresses production of Th1- and Th2-type cytokines [19,20,38].

In our model of EAM, myocarditis occurs as early as 14 days after immunization followed by aggressive cellular infiltration. Although this is not a case in mild clinical myocarditis, some clinical cases including giant cell myocarditis show similar clinical course and histopathology as this animal model. It is not clear whether the Th1 response is related to clinical myocarditis; we assume that myocarditis is mediated by the Th1 response irrespective of species including human giant cell myocarditis because of the similarity in the clinical course and histology.

In the present study, we show that ICOS/ICOSL pathway blockade suppresses cytokine production and T cell activation and thus attenuates development of EAM. These results indicate that the ICOS/ICOSL pathway blockade may have potential as a therapy for myocarditis. Further studies are needed to evaluate the clinical usefulness of this novel strategy for treatment of myocarditis.

Time for primary review 33 days.


   Acknowledgements
 
This study was supported by grants from the Japan Cardiovascular Research Foundation, a Grant-in-aid from the Japanese Ministry of Education, Science and Culture, a Grant-in-aid from the Japanese Ministry of Welfare, and the Organization for Pharmaceutical Safety and Research. We thank Ms Noriko Tamura for excellent technical assistance.


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

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