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. 2003 Dec;134(3):403–408. doi: 10.1111/j.1365-2249.2003.02312.x

Polarity of helper T cell subsets represents disease nature and clinical course of experimental autoimmune myocarditis in rats

K FUSE *, M KODAMA *, M ITO *, Y OKURA *, K KATO *, H HANAWA *, S AOKI †, Y AIZAWA *
PMCID: PMC1808899  PMID: 14632744

Abstract

The mechanisms of progression, remission and relapse of myocarditis remain unclear. To clarify these mechanisms, we focused on T helper-1 (Th1)/T helper-2 (Th2) subsets balance of peripheral lymphocytes and serum cytokine levels during disease progression in rats with experimental autoimmune myocarditis (EAM). Lewis rats were immunized with cardiac myosin on day 0. Blood samples were collected on days 0, 7, 15, 18, 21, 28, 35, 42, 49 and 56 following immunization. We examined percentages of interferon (IFN)-γ and/or interleukin (IL)-4 producing cells in stimulated peripheral CD4-positive lymphocytes using flow cytometry analysis. Serum IFN-γ, IL-2, IL-6 and IL-10 levels were measured by enzyme-linked immunosorbent assay (ELISA). The percentage of Th1/Th2 subsets in EAM on days 0, 15, 28 and 56 were 2·5 ± 0·5/0·5 ± 0·1%, 19·4 ± 3·2/1·6 ± 0·3%, 2·0 ± 0·5/22·1 ± 5·7% and 3·0 ± 0·4/1·7 ± 0·3%, respectively. Serum levels of Th1 cytokines, IFN-γ and IL-2 significantly increased in the acute phase (from day 15–18) and immediately decreased in the early recovery phase. On the other hand, serum levels of Th2 cytokine, IL-10 significantly increased in the early recovery phase (from day 24–30). These results suggest that induction of acute myocarditis might be associated with systemic Th1 dominance, while recovery is related to systemic Th2 polarity. Thus, analysis of Th1/Th2 balance in peripheral T cells may be useful in disease monitoring in patients with myocarditis and postmyocarditic dilated cardiomyopathy.

Keywords: experimental autoimmune myocarditis, helper T cell, cytokine, cytometry, Th1/Th2 balance

INTRODUCTION

A rat model of experimental autoimmune myocarditis (EAM) mimics the pathophysiology of human giant cell myocarditis. In rats, EAM is elicited about 2 weeks after injecting cardiac myosin with complete Freund's adjuvant, then gradually recovers and is followed by dilated cardiomyopathy [1–3]. Moreover, EAM is relapsed by re-immunization of the same antigen 1 month after the remission of myocarditis, which appears to have a refractory period just after remission [3]. To date, the mechanisms of progression, remission and relapse of EAM have not been disclosed. Furthermore, different susceptibility has been reported in several strains of mice and rats. EAM can be induced by cardiac myosin in A/J mice [1,4], Balb/c mice [5,6], and in Lewis rats [2,7], whereas C57BL/6 mice and Brown Norway (BN) rats are resistant to myosin-induced myocarditis. The mechanism contributing to susceptibility for EAM remains also unclear. Cytokines may play important roles in the natural mechanism of remission, relapse and production of the refractory period in EAM. Similarly, susceptibility may also be related to genetic factors including cytokine polarity after exposure to antigen.

When naïve helper T cell (Th) are stimulated by antigen-presenting cells (APCs), they differentiate into two distinct subsets: Th1 and Th2. Th1 cells secrete interferon (IFN)-γ, interleukin (IL)-2 and tumour necrosis factor (TNF)-β and promote mainly cellular immunity, whereas Th2 cells produce IL-4, IL-5, IL-10 and IL-13, primarily promoting humoral immunity [8–10]. The Th1 to Th2 balance determines the onset and outcome of a wide variety of immune disorders that include autoimmune and allergic diseases [11–14]. We have previously presented that the mRNA of Th1-related cytokines, such as IFN-γ and IL-2, were expressed throughout the acute phase and the mRNA of Th2-related cytokines, such as IL-10, were expressed on the recovery phase in the clinical course of EAM [15,16]. Similarly, in a human study, we reported that analysis of the systemic Th1/Th2 balance using flow cytometry is useful for understanding the disease activity of acute myocarditis [14]. Every cytokine has pleiotropic actions, and many cytokines share similar biological effects. Even if one cytokine is activated, counter-acting cytokines may overcome its effects. To our knowledge, most studies on cytokine production by Th cells have used immunoassays or mRNA analysis, but none have provided simultaneous information about the production of different cytokines from the same cell sources. In this study, we investigated Th cell predominance in peripheral lymphocytes of rats with EAM using flow cytometry.

MATERIALS AND METHODS

Animals

Male Lewis rats (from 8 to 10 weeks) were purchased from Charles River Japan (Kanagawa, Japan). They were bred and maintained at the Facility for Comparative Medicine and Animal Experimentation, Niigata University Graduate School of Medicine.

Antigen and immunization

Purified cardiac myosin from the ventricular muscle of pig hearts was prepared according to the procedure previously described [2] and used as an antigen. Rats were given a subcutaneous injection of 0·2 ml of antigen-complete Freund's adjuvant emulsion into their footpads on day 0.

Sacrificing and sampling

Four to seven rats were sacrificed under anaesthesia on days 0, 7, 15, 18, 21, 28, 35, 42, 49 and 56 after immunization for sampling. After thoracotomy, macroscopic findings were classified into 3 grades [2]. Blood samples were then obtained from inferior vena cava. Subsequently, hearts were weighed immediately and ventricles were collected and fixed in 10% formalin. Microscopic findings were classified into 4 grades [2]. The study protocol was approved by the guidelines on animal experimentation of our institute.

Flowcytometric analyses of intracellular cytokine synthesis

The lymphocyte fraction was obtained using the Ficoll gradient method, and the cells were re-suspended at a concentration of 1 × 106 cells/100 µl in RPMI-1640 (Nipro, Tokyo, Japan) with 10% fetal calf serum (FCS). Cells were cultured for 4 h in the presence of 50 ng/ml phorbol 12-myristate 13-acetate (Wako, Osaka, Japan) plus 1 µg/ml ionomysin (Sigma, St Louis, MO, USA) at 37°C and 5% CO2. Two hours before cell harvesting, 10 µg/ml brefeldin A (Wako) was added. Cells harvested were washed by PBS in the presence of brefeldin A (5 µg/ml) and stained using biotinylated mouse antirat CD4 antibody (Serotec, Oxford, UK), followed by Streptavidin-PerCP (Becton Dickinson, San Jose, CA, USA). Labelled cells were then fixed with 2% paraformaldehyde for 20 min Intracytoplasmic staining was performed as previously described [17]. After washing with PBS-1% FCS and incubation in saponin medium (PBS including 1% FCS and 0·1% saponin) for 10 min, cells were incubated for 30 min at room temperature with the appropriate concentration of FITC-conjugated mouse antirat IFN-γ antibody (Serotec) and PE-conjugated mouse antirat IL-4 antibody (Pharmingen, San Diego, CA, USA). After washes in PBS including 1% FCS and 0·1% saponin and one additional wash without saponin, the cells were resuspended in 500 µl PBS-1% FCS. Data was collected on 10 000 CD4 positive cells on EPICS(r) XLII System (Beckman Coulter, XL Coulter Fullerton, CA, USA) and the percentages of Th1 (IFN-γ single positive cells) and Th2 (IL-4 single positive cells) were counted by FACS.

Measurement of serum cytokine levels by enzyme-linked immunosorbent assay (ELISA)

Blood samples were spun to collect serum from rats and stored at −80°C until measurement of cytokines. Levels of IFN-γ, IL-2, IL-6 and IL-10 were measured by an immunoassay cytokine kit (Cosmo Bio, Tokyo, Japan).

Statistical analysis

Statistical comparisons of the significance among groups were performed by one-way anova followed by the Bonferroni-Dunn method. All data are presented as mean ± standard error of mean (SEM). Statistical significance was accepted at P < 0·05.

RESULTS

Clinical and histological analysis

The clinical course and histopathology of EAM is presented in Table 1 and Fig. 1. From the 15th day after immunization, discolored areas were observed on the cardiac surface. Hearts were markedly enlarged, as shown by an increase of the heart weight/body weight ratios. At the same time, inflammatory lesions spread extensively, and interstitial oedema became evident (Fig. 1a). From day 18, in addition to large amount of mononuclear cell infiltration, myocardial necrosis was observed (Fig. 1b). On day 28, most parts of the infiltrating cells disappeared and the fibrosis remained in myocardium (Fig. 1c). There were no microscopic abnormalities in the hearts of rats immunized with only complete Freund's adjuvant (Fig. 1d).

Table 1.

Clinical course of experimental autoimmune myocarditis

Day of examination Body weight (BW)(g) Heart weight (HW)(g) HW/BW × 1000 Macroscopic score Microscopic score
0 170 ± 1 0.554 ± 0.014 3.3 ± 0.4 0.0 0.0
7 172 ± 3 0.560 ± 0.025 3.3 ± 0.1 0.0 0.0
15 180 ± 3 1.035 ± 0.045 5.8 ± 0.3 2.0 3.0
18 178 ± 14 1.178 ± 0.086 6.6 ± 0.1 2.0 3.0
21 200 ± 5 1.235 ± 0.063 6.3 ± 0/2 2.0 3.0
28 210 ± 5 1.483 ± 0.081 7.1 ± 0/2 2.0 3.0
35 238 ± 9 1.353 ± 0.069 5.7 ± 0.2 2.0 2.0
42 255 ± 4 1.360 ± 0.085 5.3 ± 0.4 2.0 1.7
49 260 ± 5 1.320 ± 0.069 5.1 ± 0.5 2.0 1.3
56 261 ± 7 1.293 ± 0.065 5.0 ± 0.5 2.0 1.5
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Values are mean ± SEM. Macroscopic scores were graded as follows: 0, normal; 1, the prescence of a focal discoloured area; 2, the presence of multiple or diffuse discoloured areas on the cardiac surface. Microscopic scores were graded as follows: 0, normal; 1, presence of a few small lesions in a single section not exceeding 0.25 mm2 in size; 2, presence of multiple small lesions or a few moderately sized lesions not exceeding 6.25 mm2 in size; 3, presence of multiple moderately sized or larger lesions, with a total inflamed area exceeding 6.25 mm2.

Fig. 1.

Fig. 1

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Light microscopic images of rat hearts. a, Hearts of EAM on day 15; b, Hearts of EAM on day 18; c, Hearts of EAM on day 28; d, Hearts of rats immunized with complete Freund's adjuvant alone on day 18. (a,b,d) are Hematoxylin-eosin staining. (c) is Azan–Mallory staining. Magnification × 200.

Th1/Th2 balance in the clinical course of EAM

The time course of each Th1/Th2 cell population is shown in Figs 2 and 3. In Fig. 2, the percentage of Th1 (IFN-γ positive and IL-4 negative cells) and Th2 (IFN-γ negative and IL-4 positive cells) subsets on days 0, 15, 18, 21, 28, 35 and 56 of EAM are shown, respectively. The percentage of Th1/Th2 subsets of EAM on days 0, 15, 18, 21, 28, 35 and 56 were 2·5 ± 0·5/0·5 ± 0·1%, 5·2 ± 1·1/0·6 ± 0·1%, 19·4 ± 3·2/1·6 ± 0·3%, 10·4 ± 1·9/2·9 ± 0·6%, 3·3 ± 0·8/11·8 ± 1·4%, 2·0 ± 0·5/22·1 ± 5·7%, 1·9 ± 0·3/12·4 ± 3·1% and 3·0 ± 0·4/1·7 ± 0·3%, respectively. Th1 cells increased before the onset of EAM then Th2 cells overcome Th1 during the active or the early recovery phase (Fig. 3a,b).

Fig. 2.

Fig. 2

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Flowcytometrical profiles of Th1 (IFN-γ) and Th2 (IL-4) positive cells. 10 000 cells were acquired for each experiment. After stimulation, lymphocytes were stained with biotinylated mouse antirat CD4 antibody, FITC-conjugated mouse antirat IFN-γ antibody and PE-conjugated mouse antirat IL-4 antibody. Lymphocytes were also stained with labelled isotype antibodies as negative control. In each dot plot, the lower-right square within for fractionations showed Th1 cells (IFN-γ positive and IL-4 negative) and upper-left square showed Th2 cells (IFN-γ negative and IL-4 positive).

Fig. 3.

Fig. 3

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a, Time course of Th1 and Th2 positive cells during the clinical course of EAM. Th1 cells increased before the onset of EAM (day 7), peaked at the onset of EAM (day 15), gradually decreased. Then Th2 cells overcame Th1 during the active or the early recovery phase (day 21), peaked at day 28 then decreased. b, Time course of Th1/Th2 ratio in clinical course of EAM. *P < 0·05 versus Day 0.

Serum levels of Th1/Th2-related cytokines in EAM

We assayed the serum concentrations of cytokines using ELISA, to evaluate the correlation of Th1/Th2 cytokines between the intracellular staining and serum concentrations, Serum levels of Th1 cytokines, IFN-γ and IL-2 significantly increased in the acute phase (from day 15–18) and decreased in the early recovery phase (day 21). On the other hand, serum levels of Th2 cytokine, IL-10 significantly increased in the early recovery phase (from day 21–35) (Fig. 4).

Fig. 4.

Fig. 4

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Serum levels of representative Th1 and Th2 cytokines during the clinical course of EAM. Th1 cytokines, IFN-γ and IL-2 significantly increased in the acute phase (from day 15–18) and decreased in the early recovery phase (day 21). On the other hand, serum levels of Th2 cytokine, IL-10 significantly increased in the early recovery phase (from day 21–35). *P < 0·05 versus Day 0.

DISCUSSION

Pathogenesis of myocarditis is related to some specific cytokines production. They participate to the onset, progression and remission of myocarditis. Cytokines are important in controlling T cells response to antigens and are critical in shifting the immune response toward Th1 or Th2 pattern [13,18]. The Th1 response shifts the cytokine profile toward delayed hypersensitivity, macrophage activation, and a proinflammatory T cell response, whereas the Th2 response is associated with B cell activation and humoral immunity. Th1 cells secrete IL-2 and IFN-γ that suppress Th2 immune response. Th2 T cells secrete IL-4 and IL-10 that inhibit Th1 responses. In the pathogenesis of EAM, cytokines also play a major role. In our previous study, mRNA express-ion of Th1 cytokines increased from the onset of the disease, and subsequently, mRNA expression of Th2 cytokines could be detected in the recovery phase [15,16]. We also demonstrated that suramin, a growth factor blocker, suppressed myocardial inflammation in EAM by modulation of the Th1/Th2 cytokine environment [19]. Furthermore, remote production of IL-10, which was induced by skeletal muscle expression of plasmid DNA by electroporation, provided marked protection against EAM [20].

Cytokine production is evaluated using various methods because individual method has different specificity and sensitivity. To date cytokine production by T cells has been analysed mostly by mRNA quantification or measuring cytokine levels [21–24]. Recently, a flow cytometry method to evaluate intracellular cytokine production by individual T cells has become a standard technique [25–29]. This technique has some advantages. First, it allows the quantitative estimation of a large number of T cells. Second, it is possible to categorize the phenotype of cytokine producing cells according to intracellular cytokines and cell surface markers. Finally, the different fluorochromes allow the evaluation of the cells producing various cytokines from a similar source. Cytokines have pleiotropic actions and many cytokines share similar biological effects. Thus, to understand the pathogenesis of a disease is occasionally difficult just by evaluating cytokine concentration or the quantification of cytokine mRNA [30,31]. Thus, we studied population of cytokine producing cells in the systemic circulation using flow cytometry. This approach would reveal the relationship between immune polarity, Th1 or Th2, and nature of the disease, ongoing or healing.

Our present data clearly demonstrated that systemic T cells with Th1 function increased before the onset of EAM, then systemic T cells with Th2 function overcome Th1 T cells during the active or the early recovery phase. We have also previously demonstrated the recurrence of EAM [3]. When the second immunization with the same antigens was challenged after the first immunization, there was a resistant period to the recurrence up to about 1-month post recovery. In this study, the refractory period to recurrence agreed with the period that systemic Th2 T cells were overcoming Th1 T cells. We investigated rats immunized with complete Freund's adjuvant alone as controls. They showed increase of neither Th1 nor Th2 cells (The percentage of Th1/Th2 subsets on days 18 and 28 were 2·1 ± 0·6/0·9 ± 0·3% and 2·7 ± 0·3/1·1 ± 0·4%, respectively). Furthermore, we found that rats of the EAM-resistant and Th2 prone strain, Brown Norway (BN), did not show increase of Th1 T cells in peripheral blood after immunization of cardiac myosin (data were not shown). In addition, we also demonstrated that there was a positive correlation between number of cytokine producing cells and serum Th1/Th2 cytokine levels. These results suggest that systemic Th1/Th2 balance might regulate the onset, progression, healing and recurrence of EAM. In order to regulate the clinical course of myocarditis, it may be important to convert from Th1 polarity to Th2 polarity. Furthermore, there is a possibility that development of postmyocarditic dilated cardiomyopathy is related to the dysregulation of Th1/Th2 balance.

To date the roles of Th1/Th2 cytokines in the pathogenesis of autoimmune myocarditis are still controversial. Afanasyeva et al. [32] demonstrated that blocking IL-4 with anti-IL-4 antibody reduced the severity of autoimmune myocarditis in A/J mice. They suggest that autoimmune myocarditis is a Th2 mediated disease. In contrast, Eriksson et al. [33] demonstrated that IL-4-receptor deficient mice were still susceptible to autoimmune myocarditis. On the other hand, there are some reports suggesting that development of autoimmune myocarditis critically depend on the presence of IL-12, which is the major Th1 inducing cytokine, in mice and rats [33–36]. The Th1/Th2 balance seems to be a simplified explanation for the onset, progression, remission and recurrence of EAM, although there may be a limitation to ascribe the nature of the disease to a single theory. Th1/Th2 balance truly reflects the course of the disease and is one of the good markers for estimation of the nature of the disease in EAM. For future clinical application and to understand the aetiology of chronic myocarditis or postmyocarditic dilated cardiomyopathy, we have to investigate the whole cytokine environment, and the way that it is represented in Th1/Th2 polarity.

Acknowledgments

This study was supported in part by research grants from the Ministry of Education, Science and Culture of Japan.

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