Autoimmune Myocarditis In Mice Research Articles

Noninvasive ultrasound stimulation to treat myocarditis through splenic neuro-immune regulation

BackgroundThe cholinergic anti-inflammatory pathway (CAP) has been widely studied to modulate the immune response. Current stimulating strategies are invasive or imprecise. Noninvasive low-intensity pulsed ultrasound (LIPUS) has become increasingly appreciated for targeted neuronal modulation. However, its mechanisms and physiological role on myocarditis remain poorly defined.MethodsThe mouse model of experimental autoimmune myocarditis was established. Low-intensity pulsed ultrasound was targeted at the spleen to stimulate the spleen nerve. Under different ultrasound parameters, histological tests and molecular biology were performed to observe inflammatory lesions and changes in immune cell subsets in the spleen and heart. In addition, we evaluated the dependence of the spleen nerve and cholinergic anti-inflammatory pathway of low-intensity pulsed ultrasound in treating autoimmune myocarditis in mice through different control groups.ResultsThe echocardiography and flow cytometry of splenic or heart infiltrating immune cells revealed that splenic ultrasound could alleviate the immune response, regulate the proportion and function of CD4+ Treg and macrophages by activating cholinergic anti-inflammatory pathway, and finally reduce heart inflammatory injury and improve cardiac remodeling, which is as effective as an acetylcholine receptor agonists GTS-21. Transcriptome sequencing showed significant differential expressed genes due to ultrasound modulation.ConclusionsIt is worth noting that the ultrasound therapeutic efficacy depends greatly on acoustic pressure and exposure duration, and the effective targeting organ was the spleen but not the heart. This study provides novel insight into the therapeutic potentials of LIPUS, which are essential for its future application.Graphical

Myricetin ameliorates experimental autoimmune myocarditis in mice by modulating immune response and inhibiting MCP-1 expression

Myocarditis is defined as an inflammatory disease of the myocardium, and the autoimmune response specific to myocardium plays an important role in chronic myocarditis. Inhibiting myocardial-specific autoimmune response and inflammation is crucial to treat myocarditis. Myricetin is a plant-derived flavonoid in nature which has potent anti-inflammatory and cardiovascular protective properties. However, the pharmacological effect of myricetin in autoimmune myocarditis is undefined. It is necessary to investigate the role and potential mechanisms of myricetin in autoimmune myocarditis. Therefore, purified cardiac myosin was subcutaneously injected to mice to establish the experimental autoimmune myocarditis (EAM) model. Myricetin was solubilized in normal saline and administered everyday by gavage from the day of immunization. After 21 days of treatment, it was found that myricetin significantly alleviated myocardial injury in EAM mice. The serum anti-cardiac myosin antibody, immunoglobulin (Ig) G, IgM levels and the proportion of T helper 17 (Th17) cells were decreased and the proportion of regulatory T (Treg) cells was increased with the treatment of myricetin in EAM mice. The myosin-specific T cell proliferation was inhibited by myricetin. Meanwhile, myricetin suppressed the expressions of monocyte chemoattractant protein-1 (MCP-1), phospho (p)-p65, p-c-Jun and Act1/TRAF6/TAK1 in H9C2 cells and myocardial tissues of EAM mice. These results revealed that myricetin inhibited the autoimmune response specific to myocardium and the expression of MCP-1 in cardiomyocytes, which suggested that myricetin ameliorated autoimmune myocarditis by modulating immune response and the expression of MCP-1. Therefore, myricetin may be a promising therapeutic strategy for autoimmune myocarditis.

Genetic Ablation of Ankrd1 Mitigates Cardiac Damage during Experimental Autoimmune Myocarditis in Mice.

Myocarditis (MC) is an inflammatory disease of the myocardium that can cause sudden death in the acute phase, and dilated cardiomyopathy (DCM) with chronic heart failure as its major long-term outcome. However, the molecular mechanisms beyond the acute MC phase remain poorly understood. The ankyrin repeat domain 1 (ANKRD1) is a functionally pleiotropic stress/stretch-inducible protein, which can modulate cardiac stress response during various forms of pathological stimuli; however, its involvement in post-MC cardiac remodeling leading to DCM is not known. To address this, we induced experimental autoimmune myocarditis (EAM) in ANKRD1-deficient mice, and evaluated post-MC consequences at the DCM stage mice hearts. We demonstrated that ANKRD1 does not significantly modulate heart failure; nevertheless, the genetic ablation of Ankrd1 blunted the cardiac damage/remodeling and preserved heart function during post-MC DCM.

Clusterin is increased in morphologically normal cardiomyocytes in lymphocytic myocarditis.

Clusterin is increased in morphologically normal cardiomyocytes in lymphocytic myocarditis.

Galectin-3 deficiency enhances type 2 immune cell-mediated myocarditis in mice.

Experimental autoimmune myocarditis (EAM) is a mouse model of immune-mediated myocarditis and cardiomyopathy. The role of Galectin-3 (Gal-3), a β-galactoside-binding lectin, in autoimmune myocarditis has not been studied. Therefore, the aim of this study was to delineate the role of Gal-3 in myosin peptide-induced autoimmune myocarditis in mice. EAM was induced in relatively resistant C57BL/6J mice (wild type, WT) and in mice with a targeted deletion of Gal-3 gene (Gal-3KO) by immunization with myosin peptide MyHCα334-352. Gal-3KO mice developed more severe myocarditis and more pronounced heart hypertrophy than WT mice. Increased infiltration of CD45+ leucocytes, CD3+ T cells, F4/80+ macrophages, and eosinophils was observed in hearts of Gal-3KO mice compared to WT mice on day 21 after EAM induction. Moreover, hearts of Gal-3KO mice had more T helper type 2 (Th2) cells, alternatively activated M2 macrophages, higher amounts of IgG deposits, and higher serum levels of IL-4 and IL-33 than WT mice. Ablation of Gal-3 in Th1-dominant C57BL/6J mice that are relatively resistant to EAM resulted in more severe disease characterized by type 2 cardiac inflammation. The complex effects of Gal-3 on EAM progression might be important in the consideration of therapeutic options for the treatment of EAM.

Cholecalciterol cholesterol emulsion attenuates experimental autoimmune myocarditis in mice via inhibition of the pyroptosis signaling pathway

1,25-dihydroxyvitamin D3 and its analog (paricalcitol) have beneficial effects on multiple systems and diseases, including myocarditis. However, the therapeutic effect of cholecalciterol cholesterol emulsion (CCE) on myocarditis and the role of pyroptosis in the progress of myocarditis have not been determined. The objective of this study was to investigate the effect of CCE on experimental autoimmune myocarditis and its underlying mechanisms. The ratio of heart weight to body weight was decreased after CCE treatment compared with vehicle-treated controls. In addition, CCE improved the general status of mice, suppressed myocardial apoptosis, and inhibited the pyroptosis signaling pathway. Therefore, CCE can ameliorate experimental autoimmune myocarditis by downregulating the pyroptosis signaling pathway.

Pathogenic IL-23 signaling is required to initiate GM-CSF-driven autoimmune myocarditis in mice.

Using a mouse model of experimental autoimmune myocarditis (EAM), we showed for the first time that IL-23 stimulation of CD4(+) T cells is required only briefly at the initiation of GM-CFS-dependent cardiac autoimmunity. IL-23 signal, acting as a switch, turns on pathogenicity of CD4(+) T cells, and becomes dispensable once autoreactivity is established. Il23a(-/-) mice failed to mount an efficient Th17 response to immunization, and were protected from myocarditis. However, remarkably, transient IL-23 stimulation ex vivo fully restored pathogenicity in otherwise nonpathogenic CD4(+) T cells raised from Il23a(-/-) donors. Thus, IL-23 may no longer be necessary to uphold inflammation in established autoimmune diseases. In addition, we demonstrated that IL-23-induced GM-CSF mediates the pathogenicity of CD4(+) T cells in EAM. The neutralization of GM-CSF abrogated cardiac inflammation. However, sustained IL-23 signaling is required to maintain IL-17A production in CD4(+) T cells. Despite inducing inflammation in Il23a(-/-) recipients comparable to wild-type (WT), autoreactive CD4(+) T cells downregulated IL-17A production without persistent IL-23 signaling. This divergence on the controls of GM-CSF-dependent pathogenicity on one side and IL-17A production on the other side may contribute to the discrepant efficacies of anti-IL-23 therapy in different autoimmune diseases.

Phosphoinositide 3-kinase inhibitor LY294002 ameliorates the severity of myosin-induced myocarditis in mice

BackgroundMyocarditis, characterized by myocyte necrosis, fibrosis, and degeneration with mononuclear cell infiltration, always causes heart failure in patients. Phosphoinositide 3-kinase (PI3K) is a pivotal kinase known to regulate inflammatory responses in cardiac diseases. Although previous research has suggested that PI3K was involved in cardiac diseases such as myocardial infarction, it is still unclear whether the inhibition of PI3K is essential for the treatment of myosin-induced myocarditis. The aim of this study was to explore whether pharmacological blockade of PI3K is able to protect mice against experimental autoimmune myocarditis (EAM). Materials and methodsWe used the cardiac myosin-induced murine EAM model to investigate the therapeutic effect of PI3K inhibitor LY294002 on autoimmune myocarditis in mice. ResultsLY294002 significantly alleviated EAM injury in mice, as indicated by the reduction of cardiac necrosis, inflammatory infiltrates, and CD3+ T cells. LY294002 also decreased the expression of p-Akt upon cardiac myosin treatment in the cardiac tissue of the mice. In the present study, LY294002 resulted in a moderate reduction in absolute CD4+ cell numbers and a significant decrease in the absolute numbers of CD8+ cells. Consequently, LY294002 increased the CD4+/CD8+ ratio compared with peptide treatment alone. ConclusionThis report provides evidence that PI3K inhibitor LY294002 has potent effects against cardiac injury caused by EAM, suggesting that it has therapeutic value for the treatment of myocarditis.

Oleanolic acid alleviated pressure overload-induced cardiac remodeling.

Previous study has demonstrated that oleanolic acid (OA) possessing the anti-inflammatory and anti-oxidant properties blunted high-glucose-induced diabetic cardiomyopathy and ameliorated experimental autoimmune myocarditis in mice. However, little is known about its effects on pressure overload-induced cardiac remodeling. Herein, we investigated the effect of OA on cardiac remodeling and underlying mechanism. Mice, subjected to aortic banding (AB), were randomly assigned into control group and experimental group. OA premixed in diets was administered to mice after 3days of AB. Echocardiography and catheter-based measurements of hemodynamic parameters were performed after 8weeks' treatment of OA. Histologic examination and molecular analyses were used to assess cardiac hypertrophy and tissue fibrosis. In addition, the inhibitory effects of OA on H9c2 cardiomyocytes and cardiac primary fibroblast responded to the stimulation of AngII were also investigated. OA ameliorated the systolic and diastolic dysfunction induced by pressure overload evidenced by echocardiography and catheter-based measurements. OA also decreased the mRNA expression of cardiac hypertrophy and fibrosis markers evidenced by RT-PCR. It has been shown in our study that pressure overload activated the phosphorylations of Akt, mTOR, p70s6k, S6, GSK3β, and FoxO3a, and treatment of OA attenuated the phosphorylation of these proteins. In addition, hypertrophy of cardiomyocytes and fibrosis markers induced by AngII was inhibited by OA in vitro. Our findings uncover that OA suppressed AB-induced cardiac hypertrophy, partly by inhibiting the activity of Akt/mTOR pathway, and suggest that treatment of OA may have a benefit on retarding the progress of cardiac remodeling under long terms of pressure overload.

Endothelin receptor antagonist exacerbates autoimmune myocarditis in mice

AimsMyocarditis and subsequent dilated cardiomyopathy are major causes of heart failure in young adults. Experimental autoimmune myocarditis (EAM) is a mouse model of post-infectious myocarditis and inflammatory cardiomyopathy. The pathological role of endothelin (ET) in myocarditis has not been elucidated. Main methodsEAM was induced by immunization of cardiac myosin peptide with complete Freund's adjuvant on days 0 and 7 in BALB/c mice. An ETA/ETB dual receptor antagonist, SB209670, was administered by a continuous infusion from a subcutaneous pump for 2weeks. Key findingsAn increase in the heart-to-body weight ratio was observed in SB209670-treated mice compared with vehicle-treated mice. Heart pathology in SB209670-treated mice was remarkable for gross inflammatory infiltration, in contrast to the lesser inflammation in the hearts of vehicle-treated mice. We found that an ET blockade decreased the number of Foxp3+ regulatory T cells in the heart. The ET blockade also inhibited the expression of the suppressor of cytokine signaling 3 that plays a key role in the negative regulation of both Toll-like receptor- and cytokine receptor-mediated signaling. EAM is a CD4+ T cell-mediated disease. CD4+ T cells isolated from SB209670-treated EAM mice produced less IL-10 and more inflammatory cytokines, IL-6 and IL-17, than those isolated from vehicle-treated mice. SignificanceThe ET receptor antagonist exacerbated autoimmune myocarditis in mice. Our novel findings suggest that ET may play an important role in the regulation of inflammation in myocarditis.

Chlorogenic Acid Suppresses a Cell Adhesion Molecule in Experimental Autoimmune Myocarditis in Mice

Background: In patients with myocarditis, autoimmune disease is considered to be responsible for the pathogenesis. Previous studies revealed that plant polyphenols inhibit autoimmune myocarditis in mice. However, the effect of chlorogenic acid (CGA), one of the major polyphenol constituents of coffee beans is remain unknown. The purpose of this study is to investigate the effects of CGA on experimental autoimmune myocarditis (EAM) in mice. Methods: Balb/c mice were immunized with cardiac myosin peptides and complete Freund's adjuvant. CGA or vehicle (Cont) were administered orally from day 0 to day 21 (n=6 and 7, respectively). Mice were sacrificed on day 21. Results: We found that the CGA significantly suppressed intercellular adhesion molecule-1 (ICAM-1) expression in the EAM hearts. The suppressed ICAM-1 tended to reduce myocardial fibrosis compared to control EAM hearts. Conclusion: The findings suggest that the CGA influences inhibition of cell adhesion in myocarditis, the CGA treatment may have beneficial effects for progression of myocarditis. Keywords: Adhesion molecules, autoimmunity, Balb/c mice, coffee extract, heart, ICAM, inflammation, myocarditis, myosin, polyphenol.

Imaging of the binding of ET-1 and of linear ET-1 in rat mesenteric resistance arteries

Background: Experimental autoimmune myocarditis (EAM) is a mouse model of inflammatory cardiomyopathy. The amount of endothelin (ET) increases according to the disease progression; however, the pathological role of ET in myocarditis has not been elucidated. Methods and results: EAM was induced by immunization of cardiac myosin peptide with complete Freund's adjuvant on days 0 and 7 in BALB/c mice. ET-A/-B dual receptor antagonist SB209670 was administered by continuous infusion from a subcutaneous pump for 3 weeks. An increase in heart-to-body weight ratio was observed in SB209670-treated mice compared with vehicle-treated mice. The heart pathology in SB209670-treated mice was remarkable for gross inflammatory infiltration, in contrast to the smaller inflammation in the hearts of vehicle-treated mice. We found that ET blockade decreased the number of Foxp3+ regulatory T cells and inhibited the production of immunoregulatory cytokine IL-10 in the heart. ET blockade also inhibited the expression of suppressor of cytokine signaling 3 (SOCS3) that plays a key role in the negative regulation of both Toll-like receptor (TLR)and cytokine receptor-mediated signaling. EAM is a CD4+ T cell-mediated disease. CD4+ T cells isolated from SB209670-treated EAM mice produced less IL-10 and more inflammatory cytokines IFN-γ and IL-17 than those isolated from vehicle-treated mice. Conclusions: ET receptor antagonist exacerbated autoimmune myocarditis in mice. ET may play an important role in the regulation of inflammation in myocarditis.

The effect of myeloid differentiation factor 88 on experimental autoimmune myocarditis in mice

Objective To investigate the effect of myeloid differentiation factor88 (MyD88)on the experimental autoimmune myocarditis (EAM) in Balb/c mice.Methods Totally 90 Balb/c mice were randomly divided into three groups:MyD88 ( n =30),myocarditis ( n =30),and control ( n =30 )groups.An injection of MyD88 high expression adenovirus was carried out through vena caudalis at day 3.The mice of myocarditis group received porcine cardiac myosin at the same time and position.The mice of control group received PBS only.Serum and myocardium samples were gained at day 21.The inflammatory infiltration and the serum levels of cTnI,IgG,and IgM were examined.Results 21 days later,the inflammatory infiltration score of the myocarditis group was higher than the control group (2.39 ± 1.23 vs 0.68 ±0.65,P ＜0.05).The MyD88 group was significantly higher than the other 2 groups ( F =94.194,P ＜0.01 ),the myocarditis group ( 3.56 ± 1.34 vs 2.39 ± 1.23,P ＜ 0.05 ),and the control group ( 3.56 ±1.34 vs 0.68 ±0.65,P ＜0.01).The serum levels of cTnI,IgG,and IgM of the MyD88 group were significantly higher than the myocarditis group and the control group( P ＜0.01,P ＜0.05).Conclusions MyD88 participates in the pathogenesis of experimental autoimmune myocarditis in mice. Key words: Myeloid differentiation factor 88/metabolism; Autoimmune diseases/physiopathology/blood; Myocarditis/physiopathology/blood; Cardiac myosins; Mice

Low-Dose Inorganic Mercury Increases Severity and Frequency of Chronic Coxsackievirus-Induced Autoimmune Myocarditis in Mice

Mercury is a widespread environmental contaminant with neurotoxic impacts that have been observed over a range of exposures. In addition, there is increasing evidence that inorganic mercury (iHg) and organic mercury (including methyl mercury) have a range of immunotoxic effects, including immune suppression and induction of autoimmunity. In this study, we investigated the effect of iHg on a model of autoimmune heart disease in mice induced by infection with coxsackievirus B3 (CVB3). We examined the role of timing of iHg exposure on disease; in some experiments, mice were pretreated with iHg (200 μg/kg, every other day for 15 days) before disease induction with virus inoculation, and in others, they were treated with iHg after the acute (viral) phase of disease but before the development of dilated cardiomyopathy (DCM). iHg alone had no effect on heart pathology. Pretreatment with iHg before CVB3 infection significantly increased the severity of chronic myocarditis and DCM compared with control animals receiving vehicle alone. In contrast, treatment with iHg after acute myocarditis did not affect the severity of chronic disease. The increased chronic myocarditis, fibrosis, and DCM induced by iHg pretreatment were not due to increased viral replication in the heart, which was unaltered by iHg treatment. iHg pretreatment induced a macrophage infiltrate and mixed cytokine response in the heart during acute myocarditis, including significantly increased interleukin (IL)-12, IL-17, interferon-γ, and tumor necrosis factor-α levels. IL-17 levels were also significantly increased in the spleen during chronic disease. Thus, we show for the first time that low-dose Hg exposure increases chronic myocarditis and DCM in a murine model.

Effects of carvedilol on cardiomyocyte apoptosis in autoimmune myocarditis in mice

To observe the effects of carvedilol on the expression of Bcl-2, Bax and Fas in autoimmune myocarditis (AM). A total of 60 inbred male BALB/C mice 4 - 5 weeks of age were divided at random into 3 groups as follows: AM group (n = 20), carvedilol group (n = 20) and control group (n = 20). The mice were sacrificed after gathering blood specimens by taking out the eyeballs and hearts tissue. The histological and ultrastructural changes were observed under light microscope and electron microscope. The concentrations of cardiac troponin I (cTn I) were detected by chemiluminescence immunoassay (CLIA). Immunohistochemistry (IHC) was performed to analyze the contents of Bcl-2, Bax and Fas, TUNEL to detect the apoptotic index in myocardial cells. There were large number of lymphocyte and monocyte infiltrates under light microscope and karyopyknosis and chromatin gathered along the nuclear membrane under electron microscope in AM group. There were no inflammations and chromatin gathering in group C. Compared with control group, the Bcl-2, Bax and Fas protein expression significantly elevated in AM group (23.48 ± 2.24 vs. 6.64 ± 1.60, 26.15 ± 2.02 vs. 5.09 ± 0.85, 21.22 ± 3.62 vs. 5.86 ± 1.37, P < 0.01). The histopathologic scores (2.60 ± 0.31 vs. 2.02 ± 0.26, P < 0.05) and karyopyknosis of carvedilol group decreased as compared with AM group. The Bcl-2, Bax and Fas protein expression (17.13 ± 1.94 vs. 23.48 ± 2.24, 17.66 ± 2.62 vs. 26.15 ± 2.02, 16.79 ± 2.83 vs. 21.22 ± 3.62, P < 0.05), AI [(16.61 ± 4.67)% vs. (24.51 ± 4.70)%, P < 0.05] and contents of cTnI [(1.878 ± 0.48) ng/ml vs. (1.102 ± 0.23) ng/ml, P < 0.05] also decreased in carvedilol group compared with AM group. Carvedilol could protect against AM by alleviating cardiomyocyte apoptosis.

L‐arginine ameliorates experimental autoimmune myocarditis by maintaining extracellular matrix and reducing cytotoxic activity of lymphocytes

It was previously shown that administration of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) aggravated murine viral myocarditis by increasing myocardial virus titres. Experimental autoimmune myocarditis in mice and rats mimics human fulminant myocarditis. The effects of L-arginine, a precursor of nitric oxide, upon heart failure in experimental autoimmune myocarditis were evaluated. Dietary L-arginine (L-arginine group) and L-arginine plus N(G)-nitro-L-arginine methyl ester (L-arginine + l-NAME group) were administered to C57BL/6 mice immunized with porcine cardiac myosin over 3 weeks. An untreated myocarditis group was prepared. Cardiac damage was less in the L-arginine group compared with the other two groups, as was incidence of heart failure. In addition, extracellular matrix change was less prominent in the L-arginine group. Plasma concentrations of nitric oxide were elevated in the L-arginine group. Cytotoxic activities of lymphocytes were lower in L-arginine group than in other two groups. L-arginine treatment may be effective in preventing the development of heart failure in experimental myocarditis by maintaining extracellular matrix and reducing the cytotoxic activity of lymphocytes.

Susceptibility to autoimmune myocarditis in mice: analysis of the Eam1 susceptibility locus (129.18)

Abstract H-2sA.SW and B10.S mice are susceptible and resistant to experimental autoimmune myocarditis (EAM), respectively. Our previous studies suggested that Eam1 on chromosome 1 is a non-MHC locus conferring susceptibility to cardiac myosin-induced (EAM) in A.SW mice. B10.A Eam1 congenic mice, carrying the Eam1 locus on the EAM-resistant B10.S background, develop EAM comparable to A.SW mice, suggesting that Eam1 plays a key role in the development of disease. We have previously reported that lymph node cells (LNC) of A.SW mice are more resistant to cyslophosphamide (Cy)-induced apoptosis in comparison to B10.S mice, and a similar apoptosis-resistant phenotype is found in NOD mice has been linked to the Idd5 locus overlapping with Eam1. Therefore, we hypothesized that the mechanisms by which Eam1 mediates the susceptibility to EAM could be due to its regulatory role in apoptosis. In the current study, we found that LNC from B10.A Eam1 congenic mice present an intermediate susceptibility to Cy-induced apoptosis, i.e. the percentage of apoptotic LNC with activated caspase 3, 8, 9 in congenic mice falls in between A.SW and B10.S mice. Interestingly, data suggest that A.SW mice have significantly lower levels of apoptotic LNC following immunization with cardiac myosin in CFA than B10.S mice. Our results therefore suggest that Eam1 partially down regulates LNC apoptosis and delays the removal of self-activated T cells, thereby providing an explanation for the role of Eam1 in the susceptibility to EAM. Supported in part by NIH grant HL077611

Prevention of myosin-induced autoimmune myocarditis in mice by anti-L3T4 monoclonal antibody.

This study was aimed at studying the effect of the induction of immune tolerance to swine cardiac myosin from anti-L3T4 monoclonal antibody injection and whether the immune tolerance could protect mice with myosin-induced myocarditis from myocardial injury. Twenty-four Balb/c mice were divided into two groups at random. All of the mice were immunized with swine cardiac myosin on the 1st day, 14th, 28th, 42nd, and 52nd day. Immune tolerance was induced by triplicate injections of 400 microg anti-L3T4 McAb on the 0 day (intravenous), 1st day, and 2nd day (intraperitoneal) in McAb-treated group. In the saline-treated group, saline of the same volume as anti-L3T4 monoclonal antibody was used as a control. The sera and hearts biopsies of all mice were collected on the 58th day. The anti-cardiac myosin antibody was examined with ELISA, and pathological changes of heart were observed by light microscope. It was shown that mice immunized with swine cardiac myosin could produce anti-myosin antibody and the anti-cardiac myosin antibody was positive in most of the saline-treated group but negative in the McAb-treated group. Morphologically, myocardial degeneration, necrosis, and infiltration of inflammatory cells were found in the saline-treated group but not in the McAb-treated group. In conclusion, this study indicated that the immune tolerance to cardiac myosin was induced by the anti-L3T4 monoclonal antibody, and accordingly myocardial injury could be prevented by induction of immune tolerance.

Tenascin-C is a useful marker for disease activity in myocarditis.

Tenascin-C (TNC) is an extracellular matrix protein which appears at active sites of tissue remodelling during embryogenesis or cancer invasion. In normal heart, TNC is only present during the early stages of development but reappears in pathological states. This study examined the diagnostic value of TNC for assessing disease activity of myocarditis. Expression of TNC was examined in myosin-induced autoimmune myocarditis mouse models. Sequential changes in amount, localization and the producing cells were analysed by reverse transcriptase-polymerase chain reaction, western blotting, immunohistochemistry and in situ hybridization and compared with the histological picture. The expression of TNC was upregulated at a very early stage of myocarditis. Immunostaining was detectable before cell infiltration and myocytolysis became histologically apparent, remained during the active stage while cell infiltration and necrosis continued, and disappeared in scar tissue with healing. TNC immunostaining was always observed at the periphery of necrotic or degenerating cardiomyocytes in foci of inflammation, the expression level correlating with histological evidence of inflammatory activity. Interstitial fibroblasts were the major source of TNC, expressing the large isoform containing alternative splicing sites. These data demonstrate that TNC is a useful marker for evaluation of disease activity in myocarditis.

Interleukin-12 Receptor/STAT4 Signaling Is Required for the Development of Autoimmune Myocarditis in Mice by an Interferon-γ–Independent Pathway

Interleukin (IL)-12 exerts a potent proinflammatory effect by stimulating T-helper (Th) 1 responses. This effect is believed to be mediated primarily through the activation of STAT4 and subsequent production of interferon (IFN)-gamma. Methods and Results- We examined the role of IL-12 receptor (IL-12R) signaling in the development of murine experimental autoimmune myocarditis (EAM) induced by cardiac myosin immunization. Both IL-12Rbeta1-deficient mice and STAT4-deficient mice were resistant to the induction of myocarditis. Treatment with exogenous IL-12 exacerbated disease. We questioned whether IFN-gamma is required for the disease-promoting activity of IL-12. On the contrary, we found that IFN-gamma suppresses EAM. Lack of IFN-gamma due to either depletion with an antibody or a genetic deficiency exacerbated myocarditis. Spleens from IFN-gamma-deficient mice immunized with cardiac myosin showed increased cellularity; greater numbers of CD3+, CD4+, CD8+, and IL-2-producing cells; and heightened ability to produce cytokines on stimulation in vitro. Treatment of mice with recombinant IFN-gamma suppressed the development of myocarditis. IL-12/IL-12R/STAT4 signaling promotes the development of EAM. In contrast, IFN-gamma plays a protective role. The disease-limiting effects of IFN-gamma might be explained by its ability to control the expansion of activated T lymphocytes.