Yuji Hiraoka

Therapy With Immunoglobulin Suppresses Myocarditis in a Murine Coxsackievirus B3 Model Antiviral and Anti-inflammatory Effects

BACKGROUND The treatment of some inflammatory diseases (eg, Kawasaki disease) with immunoglobulin has been demonstrated to be effective. Accordingly, to elucidate the mechanism underlying such actions of immunoglobulin, we examined its effects on murine coxsackievirus B3 (CB3) myocarditis. METHODS AND RESULTS An in vitro study showed dose-dependent suppression of CB3 by immunoglobulin. Immunoglobulin 1 g.kg-1.d-1 IP was administered to CB3-infected C3H/He mice daily for 2 weeks, beginning simultaneously with virus inoculation in experiment 1 and on day 14 after virus inoculation in experiment 2. In both experiments, survival was higher in treated than in control mice; at the time of death, inflammatory also were reduced. Notably, in experiment 1, immunoglobulin administration completely suppressed the development of myocarditis. Serum-neutralizing antibody titers in the treated mice were significantly higher than those in untreated mice in experiment 1 but not in experiment 2. The circulating antibodies of the treated mice were primarily of exogenous origin in experiment 1 and of exogenous and endogenous origins in experiment 2. The analysis of splenic lymphocyte subsets revealed a marked decrease of the B cell population in the treated mice. CONCLUSIONS Immunoglobulin therapy completely suppressed acute CB3 myocarditis by transferring the neutralizing antibody into the host in the acute viremic stage and induced an anti-inflammatory effect in the subsequent aviremic stage; the reduction of the splenic B-cell population may be closely associated with an anti-inflammatory effect.

Cytokine and murine coxsackievirus B3 myocarditis. Interleukin-2 suppressed myocarditis in the acute stage but enhanced the condition in the subsequent stage.

BACKGROUND It has been shown that the development of coxsackievirus B3 (CB3) myocarditis is regulated by T cells and not by B cells. Interleukin-2 (IL-2) is a T-cell-derived cytokine that stimulates the growth of T cells. This study was carried out to determine the effects of IL-2 on CB3-infected BALB/c mice. METHODS AND RESULTS In two separate experiments, recombinant human IL-2 (5 x 10(4) U) was administered subcutaneously to 30 mice early (days 0 to 7) and 30 mice late (days 7 to 14) after infection with CB3. Each experiment had a control group of infected animals that did not receive IL-2. On days 7 and 10, splenic natural killer (NK) cell activity determined by 51Cr release assay and the distribution of myocardial lymphocyte subsets were compared in the treated and untreated groups. In the early treatment experiment, survival at 7 days was higher in treated compared with control animals, myocardial virus titers were lower, inflammatory cell infiltration was less (as was the severity of necrosis at the time the mice were killed), and NK cell activity was higher. However, in the late treatment experiment, survival at 14 days was lower in treated compared with control animals, and there was more infiltration, more severe necrosis, and more T-cell infiltration, but the NK cell activity did not differ significantly. In a third experiment similar to the late experiment described above but involving infected athymic nude mice, we confirmed the lack of effect of late in vivo administration of IL-2 on outcome. CONCLUSIONS IL-2 has the capacity to limit CB3 myocarditis by enhancing NK cell activity in the acute viremic stage, resulting in a reduction of cardiac pathology. However, in the subacute aviremic stage, in contrast, IL-2 exacerbates the course and severity of the disease by increasing the number of T cells infiltrating the myocardium. That is, IL-2 has differential effects on acute CB3 myocarditis. IL-2 is beneficial if treatment is given early but later in murine CB3 myocarditis.

Nitric oxide and murine coxsackievirus B3 myocarditis: Aggravation of myocarditis by inhibition of nitric oxide synthase☆

OBJECTIVES This study sought to investigate the effects of nitric oxide inhibition in a murine model of coxsackievirus B3 myocarditis. BACKGROUND Little is known about the contribution of nitric oxide to the pathophysiology of myocarditis. METHODS Antiviral activity was tested in vitro using nitric oxide inhibition by treatment with activated macrophages of NG-nitro-L-arginine methyl ester. In the in vivo experiments, NG-nitro-L-arginine methyl ester and NG-nitro-D-arginine methyl ester (both at 100 micrograms/ml) were administered to C3H/He mice early (days 0 to 14) and late (days 14 to 35) after infection with coxsackievirus B3. RESULTS In the in vitro experiments with interferon-gamma- and lipopolysaccharide-induced activated murine macrophages, treatment with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester, but not its inactive enantiomer NG-nitro-D-arginine methyl ester, restored coxsackievirus B3 titers. In the in vivo experiments in the early treatment group, myocardial virus titers were higher in NG-nitro-L-arginine methyl ester-treated than infected untreated animals, and both inflammatory cell infiltration and necrosis were more severe. In the late treatment group, more severe necrosis and more dense myocardial and perivascular fibrosis were observed in NG-nitro-L-arginine methyl ester-treated than in infected untreated animals. NG-Nitro-D-arginine methyl ester administration was ineffective. CONCLUSIONS Nitric oxide inhibition increases myocardial virus titers, resulting in the aggravation of cardiac pathology in the early stage of coxsackievirus B3 myocarditis. In the late stage, it induces more severe cardiomyopathic lesions. Nitric oxide plays a defensive role in the pathogenesis of coxsackievirus B3 myocarditis.

Clinical and experimental studies in myocarditis.

We review recent manuscripts concerning the pathogenesis as well as the treatment of myocarditis. The development of myocarditis is related to inflammatory cell infiltration, progressive and additive focal cellular necrosis, and associated reactive myocardial fibrosis. These processes may be initiated and perpetuated by autoimmune factors, including cytokines, and by alterations in the myocardial microcirculation. This has implications for the development of future therapeutic strategies for patients with myocarditis, strategies that will enable physicians to optimize medical treatment and, hopefully, to improve the prognosis.

T cell-mediated immune response enhances the severity of myocarditis in secondary cardiotropic virus infection in mice.

Abstract In this report, we showed that a previous enterovirus exposure in ordinary mice with normal T cell function, but not in T cell-deficient mice, can influence development of myocardial inflammation with a second virus exposure. Inoculation of 4-week-old male BALB/c-nu/+ (euthymic and normal T cell function) mice with amyocarditic Coxsackie virus B1 (CB1), followed by inoculation 28 days later with myocarditic variant of Coxsackie virus B3 (CB3-m) resulted in more intense myocardial inflammation and injury than was seen in BALB/c-nu/+ inoculated with CB1, followed by inoculation with non-enterovirus, i.e., encephalomyocarditis virus (EMC) or influenza A virus and in age-matched BALB/c-nu/+ mice secondary inoculated with CB3-m alone. In contrast, this phenomenon of the enhancement of the severity of myocarditis by a secondary CB3-m inoculation was not seen in BALB/c-nu/nu (athymic and T cell- deficient) mice. Interestingly, inoculation of BALB/c-nu/+ mice with CB1, followed by inoculation 28 days later with another amyocarditic variant of Coxsackie virus B3 (CB3-o), resulted in more severe myocarditis than was seen in age-matched BALB/c-nu/+ mice secondary inoculated CB3-o alone. Myocardial-activated T cells and elevated serum interleukin-6 were involved in the exacerbation of the disease during the reinfection. T cell-mediated immune responses to a conserved antigenic epitope among the enteroviruses may be involved in the exacerbation of myocardial inflammatory disease during a second enterovirus infection.

Colony-stimulating factors and coxsackievirus B3 myocarditis in mice: Macrophage colony-stimulating factor suppresses acute myocarditis with increasing interferon-α

The effects of macrophage colony-stimulating factor (M-CSF) and granulocyte colony-stimulating factor (G-CSF) on murine coxsackievirus B3 myocarditis were investigated. A total of 4 × 106 U/kg/day M-CSF and 20 μg/kg/day G-CSF were injected subcutaneously every day on day 0 to day 14 starting simultaneously with virus inoculation. Serum interferon-α was measured periodically. The survival rate of the M-CSF group was higher than that of the untreated control group (p < 0.05). On days 7 and 14 cardiac disease was significantly lower in the M-CSF group than in the untreated control group. Myocardial virus titers on day 7 in the M-CSF group were lower compared with those of the untreated control group. No significant difference was seen in the survival, cardiac disease, or myocardial virus titers between the G-CSF and the control groups. Monocyte counts on days 7 and 14 in the M-CSF group were increased compared with those in the control group. Serum interferon-α titers in the M-CSF and G-CSF groups on day 4 and those in the M-CSF group on day 7 were significantly increased in comparison with those of the untreated control group. We conclude that M-CSF but not G-CSF has the potency to limit myocardial virus titers and to reduce cardiac disease in the acute coxsackievirus B3 myocarditis. This ability is associated with an elevated interferon-α. Thus macrophages may play a defensive role in this model.

Effects of immunoglobulin upon murine myocarditis caused by influenza A virus: superiority of intact type to F(ab')2 type.

Influenza viruses play the largest role in the worldwide epidemiology of infectious diseases. Management of some inflammatory disease (eg, Kawasaki disease) with immunoglobulin has been demonstrated to be effective. We examined the effects of intact type and F(ab′)2 type of immunoglobulin preparations upon murine influenza A virus myocarditis in mice. In vitro study showed that intact type and F(ab′)2 type of immunoglobulin preparations exhibit antiviral activities against many substrains of influenza virus and other cardiotropic viruses. Dose-dependent suppression of an influenza A virus (NWS) was demonstrated by management with both intact immunoglobulin and F(ab′)2 fragments of immunoglobulin. The dose inhibiting 50% of plaques was the same between intact type and F(ab′)2 type (both 0.0002 mg/dl). Intact immunoglobulin, but not F(ab′)2 fragments of immunoglobulin, suppressed serum macrophage inflammatory protein-2 (MIP-2) productions in influenza A virus-infected macrophages in vitro, which is a murine counterpart of interleukin-8. This suppression of MIP-2 production by intact immunoglobulin treatment was blocked by a specific Fc receptor (Fc&ggr;III/II receptor) antibody pretreatment. Intact immunoglobulin or F(ab′)2 fragments of immunoglobulin were administered to virus-inoculated A/J mice intraperitoneally daily, starting simultaneously with virus inoculation (Experiment I) and 2 days after the virus inoculation (Experiment II), until 10 days after virus inoculation. In Experiment I, survival was higher in treated than in control mice; intact type and F(ab′)2 type immunoglobulins administration completely suppressed the development of myocarditis. In Experiment II, survival rate was significantly higher and myocarditis was less severe in intact immunoglobulin-treated mice, but not in F(ab′)2 fragments-treated mice compared with untreated mice. Serum neutralizing antibody titers in treated mice were significantly higher compared with untreated mice in Experiments I and II. In addition, serum MIP-2 concentrations in intact immunoglobulin-treated mice, but not in F(ab′)2 fragments-treated mice, were lower compared with untreated mice in Experiment II. Immunoglobulin therapy suppresses influenza A virus myocarditis by increasing neutralizing antibody titers and the suppression of myocardial virus activities. From the standpoint of suppression of MIP-2 concentrations, intact type is superior to F(ab′)2 type. Thus, immunoglobulin treatment may be promising for prevention of influenza virus myocarditis.

Oral administration of L-arginine prevents congestive heart failure in murine viral myocarditis.

It was previously demonstrated that administration of the nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) aggravated viral myocarditis in mice. In the current study, the effects of l-arginine, a precursor of nitric oxide, on congestive heart failure (CHF) in myocarditis were evaluated. Dietary l-arginine and l-arginine plus l-NAME (l-arginine + l-NAME group) were administered to encephalomyocarditis virus–infected BALB/c mice over 4 weeks (experiment I) and to encephalomyocarditis virus–infected DBA/2 mice from the 4th through 12th weeks after the virus inoculation (experiment II). An infected control was prepared in each experiment. In experiment I, survival was higher in the l-arginine group compared with the other two groups, and cardiac damage was less, as was incidence of CHF. In addition, extravasated fibrin was less prominent in the l-arginine group. Plasma concentrations of l-arginine and nitric oxide were elevated in the l-arginine group. In experiment II, plasma cardiomyopathic lesions in the l-arginine group were less prominent and were associated with lower plasma catecholamine and lower myocardial collagen concentrations compared with the other two groups. l-arginine treatment may be effective in preventing the development of CHF in viral myocarditis by modifying postmyocarditic architectural remodeling.

Intravenous IgG: Supertherapy for Myocarditis and Acute Dilated Cardiomyopathy

To the Editor: We read with great interest the article by McNamara et al1 on intravenous immunoglobulin (IgG) therapy for myocarditis and acute dilated cardiomyopathy (DCM). There is as yet no general agreement of effective treatment of myocarditis and acute DCM. These workers are to be congratulated for conducting a well-designed clinical study of adult patients with NYHA class III to IV heart failure, based on previously published clinical2 and experimental3 experience. We would like to stress 3 points that we consider crucial in our understanding of the significance of this treatment. The first is related to the presence of preceding or associated infectious manifestations (ie, fever, pleurodynia, myalgia, and upper respiratory tract symptoms). This strongly suggests that myocarditis is …

HLA-DR2 Antigen Linkage in Patients with Apical Hypertrophic Cardiomyopathy in Japan

Apical hypertrophic cardiomyopathy, a very common disorder in Japan, is characterized by giant negative T waves on the electrocardiogram and abnormal apical hypertrophy on the ventriculogram. This histocompatibility complexes (HLA-A, -B, -C, -DR) of 20 unrelated patients with apical hypertrophic cardiomyopathy (18 male, 2 female) were examined. There was no significant difference in the frequency of HLA-A, -B, -C antigens between patients and controls (n = 100). However, HLA-DR 2 was more frequent in patients with apical hypertrophic cardiomyopathy (65.0%) than controls (33.0%) (p < 0.01). We conclude that apical hypertrophic cardiomyopathy might be associated with genes in the HLA-DR region and that genetic factors linked to HLA play a role in the pathogenesis of this disease.