Introduction

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The therapeutic efficacy of immunoglobulin in inflammatory and autoimmune diseases has been reported (Clarkson et al., 1986; Jayne et al., 1991; Newburger et al., 1991; Wolf and Eible, 1996). The mechanisms responsible for the efficacy of this treatment are, however, unknown.

The prophylactic administration of immunoglobulin was reported to be of clinical value against some virus infections (Wolf and Eible, 1996). This effect was due to the capacity of immunoglobulin to neutralize the viruses. The successful treatment of idiopathic thrombocytopenic purpura with immunoglobulin appears to result from the blockade of Fc receptors (Clarkson et al., 1986). One possible mechanism of action of intravenous immunoglobulin in Kawasaki disease is the neutralization of a microbial toxin by immunoglobulin (Newburger et al., 1991), which binds nonspecifically to certain viable regions of the T-cell antigen receptor.

Drucker et al. (1994) reported potential benefits of intravenous immunoglobulin in the therapy of children with a recent onset of myocarditis. Subsequently, McNamara et al. (1997) reported successful treatment of adult patients with acute cardiomyopathy by immunoglobulin, which was associated with improved recovery of left ventricular function. Accordingly, clarification of the mechanisms underlying this treatment in acute cardiomyopathic patients is warranted.

We have previously demonstrated that immunoglobulin therapy suppressed murine myocarditis induced by coxsackievirus B3 (Takada et al., 1995), the most cardiotropic agent in humans. In the present study, we examined the effects of immunoglobulin upon experimental murine myocarditis induced by encephalomyocarditis virus, which is not pathogenic to humans (Barger and Craighead, 1991), and analyzed the behaviors of inflammatory cytokines.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

In Vitro Study

Immunoglobulin (Venilon) was kindly supplied by Teijin Co., Ltd. (Tokyo, Japan). Venilon is a sulfonated human immunoglobulin with Fc portion and is purified by Cohn's cold ethanol method (Takada et al., 1995). Antiviral activity against encephalomyocarditis (EMC) virus was assayed by a plaque-reduction method, as previously described (Takada et al., 1995). In brief, a serially diluted sterile solution of immunoglobulin was incubated with 100 plaque-forming units (PFU) of EMC virus at 37°C for 1 h. The reaction was stopped at 4°C for 30 min. The sample was added to confluent monolayers of African green monkey kidney (VERO) cells in six-well plastic plates. After 2 days of incubation at 37°C, the cells were fixed with acetic acid and methanol, stained with crystal violet, and the plaques were counted. Plaque formation was expressed as a percentage of the number of control plaques.

RAW 264.7 cells (murine macrophage-like cells with Fc receptor) were inoculated into six-well plates (3 × 10⁵/well) and incubated at 37°C for 24 h. The cells were maintained in Dulbecco's modified minimum essential medium with 10% fetal calf serum and then infected with EMC virus at multiplicities of infection (m.o.i.) of 0.1, 1, and 10 PFU/cells. Twenty-four hours later, a final concentration of 6 mg/ml immunoglobulin was then added to these cells (Andersson et al., 1996). From 6 to 72 h after the immunoglobulin treatment, the supernatants at m.o.i. of 10 were assayed for cytokine concentrations [tumor necrosis factor- (TNF-), interleukin (IL)-1, interferon- (INF-), and IL-6]. The dose of immunoglobulin was due to the recently published method (Andersson et al., 1996).

In Vivo Study

Infection Protocol. The virus stock of EMC virus was prepared in cultures of VERO cells in Eagle's minimum essential medium. Virus suspensions were centrifuged after the cytopathic effect had developed, and the viral stock had a titer of more than 10⁹ PFU/ml determined in tissue cultures.

Treatment Protocol. Immunoglobulin was administered intraperitoneally daily; the actual dose in each experiment was calculated from the mouse weight at the beginning of the experiment. From previous studies (Clarkson et al., 1986; Takada et al., 1995; McNamara et al., 1997), the dose of immunoglobulin used was 1 g/kg/day. Although human immunoglobulin used in this study is xenogenic to the host, immunoglobulin antigenicity between different species does not seem to be a problem (Basta et al., 1989; Weller et al., 1992).

Experiment I. Mice (n = 66) were randomized to two groups in which they received either no treatment (n = 33) or treatment with immunoglobulin (n = 33). Mice in the untreated group were injected intraperitoneally with 0.1 ml of saline during the treatment period. Beginning simultaneously with the virus inoculation, treatment was given for 14 days. The mice were observed daily and necropsy was performed immediately on those mice found dead. Thirteen mice in each group were killed on day 7 for virological and pathological studies and cytokine assay. Accordingly, the survival study covered 20 mice in each of the two groups. Mice surviving until the end of treatment period were killed, and their sera were processed for cytokine assay. The organs (lungs, liver, and heart) were weighed and the ratio of organ to body weight was calculated. The organs were processed for pathological study. Additional control groups were uninfected mice treated for 14 days with saline (n = 3) and with immunoglobulin (n = 3).

Experiment II. Altogether, 65 mice were inoculated with virus. At 14 days when treatment began, only 32 mice (49.2%) were still alive and randomized to either of two groups: no treatment (n = 16) or treatment with immunoglobulin (n = 16). Treatment was given for 14 days, i.e., until 28 days after virus inoculation. The mice were observed daily, and necropsy was carried out on those mice that died during the course of the experiment. At the end of the treatment period, the same procedure as that for experiment I was performed.

Pathological Study. Hearts were processed by standard methods, embedded in paraffin, cut into 5-µm-thick sections, and stained with hematoxylin and eosin. Myocardial lesions were graded, blinded to the respective treatment groups, to determine the severity of cellular infiltration and necrosis of the ventricles. The pathological criteria for grading the severity of infiltration and necrosis were as follows: grade 1 (mild), one or two small foci; grade 2 (mild-moderate), several large foci; grade 3 (moderate), multiple small foci or several large foci; and grade 4 (severe), multiple large foci or diffuse infiltration, and necrosis.

Virological Study. Myocardial virus titers and serum-neutralizing antibody titers were determined, as previously described (Kishimoto et al., 1988; Takada et al., 1995).

Serum Cytokine Assay. TNF-, IL-1, IFN-, macrophage inflammatory protein-2 (MIP-2), and IL-6 levels in sera were determined using antibody-sandwich enzyme-linked immunosorbent assay. In brief, anti-cytokine antibodies and biotinated antibodies were used as the capture and secondary antibodies, respectively. Color development was done by the addition of peroxidase-coupled streptavidin and substrate-chromogen (diaminobenzidine) solution before terminating the reaction with 2 M H₂SO₄. The absorbance was measured on a microplate reader. MIP-2 is considered to be a murine counterpart of IL-8 (Driscoll, 1994).

Statistical Analysis. Survival was analyzed by the Kaplan-Meier method. Data were presented as mean ± S.D. Statistical comparisons were performed by use of a two-tailed t test or analysis of variance with the Scheffé's test when appropriate. A value of p < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

In Vitro Study

The percentage of plaque formation was 96.2 ± 22.0% at an immunoglobulin concentration of 10³ mg/ml, 101.8 ± 9.3% at 10² mg/ml, 91.2 ± 17.0% at 10¹ mg/ml, and 98.2 ± 15.0% at 10⁰ mg/ml (each n = 5). There was no correlation between PFU and the immunoglobulin concentrations. Thus, immunoglobulin (Venilon) does not contain the significant amount of antibodies against EMC virus.

As shown in Table 1, significant infection dose-dependent cytokine increases were detected in the supernatants of EMC virus-infected RAW 264.7 cells. The time-dependent changes of cytokines at m.o.i. of 10 with or without immunoglobulin treatment is shown in Table 1. In general, cytokines increased with time from 12 h until 48 h. The cytokine productions were depressed by the treatment of immunoglobulin.

In Vivo Study

Mortality (Fig. 1). In experiment I, eight mice in the control group and seven in the immunoglobulin-treated group had died by day 14; the survival rate on day 14 was 60.0% (12/20) in the control group and 65.0% (13/20) in treated group. The difference was not significant.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Survival rates in experiments I and II. In experiment I, survival rate did not differ significantly between immunoglobulin-treated and untreated groups. However, in experiment II, survival rates of the immunoglobulin-treated group were higher than that of the untreated group.

Cardiac Pathology (Figs. 2 and 3; Table 2). The incidence of myocarditis was 100% in each group in both experiments. In experiment I (on days 7 and 14), both cellular infiltration and myocardial necrosis were less severe in the immunoglobulin-treated group compared with the untreated group (Fig. 2). In experiment II, both scores in the immunoglobulin-treated group were lower than those of the untreated group. In addition, pulmonary and liver congestion was microscopically less severe in the immunoglobulin-treated mice than in the untreated group.

View larger version (141K): [in this window] [in a new window]   Fig. 2.   Hematoxylin and eosin section of myocardium 7 days after virus inoculation in experiment I (untreated mouse). Severe lymphocyte infiltration in the myocardium was evident. Hematoxylin and eosin stain, 80×.

View larger version (116K): [in this window] [in a new window]   Fig. 3.   Sections of myocardium 14 days after virus inoculation in experiment I (untreated mouse). Marked cellular infiltration with extensive necrosis can be seen (left, Mayer's counterstain, 180×). Infiltrating cells are almost T cells (arrows) in the corresponding immune stain section (right, Thy 1.2 stain, 180×).

Organ Weights. There was no significant difference in organ weights between treated and untreated groups in experiment I (data not shown). In experiment II, however, the ratios of heart weight (6.6 ± 0.6 ×10³; n = 14; p < 0.01) and liver weight (48.0 ± 3.5 × 10³; n = 14; p < 0.05) to body weight were significantly less in the immunoglobulin-treated group compared with the untreated group (7.5 ± 0.8 × 10³; 50.4 ± 5.0 × 10³; each n = 7). The result may reflect less severe myocardial damage in the treated animals.

Virological Study. Myocardial virus titers of immunoglobulin-treated mice were not statistically different from those of untreated mice in experiment I (data not shown). Antibody titers on day 7 or 14 did not differ significantly between the treated and untreated groups (data not shown). Because there were no antibodies against EMC virus in the human immunoglobulin (Venilon) used in the in vitro study, neutralizing antibody activities against the virus were due to endogenous factors.

Serum Cytokine Levels (Table 3). In experiment I, TNF-, IFN-, MIP-2, and IL-6, but not IL-1, in sera were significantly decreased in the immunoglobulin-treated groups compared with the untreated groups. In experiment II, MIP-2 and IFN-, but not IL-6, in sera were significantly decreased in the treated compared with the untreated groups. Cytokine increases were significantly greater in the virus-infected mice compared with the uninfected (normal) mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

This study showed that immunoglobulin treatment sufficiently suppressed the development of heart failure in murine EMC viral myocarditis associated with the concomitant reduction of inflammatory cytokines, notwithstanding the in vitro absence of neutralizing anti-EMC viral antibodies in the immunoglobulin (Venilon). The production of cytokines was decreased in virus-infected macrophages by the treatment of immunoglobulin in vitro. Taken together with previous reports (Takada et al., 1995; Bozkurt et al., 1999; Kishimoto et al., 1999; Felix et al., 2000; Gullestad et al., 2001; McNamara et al., 1997, 2001), immunoglobulin therapy could have the potential to ameliorate congestive heart failure in myocarditis associated with the reduction of inflammatory cytokines.

There is as yet no general agreement on effective treatment for viral myocarditis. Trials with steroids, nonsteroidal anti-inflammatory drugs, immunosuppressive agents, -blockers, angiotensin-converting enzyme inhibitors, and other therapeutic modalities have been attempted. Routine treatment of myocarditis with immunosuppressive drugs did not alter the course of the disease (Mason et al., 1995). This study clearly demonstrated efficacy of immunoglobulin for myocarditis. Accordingly, we speculate that immunoglobulin might be more effective, safer, and better tolerated than corticosteroids or other immunosuppressive agents.

Immunoglobulin therapy has many merits in the treatment of autoimmune and inflammatory diseases. However, the precise mechanisms responsible for its clinical benefits are unknown. Previous studies showed several possible mechanisms of immunoglobulin action. First, immunoglobulin neutralized viruses and microbial toxins.

The prophylactic administration of immunoglobulin was reported to be of clinical value against respiratory syncytial virus in high-risk infants (Groothuis et al., 1993). In children with Kawasaki disease, the therapeutic efficiency of immunoglobulin was partially due to the neutralization of microbial toxins, which act as superantigens (Newburger et al., 1991). Second, immunoglobulin induced receptor blockade. The amelioration of idiopathic thrombocytopenic purpura and Guillian Barré by immunoglobulin therapy results from Fc receptor blockade (van der Meche et al., 1992). Third, immunoglobulin acts as a sump for activated complement components. Immunoglobulin treatment before the injection of antibody to Forssman antigen prevents Forssman shock (Basta et al., 1989). Forth, immunoglobulin acts as an anti-inflammatory agent. Fifth, immunoglobulin contains anti-idiotype antibodies. In patients with autoantibodies to Factor VIII, immunoglobulin treatment markedly reduced or removed all autoantibodies to Factor VIII (Sultan et al., 1984). Finally, exogenous immunoglobulin accelerates IgG catabolism (Yu and Lennon, 1999).

The neonatal Fc receptor, which was initially identified in neonatal intestinal epithelium, is a protective receptor that prevents the catabolism of IgG. In states of hypergammaglobulinemia, this receptor is presumably saturated, permitting the degradation of IgG to occur in proportion to its total concentration in plasma. Therefore, in autoimmune diseases mediated by pathogenic IgG, immunoglobulin treatment is effective due to acceleration of the degradation of IgG (Yu and Lennon, 1999).

The role of immunoglobulin in the therapy of myocarditis or acute dilated cardiomyopathies, however, has not been fully understood. Although idiopathic dilated cardiomyopathy is a heterogenous disorder (Report of WHO/ISFC, 1996), most patients are suspected of sharing a similar viral/autoimmune pathogenesis and may benefit from immune modulatory therapy. Our previous study had demonstrated that immunoglobulin therapy suppresses coxsackievirus B3 myocarditis by transferring the neutralizing antibody into the host in the acute stage, because immunoglobulin (Venilon) used in that study contained the neutralizing antibody against other cardiotropic viruses such as mumps, influenza, and ECHO. Thus, it is conceivable that in the acute stage immunoglobulin therapy could suppress myocarditis with other cardiotropic viral etiology via the transfer of neutralizing antibodies into the host. The present study was conducted to evaluate other effects of immunoglobulin except the neutralizing antibody activities upon murine myocarditis induced by EMC virus, because EMC virus is not pathogenic to humans (Barger and Craighead, 1991). The results clearly showed that immunoglobulin therapy could suppress myocardial necrosis with T-cell infiltrates concomitantly associated with the reduction of inflammatory cytokines.

Although there was no statistical significance found in survival rate in the early protocol (experiment I), this phase is considered to be not cardiogenic but viremic. Indeed, in the late, cardiogenic phase (experiment II), immunoglobulin treatment was sufficiently effective to the survival rate as well as cardiac pathology. However, to confirm the efficacy of immunoglobulin for myocarditis caused by human cardiotropic viruses, other experimental models of viral myocarditis with influenza and ECHO should be done before large-scale clinical trials with immunoglobulin in acute viral myocarditis are performed.

It has been suggested that cytokines also exert and important role in the development of inflammatory myocardial disease as well as congestive heart failure (Henke et al., 1992; Gullestad et al., 2001). For example, action of cytokine-mediated depression of myocardial function through up-regulation of inducible nitric-oxide synthase was reported (Kinugawa et al., 1997). Furthermore, TNF and IFN- are important mediators in the pathogenesis of myocardial inflammation in myosin-induced myocarditis (Smith and Allen, 1992) and coxsackievirus B3 myocarditis (Henke et al., 1992). IL-6 is a well known inflammatory cytokine (Henke et al., 1992). MIP-2 is a murine counterpart of IL-8, which is established to be a member of potent chemotactic factors (Driscoll, 1994). The reduction of cytokines by immunoglobulin administration was reported as a therapeutic possible mechanism in some inflammatory diseases (Wolf and Eible, 1996), because immunoglobulin itself contains antibodies against cytokines (Takei et al., 1993; Ross et al., 1997). Another mechanism for the suppression of cytokine production could probably be a direct antigen neutralization by antibodies presented in the immunoglobulin preparations (Amran et al., 1994).

Recently, suppression of cytokine-dependent T-cell proliferation by immunoglobulin treatment in in vitro experiments and in inflammatory disorders was demonstrated (Amran et al., 1994; Andersson et al., 1996; Koffman and Dalakas, 1997; Modiono et al., 1997). This immunomodulatory effect of immunoglobulin may be significant for its therapeutic actions in immune-mediated diseases and may account for the therapeutic potential in part in the present study; immunoglobulin-treated mice developed less myocardial T-cell infiltrates and improved the survival in a cytokine-dependent model of murine myocarditis. Most recently, the significance of the so-called inhibitory Fc receptors has been clarified; cross-linking Fc receptor (Barcy et al., 1995) and inhibitory Fc receptor (Samuelsson et al., 2001). The Fc portion of immunoglobulin may have the potency not only to decrease T-cell responses to antigens but also to reduce inflammatory actions via these receptors.

In clinical setting, McNamara et al. (2001) reported that for patients with recent-onset cardiomyopathy, immunoglobulin therapy does not augment the improvement of left ventricular function and that the function improved significantly during follow-up. Gullestad et al. (2001) reported that immunoglobulin therapy was effective for patients with heart failure by modulating cytokine balance. In addition, Felix et al. (2000) reported that immunoadsorption and subsequent immunoglobulin substitution improved ventricular function in patients with dilated cardiomyopathy.

In conclusion, immunoglobulin therapy could have the potential to prevent congestive heart failure in encephalomyocarditis viral myocarditis associated with the reduction of inflammatory cytokines.