Although coxsackie viruses, especially those of group B, are the most common agents responsible for viral myocarditis in humans, 1,2 the World Health Organization's yearly virus reports indicate that myocarditis are sometimes prominent in patients with influenza A virus infections. In fact, there have been reports of myocarditis associated with influenza virus infection in humans. 3,4 Influenza viruses play the largest role in the worldwide epidemiology of respiratory disease. Myocarditis associated with the infection might be more common than recognized, so it is important to understand the pathophysiology of influenza virus myocarditis.
We have previously demonstrated that immunoglobulin therapy suppressed murine coxsackievirus B3 myocarditis by an anti-viral antibody effect. 5 In clinical settings, Drucker et al 6 reported potential benefits of intravenous immunoglobulin in therapy for children with a recent onset of myocarditis. McNamara et al 7,8 reported successful treatment of adult patients with acute cardiomyopathy by immunoglobulin, which was associated with improved recovery of left ventricular function.
Although the effect of immunoglobulin against myocarditis was established, 5–8 the mechanisms and importance of the Fc portion of immunoglobulin remain nuclear. In the present study, we investigated the effects of intact type of immunoglobulin and F(ab′)2 fragments type of immunoglobulin on murine influenza virus myocarditis with the analysis of macrophage inflammatory protein-2, which is a murine counterpart of interleukin-8. The importance of Fc portion of immunoglobulin and the inhibitory Fc receptor were discussed.
Influenza viruses were obtained from Dr. H. Ochiai, Toyama Medical Pharmaceutical University, 9,10 and was harvested with the allantoic sacs of chicken embryos. Other viruses were harvested with VERO (African green monkey kidney) cells. 5 The virus fluids were stocked at −80°C until use.
Four-week-old male, inbred, certified virus-free A/J mice (Shizuoka Laboratory Animal Center) were used. The animals were cared for in accordance with the institutional policies and guidelines of Toyama Medical and Pharmaceutical University.
In Vitro Study
Immunoglobulin (Venilon) and F(ab′)2 fragments of immunoglobulin (Gamma-Venin) were kindly supplied by Teijin Co, Ltd and Aventis Co. Ltd, respectively. Anti-influenza A virus activity was assayed by the plaque formation method. 9–11 A serially diluted sterile solution of intact immunoglobulin and F(ab′)2 fragments of immunoglobulin was incubated with 100 plaque-forming units (PFU) of influenza A virus (NWS, H1N1 type) at 37°C for 1 hour. The reaction was stopped at 4°C for 30 minutes. The sample was added to confluent monolayers of Madin-Darby canine kidney (MDCK) cells in 6-well plastic plates. After 2 days of incubation at 34°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. We chose NWS, H1N1 strain of influenza virus A in in vitro and in vivo studies because the strain is cardiotropic, which was confirmed by preliminary studies.
The in vitro sensitivity of immunoglobulin preparations against different substrains of influenza virus and other viruses was tested by incubation of various dilutions of the virus in the presence of both intact type and F(ab′)2 type at a concentration of 10−2 μg/ml. The results were expressed as end-point titers of virus growth and were compared with the titers of the untreated stocks.
RAW 264.7 cells (murine macrophage-like cells with Fc receptor) were inoculated into 6-well plates (3 × 105/well) and incubated at 37°C for 24 hours. The cells were maintained in Dulbecco-modified minimum essential medium with 10% fetal calf serum and then infected with influenza A virus at multiplicities of infection (MOI) of 0.1, 1, and 10 PFU/cell. Twenty-four hours later, a final concentration of 6 mg/ml of intact immunoglobulin or F(ab′)2 fragments of immunoglobulin was added to these cells. From 2 to 36 hours after the immunoglobulin treatment, the supernatants at MOI of 1 were assayed for macrophage inflammatory protein-2 (MIP-2) concentrations. To demonstrate the direct evidence for Fc receptor involvement, a specific Fc receptor (FcγIII/II receptor) antibody, 2.4G2 (PharMinger), was pretreated (0.1, 1.0 ng/ml) 6 hours before immunoglobulin preparations administration.
In Vivo Study
The mice were infected with the virus at a dose of 5 × 103 PFU/25 μl by nasal route.
Intact immunoglobulin (Venilon, human immunoglobulin) or F(ab′)2 fragments of immunoglobulin (Gamma-Venin, human 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, the dose of the agents used was 1 g/kg/d. 5 Immunoglobulin antigenicity between different species does not seem to be a problem. 5
Mice were randomized to 3 groups in which they received no treatment (n = 25), treatment with intact immunoglobulin (n = 25), or treatment of F(ab′)2 fragments (n = 25). Mice in the untreated group were injected intraperitoneally with 0.1 mL saline during the treatment period. Beginning simultaneously with influenza A virus (NWS, H1N1 type) inoculation, treatment was given for 10 days in Experiment I. In Experiment II, treatment was begun 2 days after the virus inoculation, and was given for 8 days. The mice were observed daily, and necropsy was performed immediately on those mice found dead. Five mice in each group were killed on day 5 for virological study and for an age-matched study of cardiac pathology in Experiments I and II. Accordingly, the survival study covered 20 mice in each of the 3 groups in Experiments I and II. Mice who survived until the end of the treatment period were killed. The organs were processed for pathologic and virological studies.
Additional control groups were uninfected mice treated for 10 days with saline (n = 5), with intact immunoglobulin (n = 5), and with F(ab′)2 fragments (n = 5).
Tissues 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 pathologic criteria for grading the severity of myocardial infiltration and necrosis were as follows: grade 1 (minimal), one or two small foci; grade 2 (mild), several small foci; grade 3 (moderate), multiple small foci or several large foci; and grade 4 (severe), multiple large foci or diffuse infiltration or necrosis per low-power field. The other organs were evaluated for evidence of viral or other pathologic lesions.
Myocardial virus titers and serum-neutralizing antibody titers were determined as previously described. 5
Blood was obtained from the retro-orbital plexus, and plasma MIP-2 levels were determined by using antibody sandwich enzyme-linked immunosorption assay (ELISA). Briefly, rabbit anti-MIP-2 antibody and biotinylated anti-MIP-2 antibody were used as the capture and second-layer antibodies, respectively. Color development was continued for several minutes by addition of peroxidase-coupled streptavidin and the chromogenic substrate 3,3′-diaminobenzidine tetrahydrochloride (DAB) solution before terminating the reaction with 2 M H2SO4. The A492 was measured on a microplate reader (Bio-Rad). Five wells were used for each experimental time point to calculate the mean ± standard deviation (SD).
Survival of mice was analyzed by the Kaplan-Meier method. Statistical comparisons of plasma MIP-2 levels and histopathological data were performed by one-way analysis of variance (ANOVA). When significant differences were found, the two-tailed t test was used as a post-ANOVA test for establishing differences. Differences were considered statistically significant at P < 0.05. Results are expressed as mean ± SD.
Anti-Influenza Virus Activity In Vitro
The antiviral activity of immunoglobulin preparations was tested against different influenza virus substrains and other viruses (Table 1). In general, the intact type of immunoglobulin was more potent against the viruses tested than was the F(ab′)2 type.
The percentage of plaque formation was 97 ± 19% at intact immunoglobulin concentration of 10−5 mg/ml, 59 ± 17% at 10−4 mg/dl, and 23 ± 9% at 10−3 mg/dl (each n = 5). Linear regression analysis showed a negative correlation of Y = −88.5−37.0 log10X (r = −0.91, P < 0.01) between PFU and the logarithms of immunoglobulin concentrations. Thus, intact immunoglobulin contains anti-influenza virus activity. The percentage of plaque formation was 106 ± 24% at F(ab′)2 fragments of immunoglobulin concentration of 10−5 mg/dl, 57 ± 16% at 10−4 mg/dl, and 21 ± 8% at 10−3 mg/dl (each n = 5). Linear regression analysis showed a negative correlation of Y = −107.6−42.2 log10X (r = −0.91, P < 0.01) between PFU and the logarithms of F(ab′)2 concentrations. Thus, F(ab′)2 fragments also contain anti-influenza virus activity. The dose inhibiting 50% of plaques of intact immunoglobulin and F(ab′)2 fragments was 0.0002 mg/dl and 0.0002 mg/dl, respectively. Accordingly, the dose inhibiting 50% of plaques was the same between intact type and F(ab′)2 type.
Importance of the Fc portion
As shown in Table 2, infection dose (MOI)-dependent MIP-2 increases were detected in the supernatants of influenza virus-infected RAW 264.7 cells. The serial MIP-2 productions were depressed by the management of intact type immunoglobulin, but not by the F(ab′)2 type. The MIP-2 concentrations at 2, 14, 24, and 36 hours after the treatment were decreased by the intact type immunoglobulin treatment, but not by the F(ab′)2 type treatment. In addition, it was clearly shown that pretreatment by a specific Fc receptor antibody blocked the suppression of MIP-2 productions by intact immunoglobulin treatment (Table 2). Accordingly, the Fc portion of immunoglobulin plays an important role in the suppression of MIP-2 production on RAW 264.7 cells.
Intact Immunoglobulin and F(ab′)2 Fragments Suppress Influenza Virus Myocarditis in Experiment I
Fifteen mice in the control group, no mice in the intact immunoglobulin-treated group, and no mice in the F(ab′)2 fragments-treated group had died by day 10; the survival rate on day 10 was 25.0% (5 of 20) in the control group and 100% (20 of 20) in both treated groups (Table 3). The difference between treated (intact and F(ab′)2 fragments) and control groups was significant (P < 0.01).
In the mice killed on days 5 and 10, the scores for cellular infiltration and myocardial necrosis were lower in both immunoglobulin-treated groups than in the control group (Fig. 1). Notably, there was no myocarditis in any of both immunoglobulin-treated mice (ie, intact immunoglobulin and F(ab′)2 fragments administration completely suppressed the development of influenza virus myocarditis).
Pneumonia, probably virus-induced, was noted in all the mice in the untreated group but in no mice in the treated (intact and F(ab′)2 fragments) groups. Serum-neutralizing antibody titers on day 10 were significantly higher in both immunoglobulin-treated groups than in the untreated group. Plasma MIP-2 levels on day 5 were lower in both treated groups than in control group. No viruses were recovered from the hearts of both immunoglobulin-treated mice (ie, an exogenous antibody had neutralized influenza virus). There was no death of uninfected mice throughout the entire period. There were no abnormal pathologic changes examined in the 3 uninfected groups.
Intact Immunoglobulin, but Not F(ab′)2 Fragments, Suppresses Influenza Virus Myocarditis in Experiment II
No mice died between day 0 and day 2 after the virus innoculation; the surviving mice were divided into the 3 groups (Table 4). Thirteen mice in the control group, 5 mice in the intact immunoglobulin-treated group, and 8 mice in the F(ab′)2 fragments-treated group had died by day 10; the survival rate on day 10 was 35.0% (7 of 20) in the control group, 75.0% (15 of 20) in the intact immunoglobulin group, and 60.0% (12 of 20) in the F(ab′)2 fragments group. The difference between intact immunoglobulin, but not F(ab′)2 fragments, and control groups was significant (P < 0.05).
In the pathologic examination, the severity of myocarditis was significantly lower in the intact immunoglobulin group, but not in the F(ab′)2 fragments group, than in the control group. Serum-neutralizing antibody titers on day 10 were higher in both immunoglobulin-treated groups than in the untreated group. Plasma MIP-2 levels on day 5 were lower in the intact immunoglobulin group, but not in the F(ab′)2 fragments group, than in the control. There were no significant differences in the myocardial virus titers among the 3 groups.
The beneficial effects of immunoglobulin have been established in autoimmune and inflammatory diseases, as described previously. 12,13 Previous studies showed possible mechanisms of immunoglobulin action as follows: (i) neutralization of viruses and microbial toxins, (ii) attenuation of complement-mediated tissue damage, (iii) anti-inflammatory activities through the inhibitory Fc receptor, (iv) anti-inflammatory activities through the possession of anti-cytokine antibodies or cytokine receptor antagonists, and (v) immunomodulative activities by anti-idiotype antibodies, antibodies against membrane-associated immunologic molecules such as CD4 and CD5, or soluble forms of human leukocyte antigen class I and class II molecules in immunoglobulin. In addition, the efficiency of immunoglobulin treatment is suggested to be due to acceleration of pathogenic immunoglobulin G catabolism, because the neonatal Fc receptor (FcRn), which was initially identified in neonatal intestinal epithelium, is presumably saturated, which permits the degradation of immunoglobulin G to occur in proportion to its total concentration in plasma.
This study showed that immunoglobulin therapy completely suppressed influenza A virus myocarditis through an anti-influenza viral antibody effect associated with the reduction of plasma MIP-2 levels. Intact type and F(ab′)2 type of immunoglobulin preparations exhibited antiviral activities against many substrains of influenza virus. The anti-influenza virus activity of intact immunoglobulin was superior to that of F(ab′)2 fragments. The time-dependent production of MIP-2 was decreased in virus-infected macrophages by the management of the intact type immunoglobulin, but not by the F(ab′)2 type in vitro. The involvement of Fc receptor for this phenomenon was confirmed by the blocking experiments with a specific Fc receptor antibody.
It is well known that vaccination against influenza viruses, however, may fail to protect the host from infection, mainly because of sudden mutation of the virus antigenicity. In view of the characteristics of immunoglobulin having the potential of somatic mutation against unknown antigens, even newly appeared mutant influenza viruses could be suppressed by immunoglobulin administration. As noted previously, immunoglobulin therapy is of value in the management of autoimmune and inflammatory diseases via anti-pathogen as well as anti-inflammatory effects. 12,13 Indeed, we had already shown that immunoglobulin suppressed murine myocarditis caused by pathogenic and non-pathogenic viruses to human. 5,14 Taken together with previous reports 5,14 immunoglobulin therapy could protect against the host from various unknown virus-induced myocarditis.
Interleukin-8 (IL-8) is a monocyte/macrophage-derived peptide that belongs to a cytokine family and MIP-2 is considered to be a murine counterpart of IL-8. 9–11 This cytokine has chemotactic activity for neutrophils and lymphocytes. 15–18 For example, T-lymphocyte recruitment by IL-8 administration was reported in severe combined immunodeficiency mice and in rats with glomerulonephritis. 16,17 Accordingly, it may be of value to investigate the changes of MIP-2 in murine myocarditis. Cook et al 18 demonstrated in vivo the involvement of MIP-1, a member of the MIP-2 family, in the inflammatory response in murine coxsackievirus B3 using knockout mice. That is, homozygous MIP-1 mutant (−/−) mice were resistant to coxsackievirus-induced myocarditis compared with wild-type (+/+) controls. 18 It is considered that influenza infection may cause severe inflammatory cellular damage via increased chemotactic activities of MIP-2. 9–11 Thus, immunoglobulin therapy has much advantage for the host to reduce MIP-2 production produced by influenza virus infection, as shown in the current study. In addition, the superiority of intact immunoglobulin to F(ab′)2 fragments of immunoglobulin was demonstrated; that is, intact type, but not F(ab′)2 type, suppressed the elevation of plasma MIP-2 levels in the protocol of starting 2 days after the virus inoculation (Experiment II). Accordingly, Fc portion of immunoglobulin may play an important role in the reduction of MIP-2.
Recently, the role of inhibitory Fc receptor (Fc γ receptor II B) was demonstrated for the mechanisms of immunoglobulin in experimental autoimmune or inflammatory diseases. 19,20 In addition, increasing evidence suggested that activation of immune cells by intact immunoglobulin, for example, cytokine and free radical releases and cytotoxic activities, is related to Fc receptors.23,24 The superiority of intact type to F(ab′)2 type in view of the suppression of plasma MIP-2 levels in Experiment II might be well explained by the significant role of Fc portion of immunoglobulin, which theoretically could bind to the Fc receptor, resulting in the reduction of MIP-2.
In clinical aspects, there remains the question of whether immunoglobulin administration after approximately 1 week, when symptoms may have appeared and the virus may be difficult to grow in the myocardium, could be a useful approach.
In conclusion, immunoglobulin therapy suppresses influenza A virus myocarditis by increasing neutralizing antibody titers associated with the reduction of MIP-2 activities in mice. From the standpoint of suppression of MIP-2 concentrations, an intact type might be chosen for the treatment. This treatment may be promising for the therapy for influenza virus-induced myocarditis in spite of the antigen mutation of virus antigenicity, which is common in the virus.
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