Modification of Viral Myocarditis in Mice by Interleukin-6
Abstract Inflammatory cytokines play a key role in the myocardial injury produced by viral myocarditis. Although interleukin-6 (IL-6) reportedly possesses antiviral properties, its effect in viral myocarditis is unclear. To investigate the role of IL-6 in viral myocarditis induced by encephalomyocarditis virus (EMCV) in mice, we evaluated (1) the survival rate following IL-6 administration, (2) the viral titer in the heart, (3) viral replication in the heart by in situ hybridization, (4) histopathological changes using immunohistochemical staining, (5) neutralizing antibody against EMCV, (6) circulating interferon and tumor necrosis factor-α (TNF-α), (7) viral suppression in vitro by IL-6, and (8) natural killer (NK)–cell activity. Eight-week-old C3H/HeJ mice were injected intraperitoneally with EMCV (day 0) and were also injected subcutaneously twice daily for 4 consecutive days with 10 μg/0.1 mL of human IL-6 on day −4 (group A), day 0 (group B), or day +4 (group D) for 4 days. As a control, 0.1 mL PBS instead of IL-6 was injected on day 0 for 4 days (group C). Certain mice were killed on day 4. The myocardial virus titers, viral replication in situ, and NK-cell activity in the spleen were determined. Decreased viral titer and viral replication in the heart reduced the titer of circulating TNF-α, and lower NK-cell activity was observed in group B versus group C (control group). The titer of neutralizing antibodies against EMCV was significantly (P<.05) increased in group B compared with group C. The remaining mice were killed on days 10 and 30 after infection. The ratio of heart weight (HW) to body weight (BW) and myocardial injury in group B were reduced versus group C on days 10 and 30. The HW of group B on day 30 did not differ from the normal control group. The ratio of splenic weight to BW and the ratio of thymic weight to BW of group B increased on day 10, with expanded follicles observed in the spleen and enlargement of the medulla observed in the thymus. Immunohistochemical study revealed an increased percentage of macrophages in the heart and spleen of group B. In summary, IL-6 reduces myocardial damage in mice with viral myocarditis. Modification of immune responses together with reduction in viral replication appears to be the mechanism of the IL-6 effect. Although IL-6 is likely important in the process of viral antigen presentation, early activation of immune responses and attenuation of viral replication appear most significant, as reflected in the limited time window during which IL-6 is effective in myocarditis.
Inflammatory cytokines are involved in the pathogenesis of myocardial injury in viral myocarditis. The antiviral effects of inflammatory cytokines such as IL-1, IL-2, IL-6, and TNF-α have been studied in vitro.1 2 3 Neither IL-1 nor TNF-α had a beneficial effect on a murine model of viral myocarditis in vivo.4 5 The effect of IL-2 is controversial in viral myocarditis.6 7 The effects of IL-6 have not been studied.
IL-6 is a pleiotropic cytokine with a variety of biological activities, including the ability to stimulate B-cell differentiation,8 activate thymocytes and T cells for differentiation,9 activate macrophages,10 stimulate hepatocytes to produce acute-phase proteins,11 and activate NK cells.12 This cytokine also possesses anti-inflammatory properties.13 Whereas IL-1 and TNF-α promote inflammation and are involved in the pathogenesis of clinical disorders such as septic shock, IL-6 would be expected to produce a restorative effect by inducing the activity of immunoregulatory cells in the replication setting.
We seek to learn more about the role of cytokines in the possible prevention of the myocardial injury caused by viral myocarditis. Immunomodulatory agents have been found effective in reducing myocardial necrosis and lymphocyte infiltration in myocarditis induced in mice by the EMC virus.14 15 In the present study, we examine the protective effect of IL-6 on viral myocarditis in mice.
Materials and Methods
Eight-week-old C3H/HeJ female mice purchased from Charles River Co (Atsugi, Japan) were used in the experiments.
A myocarditic variant of EMC virus was obtained from Y. Seto, PhD (Keio University, Tokyo, Japan). Virus preparations were stored at −80°C in Eagle’s MEM supplemented with 0.1% fetal bovine serum until use.
rhIL-6, produced in Escherichia coli by recombinant DNA techniques described previously, was used (provided by Ajinomoto Co, Inc, Kanagawa, Japan).16 A previous report mentioned that human IL-6 could be active in modulating antibody responses in mice.17 Since its specific activity was 5×106 U/mg and the dose of 5×104 U per mouse was effective,16 the dose of 10 μg (5×104 U) rhIL-6 per mouse was selected in this experiment. The product contained <0.25 ng of endotoxin per milligram IL-6.
Animals were inoculated intraperitoneally with 500 plaque-forming units of EMC virus in 0.1 mL of saline.
A total of 200 mice were randomly assigned to four groups, each group consisting of 50 mice. Each mouse in groups A, B, and D received rhIL-6 at 10 μg per mouse (0.1 mL) injected subcutaneously twice daily for 4 days. Mice were injected with rhIL-6 starting on day −4 (group A, n=40), coincident to virus infection on day 0 (group B, n=40), or starting on day +4 (group D, n=40). Control mice received a corresponding volume of PBS injected subcutaneously coincident to virus (day 0) for 4 days (group C, n=40). Ten to 20 mice in each group were preselected before treatment and observed for their survival rate for 14 days. Serum IFN titers were examined on days 1, 2, 3, and 5 after infection. The neutralizing antibody titers against EMC virus were assayed on days 3, 4, 6, 9, and 12. The remaining mice were killed on days 4, 10, and 30 after inoculation, and heart viral titer and splenic NK-cell activity assays and pathological and immunohistochemical studies were conducted. In vitro IL-6 virus suppression assays were also conducted. Uninfected, untreated mice served as the NC group.
The viral titer in individual hearts was assayed in terms of the cytopathic effect, expressed as (TCID50). The heart on day 4 after inoculation was homogenized in 2 mL of Eagle’s MEM. After centrifugation, the supernatant was added to a 96-well microtiter plate containing human amnion (FL) cells in MEM containing 10% fetal calf serum and then incubated. The microtiter plate was examined daily for 5 days for the appearance of any cytopathic effect.
In Situ Hybridization
Two 5-μm transverse ventricular slices from the mouse hearts were mounted on sialinated slides, dried overnight at 60°C, and deparaffinized, permeabilized, and colorimetrically detected as previously described,18 with modification of the original methods.19
The cDNA PECq used to prepare the probe was generously provided to us by Dr Ann Palmenberg, University of Wisconsin, Madison. The probe was prepared by in vitro transcription of Sal I–linearized plasmid DNA with T7 RNA polymerase and labeled by digoxigenin. This cDNA transcribes the positive strand of the EMC virus. Therefore, we used it as a probe for the negative strand of the virus in tissue; when present, it reflects active replication.
In brief, the slides were deparaffinized, permeabilized with proteinase K, and hybridized with a digoxigenin-labeled EMC riboprobe overnight at 42°C. After a stringency washing with a 50% formamide wash buffer, the slides were blocked with lamb serum. Anti-digoxigenin antibody was then applied followed by color substrate nitro blue tetrazolium overnight. Slides were then countered with carmalum. Positive signal was seen as a purple-black precipitate.
The heart and other organs were weighed. BW was also recorded. One half of each organ was fixed in 10% buffered formalin and stained with hematoxylin-eosin; the other half was frozen in embedding compound at −120°C for immunohistochemical studies. Transverse sections of ventricular myocardium were graded for the severity of necrosis and mononuclear cell infiltration, scored from 1 to 4 as follows: grade 1, lesions involving <25% of the ventricular myocardium; grade 2, lesions involving 25% to 50% of the myocardium; grade 3, lesions involving 50% to 75% of the myocardium; and grade 4, lesions involving 75% to 100% of the myocardium. The spleen, thymus, lungs, and liver were examined grossly and microscopically. Measurements of the splenic lymphoid follicular area and thymic medullary area were expressed as a percentage of total splenic or thymic area in the long-axis sections. Tissues were evaluated blindly by an experienced pathologist who was familiar with the grading of murine viral myocarditis and had no knowledge of the study design.
The heart ventricle, thymus, and spleen of animals in groups B and C were divided into two equal cross sections along their long axes. One half of each organ was processed for immunohistochemical staining. Organs were embedded in OCT compound (Miles Laboratories). Sections 6 μm thick were cut from the frozen blocks on a cryostat at −20°C, placed on glass slides, air-dried for 1 hour, and fixed in cold methanol. Cell surface markers were demonstrated in situ by staining with 3,3′-diaminobenzidine tetrahydrochloride immunoperoxidase and by using a series of monoclonal rat alloantigenic antibodies, including rat anti-mouse Thy 1.2 (1:20, Becton Dickinson Co) to analyze T cells, goat anti-mouse IgG antibody (1:50) (Cappel Laboratories) to analyze B cells, rabbit anti-mouse heteroantiserum to asialo GM1 (1:50, WAKO Chemicals) to analyze NK cells, and Mac-1 antibody (1:100, Ortho Diagnostic) to analyze macrophages. The avidin-biotin complex immunohistochemical method used has been described elsewhere.20 Sections were counterstained with hematoxylin.
For cell counts, the sections were examined in a blinded fashion. We recorded the number of cells in each section that were stained with each antibody and the total number of nucleated inflammatory cells, and then we calculated the percentage of stained inflammatory cells. This process was repeated for each inflammatory focus, defined as an average of ≥100 infiltrating cells per focus.
Neutralizing Antibody Filtration
Volumes (0.1 mL per well) of FL cell suspensions at a concentration of 1×105 cells per milliliter, as described in relation to virus titers, were placed in each well of 96-well plastic plates and allowed to grow for 2 days at 37°C in an incubator. The sera, obtained under sterile conditions, were immobilized at 56°C for 30 minutes, and volumes of the sera, serially diluted by twofold increments, were incubated for 30 minutes at 37°C with the same volume of suspensions containing 100 TCID50 of the EMC virus. The incubated sera were then adsorbed onto FL cell monolayers for 60 minutes in a 37°C incubator, and the same volumes (50 μL) of Eagle’s MEM with 10% fetal calf serum were overlaid. Plates were observed daily for a week for signs of characteristic cytopathic effect. The highest dilution of sera that inhibited these cytopathic effects in more than one of two wells containing the same dilution of serum was determined to be the titer of neutralizing antibody against EMC virus.
Assay for Mouse IFN and TNF-α
Serum IFN was assayed by the technique of cytopathic effect inhibition described elsewhere.14 Briefly, LY cells were seeded onto a 96-well microtiter plate, and when the cells had become attached to the plate, they were treated with the test sample or the mouse IFN reference standard. Eighteen hours later, the treated cells were challenged with vesicular stomatitis virus and then incubated at 37°C under an atmosphere of 5% CO2 for an additional 48 hours. After the cells were stained with neutral red, an electrophotometer was used for evaluation. IFN activity was expressed as a reciprocal dilution resulting in 50% reduction of the neutral red uptake, and the titer of IFN was examined in murine serum on days 1, 2, 3, and 5 after viral inoculation. Mice treated with IL-6 starting on day 4 (group D) were not assayed for IFN.
Circulating TNF-α was determined by ELISA (Endogen) in the plasma of infected mice. Rat anti-mouse TNF-α monoclonal antibody was used in the ELISA assay at a concentration of 100 μL of 5 μg/mL solution in PBS, as described elsewhere.21 In brief, 50 μL of each sample was added in duplicate to the antibody-precoated plates. After 2 hours of incubation at room temperature, plates were washed five times, and 100 μL of peroxidase-conjugated goat anti-rat IgG was added to the plates and incubated for 30 minutes at room temperature. Plates were washed five times, and 100 μL of tetramethylbenzidene substrate solution was added to each well and developed in the dark for 30 minutes. Stop solution (0.18 mol/L H2SO4) was added to each well. This ELISA detects <10 pg/mL of murine TNF-α. Standard curves were simultaneously generated with murine TNF-α.
Assay for NK-Cell Cytotoxicity
NK-cell activity was assayed by the standard 51Cr-release assay as described previously.13 14 Briefly, YAC-1 tumor cells were labeled with 51Cr and diluted to a concentration of 1×105 cells per milliliter in 1640 culture medium (Roswell Park Memorial Institute) containing 10% fetal bovine serum. Spleen cells from the mice killed for study were suspended in the same medium and used as effector cells. Spleen cells and target YAC-1 cells were dispensed into a round-bottomed 96-well microtiter plate to give an effector cell–to–target cell ratio of 50:1 and 100:1, respectively, and then incubated at 37°C in a humidified chamber containing 5% CO2 for 4 hours. The cells were then harvested, and their associated radioactivity was counted with a gamma counter. The percentage of specific lysis was calculated as follows:
Viral Suppression Test
The viral titers in hearts of EMC-infected mice were determined in media containing hrIL-6. A first culture medium contained IL-6 at a concentration of 100 μg/mL; a second contained IL-6 at a concentration of 10 μg/mL. The third medium was a control medium and contained saline instead of IL-6. After preparing the culture media, supernatants from homogenized hearts of day-4 EMC-infected mice were mixed and inoculated into a plate containing FL cells, as described for the virus assay. Samples were assayed in triplicate for cytopathic effects.
Results were expressed as mean±SD. A Kaplan-Meier plot was used to determine the significance of differences in survival rate, and the Kruskal-Wallis test was used to evaluate differences in virus titer, the histological scores, circulating cytokines, and NK-cell activities. Statistical significance was considered present for values of P<.05.
Survival After IL-6 Treatment
The survival rates on day 14 were 32% in group A, 50% in group B, 25% in group C, and 17% in group D. The increase in survival in group B was statistically significant compared with that in group C (P<.05). On the other hand, survival rates in groups A and D did not differ from that in group C (Fig 1⇓). Thus, treatment with IL-6 starting on the same day as EMC virus improved the survival, although IL-6 given earlier or later was not effective in this regard.
Viral Titer in Heart
The average cardiac viral titer determined on postinoculation day 4 was 2.42±0.08 log10 TCID50/mg wet wt (n=4, mean±SD) in group A, 1.64±0.20 log10 TCID50/mg wet wt (n=4) in group B, and 2.17±0.33 log10 TCID50/mg wet wt (n=4) in group C. The viral titer in group B was significantly lower than that in group C (Fig 2⇓).
In Situ Hybridization of Virus in Myocardium
Hearts from the infected mice in all treatment groups were positive by nonradioactive in situ hybridization for the negative strand of the EMC virus. This demonstrates that there is active infection by the virus in the cardiac myocytes of these hearts. There was a greater amount of virus, located by in situ hybridization in cardiac muscle, in the untreated group of mice than in group B (Fig 3⇓).
The BW in group C on days 10 and 30 after virus inoculation was significantly (P<.01) lower than that in the NC group. The HW and HW/BW ratio in groups A, C, and D on days 10 and 30 were elevated compared with that of the NC group (P<.01). In group B, the HW and HW/BW ratio on day 10 and the HW, BW, and HW/BW ratio on day 30 were significantly (P<.01) lower than those in group C. However, the HW, BW, and HW/BW ratio in group B on day 30 did not differ significantly from those of the NC group (Table 1⇓). The HW, BW, and HW/BW ratio in group C on days 10 and 30 were elevated compared with those of the NC group (P<.01). In group B, the HW and HW/BW ratio on day 10 and the HW, BW, and HW/BW ratio on day 30 were not significantly (P<.01) different from those of the NC group (Table 1⇓).
The SpW/BW ratios on days 10 and 30 in group C did not differ from those of the NC group. The ThW/BW ratios on days 10 and 30 in group C were significantly (P<.01) lower than those in the uninfected mice. However, the SpW/BW ratio and the ThW/BW ratio in group B on day 10 were significantly (P<.01) increased versus group C. Although the LiW/BW ratio and the LuW/BW ratio on days 10 and 30 increased in group C compared with the uninfected controls, the LuW/BW ratio on day 10 and the LiW/BW ratio on day 30 were significantly reduced in group B versus group C (Table 2⇓). Although the SpW/BW ratio, LuW/BW ratio, and LiW/BW ratio on days 10 and 30 did not differ between groups A, C, and D, the ThW/BW ratio on day 10 in group A and the ThW/BW ratio on day 30 in group D were significantly (P<.05) lower than those in group C. IL-6 administration did not change the organ weights in uninfected mice compared with those observed in the NC group.
Myocardial necrosis with immune cell infiltration was observed in groups A, C, and D on days 10 and 30. However, scores of both myocardial necrosis and cellular infiltration on days 10 and 30 were significantly lower in group B than in group C (Table 1⇑).
The histopathological changes observed in groups A, C, and D included a decrease in the size of lymphoid follicles in spleens and atrophy of the thymic medulla. These changes were apparent on day 10 (Fig 4E⇓ and 4G⇓) but not on day 30. However, group B showed enlarged lymphoid follicles in the spleens and an expanded thymic medulla on day 10 (Fig 4F⇓ and 4H⇓). The livers of groups A, C, and D showed congestion on day 10, whereas the livers of group B on day 10 and day 30 showed no congestion. The lungs of groups C and D on day 10 and those of groups A, C, and D on day 30 showed congestion. The lungs of group B revealed no congestion on either day 10 or day 30.
The percentage of the splenic lymphoid follicular area was significantly higher in groups B and D than in group C. The proportion of medullary area to cortical area in the thymus was also significantly (P<.01) higher in group B than in group C (Table 3⇓). The administration of IL-6 to uninfected mice was associated with enlargement of the follicular area in the spleen but not with enlargement of the thymic medulla.
The percentage of T cells and B cells in the hearts on day 10 did not differ between groups B and C. There were a few NK cells in the hearts of both groups. However, the percentage of macrophages in the heart was significantly (P<.01) higher in group B than in group C. The percentage of macrophages in the spleen was also significantly (P<.01) higher in group B than in group C. The percentage of cells in lymphocyte subsets within the thymus was the same in both groups (Table 4⇓).
Titers of Neutralizing Antibody
Titers of neutralizing antibodies against EMC virus were significantly (P<.05) increased in group B compared with group C on day 6 after viral inoculation, whereas titers in groups A and D on day 6 were not different from those in group C. On day 9, titers in groups B and D were significantly (P<.05) higher than those in group C. On days 3, 4, and 12, there were no significant differences among the groups (Fig 5⇓).
Titers of IFN and TNF-α
Mean IFN titer on day 1 after infection was 761±204 U/mL (n=3) in group A, 857±311 U/mL (n=3) in group B, and 899±470 U/mL (n=4) in group C. On days 2, 3, and 5, there were no significant differences among the groups. Circulating TNF-α in groups A and B was significantly (P<.05) lower than in group C on days 1 and 2 after viral inoculation (Table 5⇓).
Splenic NK-cell activity was measured on day 4 after infection. The average NK-cell activity in group B was significantly (P<.01) lower than in group C. The NK-cell activity in group A did not differ from that in group C (Table 6⇓).
Viral Suppression Test
In an in vitro experiment, the addition of IL-6 did not significantly alter the viral titer at concentrations of 10 or 100 μg/mL of media. The viral titer in the first medium with IL-6 (100 μg/mL) added was 2.10±0.33 log10 TCID50/mg, that in the second medium with IL-6 (10 μg/mL) added was 2.20±0.20 log10 TCID50/mg, and that in the control medium without added IL-6 was 2.11±0.33 log10 TCID50/mg. Thus, no direct viral suppressive effect of IL-6 was observed.
In these studies, we have shown that exogenous IL-6, administered at the same time as viral inoculation in C3H/HeJ mice, improves survival rates, decreases cardiac viral titers, and reduces both myocardial necrosis and lymphocytic infiltration in mice with viral myocarditis. Accordingly, SpW/BW and ThW/BW ratios were elevated in these mice. However, the administration of IL-6 either 4 days before virus inoculation or 4 days after, the latter coincident with the peak in viremia and at the beginning of observable clinical illness,22 did not influence survival or myocardial destruction. From the overall experiment, we conclude that exogenous IL-6 has an antiviral effect on EMC viral myocarditis in this mouse model in vivo and involves the modulation of early immune responses, including those of B cells, macrophages, and NK cells.
The LuW/BW ratio reflects the degree of lung congestion; and the LiW/BW ratio, the degree of liver congestion. The simultaneous administration of IL-6 and virus (group B) leads to a significant reduction both in LuW/BW and in LiW/BW ratios, in accompaniment with a reduction of the HW/BW ratio compared with controls (group C) on day 30. The reductions of LuW/BW and LiW/BW ratios in group B are considered to reflect an improvement in congestive heart failure due to viral myocarditis. The effect of IL-6 on histopathological changes depends on the timing of administration. As noted, when administered concurrent with the virus, IL-6 led to significant reduction of myocardial necrosis and cellular infiltration, whereas earlier or later administration had no effect.
The present study also suggests that IL-6 may play an immunomodulatory role in host defense mechanisms against viral myocarditis in mice in vivo. The systemic administration of human IL-6 to mice is reported to mediate tumor regression by eliciting a T-cell response in the host, while having no direct antitumor effect.23 IL-6 also stimulates differentiation of B cells, macrophages, and NK cells in vivo.11 24 25 Early neutralizing antibody titers may be increased by IL-6, and the importance of neutralization in early antiviral host defense is well appreciated. Therefore, the antiviral role of IL-6 in mice may be to elicit these immune responses and to enhance immune cell function rather than to directly interfere with virus replication. Indeed, our in vitro study demonstrated that IL-6 did not suppress viral replication. An additional reflection of immunomodulation by IL-6 was the enlargement of lymphoid organs, including spleen and thymus, in group B. Although the precise mechanisms of IL-6 effect are not addressed in the examination of organ weights, the increased SpW/BW and ThW/BW ratios may be related to the immunomodulatory effect of IL-6.
Moreover, titers of neutralizing antibodies against EMC virus was significantly increased in group B compared with group C. The former mice survived longer than those not given IL-6. Histological examination of group B mice revealed the prominence of splenic lymphoid follicles, an increased percentage of macrophages in the spleen, and an increased area of the thymic medulla. Histologically, the lymphoid follicles included an abundance of T cells and B cells, and the thymic medulla contained T cells, B cells, and macrophages. Therefore, we propose that exogenous IL-6 exerts an antiviral effect by stimulating host immune responses, although administration for 4 days beginning on day −4 or day +4 failed to improve survival.
Earlier administration (day −4) of IL-6 may lead to a reduction in effective biological activity relevant to virus infection because of the short IL-6 half-life in vivo.25 Later administration of IL-6 may induce an excessive host immune reaction. The release of IL-6 from virus-infected myocytes has been reported to cause differentiation of cytotoxic T cells that produced myocardial injury in viral myocarditis.26 On the other hand, the later administration of IL-6 may miss the peak viral titers in blood and heart, thus failing to induce possibly higher neutralizing antibody titers when they are potentially most important. Moreover, IL-6 has been shown to activate myocyte cell surface display of CD54 and to facilitate adhesion and oxidative radical–mediated injury by activated lymphocytes in the myocardium.28 This characteristic may modify survival of this murine host with viral myocarditis. Since the beneficial effects we observed depended on the timing of IL-6 administration, this cytokine may act at more than one stage of host immune response to myocarditic EMC virus and by more than one mechanism.
IL-6 has been shown to suppress inflammation in several animal models,29 30 an effect that is attributed to the inhibition of IL-1β and TNF-α production. In recent studies, mouse strains genetically resistant to coxsackie virus B3 myocarditis developed more severe infection after the administration of either IL-1β or TNF-α.31 Moreover, anti–TNF-α antibody improved survival and myocardial lesions in a murine model of EMC viral myocarditis.5 Thus, the therapeutic effects of IL-6 may be related to its ability to inhibit the production of IL-1β and TNF-α, cytokines that play an important role in the injury of myocytes induced by viral infection. Our data showed that the serum TNF-α level in animals treated with IL-6 was significantly reduced in the early phase of viral myocarditis compared with control animals. Actually, IL-6 is reported to play a role in reducing TNF-α levels in vitro32 and in vivo.33
IL-6 has been demonstrated to be effective in the final stages of B-cell differentiation8 and is essential for the maturation of B cells to antibody-forming cells.34 Our data show that titers of neutralizing antibodies against virus were significantly increased in IL-6–treated mice (group B) on days 6 and 9, which is the early stage of viral myocarditis, compared with untreated mice. Early antibody responses and the decreased viremia are observed in resistant strains of mice infected with coxsackie virus B3 (one of the picornaviruses) myocarditis.35 On the other hand, low titers of serum-neutralizing antibody induced by immunosuppressive agents, such as cyclosporine, observed in mice in the early phase of viral myocarditis are associated with a higher mortality rate.36 Therefore, the beneficial effects of IL-6 on viral myocarditis may be due to enhanced induction of immunoglobulin production by B cells of the infected host.
IFN, NK cells, and macrophages are important in the early phase of host defense against viral infection.37 IL-6 was described previously as IFN-β2, an antiviral agent.38 However, the present study showed no significant modulation of murine IFN by IL-6 in a murine model of viral myocarditis.
IL-6 enhances NK-cell activity in vitro,12 although not in vivo.23 In the present study, IL-6 did not activate NK-cell function in the spleen on day 4. NK cells are considered to kill virus-infected myocytes in viral myocarditis, thus expressing a cytolytic factor, perforin, which permeates the virus-infected heart.39 A recent investigation has shown that an immunomodulatory agent, vesnarinone, protects against myocardial damage and inhibits NK-cell activity in EMC virus–infected mice.40 Therefore, inhibition of NK-cell activity may be related to the improvement of viral myocarditis in mice. Earlier work suggested that macrophages inhibit viral replication by early immunological processing in infected mice.41 Activated macrophages are capable of lysing virus-infected target cells in the absence of specific antibody. Indeed, our previous work showed that combination therapy with OK432 and IFN reduced myocardial damage in a murine model of EMC virus myocarditis in the setting of stimulated macrophages.14 A higher percentage of macrophages in both the inflamed myocardium and the enlarged spleen treated with IL-6 may reflect this beneficial effect of IL-6. Although we did not examine the functional activity of immune cell subsets in these organs, the enhanced number of cardiac and splenic macrophages provides a clue to the antiviral activity of IL-6.
In summary, concurrent systemic administration of human recombinant IL-6 is beneficial to mice infected with the EMC virus and with subsequent active viral myocarditis. The present study supports the observations that IL-6 promotes early adaptive immune responses in thymus, spleen, and heart muscle, and the consequent attenuation of viral replication when IL-6 is administered simultaneously with the virus. However, IL-6 treatment must be concomitant with viral exposure, making this almost useless as a future treatment modality. Additional experiments are under way to explain specifically the mechanism of IL-6 against viral myocarditis.
Selected Abbreviations and Acronyms
|ELISA||=||enzyme-linked immunosorbent assay|
|TCID50||=||tissue culture infectious doses|
|TNF||=||tumor necrosis factor|
We thank A. Okano and H. Suzuki of Ajinomoto Co, Inc, for supplying recombinant human IL-6 and Y. Nonaka for her fine technical assistance.
- Received April 6, 1995.
- Accepted January 23, 1996.
- © 1996 American Heart Association, Inc.
Mestan J, Digel W, Mittnacht S, Hillen H, Blohm D, Moller A, Jacobsen H, Kirchner H. Antiviral effects of recombinant tumor necrosis factor in vitro. Nature. 1987;323:816-819.
Rook AH, Masur H, Lane C, Frederick W, Kasahara T, Macher AM, Djen JY, Manischeurtz JF, Jackson L, Fauci AS, Quinnan GU Jr. Interleukin-2 enhances the depressed natural killer and cytomegalovirus-specific cytotoxic activities of lymphocytes from patients with the acquired immune deficiency syndrome. J Clin Invest. 1983;72:398-403.
Weissenbach J, Chernajovsky Y, Zeevi M, Shulman L, Soreq H, Nir H, Wallach D, Perricandet M, Tiollais P, Revel M. Two interferon mRNAs in human fibroblasts: in vitro translation and Escherichia coli cloning studies. Proc Natl Acad Sci U S A. 1980;77:7152-7156.
Huber SA, Pulgan J, Schultheiss P, Schwimmbeck P. Augmentation of pathogenesis of coxsackie virus B3 infection in mice by exogenous administration of interleukin-1 and interleukin-2. J Virol. 1994;68:195-206.
Yamada T, Matsumori A, Sasayama S. Therapeutic effect of anti–tumor necrosis factor-α antibody on the murine model of viral myocarditis induced by encephalomyocarditis virus. Circulation. 1994;89:846-851.
Matsumori A, Yamada T, Kawai C. Immunomodulating therapy in viral myocarditis: effects of tumor necrosis factor, interleukin-2 and anti-interleukin-2 receptor antibody in an animal model. Eur Heart J. 1991;12(suppl D):203-205.
Kishimoto C, Kuroki Y, Hiraoka Y, Ochiai H, Kurokawa M, Sasayama S. Cytokine and murine coxsackie virus B3 myocarditis: interleukins suppress myocarditis in the acute stage but enhance the condition in the subsequent stage. Circulation. 1994;89:2836-2842.
Hirano T, Yasukawa K, Harada H, Taga T, Watanabe Y, Matsuda T, Kashiwamura S, Nakajima K, Koyama K, Imamatsu A, Tsunasawa S, Sakiyama F, Matsui H, Tokuhara Y, Taniguchi T, Kishimoto T. Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature. 1986;324:73-77.
Lots M, Jirik F, Kabouridis R, Tsoukas C, Hirano T, Kishimoto T, Carson DA. BSF-2/IL-6 is costimulant for human thymocytes and T-lymphocytes. J Exp Med. 1988;140:508-513.
Mule JJ, McIntosh JK, Joblons DM, Rosenberg SA. Antitumor activity of recombinant interleukin-6 in mice. J Exp Med. 1990;171:629-635.
Gauldie JC, Richards C, Harnich D, Lansdrop P, Baumann H. Interferon β2/BSF-2 shares identity with monocyte-derived hepatocyte stimulating factor (HSF) and regulates the major acute phase protein response in liver cells. Proc Natl Acad Sci U S A. 1987;84:7251-7256.
Luger TA, Krutmann J, Kirnbaner R, Vrbarski A, Schwarz T, Klappacher G, Kock A, Mickche M, Malejczyk J, Schauer E, May LA, Sehgel PB. IFN-β2/IL-6 augments the activity of human natural killer cells. J Immunol. 1989;143:1206-1210.
Tilg H, Trehr E, Atkins MB, Dinarello CA, Mier JW. Interleukin-6 as an antiinflammatory cytokine: induction of circulating IL-1 receptor antagonist and soluble tumor necrosis factor receptor p55. Blood. 1994;83:113-118.
Yokoyama T, Kanda T, Suzuki T, Murata K. Combination therapy of OK432 and recombinant human interferon-a A/D on viral myocarditis in mice starting after infection. J Pharmacol Exp Ther. 1991;258:1114-1119.
Takatsuki F, Okano A, Suzuki C, Chieda R, Takahara Y, Hirano T, Kishimoto T, Hamuro J, Akiyama Y. Human recombinant IL-6/B cell stimulatory factor 2 augments murine antigen-specific antibody responses in vitro and in vivo. J Immunol. 1989;141:3072-3077.
Takatsuki F, Okano A, Suzuki C, Miyasaka Y, Hirano T, Kishimoto T, Ejima D, Akiyama Y. Interleukin-6 perfusion stimulates reconstitution of the immune and hematopoietic systems after 5-fluorouracil treatment. Cancer Res. 1990;50:2885-2890.
Wilson JE, Yang DC, Zeheb R, Anderson DA, Bondy GP, McManus BM. In situ hybridization for viral RNA: a comparison of manual and automated methods. FASEB J. 1995;9:A970. Abstract.
Lane JR, Neumann DA, Lafond-Walker A, Herskowitz A, Rose NR. Interleukin-1 or tumor necrosis factor can promote coxsackie B3-induced myocarditis in resistant B10A mice. J Exp Med. 1992;175:1123-1129.
Dong R, Liu P, Wee L, Butany J, Sole MJ. Verapamil ameliorates the clinical and pathological course of murine myocarditis. J Clin Invest. 1992;90:2022-2030.
Mule JJ, Custer MC, Travis WD, Rosenberg SA. Cellular mechanisms of the antitumor activity of recombinant IL-6 in mice. J Immunol. 1992;148:2622-2629.
Puri PK, Lenard P. Systemic administration of recombinant interleukin-6 in mice induces proliferation of lymphoid cells in vivo. Lymphokine Res. 1992;11:133-139.
Henke A, Mohr C, Springer H, Craebner C, Cteilzener A, Nain M, Gemsa D. Coxsackie virus B3-induced production of tumor necrosis factor-a, IL-1β and IL-6 in human monocyte. J Immunol. 1992;148:2270-2277.
Youker K, Smith CW, Miller D, Michael LH, Rossen RD, Anderson DC, Entman ML. Neutrophil adherence to isolated adult canine myocyte: induction by cardiac lymph collected during ischemia and reperfusion. J Clin Invest. 1992;89:602-609.
Barton BE, Jackson JV. Protective role of interleukin-6 in the lipopolysaccharide-galactosamine septic shock model. Infect Immun. 1993;61:1496-1500.
Lane JR, Neumann PA, Lafond-Walker A, Herskowitz A, Rose NR. Interleukin-1 or tumor necrosis factor can promote coxsackie virus B3-induced myocarditis in resistant B10 A mice. J Exp Med. 1992;175:1123-1129.
Aderka D, Le J, Vilcek J. IL-6 inhibits lipopolysaccharide-induced tumor necrosis factor production in cultured human monocytes, U937 cells, and in mice. J Immunol. 1981;143:3517-3522.
Schindler R, Mancilla J, Endres S, Ghorbani R, Clark SC, Danarello CA. Correlations and interactions in the production of interleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. Blood. 1990;75:40-44.
Muraguchi A, Hirano T, Matsuda T, Horii K, Nakajima K, Kishimoto T. The essential role of B cell stimulatory factor 2 (BSF-2/IL-6) for the terminal differentiation of B cells. J Exp Med. 1988;167:332-339.
Wolfgram LJ, Beisel KW, Herskowitz A, Rose NR. Variations in the susceptibility to coxsackie virus B3-induced myocarditis among different strains of mice. J Immunol. 1986;136:1846-1852.
Kishimoto C, Thorp KA, Abelmann WH. Immunosuppression with high doses of cyclophosphamide reduces the severity of myocarditis but increases the mortality in murine coxsackie virus B3 myocarditis. Circulation. 1990;82:982-987.
Seko Y, Shinkai Y, Kawasaki A, Yagita H, Okumura K, Takaku F, Yazaki Y. Expression of perforin in infiltrating cells in murine hearts with acute myocarditis caused by coxsackie virus B3. Circulation. 1991;84:788-795.
Matsui S, Matsumori A, Matoba Y, Uchida A. Treatment of virus-induced myocardial injury with a novel immunomodulating agent, vesnarinone. J Clin Invest. 1994;94:1212-1217.
Letvin NL, Kanffman RS, Finberg R. An adherent cell lysis virus infected targets: characterization, activation and fine specificity of the cytotoxic cell. J Immunol. 1982;129:2396-2401.