© 1996 American Heart Association, Inc.
Articles |
Modification of Viral Myocarditis in Mice by Interleukin-6
From the Department of Laboratory Medicine (T.K., I.K.) and the Second Department of Internal Medicine (R.N., S.I.), Gunma University School of Medicine, and the College of Medical Care and Technology (T.S.), Gunma University, Maebashi, Japan, and the Department of Pathology and Laboratory Medicine (J.E.W.M., D.Y., B.M.M.), University of British Columbia, St. Paul's Hospital, Vancouver, Canada.
Correspondence to Tsugiyasu Kanda, MD, Department of Laboratory Medicine, Gunma University School of Medicine, 3-39-15, Showa-machi, Maebashi 371, Japan.
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Abstract |
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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-
(TNF-), (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-, 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.
Key Words: myocarditis • interleukin-6 • host immunity • in situ hybridization
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-
have been
studied in vitro.1 2 3 Neither IL-1 nor TNF- 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- 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
Eight-week-old C3H/HeJ female mice purchased from Charles River Co (Atsugi, Japan) were used in the experiments.
Virus
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.
Chemicals
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
5x106 U/mg and the dose of 5x104 U per
mouse was effective,16 the dose of 10 µg
(5x104 U) rhIL-6 per mouse was selected in this
experiment. The product contained <0.25 ng of endotoxin per
milligram IL-6.
Infection Protocol
Animals were inoculated intraperitoneally
with 500 plaque-forming units of EMC virus in 0.1 mL of saline.
Treatment Protocol
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.
Virus Titer
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.
Pathological Examination
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.
Immunohistochemical Study
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 1x105 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-
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- was determined by ELISA (Endogen) in the plasma of
infected mice. Rat anti-mouse TNF- 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-. Standard curves were
simultaneously generated with murine TNF-.
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
1x105 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:
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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.
Statistical Methods
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.
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Results |
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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.
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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).
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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).
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Organ Weights
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).
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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.
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Pathological Findings
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.
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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.
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Immunohistochemical Findings
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).
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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).
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Titers of IFN and TNF-
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- in groups A and B was
significantly (P<.05) lower than in group C on days 1 and 2
after viral inoculation (Table 5).
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NK-Cell Activity
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).
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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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-1ß and TNF- production. In recent
studies, mouse strains genetically resistant to coxsackie virus
B3 myocarditis developed more severe infection after the
administration of either IL-1ß or TNF-.31 Moreover,
anti–TNF- 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-1ß and TNF-, cytokines that
play an important role in the injury of myocytes induced by viral
infection. Our data showed that the serum TNF- 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- 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-ß2, 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.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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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.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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