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© 1999 American Heart Association, Inc.
Integrative Physiology |
Susceptibility to Myocarditis Is Dependent on the Response of 
ß T Lymphocytes to Coxsackieviral Infection
From the Centre for Cardiovascular Research, The Toronto Hospital (M.A.O., K.A., W.-H.W., F.D., P.L.), and Ontario Cancer Institute and Amgen Institute and Departments of Medical Biophysics and Immunology (J.P., T.M.), University of Toronto, Toronto, Ontario, Canada.
Correspondence to Dr Peter P. Liu, Cardiology Research, The Toronto Hospital, EN12-324, 200 Elizabeth St, Toronto, Ontario, Canada, M5G 2C4. E-mail peter.liu{at}utoronto.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Abstract—Viral myocarditis is an important cause of heart failure and dilated cardiomyopathy. T lymphocytes are implicated in myocardial damage in murine models of coxsackievirus B3 (CVB3) myocarditis. We used knockout mice lacking CD4 (CD4-/-), CD8 (CD8-/-), both coreceptors (CD4-/-CD8-/-), or the T-cell receptor ß chain (TCRß-/-) to address the contribution of T-cell subpopulations to host susceptibility to CVB3 myocarditis. Severity of disease was magnified in CD8-/- mice but attenuated in CD4-/- mice, consistent with a pathogenic role for CD4+ lymphocytes. Elimination of both CD4 and CD8 molecules from T lymphocytes by genetic knockout better protected mice from myocarditis, demonstrating that both CD4+ and CD8+ T cells contribute to host susceptibility. The same benefit occurred in TCRß-/- mice, with prolonged survival and minimal myocardial disease observed after CVB3 infection. Elevated interferon-
and decreased tumor necrosis factor-
expression are associated with attenuated myocardial damage in
CD4-/-CD8-/- mice. These results show that
the presence of TCRß+ T cells enhances host
susceptibility to myocarditis. The severity of myocardial damage and
associated mortality are dependent on the predominant T-cell type
available to respond to CVB3 infection. One mechanism by which
CD4+ and CD8+ T-cell subsets influence the
pathogenesis of myocarditis may involve specific cytokine
expression patterns.
Key Words: myocarditis • coxsackievirus • T lymphocyte • cytokine • transgenic mice
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Viral myocarditis is an important cause of heart failure and dilated cardiomyopathy.1 2 The spectrum of disease spans from a fulminant course, including arrhythmias or sudden death to mild early symptoms, later progressing to chronic heart failure.1 2 Group B coxsackieviruses (CVB) have been associated commonly with viral myocarditis.1 Current evidence from CVB3 myocarditis in mice suggests that the acute phase of disease involves both direct, virus-mediated myocyte dysfunction and tissue injury mediated by the immune response.2 Whereas some elements of the immune response attenuate viral replication and protect the myocardium, others contribute to the development of myocardial inflammation and necrosis.2 In SCID mice, which lack both T and B cell functions, CVB3 infection induces severe disease.3 In contrast, depletion of T lymphocytes, through thymectomy, irradiation and bone marrow reconstitution or by injection of antithymocyte serum, attenuates myocarditis.4 Monoclonal antibody therapy directed against T-cell populations can modify myocarditis severity; however, depletion is not complete.5 6 A proportion of each T-cell subset remains available to participate in the immune response after antibody administration.5 6
The majority of circulating T lymphocytes express and ß chains of
the T-cell receptor (TCR) with either CD4 or CD8 coreceptor molecules,
which augment TCR signaling pathways.7 Classically, after
viral infection, the host immune system responds via activation of
TCRß+CD4+ helper T
cells and TCRß+CD8+
cytotoxic/suppressor T cells in a major histocompatibility complex
(MHC) II– and MHC I–restricted manner, respectively.7 In
addition,
TCRß+CD4–CD8–
(double negative; DN) T cells can participate in the immune
response.8
Models using transgenic knockout technology provide a highly specific
system to examine the contributions of specific subsets of T
lymphocytes to pathology.9 In this study, we have used
mice with targeted gene disruptions of CD4+,
CD8+, both CD4+ and
CD8+, or TCRß genes to examine the role of
ß T lymphocytes in the pathogenesis of viral myocarditis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Virus
The cardiovirulent strain of CVB3 was adapted by Woodruff and Woodruff 4 and passaged in the laboratory of Dr Charles Gauntt.10 Virus stock was prepared by passage through HeLa cell cultures and then titered by plaque assay and stored at –70°C. Aliquots from the same stock were used for all animals.
Mice
Transgenic knockout mice, created by disruption of the
CD4, CD8 (Ly-2), or TCRß chain gene in embryonic stem cells
through homologous recombination, have been described
previously.11 12 13 Mice heterozygous for CD4 and CD8 genes
were interbred to produce homozygotes for the
CD4-/- or CD8-/-
genotype.
CD4-/-CD8+/+ mice were
subsequently bred with
CD4+/+CD8-/- mice to
obtain CD4+/-CD8+/-
offspring.14 After mating of double heterozygotes,
CD4-/-CD8-/- offspring
were produced.14 Mice of the
CD4-/-CD8+/+ or
CD4-/-CD8+/-
genotype will be referred to as CD4-/-
throughout the text. Mice with genotypes
CD4+/+CD8-/- or
CD4+/-CD8-/- are
designated CD8-/-. No differences were found
between the 2 genotypes within each group.
CD4+/-CD8+/- (control)
mice, with both CD4 and CD8 molecules intact, served as controls. All
animals were backcrossed into an A/J strain
(H2k/k) to generate mice (fifth generation) with
uniform CVB3 susceptibility. An H2k/k haplotype
was confirmed for all models using flow cytometry. Mice were housed and
handled in an aseptic manner, including use of microisolator cages,
sterilized mouse chow and drinking water, and use of continuous laminar
airflow. Care of animals was in accordance with the policies of The
Toronto Hospital, and the protocol was approved by the Animal
Care Committee.
Experimental Protocol
CD4-/- (n=47),
CD8-/- (n=35),
CD4-/-CD8-/- (n=56),
TCRß-/- (n=16), and control (n=64) mice ages
4 to 6 weeks were inoculated intraperitoneally with
105 plaque-forming units (PFUs) of CVB3. Animals
were observed for spontaneous mortality, and a subgroup was randomly
assigned to euthanization on 4, 7, 10, 14, or 28 days after infection.
The animals who were randomized for euthanization were censored from
the mortality data.
Histopathology
Transverse midsections of hearts were fixed in 4%
paraformaldehyde and processed for hematoxylin and
eosin staining. Histopathologic grading of cellular infiltrate and
necrosis of the myocardium was on a scale of 0 to 4 as
follows: 0, absence of infiltration or necrosis; 1, limited focal areas
of infiltration or necrosis; 2, mild to moderate infiltration or
necrosis; 3, moderate infiltration or necrosis; and 4, extensive areas
of infiltration or necrosis involving the entire examined heart
tissue.15 Paraffin-embedded pancreases and livers were
examined qualitatively for evidence of inflammation and necrosis after
CVB3 infection.
Viral Titers
After aseptic removal, hearts, pancreases, livers, and spleens
were stored individually in RPMI (GIBCO-BRL) at -70°C for titer
determination. Organ samples were homogenized in 5 mL of
RPMI. After 3 freeze-thaw cycles and centrifugation at
3000 rpm for 15 minutes, virus titers were determined in duplicate by
standard plaque formation assay.
Neutralizing Antibody Titers
Neutralizing antibody titers were measured by inhibition of
viral cytopathic effect (CPE). Sera were inactivated at
56°C for 30 minutes. Serial dilutions, in 2-fold increments in
RPMI plus 10% FCS (GIBCO-BRL), were incubated for 1 hour at 37°C
with 100 PFUs CVB3-CG. Sera were adsorbed onto HeLa cell monolayers in
96-well plates for 1 hour at room temperature and then replaced with
RPMI plus 10% FCS and incubated for 48 hours at 37°C. The highest
dilution of sera that inhibited CPE, determined after staining with 1%
crystal violet in 10% formalin, was found to be the titer of
neutralizing antibody against CVB3. The positive control was
commercially produced anti-coxsackievirus antibody (Chemicon), and the
negative control was uninfected mouse serum.
Immunohistochemistry
Sections of paraffin-embedded heart tissue were deparaffinized
and rehydrated, and then endogenous peroxidase activity was
blocked in 3% hydrogen peroxide in methanol for 10 minutes. Sections
were permeabilized with 0.125% trypsin (Zymed
Laboratories) at 37°C for 10 minutes and washed in PBS with 0.05%
Tween 20 (PBST). After blocking with 10% normal goat serum (Zymed
Laboratories) in PBS for 30 minutes, samples were incubated overnight
at 4°C with a rabbit polyclonal anti-CD3 antibody (Zymed
Laboratories) to detect T cells or a monoclonal rat anti-mac-3 antibody
(1:100; PharMingen; kindly provided by Dr Marlene
Rabinovitch, The Hospital for Sick Children, Toronto,
Ontario, Canada) to detect macrophages and then washed
with PBST. Sections were incubated for 20 minutes with biotinylated
secondary antibody (goat anti-rabbit, 1:250 [GIBCO-BRL] or goat
anti-rat, 1:100 [Jackson ImmunoResearch Laboratories]) and then
rinsed with PBST. Streptavidin–horseradish peroxidase conjugate
(1:500; Jackson ImmunoResearch Laboratories) was then applied for 10
minutes. After washing, sections were developed for 5 minutes with
aminoethyl carbazole (red staining; Zymed Laboratories).
Sections of OCT-embedded frozen myocardium were fixed in acetone at 4°C for 3 minutes and then incubated in 3% hydrogen peroxide in methanol for 30 minutes. After washing with PBS, samples were blocked with 10% goat serum (Sigma) and 3% BSA (Bioshop) in PBS for 1 hour at room temperature. Samples were then incubated in rabbit anti-mouse asialo-GM1 polyclonal antibody (Cedarlane) for 1 hour at room temperature to detect natural killer (NK) cell infiltration. After washing in PBS, sections were incubated with biotinylated goat anti-rabbit secondary antibody (1:500), followed by streptavidin–horseradish peroxidase conjugate (1:500) and aminoethyl carbazole detection, as described above.
Primary antibody was replaced with normal rat serum or rabbit serum, as negative control. Additionally, sections were processed in the absence of primary antibody or control serum. Sections of normal mouse spleen and thymus were used as positive controls. All samples were counterstained with hematoxylin.
To quantify differences in infiltrating cell populations at day 10, positive cells and total infiltrating cells were counted at high power in 5 randomly selected myocardial foci for each heart examined and then expressed as percentage CD3 (for T lymphocytes)– or mac-3 (for macrophages)–positive cells. Alternatively, at day 4 the total number of asialo-GM1–positive cells in each section was expressed as the number of positive cells per high-power field (HPF; x250), with >10 HPF counted per section.
RNA Isolation and Reverse Transcriptase–Polymerase Chain
Reaction (PCR)
Hearts were snap-frozen in liquid nitrogen at the time of
euthanization. Total RNA was isolated using Trizol reagent (GIBCO-BRL)
as directed. First-strand cDNA synthesis was performed by incubation of
1 µg of RNA with 200 ng/mL random hexamers (GIBCO-BRL) at room
temperature for 10 minutes, followed by the addition of 200 units
Superscript II (GIBCO-BRL), 10 mmol/L DTT, 0.5 mmol/L of each
dNTP (Pharmacia), and first-strand buffer (GIBCO-BRL). The 20-µL
reaction was incubated at 42°C for 45 minutes, and then reverse
transcriptase was denatured at 70°C for 5 minutes.
Cytokine PCR primer sets for interferon- (IFN-), tumor
necrosis factor- (TNF-), and ß-actin were synthesized on the
basis of published sequences.16 Each 25-µL PCR reaction
contained 0.5 mmol/L of each primer pair, 100 µmol/L of
each dNTP (Pharmacia), and 0.3 units of Taq polymerase (Pharmacia) in
reaction buffer (Pharmacia). Samples were analyzed in a thermal
cycler (Perkin-Elmer Cetus) as follows: 94°C for 1 minute; 35 cycles
of 95°C for 2 minutes, 60°C for 2 minutes, and 72°C for 2
minutes; and 95°C for 1 minute, 60°C for 1 minute, and 72°C for 7
minutes. PCR products were compared using ethidium bromide-stained
agarose gel electrophoresis. Measurements of band intensity were made
under ultraviolet light with a digital camera (GelDoc 1000, Bio-Rad
Laboratories). Band intensities representing
cytokine PCR products were compared in a semiquantitative
fashion with Molecular Analyst software (version 2.1.2, Bio-Rad
Laboratories) after expression as a ratio to ß-actin for the same
cDNA sample. Splenic cDNA and the plasmid pMCQ (courtesy of Dr Cornelia
Platzer, Institut für Anatomie, Friedrich-Schiller
Universität, Jena, Germany), which contains gene fragments of
IFN-, TNF-, and ß-actin, served as positive
controls.16
Statistical Analysis
Differences in survival were evaluated by
2 analysis. Results were subjected to
ANOVA with post hoc testing using Neuman-Keuls comparisons (SuperAnova,
Abacus Concepts, Inc). Results were considered statistically
significant when the probability of a type 1 error was <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Knockout of
ß T-Cell Subsets Alters Survival After CVB3
InfectionTo examine the role of TCR
ß+ T cells in
susceptibility to CVB3 disease, animal survival was followed for 28
days after virus inoculation. Survival was only 46% in the control
group (Figure 1
). In
CD4-/- mice, a similar survival rate of 42%
was observed, but in CD8-/- mice survival
tended to be lower than control, at 30%. Outcome was significantly
improved in both
CD4-/-CD8-/- and
TCRß-/- mice, with increased survival rates
of 72% and 82%, respectively. Susceptibility to CVB3 infection was
decreased substantially by knockout of both CD4+
and CD8+ T cells, or alternatively, by knockout
of all ß T cells.
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TCRß+ T Cells Regulate Cellular Infiltration and
Necrosis in the Heart
After CVB3 infection, cardiac pathology was consistent
with observed mortalities. Widespread myocardial infiltration was seen
in controls on days 7 and 14 and remained unaltered by elimination of
CD8+ T cells (Figures 2 and 3).
Less infiltration was seen in CD4-/- mice
versus CD8-/- and control groups on day 14.
Infiltration was mild in
CD4-/-CD8-/- and
TCRß-/- mice. By day 14, myocardial necrosis
tended to be most severe in control and CD8-/-
mice in accord with the extent of infiltration observed in these groups
(Figures 2 and 3). In CD4-/- and
CD4-/-CD8-/- mice,
necrosis was less extensive at day 14. Limited myocyte necrosis was
observed at day 7 in TCRß-/- mice. The
severity of myocardial infiltration and tissue damage was most
dramatically attenuated in
CD4-/-CD8-/- and
TCRß-/- strains. In
CD4-/- mice, myocarditis was less severe. In
the CD8-/- group, with the
CD4+ T-cell subset intact, myocardial
infiltration and necrosis were severe and comparable with that of
control.
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P<0.05 vs control and P<0.01 vs
CD8-/-; 
P<0.05 vs control;
§P<0.05 vs CD8-/- and
P<0.01 vs control.No differences were found in the extent of pancreatic or liver disease among knockout groups. In the pancreas, moderate to severe acinar destruction was observed in all groups, with islet cells remaining intact. Although mild to moderate cellular vacuolation and cytolysis of hepatocytes was evident in all groups, no frank necrosis was seen. Minimal inflammatory infiltrate was found in both organs, in striking comparison with the heart.
Knockout of T-Cell Subsets Influences the Participation of Immune
Cells in CVB3 Myocarditis
Not only were differences in the extent of myocardial infiltration
observed (Figure 3
), but differences in the contribution of T
cells and macrophages to the infiltrate were identified as
well. On day 10 after infection, the proportion of T cells present
in myocarditic foci of CD4-/-,
CD8-/-, and
CD4-/-CD8-/- mice was
decreased versus controls (Table 1 and
Figure 4). Infiltration of
macrophages was also less extensive in
CD4-/- and CD8-/- mice
as compared with control mice (Table 1 and Figure 4). The
proportion of macrophages infiltrating the
myocardium was lowest in
CD4-/-CD8-/- mice.
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Infiltration of the myocardium by NK cells was evident at day 4 after infection in our T-cell knockout models. NK cell of infiltration tended to be more prominent in the myocardium of CD4-/- (2.5±0.8 cells/HPF) and CD4-/-CD8-/- mice (3.3±1.7 cells/HPF). Only 0.7±0.5 cells/HPF and 0.4±0.2 cells/HPF were found in the myocardium of CD8-/- and control mice, respectively.
The neutralizing antibody response,
representative of B cell activation, was attenuated in
some CD4-/- and
CD4-/-CD8-/- mice
(Figure 5). However, a rise in titer was
observed in the majority of these mice, suggesting that in the absence
of CD4+ T cells, neutralizing antibody can still
be produced.
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Myocardial Cytokine Expression Varies Among CD4 and CD8
Knockout Strains
To test the hypothesis that the T-cell repertoire of the
host affects the cytokine response in myocarditis, cardiac
cytokine gene expression was evaluated. On day 4, when cellular
infiltration was minimal, cytokine expression differed among
knockout groups (Figure 6). In
CD4-/- and
CD4-/-CD8-/- mice,
myocardial IFN- gene expression was increased. This difference
tended to persist until day 10. Day 4 TNF- expression was lowest in
the hearts of
CD4-/-CD8-/- mice.
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Viral Titers Do Not Correlate With Severity of Myocarditis and
Mortality in CD4 and CD8 Knockout Mice
To test whether the differences in cardiac disease were due to
differences in viral replication, cardiac CVB3 titers were
analyzed. No significant differences among genotypes
were observed, and no direct relationship between viral titers in the
heart and outcome was apparent (Figure 7). The similarity in viral titers
observed in the heart was apparent in other organs as well. No
significant differences in splenic and hepatic viral titers were found
among groups (Table 2). Pancreatic CVB3
titers were significantly lower in
CD4-/-CD8-/- mice
versus control, although this was not reflected in differences in
tissue damage. Virus was cleared from spleens, livers, and pancreases
in the majority of animals in all groups by day 7 (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T lymphocytes participate in the host immune response to viral infection. They can inhibit viral replication, but T-cell effector mechanisms can also promote host tissue damage via excess activation of cytokines and cellular cytotoxic responses.2 Gene-targeted removal of T-cell subsets alters the course of viral myocarditis, illustrating 2 key factors in the pathogenesis of myocarditis after viral infection. First, host susceptibility to myocarditis is dependent on activation of both CD4+ and CD8+
ß T
cells. Second, the predominant T-cell type available to respond to CVB3
infection determines the severity and course of myocarditis after CVB3
infection. Viral replication in the heart does not predict myocardial
damage in ß T-cell–deficient mice, underscoring the pivotal role
of the T lymphocyte in CVB3 myocarditis.
Evidence from CD4-/- and
CD8-/- knockout models demonstrates an
important pathogenic role of CD4+ T cells in
viral myocarditis. Knockout of CD4+ T cells alone
provided protection from the myocardial infiltration and necrosis seen
in control mice, but did not impact on survival. This is in contrast to
anti-CD4 monoclonal antibody therapy experiments, which had no effect
on myocarditis after CVB3 infection in A/J mice.5 The
incomplete depletion of CD4+ T cells by
monoclonal antibody therapy likely explains the differences observed.
In CD4-/- mice, total T-cell numbers are
normal, and the CD8+ cytotoxic T-cell response is
intact.12 Approximately 10% of peripheral T
cells are TCRß+DN, which can provide MHC
II–restricted help for CD8+ T cells and B
cells.12 17 Therefore, activation of the intact
CD8+ T-cell population in
CD4-/- mice may proceed in response to CVB3
infection, albeit at a less intense level, resulting in less extensive
myocardial damage. Survival was lowest in the
CD8-/- mice, whereas the severity of myocardial
disease was comparable with that observed in control mice. Animal
survival is likely inversely related to the extent of myocardial
infiltration and necrosis, such that more mice in the
CD8-/- group had severe myocardial damage that
is incompatible with life. Histopathologic examination of pancreases
and livers did not reveal any differences in inflammation or tissue
necrosis among knockout groups, which would offer an explanation other
than death caused by fulminant heart dysfunction. This is
consistent with the cardiovirulent nature of this strain of
CVB3.10 18 CD8-/- mice are unable
to produce any detectable CTL responses against viral antigens, but
they do have normal total T-cell numbers and a normal
CD4+ T-cell population that can mediate expected
levels of T-cell help and immunoglobulin class
switching.11 In conjunction with the less extensive
myocarditis observed in CD4-/- mice, the severe
disease in the CD8-/- model supports a
deleterious role for CD4+ lymphocytes in
myocarditis. Also, as suggested after monoclonal antibody depletion of
CD8+ T cells in A/J mice, removal of suppressive
CD8+ T cells may play a role.5
We found that
CD4-/-CD8-/- mice had
increased survival and decreased myocardial damage after CVB3
infection, without any impairment of viral clearance, indicating that
both CD4+ and CD8+ T cells
participate in cardiac tissue damage. Although
CD4+ and CD8+ T cells do
not develop in
CD4-/-CD8-/- mice,
other functional T-cell subsets are present. The majority of T
cells (10% of normal numbers), are TCRß+DN,
with TCR
+ T cells increased to 3% of the
total leukocyte number.14 The CD3+
cells identified in the myocardial infiltrate of
CD4-/-CD8-/- mice were
most likely TCRß+DN or
TCR+ T lymphocytes. MHC II–restricted
TCRß+DN T cells provide T-cell help in
CD4-/- mice in response to vesicular stomatitis
virus.19 Human TCRß+DN T-cell
clones can lyse target cells in a non-MHC–restricted
manner.20 Cytokine regulation of
TCRß+DN T-cell effector functions supports a
functional diversity and physiological relevance of
this T-cell population.21 TCR+
T cells can mediate both a cytotoxic response22 23 and B
cell activation with neutralizing antibody
production24 in viral infection and can influence
effector cells via cytokine production.25
Thus, in CD4-/-CD8-/-
mice, TCRß+DN and
TCR+ T cells may participate in the host
response to CVB3 infection with cytolytic capacity and cytokine
expression.
Susceptibility to myocarditis was decreased in
TCRß-/- mice, with the outcome comparable
with that observed in
CD4-/-CD8-/- mice.
TCRß-/- mice completely lack
TCRß+ T lymphocytes; however, development of
TCR+ T cells is not
impaired.13 The total thymocyte number in
TCRß-/- mice is 8% of controls, with
peripheral T cells all TCR positive.13
In the absence of TCRß+ T cells, TCR
+ T cells may participate in viral
clearance in conjunction with NK cells, macrophages, and B
cells. We have shown that removal of all
TCRß+ T cells is not required to optimally
protect the host from myocarditis. Genetic knockout of
CD4+ and CD8+ T-cell
subsets is sufficient. Therefore, the exuberant response of this T-cell
repertoire confers host susceptibility.
The elimination of specific T-cell subsets has the potential to alter
other elements of the host response to CVB3 infection, including
antibody production, cytokine expression, and NK cell
response. The detection of neutralizing antibody in all knockout groups
indicates that in the absence of CD4+ T cells,
other lymphocytes, perhaps TCRß+DN and
TCR+ T cells, can participate in B cell
activation and antibody production in CVB3-infected mice.
Neutralizing antibody is likely not an important protective factor in
these models with an A/J background, as titer does not correlate with
outcome.
Cytokines are active participants in the induction of the
immune response to viral infection and can influence both viral
replication and immune-mediated tissue damage.26 Cardiac
expression of IFN- and TNF- may be significant factors in
determining susceptibility to disease. The pattern of cytokine
expression is dependent on the specific T-cell subset available to
respond to CVB3 infection. IFN- and TNF- expression in the
myocardium on day 4 may reflect responses of cardiac
myocytes, fibroblasts, endothelial cells, and dendritic
cells. Myocardial cells may be influenced by differences in splenic
cytokine production, determined by T-cell subsets in
the knockout models.
Activation of NK cells occurs as early as day 3 after CVB3
infection27 and is compatible with the myocardial
infiltration of NK cells observed on day 4. The increased NK cell
infiltration of the myocardium in
CD4-/- and
CD4-/-CD8-/- mice in
association with elevated IFN- expression may reflect a shift in the
balance of the immune response, in favor of protective immune elements
such as IFN- and NK cells. Similar day 4 cardiac viral titers among
groups does not support widespread inhibition of viral replication by
IFN-; however, perhaps myocyte-to-myocyte spread of virus is limited
by local IFN- production, thus limiting the size and number
of myocarditic foci.
In CD4-/-CD8-/- mice,
lower cardiac TNF- expression, together with elevated IFN-, may
contribute to decreased mortality by preserving heart function despite
high viral titers. It has been shown previously that TNF-
administration to CVB3-resistant mice worsens
myocarditis,28 whereas anti-TNF- antibody treatment
before encephalomyocarditis virus infection decreases the severity of
myocarditis.29 In addition, the negative inotropic effects
associated with TNF- have been well
documented.30 31
It has been suggested that the variable responses of murine strains to immunotherapy are the result of differences in the genetic repertoire of the host.5 32 33 This may explain observations, in different murine backgrounds, that CD4 knockout mice develop severe myocarditis, whereas ß2-microglobulin knockout mice have minimal myocardial disease after CVB3 infection,34 in contrast to results shown here. Extrapolation of the present study suggests that it is in fact the complement of specific T-cell subpopulations in each host that determines susceptibility to myocarditis in mice and humans. The relevance of T-cell subset responses may have important diagnostic, prognostic, and therapeutic implications in the management of the disease.
In summary, the present study used a strategy of gene targeting to
elucidate the role of the T-cell response in the pathogenesis of
myocarditis after CVB3 infection. We have shown that the severity of
myocarditis is dependent on the responding T-cell subset. Both
CD4+ and CD8+ T lymphocytes
can contribute to myocarditis and mortality after CVB3 infection.
Distinct patterns of cytokine expression, associated with
specific T-cell populations, may alter the intensity of the
inflammatory response and severity of myocarditis. It appears that the
balance of protective and destructive immune elements can be altered by
the elimination of 1 or more T-cell subsets. Therefore, the role of the
T cell in myocarditis is complex, and the contribution of mechanisms
other than ß T cells cannot be underestimated; consequently,
elimination of the total ß T-cell population may not always be
advantageous to the host. Further investigation of the components of
the immune response to CVB3 infection may identify other important
factors of host susceptibility and lead to better understanding of the
disease and more precise targeting of therapy.
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Acknowledgments |
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This research was supported by grants from the Medical Research Council of Canada and the Heart and Stroke Foundation of Ontario. M.A.O. is a postdoctoral fellow of the Medical Research Council of Canada. P.L. is the Heart and Stroke/Polo Endowed Research Chair at the University of Toronto.
Received December 17, 1998; accepted June 30, 1999.
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