(Circulation. 2001;103:1709.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Prevention of Autoimmune Myocarditis Through the Induction of Antigen-Specific Peripheral Immune Tolerance
From the Departments of Pathology and Microbiology-Immunology and the Feinberg Cardiovascular Research Institute, Northwestern University Medical School, Chicago, Ill.
Correspondence to David M. Engman, Northwestern University Medical School, Department of Pathology, 303 E Chicago Ave, Ward 6-175, Chicago, IL 60611. E-mail d-engman{at}nwu.edu
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Abstract |
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Background—Autoimmunity to cardiac antigens, in particular cardiac myosin, has been observed in humans with myocarditis and in animals with experimental inflammatory heart disease. Current treatments for myocarditis are in many cases immunosuppressive and might lead to increased cardiac damage by reducing host defenses against infectious agents. Therefore, we sought to develop an antigen-specific approach to inhibit autoimmunity in mice with myosin-induced experimental autoimmune myocarditis.
Methods and Results—Syngeneic splenocytes, coupled with cardiac myosin by use of ethylene carbodiimide, were administered intravenously before disease induction, and the effects of this peripheral tolerization on myosin-induced myocarditis were assessed. This antigen-specific immunotherapy significantly reduced both the incidence and severity of myocarditis, with the prevention of myocyte necrosis, mononuclear cell infiltration, and fibrosis. Myosin-specific delayed-type hypersensitivity and antibody production were significantly reduced, demonstrating that peripheral tolerance affected both T- and B-cell responsiveness to the autoantigen.
Conclusions—These results suggest that the induction of antigen-specific peripheral immune tolerance may be an effective approach for the treatment of myocarditides with autoimmune involvement.
Key Words: myocarditis • myosin • lymphocytes
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Myocarditis is a primary inflammatory heart disease that is formally diagnosed by cardiac biopsy and observation of myocyte necrosis and degeneration, with adjacent mononuclear cell infiltration in the presence or absence of fibrosis.1 The most common causes of myocarditis in the United States are viral, most commonly coxsackievirus.2 Other infectious and noninfectious agents, such as bacteria, protozoans, fungi, drugs, heavy metals, and insect stings may also cause cardiac inflammation.1 3
Immunomodulatory therapies, particularly immunosuppressive
treatments, have been used in clinical trials for the treatment of
myocarditis.3 Although
immunosuppressive treatments are often useful in downregulating the
autoimmune damage in myocarditis, they may promote viral spread and
myocardial cytolysis. In fact, clinical trials and experimental studies
of murine models of viral myocarditis have revealed that steroids and
other immunosuppressive treatments are not always beneficial and may
actually increase disease severity and
mortality.4 Cytokine
inhibition with vesnarinone has been shown to downregulate
proinflammatory cytokine production, which would be deleterious during
acute viral myocarditis.5
Conversely, such treatments as interleukin-2 and interferon-
may be
effective in clearing the viral agent but may promote the development
of cardiac
autoimmunity.3
Experimental autoimmune myocarditis (EAM) has proved to be a
highly useful animal model of inflammatory heart disease. Myocarditis
is induced on immunization of susceptible strains of rats and mice with
the cardiac myosin -heavy
chain.6 Interestingly, immune
responses against cardiac antigens, cardiac myosin in particular, have
been observed in human inflammatory heart
disease,7 making myosin a
relevant antigen for disease induction in the mouse model. EAM is
histologically similar to human myocarditis, with myocyte swelling and
necrosis accompanied by mononuclear cell infiltration and fibrosis. EAM
is a T cell–mediated disease, requiring both CD4+ and CD8+
subsets,8 9 10 11
whereas B cells are not vital for antigen presentation in EAM and
autoantibodies are not necessary for the progression of
myocarditis.12
We wish to develop treatments to be used immediately on the suspicion of myocarditis without the necessity of determining the presence or absence of an infectious agent. Antigen-specific immunosuppression would allow for the inhibition of autoimmune damage without adversely affecting the beneficial immune response mounted against the infectious agent that initiated the damage. We and others have successfully used intravenous administration of syngeneic splenocytes covalently coupled to antigen with ethylene carbodiimide (ECDI) to prevent and, more importantly, to treat a number of autoimmune diseases in animal models, such as experimental autoimmune encephalomyelitis, neuritis, uveitis, and thyroiditis,13 14 15 16 and initial application of this approach to humans has been successful.17 In this report, we demonstrate that coupled-cell tolerance is effective for prevention of myocarditis in this mouse model, suggesting the usefulness of antigen-specific immunotherapy in treating myocarditis.
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Methods |
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Experimental Animals
Male A/J strain mice (n=142; Jackson Laboratories, Bar Harbor, Me) were 6 to 8 weeks of age at initiation of the experiments. Mice were anesthetized by a single injection of 60 mg/kg sodium pentobarbital IP for each experimental manipulation. The use and care of mice were in accordance with the guidelines of the Center for Experimental Animal Research at Northwestern University.
Preparation of Myosin
Cardiac myosin heavy chains were purified according
to the method of Shiverick et
al.18 Protein concentration
was determined by comparing dilutions of the purified myosin solution
with known concentrations of purified rabbit myosin heavy chain
standards (Sigma) by
SDS-PAGE.19
Preparation of Myosin-Coupled
Splenocytes
Splenocytes were purified from naïve mice and
incubated at 37°C for 10 minutes in Tris-NH4Cl
(0.017 mol/L Tris free base, 0.14 mol/L NH4Cl,
pH 7.2) to lyse erythrocytes, followed by 2 washes in HBSS (Gibco/BRL).
The splenocytes were then coupled with purified cardiac myosin with the
cross-linking chemical ECDI (Calbiochem) in the following manner. The
splenocytes were washed in saline (0.15 mol/L NaCl, pH 7.2), pelleted
at 1500g for 10 minutes in a
50-mL conical tube, and resuspended at a final concentration of
5x108 cells/mL in cold saline containing 1
mg/mL purified cardiac myosin or 1 mg/mL BSA (Sigma). The coupling
reaction was initiated by the addition of 0.5 mL of freshly prepared
ECDI (150 mg/mL in saline) per mL of the cell suspension and incubated
for 1 hour on ice with periodic gentle inversion. The cells were washed
3 times with HBSS and maintained at 4°C until injection into
mice.
Induction of Peripheral Tolerance
Tolerance was induced by the intravenous injection of
7.5x107 coupled splenocytes in 0.5 mL HBSS.
Seven days after injection, the mice then received the normal myosin
immunization protocol described below.
Induction of Autoimmune Myocarditis
Mice were immunized subcutaneously in 3 sites on the
dorsal flank in a 100-µL volume containing 300 µg of purified
cardiac myosin diluted 1:1 in complete Freund’s adjuvant (Difco).
Seven days later, the mice were boosted with another 300 µg myosin in
the same manner.
Histological Evaluation of Disease
Hearts were excised, fixed in 10% buffered formalin,
and embedded in paraffin. Hearts were cut in a plane such that both
atria and ventricles were present in each section. Four or more
sections per heart were stained with hematoxylin and eosin or Masson’s
trichrome and examined by microscopy. Evaluation of the heart sections
was performed blind. Disease severity was graded as follows: normal
(0), no infiltrate; mild (1), <10% of the tissue section involved;
moderate (2), 10% to 25% of the tissue section involved; and severe
(3), >25% of the tissue section involved.
Measurement of Antigen-Specific
Delayed-Type Hypersensitivity
Delayed-type hypersensitivity (DTH) responses were
quantified with an ear-swelling assay 21 days after
immunization. Mice were anesthetized, and prechallenge ear thickness
was measured with a Mitutoyo model 7326 engineer’s micrometer
(Mitutoyo MTI Corp). Antigen (10 µg) in a 10-µL volume of DTH
buffer (0.15 mol/L
K2HPO4, 0.01 mol/L
Na4P2O7,
0.3 mol/L KCl, pH 6.8) was then injected intradermally into the dorsal
surface of the ear with a 100-µL Hamilton syringe fitted with a
30-gauge needle. Ear swelling, resulting from mononuclear cell
infiltration, was then measured 24 hours later and expressed in units
of 10-4
in.
Serological Analysis
A myosin-specific ELISA was developed to analyze the
isotype specificities of myosin-specific antibodies. After each
incubation, the plates were washed 5 times with 1x PBST (1x PBS,
0.05% Tween-20). Ninety-six–well plates were coated with 2.5 µg/mL
purified myosin overnight at room temperature and then blocked for 2
hours with 5% normal goat serum, 2% BSA in PBST. Mouse sera were
diluted at 1:1000 in the blocking agent and incubated on plates for 2
hours at room temperature. The biotin-conjugated, isotype-specific
secondary antibodies (Caltag Laboratories) were added at a dilution of
1:15 000 and allowed to adsorb for 1 hour. Peroxidase-labeled
streptavidin (KPL Laboratories) was incubated at a dilution of 1:4000
for 30 minutes, and the plates were developed with TMB developing
solution (KPL Laboratories) according to the manufacturer’s
instructions. Each serum was analyzed separately, and the data were
compiled for each group. Each plate contained the same myosin-positive
(from a severely diseased EAM mouse) and -negative sera for
normalization.
Statistical Analyses
All values are expressed as mean±SEM. The
statistical significance of DTH, disease severity, and antibody
isotypes was analyzed by 1-way ANOVA followed by post hoc Bonferroni
analysis. The statistical significance of disease incidence was
analyzed with Pearson’s 
2. Probability
values of P<0.05 were
considered significant in this
study.
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Results |
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Induction of Myosin-Specific Tolerance in EAM
A/J mice, when immunized with syngeneic cardiac myosin, develop severe myocarditis 21 days after immunization. In the present study, this immunization protocol was coupled with a tolerogenic protocol in an attempt to inhibit the autoimmune response, thereby reducing cardiac inflammation (Figure 1
). Mice received an intravenous injection of
7.5x107 naïve, syngeneic splenocytes that
had been coupled with cardiac myosin or BSA (sham control) with the
cross-linking agent ECDI (day -7). One week later, the mice were
immunized subcutaneously with 300 µg cardiac myosin in complete
Freund’s adjuvant (day 0), followed by a boost 7 days later with the
same antigen (day 7). At day 21, the effect of the tolerization
protocol on the development of myocarditis and on myosin-specific
immunity was analyzed by a number of methods: cardiac histology,
DTH, and myosin-specific antibody
production.
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Myosin Tolerization Prevents the Development of
Myosin-Induced Myocarditis
On day 21 postimmunization, hearts were excised for
histological examination, and sections were examined after staining
with hematoxylin and eosin and Masson’s trichrome
(Figure 2). Myosin tolerization was highly effective at
preventing both the incidence and severity of myocarditis
(Figure 2 and
Table).
Hearts from most of the myosin-tolerized mice (13 of 22) displayed no
inflammatory cell infiltration or fibrosis
(Figure 2A and 2E), comparable to heart tissue from
PBS-immunized controls
(Figure 2D and 2H). In 9 animals, tolerance was not complete,
with 2 of 9 mice demonstrating severe myocarditis and 7 of 9
demonstrating mild inflammation (data not shown). This is in striking
contrast to the hearts from sham-tolerized
(Figure 2B and 2F) and nontolerized
(Figure 2C and 2G) animals, which displayed massive
mononuclear cell infiltration, myocyte swelling and necrosis, and
fibrosis. All cardiac sections were scored for disease (scores ranged
from 0, normal, to 3, most severe; see Methods for details), and the
results are presented in the
Table.
The incidence of disease in the myosin-tolerized group was not
significantly different from that of the PBS control group but was
different from those of the sham
(P<0.002) and nontolerized
(P<0.003) groups. The
myosin-tolerized mice (1.4/3.0) had a significantly reduced disease
severity compared with sham (2.1/3.0,
P<0.02) and nontolerized
(2.2/3.0, P<0.002) mice, while
not being significantly different from the PBS control group. Thus,
myosin-specific tolerization was effective in decreasing disease
incidence and severity in most of the mice that did develop
disease.
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Inhibition of Myocarditis Through
Antigen-Specific Tolerance Is Due to the Inhibition of Myosin-Specific
Cellular Immunity
We have hypothesized that this treatment induces
antigen-specific T-cell
anergy.20 21
Therefore, we analyzed T-cell immunity to the tolerizing antigen by
measuring the myosin-specific DTH elicited in each treatment group
(Figure 3). Myosin-specific DTH was significantly diminished
in the myosin-tolerized animals compared with both the nontolerized
(P<0.0005) and sham-tolerized
(P<0.0005) groups. Responses
were not significantly different between the myosin-tolerized and PBS
control groups or between the sham-tolerized and nontolerized groups.
These results indicate that T cells have been affected by the
tolerization protocol in an antigen-specific manner and that the
reduction in DTH, and disease, was not simply due to the introduction
of ECDI-treated splenocytes in this model.
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Antigen-Specific Tolerization Reduces
Myosin-Specific Antibody Production in EAM
An isotype-specific ELISA was developed to determine
the effect of myosin tolerization on myosin-specific antibody
production. Myosin-tolerized mice produced very little myosin-specific
antibody of any isotype
(Figure 4A). Although the antibody levels were higher than
those detected in PBS controls
(Figure 4D), they were much lower than those present in the
sham-tolerized
(Figure 4B) and nontolerized
(Figure 4C) groups. Myosin-specific total IgG and IgG1 were
significantly lower in the myosin-tolerized mice than in the
nontolerized (P<0.002 and
0.03, respectively) and sham-tolerized
(P<0.0003 and 0.002,
respectively) groups. None of the other antibody isotypes were
significantly different in myosin-tolerized mice and control groups.
Interestingly, the myosin-specific antibody isotypes produced by
animals with myocarditis suggest that both Th1 (IgG2a)– and Th2
(IgG1)–type T-cell responses are
present.22
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Discussion |
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A wide variety of experimental methods of immunosuppression have been used to treat myocarditis and other autoimmune diseases, including administration of neutralizing antibody directed against T cells and antigen-presenting cells, such as anti-CD4,8 10 anti-CD8,8 10 and anti–major histocompatibility complex class II.23 Neutralization of proinflammatory cytokines such as tumor necrosis factor-
24 or trafficking
molecules such as chemokines like macrophage inflammatory
protein-125 have been
used with some success. Although these are useful in downregulating
damage due to autoimmunity, there may be adverse consequences in
downregulating immune responses directed against an infectious
agent.4 Conversely,
treatments such as administration of recombinant interleukin-2 and
interferon- may aid in viral clearance but might promote autoimmune
cardiac damage.3 All of these
approaches suffer in part from a lack of specificity and do not
overcome the challenges posed by infection-induced, organ-specific
inflammatory diseases.
The goal of the present study was to inhibit only
self-directed immune responses by restoring autoantigen-specific
peripheral tolerance. Our tolerization protocol was highly successful
in inhibiting cardiac inflammation induced by myosin immunization.
Myofibrillar swelling and necrosis and reparative fibrosis were absent
in the tolerized mice
(Figure 2
and
Table).
Coupled-cell tolerance has been shown to downregulate both cellular and
humoral immune responses as measured by DTH and antibody
production.26 In the
Theiler’s murine encephalomyelitis virus model of multiple sclerosis,
however, tolerance results in immune deviation in which the antibody
isotypes produced switch from IgG2a and IgG2b, reflecting a Th1-type
T-cell response, to IgG1, corresponding to a Th2-type
response.27 In the present
study, myosin-specific DTH was significantly decreased upon
tolerization
(Figure 3), and anti-myosin total IgG and IgG1 antibodies
were significantly decreased
(Figure 4). We performed all antibody analyses at 21 days
after immunization, but future studies will also address responses
during the entire disease course and when tolerance treatment is
administered after disease onset. Studies in intravenous and nasal
tolerization have also reported a decrease in antibodies
directed against the tolerizing
antigen,28 29
which is believed to be due to inhibition of T-cell help in tolerized
animals.30 31
Although autoreactive B cells are present, autoantibody is not detected
unless T-cell tolerance is overcome, as is true in EAM and is
hypothesized to occur in human myocarditis. Clearly, autoantibodies to
a number of cardiac antigens, including myosin, have been detected in
humans with myocarditis,7 and
it is possible that a pathogen or other damage-inducing event, such as
myocardial infarction, could cause the breakdown in T-cell tolerance to
self-cardiac antigens.
Among the possible mechanisms underlying peripheral tolerization are (1) direct anergy/deletion of Th1 lymphocytes20 21 32 ; (2) immune deviation, in which activation of Th2 lymphocytes occurs instead of the normal Th1 inflammatory cell activation27 ; and (3) cytotoxic T-lymphocyte antigen 4 regulation of T-cell function by modulation of the threshold of T-cell activation, in which the strength of the signal by antigen stimulus must be much greater on cells expressing more molecules of cytotoxic T-lymphocyte antigen 4 or nonresponsiveness is maintained (T.N. Eagar et al, unpublished data, 2000). Unfortunately, it is not currently possible to analyze myosin-specific T-cell responses in vitro, because myosin is toxic to the cells23 and/or may induce their apoptosis (L.M.G., unpublished data, 2000). Our results support the theory that the treatment results in the antigen-specific nonresponsiveness of T cells, specifically Th1 T cells, responsible for the strong myosin-specific DTH. Myosin-specific antibody responses were also suppressed by the treatment, suggesting that tolerization also inhibits T-cell help for myosin-specific B-cell responses.30 31
We chose the EAM model to evaluate the effectiveness of coupled-cell tolerance to treat myocarditis because it separates cardiac autoimmunity from the complicating immune responses directed against an infectious, disease-inducing agent. There are a number of murine models of myocarditis that can be used to study the efficacy of this tolerance protocol in the presence of an infectious agent. These models include protozoans, such as Trypanosoma cruzi, and viruses, such as coxsackieviruses and encephalomyocarditis virus.33 EAM and the infectious models are very similar in a number of ways, including the presence of myosin-specific autoantibodies, myocyte necrosis, and inflammatory cell infiltrate. It is interesting to note that strains of mice susceptible to coxsackievirus-induced myocarditis are also susceptible to cardiac myosin–induced myocarditis.2
In conclusion, antigen-specific peripheral tolerance induction provides a powerful tool for dissecting the mechanisms involved in cardiac autoimmunity. Although myosin-specific tolerization was used to prevent EAM in this report, it should be emphasized that tissue homogenates can be used as well.13 Thus, specific knowledge of the target proteins in an autoimmune response is not a prerequisite for administration of the therapy. It will be of interest to test the approach in other models of myocarditis, including Chagas disease and coxsackievirus myocarditis, and in cardiac allograft rejection. The results reported here indicate the usefulness of this method and the need to use its efficacy in infectious forms of myocarditis.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health. Dr Engman is an Established Investigator of the American Heart Association. We thank Dr A. Rademaker for his advice on statistical analysis. We thank the members of the Miller and Karpus laboratories for their advice and guidance in the experimental plan and data interpretation present in this article.
Received September 11, 2000; revision received October 13, 2000; accepted October 18, 2000.
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