(Circulation. 2001;103:18.)
© 2001 American Heart Association, Inc.
Brief Rapid Communications |
Lethal Autoimmune Myocarditis in Interferon-
Receptor–Deficient Mice
Enhanced Disease Severity by Impaired Inducible Nitric Oxide Synthase Induction
From Medicine A (F.F., U.W.), the Department of Pathology (M.O.K., P.S.), and the Division of Clinical Immunology, Department of Medicine (H.P.E.), University Hospital, Zurich, Switzerland, and the Medical Intensive Care Unit, University Hospital, Basel, Switzerland (U.E., R.B., S.M.).
Correspondence to Urs Eriksson, MD, Medical ICU, University Hospital, Petersgraben 5, CH-4031 Basel, Switzerland. klinerr@usz.unizh.ch.
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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Background—Interferon-
(IFN-) is an essential cytokine in the regulation of inflammatory
responses in autoimmune diseases. Little is known about its role in
inflammatory heart disease.
Methods and Results—We
showed that IFN- receptor–deficient mice
(IFN-R–/–) on a BALB/c
background immunized with a peptide derived from cardiac 
-myosin
heavy chain develop severe myocarditis with high mortality. Although
myocarditis subsided in wild-type mice after 3 weeks,
IFN-R–/– mice showed persistent
disease. The persistent inflammation was accompanied by vigorous in
vitro CD4 T-cell responses and impaired inducible nitric oxide synthase
expression, together with evidence of impaired nitric oxide production
in IFN-R–/– hearts. Treatment of
wild-type mice with the nitric oxide synthetase inhibitor
N-nitro-l-arginine-methyl-ester
enhanced in vitro CD4 T-cell proliferation and prevented healing of
myocarditis.
Conclusions—Our data
provide evidence that IFN- protects mice from lethal autoimmune
myocarditis by inducing the expression of inducible nitric oxide
synthase followed by the downregulation of T-cell
responses.
Key Words: interferons • mice • autoimmunity • myocarditis • myosin • nitric oxide synthase
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Cardiomyopathy may result from a long-term autoimmune reaction to cardiac tissue.1 Experimental autoimmune myocarditis can be induced in mice of the BALB/c strain by immunization with
-myosin-heavy-chain–derived peptides, and it is
mediated mainly by CD4+ T-helper
cells.2 3
Inflammatory cytokines like interferon- (IFN-) and tumor necrosis
factor- (TNF-) are critically involved in the pathogenesis of
various CD4+ T-cell–mediated autoimmune diseases. Mice lacking the
receptor for TNF- are protected from autoimmune
myocarditis,4 but the role of
IFN- in autoimmune myocarditis is, as yet, unknown.
Nitric oxide (NO) is an immune regulator and an effector
molecule mediating tissue
injury.5 6 Its
formation is catalyzed by NO synthases (NOS). NOS are constitutively
expressed in neuronal (nNOS) and endothelial (eNOS) cells, but they
also exist as inducible isoforms (iNOS). IFN- and TNF- can
trigger iNOS expression in macrophages, cardiac endothelial cells, and
cardiac
myocytes.5 6
Enhanced iNOS expression has been found in the myocardium of patients
with dilative
cardiomyopathy.7 However, the
role of iNOS in the pathogenesis of murine autoimmune myocarditis is
not clear.6
To understand the role of IFN- in experimental autoimmune
myocarditis, we compared disease prevalence, severity, immune
responses, and iNOS expression in IFN- receptor–deficient
(IFN-R–/–) mice with wild-type (WT)
control mice.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Mice
Breeding pairs of IFN-
R–/– mutant
mice8 backcrossed for >10
generations on BALB/c background were a generous gift from Prof Jacques
Louis (Institut de Biochimie, Lausanne, Switzerland). BALB/c mice were
purchased from Biological Research Laboratories Ltd, Füllinsdorf,
Switzerland. Male mice were immunized at 8 to 10 weeks of age. All
experiments involving mice were performed in accordance with Swiss
federal legislation and were approved by the local
authorities.
Induction of Experimental Autoimmune
Myocarditis
Mice were immunized with a heart muscle–specific,
-myosin-heavy-chain–derived peptide (myh 614 to 634;
Ac-SLKLMATLFSTYASADTGDSGKGKGKGGK KG-OH; designated
MA30) together with complete Freund’s adjuvant.
Control mice received complete Freund’s adjuvant
only.3
Histopathology and Immunohistochemistry
Hematoxylin- and eosin-stained heart sections were
evaluated on a semiquantitative scale using severity scores from 0 to 4
(0, no inflammatory infiltrates; 1, small foci of inflammatory cells
between myocytes; 2, larger foci of >100 inflammatory cells; 3, >10%
of a cross-section involved; and 4, >30% of a cross-section
involved). Immunohistochemical staining was performed with an anti-iNOS
antibody (1:200 rabbit polyclonal, Cat No N32030, Transduction
Laboratories) and an anti-nitrotyrosine antibody (1:100 rabbit
polyclonal, Cat No 06 to 284, Upstate) according to standard
procedures.
Proliferation Assays
CD4+ T-cells were purified from splenocytes by
depletion with commercially available specific antibodies coupled to
magnetic beads (MACS, Miltenyi Biotech GmbH). CD4 T-cells and
irradiated (2000 rad) syngenic antigen-presenting cells were
pulsed with 50 µg/mL MA30 or 100 µg/mL
ovalbumin as unspecific control antigen. Proliferative responses were
assessed by measuring [3H]methylthymidine
incorporation after 72 hours of culture in serum-free medium with 1
mmol/L of the NOS inhibitor
N-nitro-l-arginine-methyl-ester
(L-NAME; Sigma, No N5751) or the biologically inactive D-NAME.
To generate NO in cell cultures, we used the NO donor
S-nitroso-N-acetyl-penicillamine (SNAP; Sigma, No. N3398) at 0.5
mmol/L.
In Vivo Blocking of NO Production
NO production was blocked in vivo by treating mice
intraperitoneally with 10 mg/kg body weight of L-NAME from day 0 until
they were killed. Control mice received phosphate-buffered saline
only.4
Statistics
The Mann-Whitney U test was used to evaluate severity
scores and heart weights. Proliferative responses were compared using
2-way ANOVA followed by the unpaired
t-test with Bonferroni’s
correction. Dichotomous data were analyzed by Fisher’s exact test.
Differences for which P<0.05
are indicated in the tables and figures. Myocarditis prevalence refers
to the number of diseased mice compared with the number of immunized
mice still alive at the days
indicated.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Disease prevalence and severity of autoimmune myocarditis were higher in IFN-
R–/–
mice than WT controls
(Table 1
). Hearts from
IFN-R–/– mice were enlarged and
entirely infiltrated with histiocytes, lymphocytes, and numerous
neutrophils and eosinophils
(Figure 1). In addition, rare giant cells were observed.
Although WT mice almost completely recovered from disease, most
MA30-immunized
IFN-R–/– mice died from severe
myocarditis within 35 days
(Table 2 and
Figure 2a). IFN-R–/– control
mice immunized with complete Freund’s adjuvant only had neither
myocarditis nor were they different from WT mice with respect to
mortality.
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Given that experimental autoimmune myocarditis is mainly a
CD4 T-cell–mediated
disease,1 2 we
compared the in vitro CD4+ T-helper cell responses of
IFN-R–/– and WT mice.
IFN-R–/– CD4+ T-cells showed higher
proliferation indices than WT CD4 T-cells
(Figure 2b
). These proliferative responses persisted in CD4
T-cells isolated from IFN-R–/– mice for
up to 35 days after immunization, whereas proliferation of WT CD4+
T-cells decreased after 3 weeks. This suggests that an ongoing
reduction in the number of antigen-specific CD4+ T-cells parallels
recovery from disease in WT mice.
Given that IFN- is a potent inducer of iNOS expression
and that NO reversibly impairs T-cell
proliferation9 and
contributes to the elimination of antigen-specific activated
T-cells,10 a causal
relationship between increased severity of myocarditis in
IFN-R–/– mice and impaired iNOS
induction is possible. To test this hypothesis, iNOS expression was
assessed in the hearts of diseased mice by immunohistochemistry. As
shown in
Figure 1, iNOS was not detectable in diseased
IFN-R–/– hearts, but it was abundant in
the inflammatory mononuclear infiltrates of WT mice. In contrast to
inflamed WT hearts, nitrosylated tyrosine residues were not detectable
in the heart tissue of IFN-R–/– mice,
indicating an absence of NO-derived peroxynitrite (data not shown).
Therefore, we concluded that NO production in
IFN-R–/– hearts is impaired due to a
failure of iNOS expression.
To test the immunomodulating role of NO in vivo, WT mice
were treated with the NOS inhibitor L-NAME. Disease severity and
prevalence were not significantly enhanced in L-NAME–treated mice
after 21 days, but myocarditis persisted for up to 35 days after
immunization, which was comparable to the duration seen in
IFN-R–/– mice
(Tables 1 and 2). In contrast to WT CD4 T-cells,
proliferative responses of CD4 T-cells isolated from L-NAME–treated WT
mice were higher and closer to the responses observed for
IFN-R–/– CD4 T-cells
(Figure 2c). The addition of the NO-generating agent SNAP to
culture wells reduced proliferation in a dose-dependent manner in CD4
T-cells from IFN-R–/–, WT, and
L-NAME–treated WT mice. These findings suggest that impaired NO
generation in vivo results in an increased number of antigen-specific
CD4 T-cells. In addition, NO can directly downregulate the
proliferation of CD4 T-cells, independent of
IFN-.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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IFN-
confines murine experimental autoimmune
myocarditis and protects mice from lethal disease. Persistent
myocarditis parallels an ongoing CD4 T-cell response in
IFN-R–/– knockout mice. As was recently
shown, IFN- reversibly inhibits T-cell
proliferation,9 and it may be
essential to a mechanism eliminating activated CD4 T-cells by
apoptosis.10 Our data
suggest that IFN- downregulates CD4 T-cells in autoimmune
myocarditis. This mechanism is probably mediated by NO, as documented
by the impaired iNOS expression in
IFN-R–/– mice and by prior data
published by our group.9 The
observation that WT mice treated with the NOS inhibitor L-NAME develop
prolonged myocarditis and vigorous CD4-T-cell responses support this
hypothesis. However, L-NAME treatment did not significantly increase
myocarditis severity. This could be explained by the fact that the
L-NAME treatment used in our protocol does not completely inhibit the
generation of NO in vivo, despite the fact that iNOS cannot be detected
by immunohistochemistry in L-NAME–treated mice (data not shown). To
specifically assess the in vivo role of iNOS in myocarditis and T-cell
regulation, further experiments with iNOS-deficient mice should be
performed. Our findings suggest that IFN-, although known to be a
proinflammatory cytokine, may induce a negative feedback mechanism
through the production of NO that inhibits CD4-cell responses. Thus
IFN- and NO should be evaluated as therapeutic agents in established
myocarditis. In addition, IFN-R–/– mice
may be a useful model for the elucidation of inflammation mechanisms in
rapidly progressive
myocarditis.
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Acknowledgments |
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We thank M. Baucamp and K. Schönheinz for expert technical assistance.
Received September 12, 2000; revision received October 24, 2000; accepted October 26, 2000.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1. 1. Caforio AL. Role of autoimmunity in dilated cardiomyopathy. Br Heart J. 1994;72(suppl):S30–S34.
2.
Bachmaier K, Neu N,
de-la Maza ML, et al. Chlamydia infections and heart disease linked
through antigenic mimicry.
Science. 1999;283:1335–1339.
3. Penninger JM, Pummerer C, Liu P, et al. Cellular and molecular mechanisms of murine autoimmune myocarditis. APMIS. 1997;105:1–13.[Medline] [Order article via Infotrieve]
4.
Bachmaier K,
Pummerer C, Kozieradzki I, et al. Low-molecular-weight tumor necrosis
factor receptor p55 controls induction of autoimmune heart disease.
Circulation. 1997;95:655–661.
5. Sahrbacher UC, Lechner F, Eugster HP, et al. Mice with an inactivation of the inducible nitric synthase gene are susceptible to experimental autoimmune encephalomyelitis. Eur J Immunol. 1998;28:1332–1338.[Medline] [Order article via Infotrieve]
6. Bachmaier K, Neu N, Pummerer C, et al. iNOS expression and nytrotyrosine formation in the myocardium in response to inflammation is controlled by the interferon regulatory transcription factor 1. Circulation. 1997;96:585–591.
7. De Belder AJ, Radomski MW, Why HJ, et al. Nitric oxide synthase activities in human myocardium. Lancet. 1993;341:84–85.[Medline] [Order article via Infotrieve]
8.
Huang S, Hendriks
W, Althage A, et al. Immune response in mice that lack the
interferon-gamma receptor.
Science. 1993;259:1742–1745.
9.
Bingisser RM,
Tilbrook PA, Holt PG, et al. Macrophage-derived nitric oxide regulates
T cell activation via reversible disruption of the Jak3/STAT5 signaling
pathway. J Immunol. 1998;160:5729–5734.
10.
Chu CQ, Wittmer
S, Dalton DK. Failure to suppress the expansion of activated CD4 T-cell
population in interferon-deficient mice leads to exacerbation of
experimental autoimmune encephalomyelitis.
J Exp Med. 2000;192:117–122.
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