Induction of Autoimmune Myocarditis in Interleukin-2–Deficient Mice
Background Interleukin (IL)-2 is an important growth and survival factor for T cells and plays a crucial role in inflammation. Myosin-induced myocarditis is strictly dependent on activated T cells and is a model for postinfectious inflammatory heart disease in humans. To explore the role of IL-2 in myocarditis, we injected mice genetically deficient for IL-2 with cardiac myosin. Because it is conceivable that the lack of IL-2 either promotes or ameliorates the disease, we selected mouse strains that differ in their susceptibility to cardiac myosin–induced myocarditis.
Methods and Results Mice from a susceptible strain (C3H) that were rendered IL-2 deficient by gene targeting (IL-2−/− mice) and littermate controls were immunized twice with purified cardiac myosin at a 7-day interval. Three weeks after the first immunization, hearts were obtained for histopathological and immunohistochemical analysis. Sera were tested for autoantibodies to the cardiac myosin isoform by enzyme-linked immunosorbent assay. The majority of C3H IL-2−/− mice developed severe myocarditis accompanied by high-titer myosin autoantibodies. In C57BL/6 mice, which develop only little myocarditis on myosin immunization, lack of IL-2 did not increase susceptibility to the disease. Moreover, the composition of the inflammatory infiltrate in C3H IL-2−/− mice was virtually identical to that seen in the wild-type strain.
Conclusions Our data provide the first genetic evidence that in cardiac myosin–immunized mice, IL-2 has no essential role for the development of autoimmune heart disease and the generation of myosin autoantibodies.
Clinical and experimental findings suggest that chronic forms of inflammatory heart disease are mediated by a postinfectious autoimmune response.1 2 3 In genetically predisposed mice, myocarditis can be triggered by inoculation with CVB3.4 However, the ongoing inflammatory process appears to be driven by an immune response to myocardial “self” antigens that are exposed to the immune system on the occurrence of viral myonecrosis. On the basis of this hypothesis, we established a virus-free model of autoimmune myocarditis in which cardiac inflammation is induced by immunization with cardiac myosin.5 Because of the availability of a defined autoantigen and genetically defined mouse strains, this model system is particularly suitable for studying the immunopathology of postinfectious inflammatory heart disease.
In CVB3-infected mice, IL-2 exacerbates the disease severity in the subacute aviremic stage.6 Furthermore, treatment regimens that inhibit production of IL-2 and other cytokines have been used in myocarditis patients.1 Cardiac myosin–induced myocarditis is T-cell mediated and mimics the subacute phase of viral heart disease. Therefore, we were interested in whether the lack of this cytokine ameliorates the disease in IL-2 gene–deficient mice. Conversely, lack of IL-2 could increase susceptibility to the disease, and therefore a mouse strain that is normally resistant to myosin-induced heart disease was also tested.
Mice. C3H and C57BL/6 mice were rendered IL-2 deficient by targeted disruption of the IL-2 gene.7 Mice homozygous for the IL-2 defect (IL-2−/− mice) were obtained by breeding of heterozygous parents. IL-2 deficiency in individual mice was confirmed by polymerase chain reaction.7 For all experiments, heterozygous (IL-2+/− mice) and wild-type (IL-2+/+) littermates were used as controls.
Antigen preparation and immunization. Murine cardiac myosin was purified as described previously.5 To induce myocarditis, 4-week-old mice were immunized twice at a 7-day interval with 150 μg of cardiac myosin emulsified in FCA.5 Littermate controls were injected with buffer in FCA. On day 21 after the first immunization, blood was obtained for serological analysis and hearts were removed for histological examination.
Histopathology. Transverse sections from paraffin-embedded tissues were obtained at several levels and stained with hematoxylin and eosin. The severity of myocarditis was determined according to a previously described scoring system5 ranging from 0 to 4 (1 corresponds to infiltration of <5% of at least one histological cross section; 2, 5% to 10%; 3, 10% to 20 %; and 4, >20%).
Immunohistochemistry. Frozen heart tissue sections were stained for CD3, CD4, CD8, macrophages (Mac-1), and MHC class II molecules by use of monoclonal antibodies and immunoperoxidase as described previously.8 Infiltrated areas of heart tissue sections were evaluated by determining the proportion of immunoperoxidase-stained cells to the total number of infiltrating mononuclear cells. At least 1000 infiltrating cells were counted for each staining.
ELISA. IgG-autoantibody titers were determined by ELISA with cardiac myosin used as the test antigen.5
Induction of Autoimmune Myocarditis in Mice Lacking IL-2
C3H and C57BL/6 IL-2−/− mice as well as IL-2+/− and IL-2+/+ controls were twice injected with cardiac myosin. The susceptibility to cardiac myosin–induced myocarditis maps to certain MHC haplotypes.5 Therefore, C3H (H-2s) was selected as a highly susceptible strain and C57BL/6 (H-2b) as a strain with low susceptibility. As expected, C3H mice with normal IL-2 production and their IL-2+/− littermates developed autoimmune myocarditis at high prevalence and severity. Surprisingly, C3H IL-2−/− mice developed the disease at similar prevalence and severity (Table 1⇓). Similar to IL-2+/+ mice,8 the inflammatory infiltrate in IL-2−/− mice was diffuse and interstitial, and myocyte necrosis was not prominent (not shown). In cardiac myosin–immunized C57BL/6 IL-2+/+ mice, myocarditis occurred at minimal prevalence and severity, and IL-2 deficiency did not increase susceptibility to the disease (Table 1⇓).
Inflammatory Cells and MHC Class II Expression in Heart Tissue
To determine whether IL-2 deficiency leads to alterations of the inflammatory infiltrate, heart tissue sections from myosin-immunized IL-2+/+ and IL-2−/− mice were tested for the presence of T-cell subsets, macrophages, and expression of MHC class II molecules (Table 2⇓). In agreement with previous results,8 the inflammatory heart infiltrate of IL-2+/+ mice was composed of macrophages (Mac-1, ≈80%) and T cells (≈20%). The T cells consisted of CD4+ and, to a lesser extent, CD8+ cells. Furthermore, MHC class II expression was detectable within the inflammatory infiltrate and on adjacent interstitial cells but not on myofibrils and endothelial cells. Immunohistochemical analyses of heart tissues from IL-2−/− mice showed similar results.
Production of High-Titer Anti-Myosin Autoantibodies in the Absence of IL-2
As shown previously, cardiac myosin–induced myocarditis is associated with high-titer IgG autoantibodies against cardiac myosin.5 The Figure⇓ shows titers of these autoantibodies in mice injected with cardiac myosin. C3H IL-2−/− mice with myocarditis developed high-titer anti-myosin autoantibodies to an extent that was comparable to heterozygous and normal littermate controls. Myosin-immunized C57BL/6 IL-2−/− mice and FCA-injected control mice did not develop significant autoantibody levels.
The IL-2 system has been studied extensively in the context of T-cell proliferation and has become a paradigm of how interleukins function in the development and regulation of the immune system. IL-2, itself a product of activated T cells, also stimulates a variety of other cell types including B cells, monocytes, activated killer cells, and natural killer cells,9 all of which are potential mediators of cardiac tissue damage.10
This is the first study that demonstrates on a genetic basis that a T-cell–dependent autoimmune disease, ie, experimental myocarditis, and production of autoantibodies are inducible in IL-2–deficient mice. The similarity between the inflammatory heart infiltrate and cardiac MHC class II expression in both wild-type and IL-2−/− mice suggests that the immunopathologic mechanisms are not altered by disruption of the IL-2 gene. Myosin-induced myocarditis is CD4+ T-cell mediated and strictly dependent on MHC class II expression.11 MHC class II expression on interstitial cells of the heart tissue is a prerequisite for target recognition by autoreactive T cells and is induced by tumor necrosis factor.12 13 CD8+ cells are not involved in the induction but might be important for the perpetuation of the disease.8 Our data do not preclude that IL-2 has a role in myosin-induced myocarditis in normal mice and that in the absence of this cytokine, the disease may be mediated via alternative pathways. Regardless of whether this is the case, our study clearly demonstrates that IL-2 is not essential for the development of autoimmune heart disease. Because IL-2−/− mice are prone to develop spontaneous autoimmune diseases due to a disturbed immunoregulation,7 14 it was conceivable that the lack of IL-2 could render a normally resistant mouse strain susceptible to autoimmune myocarditis. However, a disease-promoting effect was not seen in C57BL/6 IL-2−/− mice, suggesting that IL-2 does not control disease susceptibility in this model.
As mentioned above, cardiac myosin–induced myocarditis closely mimics autoimmune-mediated myocarditis seen after infection of susceptible mice with CVB3. In CVB3-infected mice, it has been shown that administration of IL-2 had beneficial effects during the viremic stage of the disease by limiting myocardial virus replication. However, this cytokine was deleterious when administered during the second, aviremic stage.6 It has been suggested that IL-2 exacerbates the severity of myocarditis by increasing the number of infiltrating T cells. Additionally, it has been shown that IL-2 has an indirect cytolytic effect on cardiac myocytes by stimulating cytotoxic lymphocytes.15 Therefore, it appeared that IL-2 is a key cytokine in mediating autoimmune damage of the myocardial tissue. However, in the present study, genomic lack of IL-2 did not influence the development of severe myocarditis, thereby suggesting other cytokine pathways for promoting the disease. IL-2–deficient mice can generate almost normal cytotoxic T-lymphocyte responses and reject allografts.16 Recently, the cytokine IL-15 was shown to stimulate cells through the β and γ subunits of the IL-2 receptor,17 and therefore a role for IL-15 cannot be excluded in the myocarditis model.
Immunosuppressive treatments with cyclosporine and corticosteroids lead to inhibited production of several cytokines, including IL-2, and have been used in myocarditis patients.1 On the other hand, there are reports of deleterious effects of cyclosporine in experimental viral myocarditis.18 19 The immunopathology of cardiac myosin–induced myocarditis cannot be generally extrapolated to the situation in humans, in whom a wide spectrum of pathology is observed. However, our finding that IL-2 is not essential for the induction of this form of experimental autoimmune heart disease suggests that treatments that block IL-2 production may not be effective in all forms of myocarditis.
Selected Abbreviations and Acronyms
|CD||=||cluster of differentiation|
|CVB3||=||coxsackie B3 virus|
|ELISA||=||enzyme-linked immunosorbent assay|
|FCA||=||Freund’s complete adjuvant|
|IL-2−/−||=||mice homozygous for interleukin-2 deficiency|
|IL-2+/−||=||mice heterozygous for interleukin-2 deficiency|
|MHC||=||major histocompatibility complex|
The excellent technical assistance of Hildegard Haber is gratefully acknowledged. This work was supported by the Austrian FWF, Project P 8960, and by the Deutsche Forschungsgemeinschaft (Ho 493/10-1).
- Received December 2, 1996.
- Revision received February 10, 1997.
- Accepted February 13, 1997.
- Copyright © 1997 by American Heart Association
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