Interleukin-6–Deficient Mice Resist Development of Autoimmune Myocarditis Associated With Impaired Upregulation of Complement C3
From the Department of Internal Medicine (U.E., S.C.M.), University Hospital, Basel; Departments of Pathology (M.O.K.) and Clinical Immunology (A.F., H.-P.E.), University Hospital, Zurich; and Molecular Biomedicine (N.S., M.K.), Swiss Federal Institute of Technology, Zurich, Switzerland.2q, http://www.100md.com
Abstract2q, http://www.100md.com
Background— Interleukin (IL)-6 regulates various aspects of the immune response. In the context of heart diseases, it has been recognized as a prognostic factor for dilated cardiomyopathy, which often results from myocarditis.2q, http://www.100md.com
Methods and Results— Using IL-6–deficient mice, we studied the role of IL-6 in a model of autoimmune myocarditis resulting from immunization with a peptide derived from cardiac {alpha}2q, http://www.100md.com
-myosin. Prevalence and severity of myocarditis were markedly reduced in the absence of IL-6. CD4+ T cells from immunized IL-6–deficient mice proliferated poorly on restimulation with specific antigen in vitro and did not mediate disease on adoptive transfer into IL-6–competent RAG-2–deficient mice, which otherwise lack B cells and T cells. Production of complement C3, a crucial factor for the development of myocarditis, was strongly upregulated in IL-6+/+ but not in IL-6–deficient mice after immunization.
Conclusions— Our results demonstrate that IL-6 is required for the expansion of autoimmune CD4+ T cells and the pathogenesis of autoimmune myocarditis, possibly by upregulation of complement C3.wdc, 百拇医药
Key Words: interleukins myocarditis cardiomyopathywdc, 百拇医药
Introductionwdc, 百拇医药
Infections, surgery, myocardial infarction, and the systemic inflammatory response syndrome can cause cardiac inflammation. Acute myocarditis may initiate a chronic immune-mediated inflammation of the heart and result in idiopathic cardiomyopathy.1wdc, 百拇医药
Experimental autoimmune myocarditis is a model for CD4+ T cell–mediated inflammatory heart disease and can be induced in susceptible mice (eg, BALB/c, A/J) by immunization with cardiac myosin and peptides derived from it.2 Cytokines like tumor necrosis factor (TNF)-{alpha}wdc, 百拇医药
/TNFR13 and interleukin (IL)-124,5 are essential for disease induction, whereas interferon (INF)- protects from myocarditis.6
IL-6 is a multifunctional cytokine that controls viral, bacterial, and fungal infection7–10 and the pathogenesis of various diseases.11,12 Furthermore, in the context of autoimmunity, IL-6 is involved in development of experimental allergic encephalitis13,14 and experimental arthritis.15,16r, 百拇医药
Several studies point toward a role for IL-6 in heart disease. It has been shown that elevated plasma concentrations of IL-6 predict a poor prognosis in congestive heart failure secondary to idiopathic dilated cardiomyopathy.17 Furthermore, its serum levels are associated with an increased future risk of myocardial infarction among healthy men.18 We addressed the role of IL-6 in cardiac inflammation by using the experimental autoimmune myocarditis model and knockout mice.r, 百拇医药
Methodsr, 百拇医药
Mice, Immunization, and Treatment Protocolsr, 百拇医药
IL-6–deficient mice (generated by M. Kopf7), RAG-2–deficient mice lacking both B cells and T cells (purchased from BRL, Fuellinsdorf, Switzerland), and µMT-deficient mice lacking B cells (generated by K. Rajewsky et al19) (all >8 generations backcrossed to BALB/c) and IL-6+/+ BALB/c mice (wild-type; purchased from BRL, Fuellinsdorf, Switzerland) were immunized at 8 to 10 weeks of age with a peptide derived from {alpha}
-myosin heavy chain (Myhc-{alpha}{%(f0, 百拇医药
614-634 Ac-SLKLMATLFSTYASAD-OH), as described.2 No deaths were observed after immunization. For TNF-{alpha}{%(f0, 百拇医药
treatment, mice were injected intraperitoneally with 300 ng of synthetic TNF-{alpha}{%(f0, 百拇医药
peptide (ICN) every second day until day 21. Controls received saline only. All experiments were performed in accordance with Swiss federal legislation and were approved by the local authorities.{%(f0, 百拇医药
Histopathology and Immunohistochemistry{%(f0, 百拇医药
Hearts were assessed on routine paraffin sections and scored on a semiquantitative scale (0 indicated 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; 4, >30% of a cross-section involved).{%(f0, 百拇医药
For immunohistochemistry, samples were incubated with antibodies against CD106 (vascular cellular adhesion molecule [VCAM], Serotec, Oxford, UK), CD54 (intracellular adhesion molecule [ICAM], Serotec), CD11c (N418), MHC class II, MHC class I (M1/42), CD3 (KT3-1.1), CD4 (YTS 191), and CD8 (YTS169), followed by goat anti-rat Ig (Caltag, South San Francisco, Calif) or rabbit anti-hamster Ig (Jackson ImmunoResearch) and appropriate alkaline phosphatase (AP)-labeled third antibodies.
Measurement of Specific Antibody Responses and Complement C38z, http://www.100md.com
Antibody responses directed against Myhc-{alpha}8z, http://www.100md.com
614-634 were assessed by ELISA as described4 using AP-labeled goat anti-mouse IgG1, IgG2a, and IgG2b antibodies (Southern Biotechnology Associates). Serum C3 levels were measured by ELISA as described.20 Titers were determined at half maximum OD405nm.8z, http://www.100md.com
Cytokine Measurements8z, http://www.100md.com
Twenty-one days after immunization, cytokines were measured in supernatants of lymph node cells cultured with 20 µg/mL of Myhc-{alpha}8z, http://www.100md.com
614-634 for 20 hours (TNF-{alpha}) or 48 hours (IL-2, IL-4, IL-10, IL-1ß) using commercially available ELISA kits. For intracellular staining, cells were restimulated for 72 hours with Myhc-{alpha}8z, http://www.100md.com
614-634 before adding PMA (10-7 mol/L) and ionomycin (1 µg/mL) for 4 hours. Two hours before harvesting, Brefeldin A (10 µg/mL) was added. Cells were stained with APC-labeled anti-CD4 mAb and fixed with 2% paraformaldehyde. Subsequently, cytokines were stained with FITC-labeled anti–IFN- and PE-labeled anti–IL-4 in a 1% BSA/PBS solution containing 0.5% saponin for permeabilization before analysis by flow cytometry.
CD4+ Cell Proliferation Assaysv(|4u, 百拇医药
CD4+ T cells were purified from spleens using magnetic beads as described4 and restimulated on irradiated syngenic splenocytes pulsed with 10 µg/mL of Myhc-{alpha}v(|4u, 百拇医药
or ovalbumin as unspecific control antigen. Proliferation was measured by [3H]methyl-thymidine incorporation.v(|4u, 百拇医药
Adoptive Transferv(|4u, 百拇医药
CD4+ T cells were isolated and cultured for 96 hours on antigen-pulsed APC in the presence of 50 U/mL of recombinant murine IL-2. T cells 1x107 (>90% CD4+) were transferred intravenously into RAG-2-deficient mice. Recipients were killed after 14 days.v(|4u, 百拇医药
Statisticsv(|4u, 百拇医药
The Mann-Whitney U test was used for the evaluation of severity scores and heart weights. Proliferation responses and cytokine levels were compared using ANOVA and the unpaired t test. Dichotomous data were analyzed by Fisher’s exact test. Myocarditis prevalence refers to the number of diseased mice compared with the number of immunized mice still alive at the days indicated.
Results+m%1p+%, 百拇医药
IL-6–Deficient Mice Are Protected From Autoimmune Myocarditis+m%1p+%, 百拇医药
IL-6-/- and IL-6+/+ mice were immunized with Myhc-{alpha}+m%1p+%, 百拇医药
16. IL-6+/+ mice developed severe myocarditis, which peaked at day 21, as described previously.4,6 In contrast, disease prevalence and severity were strongly reduced in IL-6-/- mice (, ). Moreover, immunohistochemistry showed reduced expression of MHC class II, VCAM-1, and ICAM-1 molecules () and low numbers of CD3-positive cells (not shown) in the hearts of IL-6–deficient mice compared with controls.+m%1p+%, 百拇医药
fig.ommitted+m%1p+%, 百拇医药
IL-6–deficient mice are protected from autoimmune myocarditis. Inflammatory infiltrates are present in IL-6+/+ but not IL-6-/- hearts 21 days after immunization. In IL-6+/+ mice, VCAM-1 is upregulated along capillaries of infiltrated myocardium, whereas IL-6-/- mice show only constitutive VCAM-1 expression. ICAM-1 and MHC II are present on inflammatory cells or capillaries of infiltrated myocardium, whereas in IL-6–deficient mice, only constitutive expression of ICAM-1 on dendritic cells and blood vessels and MHC II on dendritic cells are seen (x480 original magnification).
fig.ommittedge, http://www.100md.com
TABLE 1. Myocarditis Prevalence and Severity in IL-6+/+, IL-6-/-, and B Cell–Deficient (µMT) Mice at Different Times After Immunizationge, http://www.100md.com
Reduced Myosin Autoantibodies in IL-6–Deficient Micege, http://www.100md.com
IL-6+/+ mice showed high autoantibody levels against Myhc-{alpha}ge, http://www.100md.com
16 of the subclasses IgG1>IgG2b>IgG2a. In contrast, IgG1, IgG2b, and IgG2b autoantibody levels were significantly reduced in IL-6–deficient mice (). Thus, disease severity correlated with the magnitude of the autoantibody response in IL-6+/+ and IL-6-/- mice. Because IL-6 promotes B cell responses, we asked whether impaired antibody responses contribute to disease resistance. We therefore immunized B cell–deficient (µMT) mice with Myhc-{alpha}ge, http://www.100md.com
. As shown in , mice lacking B cells develop myocarditis indistinguishable from IL-6+/+ controls, demonstrating that antibodies play no role in this disease model. In addition, there was no difference in the course of myocarditis between IL-6+/+ and B cell–deficient mice up to 4 weeks after immunization (not shown). Moreover, our data suggest that the reduced autoantibody response was not responsible for protection from myocarditis in IL-6–deficient mice.
fig.ommitted2@;:9fi, http://www.100md.com
Reduced antibody responses in IL-6–deficient mice. Sera were taken from IL-6+/+ mice (filled bars) and IL-6-/- mice (open bars) 21 days after the first immunization. Titers of Myhc-{alpha}2@;:9fi, http://www.100md.com
–specific IgG1, IgG2a, and IgG2b antibody responses were measured by ELISA. Values indicate mean±SD of 5 individual mice. P values were calculated using the Mann-Whitney U test.2@;:9fi, http://www.100md.com
Inefficient Induction of Autoreactive CD4+ T Cells in the Absence of IL-62@;:9fi, http://www.100md.com
Because autoimmune myocarditis is CD4+ T cell–mediated, we investigated the role of IL-6 for the induction and Th1/Th2 subset polarization of antigen-specific CD4+ T cells. Twenty-one days after immunization, IL-6-/- CD4+ T cells showed reduced proliferation compared with IL-6+/+ CD4+ T cells on in vitro stimulation (A). Intracellular staining of IL-4 and IFN- in CD4+ T cells of restimulated lymph node cells revealed a similar Th1/Th2 cell ratio with only a minimal increase of IL-4 producing CD4+ T-cells in IL-6-/- mice (B). Furthermore, cytokine measurement in the supernatants of these cell cultures showed no significant differences in IL-4 and IL-10 production, although IFN- levels were slightly reduced for IL-6–deficient lymph node cells. (C). Interestingly, supernatants of IL-6-/- cultures contained reduced TNF-{alpha}
, whereas levels of IL-1ß were comparable to IL-6+/+ cultures (D). To additionally investigate the function of autoimmune CD4+ T cells in vivo, we purified CD4+ T cells from immunized IL-6-/- and IL-6+/+ mice at day 21, restimulated with Myhc-{alpha})r69x5a, 百拇医药
614-634, and injected them into recipients devoid of endogenous B cells and T cells (RAG-2–deficient mice). Adoptive transfer of CD4+ T cells from diseased IL-6+/+ mice induced myocarditis in RAG-2–deficient recipients (). In contrast, transfer of IL-6-/- CD4+ T cells failed to induce disease in RAG-2–deficient mice, which are otherwise competent to produce IL-6. Thus, protection from myocarditis in IL-6–deficient mice results from inefficient induction of autoreactive CD4+ T cells. Additionally, reduced TNF-{alpha})r69x5a, 百拇医药
production may prevent myocarditis development in the absence of IL-6.)r69x5a, 百拇医药
fig.ommitted
Reduced CD4+ T cell responses in IL-6–deficient mice. CD4+ T cells were isolated at day 21 and (A) cultured for 72 hours with irradiated (2000 rad) splenocytes in the absence or presence of 10 µg/mL Myhc-{alpha}j, http://www.100md.com
before measurement of [3H] thymidine incorporation. Values indicate mean±SD of 5 to 6 mice. B, Lymph node cells were stimulated with peptide for 72 hours and subsequently with PMA and ionomycin for 4 hours in the presence of Brefeldin for the last 2 hours to retain cytokines in the cytoplasm. Cells were then stained with APC-labeled anti-CD4 followed by fixation, permeabilization, and intracellular staining with PE-labeled IL-4 and FITC-labeled anti–IFN-. Cells were analyzed by 3-color flow cytometry. Dot plots are shown from individual samples that are representative for the group. Results are expressed as a percentage of lymph node cells gated on CD4+ T cells. Values in brackets indicate averages of the group. C and D, IFN-, IL-4, IL-10, and IL-1ß levels were measured after 40 hours and TNF-{alpha}j, http://www.100md.com
levels after 20 hours in supernatants of antigen-restimulated lymph node cells. Values indicate mean±SD. P values were calculated using ANOVA and the unpaired t test.
fig.ommitted)(h, 百拇医药
Myocarditis/Epicarditis Prevalence After Adoptive Transfer of Antigen-Restimulated CD4+ T Cells in RAG-/- (Ad) Mutant Mice)(h, 百拇医药
TNF-{alpha})(h, 百拇医药
Does Not Restore Disease Susceptibility of IL-6–Deficient Mice)(h, 百拇医药
TNFR1 is crucial for the development of autoimmune myocarditis.3 Given the markedly reduced TNF-{alpha})(h, 百拇医药
levels within lymphocyte cultures from IL-6–deficient mice, we speculated that TNF-{alpha})(h, 百拇医药
treatment may reverse myocarditis resistance of IL-6–deficient mice. Immunized IL-6–deficient mice were injected with a synthetic peptide fragment mediating TNF-{alpha})(h, 百拇医药
bioactivity according to an established protocol.21 Compared with saline, injection of the TNF-{alpha})(h, 百拇医药
–derived peptide upregulated MHC class II and adhesion molecules in the hearts of IL-6+/+ and IL-6–deficient mice within 24 hours (not shown). However, TNF-{alpha}
peptide treatment failed to restore disease susceptibility in IL-6–deficient mice (). Moreover, there was no change in the cytokine production pattern of in vitro restimulated lymphocytes (not shown) after TNF-{alpha}3y3h9vs, 百拇医药
treatment. These data suggest that the impaired production of TNF-{alpha}3y3h9vs, 百拇医药
is not responsible for myocarditis resistance of IL-6-/- mice.3y3h9vs, 百拇医药
IL-6 Is Required for C3 Upregulation After Immunization3y3h9vs, 百拇医药
Because complement C3 is essential for development of myocarditis22 and IL-6 is a major regulator of the acute phase response including C3,7,23 we assessed C3 levels in sera of immunized mice. Immunization with Myhc-{alpha}3y3h9vs, 百拇医药
16 in adjuvant resulted in a strong increase of C3 serum levels in IL-6+/+ mice within 24 hours (). In contrast, no increase was observed in IL-6–deficient mice. Thus, impaired upregulation of complement is associated with protection against myocarditis in IL-6–deficient mice.
fig.ommitted#y+6o|, 百拇医药
Reduced C3 levels in sera of IL-6–deficient mice. Groups of mice (n=6 per group) were immunized and bled at indicated days. C3 serum levels were measured by ELISA as described.20 Values indicate mean±SD of IL-6+/+ mice (filled columns) and IL-6–deficient mice (open columns). P values were calculated using the Mann-Whitney U test.#y+6o|, 百拇医药
Discussion#y+6o|, 百拇医药
Together with indirect clinical evidence,17,18 our data demonstrate a critical role for IL-6 in the pathogenesis of inflammatory heart disease.#y+6o|, 百拇医药
Immunized IL-6–deficient mice showed a reduction in innate and adaptive immune responses linked to myocarditis susceptibility, such as expression of adhesion molecules (ie, ICAM-1 and VCAM-1), inflammatory responses (ie, C3 and TNF-{alpha}), levels of autoantibodies, and proliferation of autoreactive CD4+ T cells.#y+6o|, 百拇医药
Heart ICAM-1 and VCAM-1 expression can be induced by IL-1ß and TNF-{alpha}
and by bacterial components leading to accumulation of inflammatory cells in the heart and contractile dysfunction.24 Moreover, increased ICAM-1 expression has been implicated in the pathogenesis of viral myocarditis.25 Reduced ICAM-1 and VCAM-1 expression may reflect impaired activation of endothelial cells and result in less-efficient recruitment of autoreactive T cells to the heart of IL-6–deficient mice. IL-6 has been suggested to contribute indirectly to the activation of endothelial cells, which lack the IL-6 receptor (IL-6R). Thus, expression of ICAM-1, VCAM-1, and E-selectin on endothelial cells is mediated by transactivation mediated by soluble IL-6R released from stimulated neutrophils.26 Reduced expression of adhesion molecules was also observed in IL-6–deficient mice resisting experimental allergic encephalomyelitis.13 Interestingly, triggering of a TNFR1-dependent pathway by staphylococcus enterotoxin B treatment restored adhesion molecule expression and overcame disease resistance of IL-6–deficient mice in the experimental allergic encephalomyelitis model.13 Because TNF-{alpha}
is a critical factor for the development of Coxsackie virus–induced myocarditis21 and TNFR1 is crucial for autoimmune myocarditis,3 we reasoned that impaired TNF-{alpha}\]+oyz, 百拇医药
production may contribute to myocarditis resistance of IL-6-/- mice. However, TNF-{alpha}\]+oyz, 百拇医药
(Table 1) treatment did not render IL-6–deficient mice susceptible. Thus, impaired production of TNF-{alpha}\]+oyz, 百拇医药
and expression of adhesion molecules seem not to be responsible for protection from myocarditis in IL-6–deficient mice.\]+oyz, 百拇医药
IL-6 is required for efficient antibody responses after infection with viruses27 and after immunization with antigen.23 In keeping with these results, we found that IL-6–deficient mice showed reduced levels of autoantibodies. Although autoimmune myocarditis is associated with autoantibody formation, B cells were shown to be dispensable for disease development after immunization of BALB/c mice with cardiac myosin whole protein.28 Nevertheless, it remained possible that B cells play an important role after immunization with the myosin peptide used in our immunization regimen, because of their higher density of MHC class II myosin peptide complexes. This possibility was excluded by showing that disease severity and mortality did not differ between B cell–deficient (µMT) mice and IL-6+/+ mice after immunization. This finding does not preclude a role of autoantibodies in a delayed cardiac dysfunction, in human cardiomyopathy, or in other mouse models of myocarditis, including viral myocarditis or autoimmune myocarditis in DBA/2 mice, which have been shown to develop disease after transfer of autoantibodies.29,30 Nevertheless, our data suggest that reduced IgG autoantibody production is not responsible for resistance to autoimmune myocarditis in IL-6–deficient mice on a BALB/c background.
Autoimmune myocarditis is dependent on CD4+ T cells. We found that the proliferation of autoreactive CD4+ T cells was impaired in IL-6–deficient mice. Furthermore, IL-6-/- CD4+ T cells, in contrast to IL-6+/+ CD4+ T cells, failed to mediate disease on adoptive transfer. Defective T cell responses in IL-6–deficient mice have also been described in other models of autoimmune disease. In some reports, reduced CD4+ T cell proliferation was associated with a reduction of both IL-4 and IFN- responses,15 whereas other studies demonstrated a shifted Th1:Th2 ratio with decreased IFN- and increased IL-4 production in the absence of IL-6. The Th1/Th2 paradigm is still widely used to explain autoimmunity with IFN- promoting and IL-4 protecting against disease. However, it has been shown previously that autoimmune myocarditis is exacerbated in IFN-–deficient mice,4,6 whereas disease prevalence at the peak of the response is reduced in IL-4R–deficient mice with an abrogated IL-4 and IL-13 pathway4 or on injection of anti–IL-4 monoclonal antibody.31 IL-6–deficient mice display a slightly increased IL-4 to IFN- ratio, which, considering the phenotype of the respective knockouts, cannot explain resistance to myocarditis. Therefore, our data suggest that IL-6 is involved in the activation or maintenance of autoimmune CD4+ T cells independent of Th1/Th2 polarization. Interestingly, in experimental colitis induced by adoptive transfer of congenic CD62L+CD45RBhigh CD4+ T cells into SCID (severe combined immunodeficiency) mice, neutralization of IL-6 resulted in apoptosis of lamina propria cells and reduced disease severity.12 Our findings of reduced proliferation do not exclude the possibility of enhanced apoptosis of myosin-specific IL-6-/- CD4+ T cells.
We have previously demonstrated that impaired complement C3 production is responsible for defective germinal center development and B-cell responses in immunized IL-6–deficient mice.23 Moreover, we have reported that C3 is essential for the activation and recruitment of both CD4+ and CD8+ T cells after pulmonary virus infection,32 which is also defective in IL-6–deficient mice (N. Schmitz et al, unpublished data, 2001). Consistent with these data, we describe here that IL-6–deficient mice fail to upregulate C3 production associated with impaired CD4+ T cell responses after immunization with Myhc-{alpha}at, 百拇医药
614-634. Recently, autoimmune myocarditis has been shown to depend on C3 and the complement receptors CD21 and CD35.22 Depletion of C3 during the inductive phase but not during the progression of inflammation (effector phase) prevented disease. Furthermore, the absence of the complement receptors CD21 (CR2) and CD35 (CR1) prevented myocarditis. Protection from myocarditis in C3-depleted and CR1/2-deficient mice was associated with reduced production of proinflammatory cytokines. In addition, CD4+ T cells from CR1/2-deficient mice were less responsive to antigen activation, as indicated by reduced expression of CD25 and CD69.22 Thus, the protection from myocarditis observed for IL-6–deficient and C3-deficient mice may be related. We suggest that impaired generation of complement is a key factor for disease prevention in the absence of IL-6. Despite the fact that complement levels do not increase in IL-6–deficient mice after immunization, baseline concentrations in the serum were in the normal range but seem insufficient to activate CD4+ T cells and autoimmune disease. Thus, it seems that IL-6 regulates C3 production locally in the microenvironment of lymphatic tissue,23 where CD4+ T cells interact with B cells and antigen-presenting cells.
In conclusion, we provide genetic evidence that IL-6 is crucial for the development of autoimmune heart disease. IL-6 bridges innate and adaptive immune responses by regulation of complement C3. We suggest that elevated IL-6 levels in patients with cardiomyopathy or after myocardial infarction are not merely prognostic surrogate markers but contribute to cardiac damage by triggering pathways resulting in autoreactivity. Therefore, blocking the IL-6 pathway may be beneficial in the treatment of inflammatory heart disease.'e4-, 百拇医药
Acknowledgments'e4-, 百拇医药
This work was supported by the Swiss Heart Foundation and by the Helmut Horten Foundation, Lugano, Switzerland.'e4-, 百拇医药
Received July 23, 2002; revision received September 17, 2002; accepted September 18, 2002.'e4-, 百拇医药
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Malkiel S, Factor S, Diamond B. Autoimmune myocarditis does not require B cells for antigen presentation. J Immunol. 1999; 163: 5265–5268.6q*:?4w, 百拇医药
Kuan AP, Chamberlain W, Malkiel S, et al. Genetic control of autoimmune myocarditis mediated by myosin-specific antibodies. Immunogenetics. 1999; 49: 79–85.6q*:?4w, 百拇医药
Kuan AP, Zuckier L, Liao L, et al. Immunoglobulin isotype determines pathogenicity in antibody-mediated myocarditis in naive mice. Circ Res. 2000; 86: 281–285.6q*:?4w, 百拇医药
Afanasyeva M, Wang Y, Kaya Z, et al. Experimental autoimmune myocarditis in A/J mice is an interleukin-4–dependent disease with a Th2 phenotype. Am J Pathol. 2001; 159: 193–203.6q*:?4w, 百拇医药
Kopf M, Abel B, Gallimore A, et al. The complement component C3 promotes T cell priming and lung migration to control acute influenza virus infection. Nat Med. 2002; 8: 373–378.(Urs Eriksson MD Michael O. Kurrer MD Nicole Schmitz Stephan C. Marsch MD Adriano Fontana MD Hans-Pie)
Abstract2q, http://www.100md.com
Background— Interleukin (IL)-6 regulates various aspects of the immune response. In the context of heart diseases, it has been recognized as a prognostic factor for dilated cardiomyopathy, which often results from myocarditis.2q, http://www.100md.com
Methods and Results— Using IL-6–deficient mice, we studied the role of IL-6 in a model of autoimmune myocarditis resulting from immunization with a peptide derived from cardiac {alpha}2q, http://www.100md.com
-myosin. Prevalence and severity of myocarditis were markedly reduced in the absence of IL-6. CD4+ T cells from immunized IL-6–deficient mice proliferated poorly on restimulation with specific antigen in vitro and did not mediate disease on adoptive transfer into IL-6–competent RAG-2–deficient mice, which otherwise lack B cells and T cells. Production of complement C3, a crucial factor for the development of myocarditis, was strongly upregulated in IL-6+/+ but not in IL-6–deficient mice after immunization.
Conclusions— Our results demonstrate that IL-6 is required for the expansion of autoimmune CD4+ T cells and the pathogenesis of autoimmune myocarditis, possibly by upregulation of complement C3.wdc, 百拇医药
Key Words: interleukins myocarditis cardiomyopathywdc, 百拇医药
Introductionwdc, 百拇医药
Infections, surgery, myocardial infarction, and the systemic inflammatory response syndrome can cause cardiac inflammation. Acute myocarditis may initiate a chronic immune-mediated inflammation of the heart and result in idiopathic cardiomyopathy.1wdc, 百拇医药
Experimental autoimmune myocarditis is a model for CD4+ T cell–mediated inflammatory heart disease and can be induced in susceptible mice (eg, BALB/c, A/J) by immunization with cardiac myosin and peptides derived from it.2 Cytokines like tumor necrosis factor (TNF)-{alpha}wdc, 百拇医药
/TNFR13 and interleukin (IL)-124,5 are essential for disease induction, whereas interferon (INF)- protects from myocarditis.6
IL-6 is a multifunctional cytokine that controls viral, bacterial, and fungal infection7–10 and the pathogenesis of various diseases.11,12 Furthermore, in the context of autoimmunity, IL-6 is involved in development of experimental allergic encephalitis13,14 and experimental arthritis.15,16r, 百拇医药
Several studies point toward a role for IL-6 in heart disease. It has been shown that elevated plasma concentrations of IL-6 predict a poor prognosis in congestive heart failure secondary to idiopathic dilated cardiomyopathy.17 Furthermore, its serum levels are associated with an increased future risk of myocardial infarction among healthy men.18 We addressed the role of IL-6 in cardiac inflammation by using the experimental autoimmune myocarditis model and knockout mice.r, 百拇医药
Methodsr, 百拇医药
Mice, Immunization, and Treatment Protocolsr, 百拇医药
IL-6–deficient mice (generated by M. Kopf7), RAG-2–deficient mice lacking both B cells and T cells (purchased from BRL, Fuellinsdorf, Switzerland), and µMT-deficient mice lacking B cells (generated by K. Rajewsky et al19) (all >8 generations backcrossed to BALB/c) and IL-6+/+ BALB/c mice (wild-type; purchased from BRL, Fuellinsdorf, Switzerland) were immunized at 8 to 10 weeks of age with a peptide derived from {alpha}
-myosin heavy chain (Myhc-{alpha}{%(f0, 百拇医药
614-634 Ac-SLKLMATLFSTYASAD-OH), as described.2 No deaths were observed after immunization. For TNF-{alpha}{%(f0, 百拇医药
treatment, mice were injected intraperitoneally with 300 ng of synthetic TNF-{alpha}{%(f0, 百拇医药
peptide (ICN) every second day until day 21. Controls received saline only. All experiments were performed in accordance with Swiss federal legislation and were approved by the local authorities.{%(f0, 百拇医药
Histopathology and Immunohistochemistry{%(f0, 百拇医药
Hearts were assessed on routine paraffin sections and scored on a semiquantitative scale (0 indicated 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; 4, >30% of a cross-section involved).{%(f0, 百拇医药
For immunohistochemistry, samples were incubated with antibodies against CD106 (vascular cellular adhesion molecule [VCAM], Serotec, Oxford, UK), CD54 (intracellular adhesion molecule [ICAM], Serotec), CD11c (N418), MHC class II, MHC class I (M1/42), CD3 (KT3-1.1), CD4 (YTS 191), and CD8 (YTS169), followed by goat anti-rat Ig (Caltag, South San Francisco, Calif) or rabbit anti-hamster Ig (Jackson ImmunoResearch) and appropriate alkaline phosphatase (AP)-labeled third antibodies.
Measurement of Specific Antibody Responses and Complement C38z, http://www.100md.com
Antibody responses directed against Myhc-{alpha}8z, http://www.100md.com
614-634 were assessed by ELISA as described4 using AP-labeled goat anti-mouse IgG1, IgG2a, and IgG2b antibodies (Southern Biotechnology Associates). Serum C3 levels were measured by ELISA as described.20 Titers were determined at half maximum OD405nm.8z, http://www.100md.com
Cytokine Measurements8z, http://www.100md.com
Twenty-one days after immunization, cytokines were measured in supernatants of lymph node cells cultured with 20 µg/mL of Myhc-{alpha}8z, http://www.100md.com
614-634 for 20 hours (TNF-{alpha}) or 48 hours (IL-2, IL-4, IL-10, IL-1ß) using commercially available ELISA kits. For intracellular staining, cells were restimulated for 72 hours with Myhc-{alpha}8z, http://www.100md.com
614-634 before adding PMA (10-7 mol/L) and ionomycin (1 µg/mL) for 4 hours. Two hours before harvesting, Brefeldin A (10 µg/mL) was added. Cells were stained with APC-labeled anti-CD4 mAb and fixed with 2% paraformaldehyde. Subsequently, cytokines were stained with FITC-labeled anti–IFN- and PE-labeled anti–IL-4 in a 1% BSA/PBS solution containing 0.5% saponin for permeabilization before analysis by flow cytometry.
CD4+ Cell Proliferation Assaysv(|4u, 百拇医药
CD4+ T cells were purified from spleens using magnetic beads as described4 and restimulated on irradiated syngenic splenocytes pulsed with 10 µg/mL of Myhc-{alpha}v(|4u, 百拇医药
or ovalbumin as unspecific control antigen. Proliferation was measured by [3H]methyl-thymidine incorporation.v(|4u, 百拇医药
Adoptive Transferv(|4u, 百拇医药
CD4+ T cells were isolated and cultured for 96 hours on antigen-pulsed APC in the presence of 50 U/mL of recombinant murine IL-2. T cells 1x107 (>90% CD4+) were transferred intravenously into RAG-2-deficient mice. Recipients were killed after 14 days.v(|4u, 百拇医药
Statisticsv(|4u, 百拇医药
The Mann-Whitney U test was used for the evaluation of severity scores and heart weights. Proliferation responses and cytokine levels were compared using ANOVA and the unpaired t test. Dichotomous data were analyzed by Fisher’s exact test. Myocarditis prevalence refers to the number of diseased mice compared with the number of immunized mice still alive at the days indicated.
Results+m%1p+%, 百拇医药
IL-6–Deficient Mice Are Protected From Autoimmune Myocarditis+m%1p+%, 百拇医药
IL-6-/- and IL-6+/+ mice were immunized with Myhc-{alpha}+m%1p+%, 百拇医药
16. IL-6+/+ mice developed severe myocarditis, which peaked at day 21, as described previously.4,6 In contrast, disease prevalence and severity were strongly reduced in IL-6-/- mice (, ). Moreover, immunohistochemistry showed reduced expression of MHC class II, VCAM-1, and ICAM-1 molecules () and low numbers of CD3-positive cells (not shown) in the hearts of IL-6–deficient mice compared with controls.+m%1p+%, 百拇医药
fig.ommitted+m%1p+%, 百拇医药
IL-6–deficient mice are protected from autoimmune myocarditis. Inflammatory infiltrates are present in IL-6+/+ but not IL-6-/- hearts 21 days after immunization. In IL-6+/+ mice, VCAM-1 is upregulated along capillaries of infiltrated myocardium, whereas IL-6-/- mice show only constitutive VCAM-1 expression. ICAM-1 and MHC II are present on inflammatory cells or capillaries of infiltrated myocardium, whereas in IL-6–deficient mice, only constitutive expression of ICAM-1 on dendritic cells and blood vessels and MHC II on dendritic cells are seen (x480 original magnification).
fig.ommittedge, http://www.100md.com
TABLE 1. Myocarditis Prevalence and Severity in IL-6+/+, IL-6-/-, and B Cell–Deficient (µMT) Mice at Different Times After Immunizationge, http://www.100md.com
Reduced Myosin Autoantibodies in IL-6–Deficient Micege, http://www.100md.com
IL-6+/+ mice showed high autoantibody levels against Myhc-{alpha}ge, http://www.100md.com
16 of the subclasses IgG1>IgG2b>IgG2a. In contrast, IgG1, IgG2b, and IgG2b autoantibody levels were significantly reduced in IL-6–deficient mice (). Thus, disease severity correlated with the magnitude of the autoantibody response in IL-6+/+ and IL-6-/- mice. Because IL-6 promotes B cell responses, we asked whether impaired antibody responses contribute to disease resistance. We therefore immunized B cell–deficient (µMT) mice with Myhc-{alpha}ge, http://www.100md.com
. As shown in , mice lacking B cells develop myocarditis indistinguishable from IL-6+/+ controls, demonstrating that antibodies play no role in this disease model. In addition, there was no difference in the course of myocarditis between IL-6+/+ and B cell–deficient mice up to 4 weeks after immunization (not shown). Moreover, our data suggest that the reduced autoantibody response was not responsible for protection from myocarditis in IL-6–deficient mice.
fig.ommitted2@;:9fi, http://www.100md.com
Reduced antibody responses in IL-6–deficient mice. Sera were taken from IL-6+/+ mice (filled bars) and IL-6-/- mice (open bars) 21 days after the first immunization. Titers of Myhc-{alpha}2@;:9fi, http://www.100md.com
–specific IgG1, IgG2a, and IgG2b antibody responses were measured by ELISA. Values indicate mean±SD of 5 individual mice. P values were calculated using the Mann-Whitney U test.2@;:9fi, http://www.100md.com
Inefficient Induction of Autoreactive CD4+ T Cells in the Absence of IL-62@;:9fi, http://www.100md.com
Because autoimmune myocarditis is CD4+ T cell–mediated, we investigated the role of IL-6 for the induction and Th1/Th2 subset polarization of antigen-specific CD4+ T cells. Twenty-one days after immunization, IL-6-/- CD4+ T cells showed reduced proliferation compared with IL-6+/+ CD4+ T cells on in vitro stimulation (A). Intracellular staining of IL-4 and IFN- in CD4+ T cells of restimulated lymph node cells revealed a similar Th1/Th2 cell ratio with only a minimal increase of IL-4 producing CD4+ T-cells in IL-6-/- mice (B). Furthermore, cytokine measurement in the supernatants of these cell cultures showed no significant differences in IL-4 and IL-10 production, although IFN- levels were slightly reduced for IL-6–deficient lymph node cells. (C). Interestingly, supernatants of IL-6-/- cultures contained reduced TNF-{alpha}
, whereas levels of IL-1ß were comparable to IL-6+/+ cultures (D). To additionally investigate the function of autoimmune CD4+ T cells in vivo, we purified CD4+ T cells from immunized IL-6-/- and IL-6+/+ mice at day 21, restimulated with Myhc-{alpha})r69x5a, 百拇医药
614-634, and injected them into recipients devoid of endogenous B cells and T cells (RAG-2–deficient mice). Adoptive transfer of CD4+ T cells from diseased IL-6+/+ mice induced myocarditis in RAG-2–deficient recipients (). In contrast, transfer of IL-6-/- CD4+ T cells failed to induce disease in RAG-2–deficient mice, which are otherwise competent to produce IL-6. Thus, protection from myocarditis in IL-6–deficient mice results from inefficient induction of autoreactive CD4+ T cells. Additionally, reduced TNF-{alpha})r69x5a, 百拇医药
production may prevent myocarditis development in the absence of IL-6.)r69x5a, 百拇医药
fig.ommitted
Reduced CD4+ T cell responses in IL-6–deficient mice. CD4+ T cells were isolated at day 21 and (A) cultured for 72 hours with irradiated (2000 rad) splenocytes in the absence or presence of 10 µg/mL Myhc-{alpha}j, http://www.100md.com
before measurement of [3H] thymidine incorporation. Values indicate mean±SD of 5 to 6 mice. B, Lymph node cells were stimulated with peptide for 72 hours and subsequently with PMA and ionomycin for 4 hours in the presence of Brefeldin for the last 2 hours to retain cytokines in the cytoplasm. Cells were then stained with APC-labeled anti-CD4 followed by fixation, permeabilization, and intracellular staining with PE-labeled IL-4 and FITC-labeled anti–IFN-. Cells were analyzed by 3-color flow cytometry. Dot plots are shown from individual samples that are representative for the group. Results are expressed as a percentage of lymph node cells gated on CD4+ T cells. Values in brackets indicate averages of the group. C and D, IFN-, IL-4, IL-10, and IL-1ß levels were measured after 40 hours and TNF-{alpha}j, http://www.100md.com
levels after 20 hours in supernatants of antigen-restimulated lymph node cells. Values indicate mean±SD. P values were calculated using ANOVA and the unpaired t test.
fig.ommitted)(h, 百拇医药
Myocarditis/Epicarditis Prevalence After Adoptive Transfer of Antigen-Restimulated CD4+ T Cells in RAG-/- (Ad) Mutant Mice)(h, 百拇医药
TNF-{alpha})(h, 百拇医药
Does Not Restore Disease Susceptibility of IL-6–Deficient Mice)(h, 百拇医药
TNFR1 is crucial for the development of autoimmune myocarditis.3 Given the markedly reduced TNF-{alpha})(h, 百拇医药
levels within lymphocyte cultures from IL-6–deficient mice, we speculated that TNF-{alpha})(h, 百拇医药
treatment may reverse myocarditis resistance of IL-6–deficient mice. Immunized IL-6–deficient mice were injected with a synthetic peptide fragment mediating TNF-{alpha})(h, 百拇医药
bioactivity according to an established protocol.21 Compared with saline, injection of the TNF-{alpha})(h, 百拇医药
–derived peptide upregulated MHC class II and adhesion molecules in the hearts of IL-6+/+ and IL-6–deficient mice within 24 hours (not shown). However, TNF-{alpha}
peptide treatment failed to restore disease susceptibility in IL-6–deficient mice (). Moreover, there was no change in the cytokine production pattern of in vitro restimulated lymphocytes (not shown) after TNF-{alpha}3y3h9vs, 百拇医药
treatment. These data suggest that the impaired production of TNF-{alpha}3y3h9vs, 百拇医药
is not responsible for myocarditis resistance of IL-6-/- mice.3y3h9vs, 百拇医药
IL-6 Is Required for C3 Upregulation After Immunization3y3h9vs, 百拇医药
Because complement C3 is essential for development of myocarditis22 and IL-6 is a major regulator of the acute phase response including C3,7,23 we assessed C3 levels in sera of immunized mice. Immunization with Myhc-{alpha}3y3h9vs, 百拇医药
16 in adjuvant resulted in a strong increase of C3 serum levels in IL-6+/+ mice within 24 hours (). In contrast, no increase was observed in IL-6–deficient mice. Thus, impaired upregulation of complement is associated with protection against myocarditis in IL-6–deficient mice.
fig.ommitted#y+6o|, 百拇医药
Reduced C3 levels in sera of IL-6–deficient mice. Groups of mice (n=6 per group) were immunized and bled at indicated days. C3 serum levels were measured by ELISA as described.20 Values indicate mean±SD of IL-6+/+ mice (filled columns) and IL-6–deficient mice (open columns). P values were calculated using the Mann-Whitney U test.#y+6o|, 百拇医药
Discussion#y+6o|, 百拇医药
Together with indirect clinical evidence,17,18 our data demonstrate a critical role for IL-6 in the pathogenesis of inflammatory heart disease.#y+6o|, 百拇医药
Immunized IL-6–deficient mice showed a reduction in innate and adaptive immune responses linked to myocarditis susceptibility, such as expression of adhesion molecules (ie, ICAM-1 and VCAM-1), inflammatory responses (ie, C3 and TNF-{alpha}), levels of autoantibodies, and proliferation of autoreactive CD4+ T cells.#y+6o|, 百拇医药
Heart ICAM-1 and VCAM-1 expression can be induced by IL-1ß and TNF-{alpha}
and by bacterial components leading to accumulation of inflammatory cells in the heart and contractile dysfunction.24 Moreover, increased ICAM-1 expression has been implicated in the pathogenesis of viral myocarditis.25 Reduced ICAM-1 and VCAM-1 expression may reflect impaired activation of endothelial cells and result in less-efficient recruitment of autoreactive T cells to the heart of IL-6–deficient mice. IL-6 has been suggested to contribute indirectly to the activation of endothelial cells, which lack the IL-6 receptor (IL-6R). Thus, expression of ICAM-1, VCAM-1, and E-selectin on endothelial cells is mediated by transactivation mediated by soluble IL-6R released from stimulated neutrophils.26 Reduced expression of adhesion molecules was also observed in IL-6–deficient mice resisting experimental allergic encephalomyelitis.13 Interestingly, triggering of a TNFR1-dependent pathway by staphylococcus enterotoxin B treatment restored adhesion molecule expression and overcame disease resistance of IL-6–deficient mice in the experimental allergic encephalomyelitis model.13 Because TNF-{alpha}
is a critical factor for the development of Coxsackie virus–induced myocarditis21 and TNFR1 is crucial for autoimmune myocarditis,3 we reasoned that impaired TNF-{alpha}\]+oyz, 百拇医药
production may contribute to myocarditis resistance of IL-6-/- mice. However, TNF-{alpha}\]+oyz, 百拇医药
(Table 1) treatment did not render IL-6–deficient mice susceptible. Thus, impaired production of TNF-{alpha}\]+oyz, 百拇医药
and expression of adhesion molecules seem not to be responsible for protection from myocarditis in IL-6–deficient mice.\]+oyz, 百拇医药
IL-6 is required for efficient antibody responses after infection with viruses27 and after immunization with antigen.23 In keeping with these results, we found that IL-6–deficient mice showed reduced levels of autoantibodies. Although autoimmune myocarditis is associated with autoantibody formation, B cells were shown to be dispensable for disease development after immunization of BALB/c mice with cardiac myosin whole protein.28 Nevertheless, it remained possible that B cells play an important role after immunization with the myosin peptide used in our immunization regimen, because of their higher density of MHC class II myosin peptide complexes. This possibility was excluded by showing that disease severity and mortality did not differ between B cell–deficient (µMT) mice and IL-6+/+ mice after immunization. This finding does not preclude a role of autoantibodies in a delayed cardiac dysfunction, in human cardiomyopathy, or in other mouse models of myocarditis, including viral myocarditis or autoimmune myocarditis in DBA/2 mice, which have been shown to develop disease after transfer of autoantibodies.29,30 Nevertheless, our data suggest that reduced IgG autoantibody production is not responsible for resistance to autoimmune myocarditis in IL-6–deficient mice on a BALB/c background.
Autoimmune myocarditis is dependent on CD4+ T cells. We found that the proliferation of autoreactive CD4+ T cells was impaired in IL-6–deficient mice. Furthermore, IL-6-/- CD4+ T cells, in contrast to IL-6+/+ CD4+ T cells, failed to mediate disease on adoptive transfer. Defective T cell responses in IL-6–deficient mice have also been described in other models of autoimmune disease. In some reports, reduced CD4+ T cell proliferation was associated with a reduction of both IL-4 and IFN- responses,15 whereas other studies demonstrated a shifted Th1:Th2 ratio with decreased IFN- and increased IL-4 production in the absence of IL-6. The Th1/Th2 paradigm is still widely used to explain autoimmunity with IFN- promoting and IL-4 protecting against disease. However, it has been shown previously that autoimmune myocarditis is exacerbated in IFN-–deficient mice,4,6 whereas disease prevalence at the peak of the response is reduced in IL-4R–deficient mice with an abrogated IL-4 and IL-13 pathway4 or on injection of anti–IL-4 monoclonal antibody.31 IL-6–deficient mice display a slightly increased IL-4 to IFN- ratio, which, considering the phenotype of the respective knockouts, cannot explain resistance to myocarditis. Therefore, our data suggest that IL-6 is involved in the activation or maintenance of autoimmune CD4+ T cells independent of Th1/Th2 polarization. Interestingly, in experimental colitis induced by adoptive transfer of congenic CD62L+CD45RBhigh CD4+ T cells into SCID (severe combined immunodeficiency) mice, neutralization of IL-6 resulted in apoptosis of lamina propria cells and reduced disease severity.12 Our findings of reduced proliferation do not exclude the possibility of enhanced apoptosis of myosin-specific IL-6-/- CD4+ T cells.
We have previously demonstrated that impaired complement C3 production is responsible for defective germinal center development and B-cell responses in immunized IL-6–deficient mice.23 Moreover, we have reported that C3 is essential for the activation and recruitment of both CD4+ and CD8+ T cells after pulmonary virus infection,32 which is also defective in IL-6–deficient mice (N. Schmitz et al, unpublished data, 2001). Consistent with these data, we describe here that IL-6–deficient mice fail to upregulate C3 production associated with impaired CD4+ T cell responses after immunization with Myhc-{alpha}at, 百拇医药
614-634. Recently, autoimmune myocarditis has been shown to depend on C3 and the complement receptors CD21 and CD35.22 Depletion of C3 during the inductive phase but not during the progression of inflammation (effector phase) prevented disease. Furthermore, the absence of the complement receptors CD21 (CR2) and CD35 (CR1) prevented myocarditis. Protection from myocarditis in C3-depleted and CR1/2-deficient mice was associated with reduced production of proinflammatory cytokines. In addition, CD4+ T cells from CR1/2-deficient mice were less responsive to antigen activation, as indicated by reduced expression of CD25 and CD69.22 Thus, the protection from myocarditis observed for IL-6–deficient and C3-deficient mice may be related. We suggest that impaired generation of complement is a key factor for disease prevention in the absence of IL-6. Despite the fact that complement levels do not increase in IL-6–deficient mice after immunization, baseline concentrations in the serum were in the normal range but seem insufficient to activate CD4+ T cells and autoimmune disease. Thus, it seems that IL-6 regulates C3 production locally in the microenvironment of lymphatic tissue,23 where CD4+ T cells interact with B cells and antigen-presenting cells.
In conclusion, we provide genetic evidence that IL-6 is crucial for the development of autoimmune heart disease. IL-6 bridges innate and adaptive immune responses by regulation of complement C3. We suggest that elevated IL-6 levels in patients with cardiomyopathy or after myocardial infarction are not merely prognostic surrogate markers but contribute to cardiac damage by triggering pathways resulting in autoreactivity. Therefore, blocking the IL-6 pathway may be beneficial in the treatment of inflammatory heart disease.'e4-, 百拇医药
Acknowledgments'e4-, 百拇医药
This work was supported by the Swiss Heart Foundation and by the Helmut Horten Foundation, Lugano, Switzerland.'e4-, 百拇医药
Received July 23, 2002; revision received September 17, 2002; accepted September 18, 2002.'e4-, 百拇医药
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