Identification of cardiac myosin peptides capable of inducing autoimmune myocarditis in BALB/c mice.

Immunization with cardiac myosin induces T cell-mediated myocarditis in genetically predisposed mice and serves as a model for autoimmune heart disease. This study was undertaken to identify pathogenic epitopes on the myosin molecule. Our approach was based on the comparison of the pathogenicity between cardiac (alpha-)myosin and soleus muscle (beta-)myosin. We show that alpha-myosin is the immunodominant isoform and induces myocarditis at high severity and prevalence whereas beta-myosin induces little disease. Therefore the immunodominant epitopes of alpha-myosin must reside in regions of different amino acid sequence between alpha- and beta-myosin isoforms. Cardiac myosin peptides corresponding to these regions of difference were synthesized and tested for their ability to induce inflammatory heart disease. Three pathogenic peptides were identified. One peptide that is located in the head portion of the molecule induced severe myocarditis, whereas two others that reside in the rod portion possessed only minor pathogenicity. The identification of pathogenic epitopes on the cardiac myosin molecule will allow detailed studies on the recognition of this antigen by the immune system and might be used to downmodulate ongoing heart disease.

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.

Cardiac myosin-induced myocarditis: target recognition by autoreactive T cells requires prior activation of cardiac interstitial cells.

Immunization with cardiac myosin causes T cell-mediated myocarditis in genetically predisposed mice and serves as a model for autoimmune heart disease. The normal heart is not susceptible to T cells autoreactive with cardiac myosin; therefore, we investigated the conditions that are required to facilitate recognition of the target tissue. A.SW mice were immunized with cardiac myosin on Days 0 and 7. Major histocompatibility antigen (MHC Ag) and intercellular adhesion molecule-1 (ICAM-1) expression in the heart tissue was investigated by immunohistochemical techniques shortly before disease onset (ie, on Day 9). At this time point, cardiac interstitial cells expressing class II but not class I MHC Ag were significantly increased. In addition, endothelial ICAM-1 expression was strongly up-regulated. Myofibers did not show expression of these markers, and T cells were virtually absent. Because lipopolysaccaride (LPS) induced a similar distribution of class II MHC Ag and ICAM-1 in the myocardial tissue and because these molecules could be crucial to disease onset, we determined whether treatment with this immunomodulator renders the heart susceptible to passively transferred myosin-reactive T cells. We found that concanavalin A-activated spleen cells from myosin-immunized donors induced myocarditis in LPS-primed recipients, whereas normal mice were resistant to the injection of such cells. Increased class II MHC Ag expression after LPS-treatment was mediated by TNF because LPS-primed mice genetically lacking the TNF receptor failed to increase class II MHC Ag expression in the heart tissue. In summary, these results suggest that in cardiac myosin-induced myocarditis, expression of interstitial class II MHC Ag and/or endothelial ICAM-1 is a prerequisite for target organ recognition by autoreactive T cells.