Cytolytic cross-reactive antibodies directed against the cardiac membrane and viral proteins in coxsackievirus B3 and B4 myocarditis. Characterization and pathogenetic relevance

Abstract

Background: Coxsackievirus B3 and B4 (CVB) myocarditis was assessed by a more than twofold change in titer of the microneutralization tests against enteroviruses within 3 weeks in all patients, by an endomyocardial biopsy indicative of active myocarditis in eight cases, and by pericardial effusion and acute cardiomegaly in two patients. In all endomyocardial biopsies, immunoglobulin binding to the sarcolemma and to the interstitial tissue was demonstrated irrespective of an infiltrate being at the same focus or not. IgG binding was found in nine, IgM and IgA in seven, C3 in C1q in three, and C5b9 in three of 10 patients. In addition, circulating antimyolemmal antibodies (AMLAs) were demonstrated regularly.

Methods and results: In this study, for the first time adult human myocytes isolated from atrial appendages during open-heart surgery were used as antigen in the indirect immunofluorescence test: nine of 10 sera of patients with CVB myocarditis demonstrated AMLAs of the homologous type in titers of 1:40-1:320, whereas eight of 10 reacted with rat myocytes (heterologous type) only. Circulating AMLAs fixed complement component C4 in the majority of cases. During the in vitro assay of antibody-mediated cytolysis with vital heart cells, fixation of components C3, C4 to the myolemma in all, of C1q in seven, and of the C3b9 complex in eight of 10 sera was demonstrated after addition of a fresh complement source, indicating the potential of a complement-mediated cytolysis being operative. In vitro cardiocytolysis of isolated adult rat heart cells is present in the untreated sera of patients with enteroviral myocarditis and is abolished after adsorption of sera with CVB and with isolated rat heart cells. This indicates functional cross-reactivity of the antimembrane antibodies. To analyze further the cross-reactive epitopes, sodium dodecyl sulfate gel electrophoresis of human and rat sarcolemma and consecutive immunoblots were performed. Cross-reactivity between viral (CVB) and sarcolemmal epitopes could be demonstrated to bands of 220 kd in 10%, 110 kd in 50%, 48 kd in 40%, 35 kd in 40%, and 31 and 28 kd in 30% each. Cardiospecific non-cross-reactive epitopes for antisarcolemmal antibodies or AMLAs were membrane proteins of 90 kd and 78 kd in 50%, 72 kd in 90%, 67 kd in 40%, and 45 kd in 50%. Virus-specific antibody binding sites for sera included proteins of 33 and 34 kd.

Conclusions: Western blot analysis of sera incubated with cardiac membranes or enteroviral proteins demonstrated that the antibodies are directed to defined epitopes of the sarcolemma. Some antibodies were cross-reactive to enteroviral proteins, indicating that enteroviral infection may be the etiological trigger of an autoreactive myocarditis. The cytolytic property of the patients' sera in vitro suggests in addition that humoral autoreactivity and antigenic mimicry are major pathogenetic principles operative in human enteroviral myocarditis and its sequelae.