Search

There are two major haplotypes of signal lymphocytic activation molecule (Slam) in inbred mouse strains, with the Slam haplotype 1 expressed in C57Bl/6 mice and the Slam haplotype 2 expressed in most other commonly used inbred strains, including 129 mice. Because signaling through Slam family receptors can affect innate immunity [natural killer T cell (NKT) and γ-δ T-cell receptor], and innate immunity can determine susceptibility to coxsackievirus B3 (CVB3) infection, the present study evaluated the response of C57Bl/6 and congenic B6.129c1 mice (expressing the 129-derived Slam locus) to CVB3. CVB3-infected C57Bl/6 male mice developed increased myocarditis but reduced hepatic injury compared with infected B6.129c1 mice. C57Bl/6 mice also had increased γδ(+) and CD8(+)interferon-γ(+) cells but decreased numbers of NKT (T-cell receptor β chain + mCD1d tetramer(+)) and CD4(+)FoxP3(+) cells compared with B6.129c1 mice. C57Bl/6 mice were infected with CVB3 and treated with either α-galactosylceramide, an NKT cell-specific ligand, or vehicle (dimethyl sulfoxide/PBS). Mice treated with α-galactosylceramide showed significantly reduced myocarditis. Liver injuries, as determined by alanine aminotransferase levels in plasma, were increased significantly, confirming that NKT cells are protective for myocarditis but pathogenic in the liver., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Ongoing coxsackievirus B3 (CVB3) myocarditis is characterized by persistence of viral RNA and chronic inflammation primarily mediated by macrophages and T cells. Activated macrophages produce anti-viral effector molecules comprising reactive nitrogen intermediates; however, reactive nitrogen intermediates also contribute to host tissue damage. Controlled activation of macrophages depends on interferon (IFN)-gamma and interleukin (IL)-10. To evaluate mechanisms involved in CVB3-induced pathogenesis of myocarditis, we determined the relationship of inducible nitric-oxide synthase (iNOS) mRNA expression with IFN-gamma and IL-10 secretion during CVB3 infection in different mouse strains. We found in susceptible A.BY/SnJ mice that develop ongoing myocarditis, a low and delayed IFN-gamma secretion and highly diminished IL-10 production compared with resistant C57BL/6 mice. Consequently, iNOS mRNA synthesis was delayed but clearly prolonged in susceptible mice. IL-10 gene-deficient mice confirmed the regulatory role of IL-10 in the outcome of CVB3 myocarditis. These mice did not establish a persistent cardiac infection and revealed IFN-gamma secretion kinetics similar to resistant mice but showed a slightly elongated cardiac iNOS mRNA expression resulting in extended myocarditis. We conclude that coordinated secretion of IFN-gamma and IL-10 is crucial for the effective resolution of CVB3 myocarditis. Moreover, lack of regulatory IL-10 leads to uncontrolled iNOS mRNA production, thus contributing to ongoing myocardial injury.

To gain insight into the strategies of the immune system to confer resistance against the development of chronic coxsackievirus B3 (CVB3) myocarditis we compared the course of the disease in C57BL/6 mice, beta2-microglobulin knockout (beta2m(-/-)) mice, and perforin-deficient (perforin(-/-)) mice. We found that perforin(-/-) mice as well as immunocompetent C57BL/6 mice reveal a resistant phenotype with complete elimination of the virus from the heart in the course of acute myocarditis. In contrast, myocardial CVB3 infection of beta2m(-/-) mice was characterized by a significantly higher virus load associated with a fulminant acute inflammatory response and, as a consequence of virus persistence, by the development of chronic myocarditis. Interferon-gamma secretion of stimulated spleen cells was found to be significantly delayed in beta2m(-/-) mice compared to perforin(-/-) mice and C57BL/6 control mice during acute myocarditis. In addition, generation of virus-specific IgG and neutralizing antibodies were found to be significantly decreased in beta2m(-/-) mice during acute infection. From these results we conclude that protection against the development of chronic myocarditis strongly depends on the expression of beta2m, influencing the catabolism of IgG as well as the production of protective cytokines, such as interferon-gamma. Moreover, CVB3-induced cardiac injury and prevention of chronic myocarditis was found to be unrelated to perforin-mediated cytotoxicity in our model system.

Viral myocarditis is remarkably common, being detected in approximately 1% of unselected asymptomatic individuals. Many cases are attributable to enteroviral infection, and in particular to coxsackievirus B3. The underlying pathogenesis is controversial, but most studies admit the important immunopathological role of infiltrating CD8+ (cytotoxic) T lymphocytes (CTLs). We have previously shown that CTLs play conflicting roles in coxsackievirus B (CVB) myocarditis; they assist in controlling virus replication, but also are instrumental in causing the extensive inflammatory disease, which often results in severe myocardial scarring. A role for perforin, the major CTL cytolytic protein, in CVB myocarditis has been suggested, but never proven. In the present study we use perforin knockout (PKO) mice to show that perforin plays a major role in CVB infection; in broad terms, perforin is important in immunopathology, but not in CVB clearance. For example, PKO mice are better able to withstand a normally lethal dose of CVB (100% survival of PKO mice compared with 90% death in +/+ littermates). In addition, PKO mice given a nonlethal dose of CVB develop only a mild myocarditis, whereas their perforin+ littermates have extensive myocardial lesions. The myocarditis in PKO mice resolves more quickly, and these mice show minimal histological sequelae; in contrast, late in disease the perforin+ mice develop severe myocardial fibrosis. PKO mice, despite lacking this major CTL effector function, can control the infection and eradicate the virus; growth kinetics and peak CVB titers are indistinguishable in PKO and perforin+ mice. Therefore, the immunopathological and antiviral effects of CTLs can be uncoupled by ablation of perforin; this offers a promising target for therapy of myocarditis. Furthermore, we evaluate the possible roles of apoptosis, and of chemokine expression, in CVB infection. In perforin+ mice, apoptotic cells are detected within the inflammatory infiltrate, whereas in their PKO counterparts, apoptotic myocyte nuclei are seen. Chemokine expression in both PKO and perforin+ mice precedes and parallels the course of myocarditis. Several chemokines are detectable earlier in PKO mice than in perforin+ mice, but PKO mice show reduced peak levels, and chemokine expression decays sooner. In particular, MIP-1alpha expression is barely detectable at any time point in PKO mice, but it is readily identified in perforin+ animals, peaking just before the time of maximal myocarditis; this is particularly interesting, given that MIP-1alpha knockout mice are resistant to CVB myocarditis, but remain able to control viral infection. Thus, the chemokine pathway offers a second route of intervention to diminish myocarditis and its sequelae, while permitting the host to eradicate the virus.

Group B Coxsackieviruses are a common cause of myocarditis. To detect the viral genome and its localization in the myocardium, we examined C3H/He mice with Coxsackievirus B3 (CVB3) myocarditis on days 5, 8, and 14 after inoculation by the reverse transcriptase polymerase chain reaction and by in situ hybridization. Sense and antisense CVB3 RNA were detected in the myocardium of all mice up to day 14 by reverse transcriptase polymerase chain reaction. Light microscopic in situ hybridization with a cDNA probe for CVB3 showed clusters of positive signals in the areas of myocardial necrosis and cell infiltration. With electron microscopic in situ hybridization, CVB3 RNA was detected in the cytoplasm of cardiocytes, between the myofibrils, near the mitochondria, and in tubular or vesicular structures. Viral RNA was also detected in necrotic debris, in the cytoplasm of macrophages, and in the cytoplasm of interstitial fibroblasts. These findings suggest that CVB3 RNA is replicated in the cytoplasm of cardiocytes, transferred into tubular or vesicular structures, released into the interstitium, and phagocytosed by macrophages. Some positive signals were also detected in the cytoplasm of cardiocytes showing close contact with infiltrating lymphocytes, suggesting that the lymphocytes recognized virus-infected cardiocytes and caused cell-mediated immune cardiocyte damage.

The function of apolipoprotein J (apoJ) is unknown, but it has been hypothesized to be cytoprotective. In the normal heart, abundant apoJ mRNA and protein are expressed in atrial myocytes; no expression is detected in ventricular myocytes. To provide clues about the role of apoJ in the heart, the response of apoJ to heart disease, including three models of myocarditis and two models of in vivo pressure overload hypertrophy, were examined. In the disease model studied extensively, myosin-induced myocarditis, in situ hybridization detected induction of apoJ mRNA in ventricular myocytes immediately before histological evidence of injury. ApoJ message in ventricular myocytes reached high levels as myocarditis became more severe. Evidence of early apoJ induction, before inflammation and injury, also occurred in viral myocarditis. ApoJ mRNA was not present in the inflammatory or interstitial cells during myocarditis. In areas of severe inflammation and myocardial fiber degeneration, apoJ showed a gradient of expression, with highest levels in myocytes immediately surrounding the lesion and diminishing with increasing distance. ApoJ protein also accumulated in myocytes at the interface between degenerated myocardial tissue and the surrounding cardiac tissue. During cardiac hypertrophy that occurred without associated inflammation or cell damage, ventricular apoJ mRNA was not detected. When ischemic damage accompanied hypertrophy, apoJ was induced in the ventricular myocytes near the lesion borders. The correlation of apoJ induction with ventricular tissue damage, but not hypertrophy, suggests that apoJ is a repair response protein. We propose that apoJ functions to limit tissue injury and/or promote tissue remodeling.

In adolescent CD-1 male mice inoculated with a myocarditic coxsackievirus B3 (CVB3m) acute focal lesions containing necrotic myocytes, infiltrating mononuclear cells, and fibroblasts develop. With the use of an in situ immune autoradiographic method with rat monoclonal antibodies (MAb) and an 35S-labeled antibody, viral antigens were detected outside of lesions. Macrophages, T lymphocytes, and natural killer (NK) cells were identified within myocarditic lesions during the acute phase of the disease. Macrophages detected by anti-Mac-1 MAb were in focal areas within myocarditic lesions on Days 4-7 after inoculation. T lymphocytes were detected in myocarditic lesions on Days 4-10, with MAb to Thy-1 and Lyt-1 antigens showing diffuse reaction patterns, suggesting random distribution of these cells in lesions. Focal areas of reactivity were detected with MAbs to L3T4 and Lyt-2 antigens, suggesting clusters of helper and cytotoxic/suppressor T lymphocytes, respectively. NK cells were presumptively detected by asialo GM1 surface marker in lesions at all times. The presence of activated NK cells in lesions was confirmed by assay of mechanically dissociated heart tissues on Day 8. These data describe the temporal sequence and identity of leukocytes entering into CVB3-induced focal myocarditic lesions during the acute phase of disease in CD-1 mice.