Variation in susceptibility of Balb/c mice to coxsackievirus group B type 3-induced myocarditis with age

The severity of cardiac lesions in coxsackievirus group B, type 3 (CVB3)-infected Balb/c mice depends upon both the age and the sex of the animal at the time of viral inoculation. Suckling animals (1-3 weeks old) of either sex develop few cardiac lesions. Thereafter, males rapidly demonstrate increasing disease susceptibility peaking at 16-18 weeks old and then decreasing susceptibility from 20 to 40 weeks of age. Female susceptibility increases much more gradually and myocarditis in this sex never reaches maximal levels as seen in males. Increased susceptibility correlates with virus concentrations in the heart and anti-CVB3 titers in the serum. Cardiac injury is dependent on functional T lymphocytes since treatment of the animals with rabbit anti-mouse thymocyte serum abrogates inflammation and myocyte necrosis. Sex-associated steroid hormones influence both virus concentrations and immune responses in mice and are probably responsible for variations in disease susceptibility throughout the animal's life.     

Aggravation of coxsackievirus, group B, type 3-induced myocarditis and increase in cellular immunity to myocyte antigens in pregnant Balb/c mice and animals treated with progesterone

Male Balb/c mice inoculated with a heart-adapted variant of coxsackievirus, group B, type 3 (CVB3M) develop severe myocarditis characterized by extensive focal lesions of inflammatory cells and necrosis of the myocardium. Females generally develop minimal myocarditis except when infected during the first and third trimesters of pregnancy. Enhanced myocarditis is usually accompanied by elevations in virus concentrations in the heart, virus-specific antibody titers, and lymphocyte mediated cytolytic activity to both uninfected and CVB3M-infected myocytes in vitro. As previously shown in males, T-lymphocyte-depleted pregnant female mice inoculated with the virus do not develop significant myocarditis indicating that immune rather than virus-mediated myocyte damage is important in myocarditis. Progesterone increases during gestation reaching maximum concentrations during the third week when heart disease is most severe. Administration of progesterone to castrated male and female mice prior to virus inoculation resulted in increased virus concentrations, cellular and humoral CVB3M-specific immunity, and myocarditis. Two hypotheses for exacerbation of the disease with elevated progesterone concentrations have been postulated: the hormone either indirectly increases cellular immune responses by enhancing virus replication, or independently enhances both T-cell responses and virus replication.     

Involvement of natural killer cells in coxsackievirus B3-induced murine myocarditis

The role of natural killer cells in the temporal development of coxsackievirus B3-induced myocarditis in adolescent CD-1 male mice was examined. Inoculation of purified CVB3m induced maximum NK cell activity in the splenic populations at 3 days postinoculation (p.i.) as assessed by lysis of YAC-1 cells; maximum virus titers in heart tissues were also found at day 3 p.i. Mice depleted of NK cells after injection of anti-asialo GM1 antiserum i.v. had decreased NK cell activity, increased CVB3m titers in heart tissues, and exacerbated myocarditis. Although lesion number was not increased in heart tissues of the latter mice, lesions in these mice exhibited increased myocyte degeneration and dystrophic calcification above that found in lesions of mice inoculated with CVB3m only. No alteration in interferon titers were observed in CVB3m-infected mice treated with anti-asialo GM1 antiserum as compared with normal CVB3m-infected mice. Measurements of splenic NK cell activity in mice inoculated with doses of 10(2) to 10(8) PFU of CVB3m per mouse or UV-irradiated virus suggest that replication of CVB3m is required for NK cell activation. An amyocarditic variant of CVB3m (ts5R) was shown to replicate in heart tissues and to elicit NK cell activity comparable to that elicited by CVB3m. Therefore, the data suggest that NK cell activation depends on virus replication and that these cells provide some protection against CVB3m-induced myocarditis by limiting virus replication in heart tissues.     

Demonstration of suppressor cells in coxsackievirus group B, type 3 infected female Balb/c mice which prevent myocarditis

Coxsackievirus group B type 3 (CVB3) induces myocarditis in male Balb/c mice but produces little cardiac injury in females. Males develop cytolytic T lymphocytes (CTL) reactive to heart antigens which primarily cause the inflammation and cardiac injury observed in the disease. Infected female mice lack this CTL response because they rapidly produce suppressor cells inhibiting both cellular immunity and cardiac inflammation. Four lines of evidence demonstrate suppressor cells in females. First, females develop myocarditis when treated with low-dose cyclophosphamide under conditions known to preferentially eliminate suppressor cells but not other immune cells. Second, lymphocytes obtained from females at various times after infection prevent myocarditis when adoptively transferred into CVB3-infected males. Virus concentrations in the hearts of males receiving immune female cells and control males were equivalent. Thus protection did not result from accelerated virus elimination in recipient males. Third, CTL from CVB3 infected male mice could induce myocarditis in infected T-lymphocyte depleted but not in intact females suggesting the presence of an inhibitory T cell in the intact animals. Finally, male lymphocytes cultured on heart cell monolayers for 5 days generate significant cytolytic activity to myocyte targets. CTL generation could be inhibited by co-culture of the male cells with immune female lymphocytes. Nonimmune female cells were not inhibitory.     

Carvedilol has stronger anti-inflammation and anti-virus effects than metoprolol in murine model with coxsackievirus B3-induced viral myocarditis

Aims

This study aims to compare the effects of carvedilol and metoprolol in alleviating viral myocarditis (VMC) induced by coxsackievirus B3 (CVB3) in mice.

Methods

A total of 116 Balb/c mice were included in this study. Ninety-six mice were inoculated intraperitoneally with CVB3 to induce VMC. The CVB3 inoculated mice were evenly divided into myocarditis group (n = 32), carvedilol group (n = 32) and metoprolol group (n = 32). Twenty mice (control group) were inoculated intraperitoneally with normal saline. Hematoxylin and eosin staining and histopathologic scoring were used to investigate the effects of carvedilol and metoprolol on myocardial histopathologic changes on days 3 and 5. In addition, serum cTn-I levels, cytokine levels and virus titers were determined using chemiluminescence immunoassay, enzyme-linked immunosorbent assay and plaque assay, respectively, on days 3 and 5. Finally, the levels of phosphorylated p38MAPK were studied using immunohistochemical staining and Western blotting on day 5.

Results

Carvedilol had a stronger effect than metoprolol in reducing the pathological scores of VMC induced by CVB3. Both carvedilol and metoprolol reduced the levels of cTn-I, but the effect of carvedilol was stronger. Carvedilol and metoprolol decreased the levels of myocardial pro-inflammatory cytokines and increased the expression of anti-inflammatory cytokine, with the effects of carvedilol being stronger than those of metoprolol. Carvedilol had a stronger effect in reducing myocardial virus concentration compared with metoprolol. Carvedilol was stronger than metoprolol in decreasing the levels of myocardial phosphorylated p38MAPK.

Conclusions

In conclusion, carvedilol was more potent than metoprolol in ameliorating myocardial lesions in VMC, probably due to its stronger modulation of the balance between pro- and anti-inflammatory cytokines by inhibiting the activation of p38MAPK pathway through β1- and β2-adrenoreceptors.     

Role of CD1d in coxsackievirus B3-induced myocarditis

The myocarditic (H3) variant of Coxsackievirus B3 (CVB3) causes severe myocarditis in BALB/c mice and BALB/c mice lacking the invariant J alpha 281 gene, but minimal disease in BALB/c CD1d(-/-) animals. This indicates that CD1d expression is important in this disease but does not involve the invariant NKT cell often associated with CD1d-restricted immunity. The H3 variant of the virus increases CD1d expression in vitro in neonatal cardiac myocytes whereas a nonmyocarditic (H310A1) variant does not. V gamma 4(+) T cells show increased activation in both H3-infected BALB/c and J alpha 281(-/-) mice compared with CD1d(-/-) animals. The activated BALB/c V gamma 4(+) T cells from H3-infected mice kill H3-infected BALB/c myocytes and cytotoxicity is blocked with anti-CD1d but not with anti-MHC class I (K(d)/D(d)) or class II (IA/IE) mAbs. In contrast, H3 virus-infected CD1d(-/-) myocytes are not killed. These studies demonstrate that CD1d expression is essential for pathogenicity of CVB3-induced myocarditis, that CD1d expression is increased early after infection in vivo in CD1d(+) mice infected with the myocarditic but not with the nonmyocarditic CVB3 variant, and that V gamma 4(+) T cells, which are known to promote myocarditis susceptibility, appear to recognize CD1d expressed by CVB3-infected myocytes.     

Hormonal regulation of CD4(+) T-cell responses in coxsackievirus B3-induced myocarditis in mice

Coxsackievirus B3 infection causes significant cardiac inflammation in male, but not female, B1.Tg.Ealpha mice. This gender difference in disease susceptibility correlates with selective induction of CD4(+) Th1 (gamma interferon-positive) cell responses in animals with testosterone, whereas estradiol promotes preferential CD4(+) Th2 (interleukin-4 positive [IL-4(+)]) cell responses. Differences in immune deviation of CD4(+) T cells cannot be explained by variation in B7-1 or B7-2 expression. Infection significantly upregulated both molecules, but no differences were detected between estradiol- and testosterone-treated groups. Significantly increased numbers of activated (CD69(+)) T cells expressing the gammadelta T-cell receptor were found in male and testosterone-treated male and female mice. In vivo depletion of gammadelta+ cells by using monoclonal antibodies inhibited myocarditis and resulted in a shift from a Th1 to Th2 response phenotype. Taken together, our results indicate that testosterone promotes a CD4(+) Th1 cell response and myocarditis by promoting increased gammadelta+ cell activation.     

Protective effects of losartan in mice with chronic viral myocarditis induced by coxsackievirus B3

AimTo investigate whether losartan has protective effects in mice with chronic viral myocarditis induced by coxsackievirus B3 (CVB3).Main methodsThirty two male Balb/c mice were intraperitoneally injected with CVB3 (10 × TCID₅₀) to induce chronic viral myocarditis (CVM). Losartan at 12.5 mg/kg (n = 16) or normal saline (n = 16) were orally administered daily for 28 days to these mice. Uninfected mice (n = 6) were used as controls. On day 29, all mice underwent anesthesia and echocardiography prior to sacrifice. Serum IL-17, IL-4, IFN-γ and TNF-α levels were measured by enzyme-linked immunosorbent assay, and cardiac tissues were histologically examined after hematoxylin & eosin staining. In addition, the effect of losartan on the virus titers in primary cultured neonatal rat cardiomyocytes infected with CVB3 was measured on Hep-2 cells at 72 h post infection.Key findingsMice infected with CBV3 had significantly increased mortality, heart/body weight ratios, necrosis and inflammatory scores and decreased cardiac ejection fractions, compared with the controls (all P < 0.05). Losartan significantly decreased mortality from 40.0% to 12.5%, heart/body weight ratios from 7.08 ± 2.17 to 4.15 ± 0.99, and necrosis and inflammatory scores from 3.33 ± 0.50 to 2.50 ± 0.65 (all P < 0.05), and increased ejection fractions from 55.80 ± 9.25 to 72.31 ± 12.15 (P < 0.05). Losartan significantly enhanced IL-4, and decreased IFN-γ, TNF-α and IL-17 (all P < 0.05). In the in vitro experiment, losartan had no influence on virus titers.SignificanceLosartan protects mice against CVB3-induced CVM, most likely through upregulating Th2 responses, and down-regulating Th1 and Th17 responses.     

Differential effects of NF-{kappa}B and p38 MAPK inhibitors and combinations thereof on TNF-{alpha}- and IL-1{beta}-induced proinflammatory status of endothelial cells in vitro

Endothelial cells actively participate in inflammatory events by regulating leukocyte recruitment via the expression of inflammatory genes such as E-selectin, VCAM-1, ICAM-1, IL-6, IL-8, and cyclooxygenase (COX)-2. In this study we showed by real-time RT-PCR that activation of human umbilical vein endothelial cells (HUVEC) by TNF- and IL-1 differentially affected the expression of these inflammatory genes. Combined treatment with TNF- and IL-1 resulted in nonadditive, additive, and even synergistic induction of expression of VCAM-1, IL-8, and IL-6, respectively. Overexpression of dominant-negative inhibitor B protein blocking NF-B signaling confirmed a major role of this pathway in controlling both TNF-- and IL-1-induced expression of most of the genes studied. Although dexamethasone exerted limited effects at 1 µM, the thioredoxin inhibitor MOL-294, which regulates the redox state of NF-B, mainly inhibited adhesion molecule expression. Its most pronounced effect was seen on VCAM-1 mRNA levels, especially in IL-1-activated endothelium. One micromolar RWJ-67657, an inhibitor of p38 MAPK activity, diminished TNF-- and IL-1-induced expression of IL-6, IL-8, and E-selectin but had little effect on VCAM-1 and ICAM-1. Combined treatment of HUVEC with MOL-294 and RWJ-67657 resulted in significant blocking of the expression of E-selectin, IL-6, IL-8, and COX-2. The inhibitory effects were much stronger than those observed with single drug treatment. Application of combinations of drugs that affect multiple targets in activated endothelial cells may therefore be considered as a potential new therapeutic strategy to inhibit inflammatory disease activity. inflammatory gene expression; anti-inflammatory drugs; pharmacology; combination treatment     

Estimation of ^{3}J_{HN\hbox{-}H\alpha} and ^{3}J_{H\alpha\hbox{-}H\beta} coupling constants from heteronuclear TOCSY spectra

(3)J proton-proton coupling constants bear information on the intervening dihedral angles. Methods have been developed to derive this information from NMR spectra of proteins. Using series expansion of the time dependent density matrix, and exploiting the simple topology of amino acid spin-systems, formulae for estimation of (3)J(HN-Halpha) and (3)J(Halpha-Hbeta) from HSQC-TOCSY spectra are derived. The results obtained on a protein entailing both alpha-helix and beta-sheet secondary structure elements agree very well with J-coupling constants computed from the X-ray structure. The method compares well with existing methods and requires only 2D spectra which would be typically otherwise recorded for structural studies.     

The role of CD8+ T lymphocytes in coxsackievirus B3-induced myocarditis.

Coxsackievirus infections have previously been shown to cause acute or chronic myocarditis in humans, and several mouse models have been established to study the pathology of this disease. Myocardial injury may result from direct viral effects and/or may be immune mediated. To determine the relative roles of these processes in pathogenesis, we have compared coxsackievirus B3 (CVB3) infections of normal and immuno-compromised transgenic knockout (ko) mice. CVB3 was able to infect all strains used (C57BL/6, CD4ko, and beta-microglobulin ko [beta 2Mko]), and following intraperitoneal injection, two disease processes could be distinguished. First, the virus caused early (3 to 7 days postinfection) death in a viral dose-dependent manner. Immunocompetent C57BL/6 mice were highly susceptible (50% lethal dose = 70 PFU), while immunodeficient transgenic ko mice were less susceptible, showing 10- and 180-fold increases in the 50% lethal dose (for CD4ko and beta 2Mko mice, respectively). Second, a histologic examination of surviving CD4ko mice at 7 days postinfection revealed severe myocarditis; the inflammatory infiltrate comprised 40 to 50% macrophages, 30 to 40% NK cells, and 10 to 20% CD8+ T lymphocytes. The infiltration resolved over the following 2 to 3 weeks, with resultant myocardial fibrosis. In vivo depletion of CD8+ T lymphocytes from these CD4ko mice led to a marked reduction in myocarditis and an increase in myocardial virus titers. beta 2Mko mice, which lack antiviral CD8+ T cells, are much less susceptible to early death and to the development of myocarditis. We conclude that our data support a strong immunopathologic component in CVB3-induced disease and implicate both CD4+ and CD8+ T cells. Compared with immunocompetent animals, (i) mice lacking CD4+ T cells (CD4ko) were more resistant to virus challenge, and (ii) mice lacking CD8+ T cells (beta 2Mko and in vivo-depleted CD4ko) showed enhanced survival and a reduced incidence of the later myocarditis. Nevertheless, the picture is complex, since (iii) removal of the CD4+ component, while protecting against early death, greatly magnified the severity of myocarditis, and (iv) removal of the CD8+ cells from CD4ko mice, although protecting against early death and later myocarditis, led to markedly increased virus titers in the heart. These data underscore the complex balance between the costs and benefits of effective antiviral immune responses.     

Antiviral effects of sophoridine against coxsackievirus B3 and its pharmacokinetics in rats

Coxsackievirus B3 (CVB3) is a major pathogen for acute and chronic viral myocarditis. The aim of this study was to investigate the antiviral effects of sophoridine, an alkaloid extracted from Chinese medicinal herb, Sophora flavescens, against CVB3, and the underlying pharmacokinetics. First, we determined the antiviral effects of sophoridine against CVB3 in in vitro (primarily cultured myocardial cells), in vivo (BALB/c mice) and serum pharmacological experiments. Then, we determined the pharmacokinetic behavior in serum samples of SD rats after oral administration by HPLC. Finally, we determined the effects of sophoridine on the production of cytokines in a murine viral myocarditis model by measuring mRNA expression of some important cytokines in hearts of infected BALB/c mice by RT-PCR. We found that sophoridine exhibited obvious antiviral effects both in vitro and in vivo, and serum samples obtained from rats with oral administration of sophoridine reduced the virus titers in infected myocardial cells. The serum concentration profile correlated closely with antiviral activity profile. Moreover, sophoridine significantly enhanced mRNA expression of IL-10 and IFN-gamma, but decreased TNF-alpha mRNA expression. In conclusion, sophoridine possesses antiviral activities against CVB3, by regulating cytokine expression, and it is likely that sophoridine itself, not its metabolites, is mainly responsible for the antiviral activities. Therefore, sophoridine may represent a potential therapeutic agent for viral myocarditis.     

Blockade of interleukin-17A protects against coxsackievirus B3-induced myocarditis by increasing COX-2/PGE2 production in the heart

The Th17/interleukin (IL)-17 axis controls inflammation and might be important in the pathogenesis of experimental autoimmune myocarditis (EAM) and other autoimmune diseases. However, the mechanism underlying the increased Th17 cell response in coxsackievirus-induced myocarditis remains unclear. This study aimed to elucidate the regulatory mechanisms affected by blocking IL-17A responses in acute virus-induced myocarditis (AVMC) mice. The results showed that IL-17A and COX-2 proteins were significantly increased in the cardiac tissue of acute myocarditis, as were Th17 cells in the spleen. Using anti-mouse IL-17Ab to block IL-17A on day 7 of the viral myocarditis led to decreased expressions of cardiac tumor-necrosis factor alpha, IL-17A and transforming growth factor beta in AVMC mice compared to isotype control mice. COX-2 and prostaglandin E2 proteins were dramatically elevated, followed by marked reductions in CVB3 replication and myocardial injury. These results hint that the Th17/IL-17 axis is intimately associated with viral replication in acute myocarditis via induction of COX-2 and prostaglandin E2.     

Cardiac injury in myocarditis induced by Coxsackievirus group B, type 3 in Balb/c mice is mediated by Lyt 2 + cytolytic lymphocytes

Male Balb/c mice inoculated with a heart-adapted variant of Coxsackievirus, group B, type 3 (CVB3) develop severe myocarditis 7 days later. The lesions are characterized by mononuclear cell inflammation and myocyte necrosis. Infected T-lymphocyte-deficient mice show either minimal or no cardiac injury, although virus concentrations in the hearts of T-cell-deficient and -sufficient animals are similar. Adoptive transfer of 2 X 10(6) CVB3 immune Thy 1+ cells into CVB3-infected T-cell-deficient mice effectively restored myocarditis to levels observed in intact animals. Similar reconstitution with immune Ig+ cells or serum resulted in only a minimal increase in cardiac injury. To determine whether T-lymphocyte-dependent humoral or cellular immunity was responsible for myocarditis. T lymphocytes were obtained from Balb/c mice 6 days after infection with CVB3, separated into Lyt 1+2- (helper) and Lyt 1-2+ (cytolytic/suppressor) cell populations, and 2 X 10(6) of the enriched helper and cytolytic cells were adoptively transfused into infected T-cell-deficient recipients. Animals receiving the immune Lyt2+ cells developed severe myocarditis, had cytolytic T lymphocytes to both CVB3-infected and uninfected myocytes, but lacked a detectable IgG antibody response. Recipients of the Lyt 1+ cells failed to develop either myocarditis or cytolytic T cells but had normal serum IgG antibody titers to the virus. These results demonstrate that cardiac myocarditis is the product of cellular immune mechanisms.     

Cytokine production by Vgamma(+)-T-cell subsets is an important factor determining CD4(+)-Th-cell phenotype and susceptibility of BALB/c mice to coxsackievirus B3-induced myocarditis

Two coxsackievirus B3 (CVB3) variants (H3 and H310A1) differ by a single amino acid mutation in the VP2 capsid protein. H3 induces severe myocarditis in BALB/c mice, but H310A1 is amyocarditic. Infection with H3, but not H310A1, preferentially activates Vgamma4 Vdelta4 cells, which are strongly positive for gamma interferon (IFN-gamma), whereas Vgamma1 Vdelta4 cells are increased in both H3 and H310A1 virus-infected animals. Depletion of Vgamma1(+) cells using monoclonal anti-Vgamma1 antibody enhanced myocarditis and CD4(+)-, IFN-gamma(+)-cell responses in both H3- and H310A1-infected mice yet decreased the CD4(+)-, IL-4(+)-cell response. Depleting Vgamma4(+) cells suppressed myocarditis and reduced CD4(+) IFN-gamma(+) cells but increased CD4(+) IL-4(+) T cells. The role of cytokine production by Vgamma1(+) and Vgamma4(+) T cells was investigated by adoptively transferring these cells isolated from H3-infected BALB/c Stat4 knockout (Stat4ko) (defective in IFN-gamma expression) or BALB/c Stat6ko (defective in IL-4 expression) mice into H3 virus-infected wild-type BALB/c recipients. Vgamma4 and Vgamma1(+) T cells from Stat4ko mice expressed IL-4 but no or minimal IFN-gamma, whereas these cell populations derived from Stat6ko mice expressed IFN-gamma but no IL-4. Stat4ko Vgamma1(+) cells (IL-4(+)) suppress myocarditis. Stat6ko Vgamma1(+) cells (IFN-gamma(+)) were not inhibitory. Stat6ko Vgamma4(+) cells (IFN-gamma(+)) significantly enhanced myocarditis. Stat4ko Vgamma4(+) cells (IL-4(+)) neither inhibited nor enhanced disease. These results show that distinct gammadelta-T-cell subsets control myocarditis susceptibility and bias the CD4(+)-Th-cell response. The cytokines produced by the Vgamma subpopulation have a significant influence on the CD4(+)-Th-cell phenotype.     

QiHong prevents death in coxsackievirus B3 induced murine myocarditis through inhibition of virus attachment and penetration

Viral myocarditis affects about 5% to 20% of the population. So far, there are not many effective antiviral treatments available. QiHong, the combination of the extracts from Astragali (Huangqi), Rhadiola rosea (Hongjingtian), and Sophora flavescens (Kushen), was developed based on laboratory research. The aim of this study was to investigate the effect and mechanism of QiHong on coxsackievirus B3 (CVB3)-induced myocarditis. The antiviral activity of QiHong in vitro was evaluated on HeLa and Vero cells infected by CVB3. Ribavirin was chosen as positive control. Our results showed that QiHong possessed potent antiviral effects on CVB3 by sodium 3'-[1-(phenylamino-carbonyl)-3, 4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid and plaque-forming assay (50% inhibitory concentrations [IC50] were 7.16 +/- 0.8 microg/ml and 2.63 +/- 0.5 microg/ml, respectively). The 50% cytotoxicity concentration (CC50) was 16-fold higher in QiHong-treated cells than in ribavirin-treated cells. Time course studies demonstrated that the antiviral effect of QiHong was mainly found during 0-4 hrs of infection, and it blocked the attachment and penetration of CVB3 into cells. In vivo 4-week-old male Balb/C mice were used and inoculated intraperitoneally with CVB3 suspension or normal saline. At 48 hrs after inoculation, the infected mice were gavaged with QiHong or ribavirin. On Day 6, myocardial virus titers were significantly lower in the QiHong-treated group than in the viral-infected groups. On Day 14, QiHong significantly ameliorated CVB3-induced myocardium necrosis; on Day 28, QiHong treatment increased survival rate 4-fold compared with CVB3-infected controls (64% vs. 16%; P < 0.05). The results showed that QiHong is a very promising potent antiviral agent with a highly significant favorable effect on survival and pathologic changes in CVB3-induced myocarditis with less toxicity than ribavirin. The antiviral activity of QiHong is at least partially due to an inhibitory effect on virus attachment and penetration.     

Pancreatic expression of interferon-gamma protects mice from lethal coxsackievirus B3 infection and subsequent myocarditis

Cardiovascular disease is one of the leading causes of death worldwide, and has been associated with many environmental risk factors. Recent evidence has indicated the involvement of pathogens such as viruses as causative agents, and specifically identified the coxsackievirus B serogroup as the leading culprit. Not only has coxsackievirus B3 (CB3) been identified from patients with cardiovascular disease, but also infection of mice with CB3 strains can reproduce human clinical heart disease in rodents. Several mechanisms have been proposed in an attempt to distinguish between pathology mediated by direct viral destruction of cardiac muscle cells or by the virus-induced immune response directed at infected myocytes or at 'mimicked' epitopes shared between viral and cardiac antigens. To distinguish between these mechanisms, we infected a unique mouse that diminishes the extent of infection and spread of the virus, but allows complete immunity to the virus. Transgenic mice expressing interferon-gamma in their pancreatic beta cells failed to develop CB-3-induced myocarditis. This work challenges the idea of the function of the immune response and 'molecular mimicry' in the CB-3-induced autoimmune myocarditis model, and instead favors the idea of virus-mediated damage. These results emphasize the benefit of reducing the level of viremia early during infection, thereby reducing the incidence of virus-mediated heart damage and autoimmunity.     

Roles of tumor necrosis factor alpha (TNF-alpha) and the p55 TNF receptor in CD1d induction and coxsackievirus B3-induced myocarditis

Giving C57BL/6 mice 10(4) PFU of coxsackievirus B3 (H3 variant) fails to induce myocarditis, but increasing the initial virus inoculum to 10(5) or 10(6) PFU causes significant cardiac disease. Virus titers in the heart were equivalent at days 3 and 7 in mice given all three virus doses, but day 3 titers in the pancreases of mice inoculated with 10(4) PFU were reduced. Tumor necrosis factor alpha (TNF-alpha) concentrations in the heart were increased in all infected mice, but cytokine levels were highest in mice given the larger virus inocula. TNF-alpha(-/-) and p55 TNF receptor-negative (TNFR(-/-)) mice developed minimal myocarditis compared to B6;129 or C57BL/6 control mice. p75 TNFR(-/-) mice were as disease susceptible as C57BL/6 animals. No significant differences in virus titers in heart or pancreas were observed between the groups, but C57BL/6 and p75 TNFR(-/-) animals showed 10-fold more inflammatory cells in the heart than p55 TNFR(-/-) mice, and the cell population was comprised of high concentrations of CD4(+) gamma interferon-positive and Vgamma4(+) cells. Cardiac endothelial cells isolated from C57BL/6 and p75 TNFR(-/-) mice upregulate CD1d, the molecule recognized by Vgamma4(+) cells, but infection of TNF(-/-) or p55 TNFR(-/-) endothelial cells failed to upregulate CD1d. Infection of C57BL/6 endothelial cells with a nonmyocarditic coxsackievirus B3 variant, H310A1, which is a poor inducer of TNF-alpha, failed to elicit CD1d expression, but TNF-alpha treatment of H310A1-infected endothelial cells increased CD1d levels to those seen in H3-infected cells. TNF-alpha treatment of uninfected endothelial cells had only a modest effect on CD1d expression, suggesting that optimal CD1d upregulation requires both infection and TNF-alpha signaling.     

Dual functions of murine gammadelta cells in inflammation and autoimmunity in coxsackievirus B3-induced myocarditis: role of Vgamma1+ and Vgamma4+ cells

Coxsackieviruses are a cause of clinical myocarditis. Both virus replication and host defense mechanisms, including virus-induced autoimmunity, mediate heart injury and cardiac dysfunction. Vgamma4+ cells kill infected cardiocytes and virus-specific CD4+ Th2 cells through Fas-dependent apoptosis and CD1d. The CD4+ Th1 response is necessary for activation of the autoimmune CD8+ T cells, which kill uninfected cardiocytes through perforin-dependent mechanisms.     

V gamma 1+ T cells suppress and V gamma 4+ T cells promote susceptibility to coxsackievirus B3-induced myocarditis in mice

Coxsackievirus B3 infections of C57BL/6 mice, which express the MHC class II IA but not IE Ag, results in virus replication in the heart but minimal myocarditis. In contrast, Bl.Tg.Ealpha mice, which are C57BL/6 mice transgenically induced to express IE Ag, develop significant myocarditis upon Coxsackievirus B3 infection. Despite this difference in inflammatory damage, cardiac virus titers are similar between C57BL/6 and Bl.Tg.Ealpha mice. Removing gammadelta T cells from either strain by genetic manipulation (gammadelta knockout(ko)) changes the disease phenotype. C57BL/6 gammadelta ko mice show increased myocarditis. In contrast, Bl.Tg.Ealpha gammadelta ko mice show decreased cardiac inflammation. Flow cytometry revealed a difference in the gammadelta cell subsets in the two strains, with Vgamma1 dominating in C57BL/6 mice, and Vgamma4 predominating Bl.Tg.Ealpha mice. This suggests that these two Vgamma-defined subsets might have different functions. To test this possibility, we used mAb injection to deplete each subset. Mice depleted of Vgamma1 cells showed enhanced myocarditis, whereas those depleted of Vgamma4 cells suppressed myocarditis. Adoptively transfusing enriched Vgamma4(+) cells to the C57BL/6 and Bl.Tg. Ealpha gammadelta ko strains confirmed that the Vgamma4 subset promoted myocarditis. Th subset analysis suggests that Vgamma1(+) cells biased the CD4(+) T cells to a dominant Th2 cell response, whereas Vgamma4(+) cells biased CD4(+) T cells toward a dominant Th1 cell response.