[The mechanisms responsible for the therapeutic effects of anti-Fas ligand antibody on viral myocarditis in mice].
OBJECTIVE
Viral myocarditis (VM) is one of the most common acquired myocardial diseases in children. However, its pathogenesis is not clear. Recent studies indicate that the cytotoxicity mediated by cytotoxic T lymphocyte (CTL) plays an important role in the development of myocardial injury involved in VM. Apoptosis mediated by Fas/FasL pathway is an essential mechanism of target cells damage by CTL. In this study, the authors investigated the regulatory effects of neutralizing anti-Fas ligand (anti-FasL) antibody on apoptosis and caspase-3 expression in experimental coxsackievirus B3 myocarditis and the role of the CTL mediated apoptosis in myocardium through Fas/FasL pathway in the development of VM.


METHODS
A total of 80 BALB/c mice were used in the experiments. They were divided randomly into the following groups: normal control group (Gr1), CVB3 control group (Gr2), IgG control group (Gr3) and anti-FasL antibody therapy group (Gr4). The mice in Gr2, Gr3 and Gr4 were inoculated with 0.15 ml of TCID(50) 10(9)/ml coxsackie virus B3 (CVB3) and the mice in Gr1 with 0.15 ml of Eagle reagent. The mice in Gr3 and Gr4 were inoculated with IgG (0.1 mg/kg) and FasL antibody (0.1 mg/kg) on days 0 and days 3 after inoculation (p.i.), respectively. Eight mice in each group were sacrificed on day 10 p.i. Histopathological studies and terminal transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays were used to quantify inflammation, necrosis and apoptosis in myocardium. The expression of active caspase-3 in myocardium was determined by immunohistochemistry. Caspase-3 mRNA and CVB3 mRNA were analyzed by reverse-transcription polymerase chain reaction (RT-PCR).


RESULTS
(1) Caspase-3 activation and apoptosis were seen in the myocardium of mice with myocarditis. They had a significantly positive correlation with the changes of myocardial histopathologic scores (r = 0.81, P < 0.05; r = 0.73, P < 0.05). (2) The pathologic scores, average percentages of apoptotic cardiomyocytes, expression of active caspase-3 (protein and mRNA) and expression of CVB3 mRNA in myocardium of mice in Gr4, were significantly reduced compared to those in the myocardium of mice in Gr2 (P < 0.01, P < 0.01, P < 0.01, P < 0.01, and P < 0.05, respectively) and Gr3 (P < 0.01, P < 0.01, P < 0.05, P < 0.01, and P < 0.05, respectively).


CONCLUSIONS
Myocytic apoptosis is a key mechanism responsible for myocardial damage in viral myocarditis. Anti-FasL antibody can effectively reduce expression of active caspase-3 protein and mRNA, viral replication, cardiomyocytic apoptosis and myocardial injury in the experimental CVB3 myocarditis.
Chang
Hong
H
Department of Pediatrics, Shandong University Shandong Provincial Hospital, Jinan 250021, China.
Han
Bo
B
Han
Xiu-zhen
XZ
D000818:Animals; D057134:Antibodies, Neutralizing / Q000627:therapeutic use*; D017209:Apoptosis; D053148:Caspase 3 / Q000276:immunology; D003384:Coxsackievirus Infections / Q000188:drug therapy* / Q000276:immunology; D029822:Enterovirus B, Human / Q000502:physiology; D053222:Fas Ligand Protein / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009205:Myocarditis / Q000188:drug therapy* / Q000276:immunology / Q000821:virology; D009206:Myocardium / Q000276:immunology / Q000473:pathology; D014779:Virus Replication
D057134:Antibodies, Neutralizing; D053222:Fas Ligand Protein; D053148:Caspase 3