Effects Of Intervention With CTLA4-Ig And Anti-CD40l Monoclonal Antibody On Mice With Experimental Autoimmune Myocarditis

Objective:CD28 is the primary co-stimulatory signal, but additional pathways such as CD40L has selective role in T cell activation, Here we investigated the effects of CD28 and CD40L blockade by CTLA4-Ig and anti-CD40L monoclonal antibody on mice with experimental autoimmune and its mechanisms.Methods:Sixty Balb/c mice were random divided into two groups: (1) experimental group consisted 50 mice. Genetically predisposed Balb/C mice were immunized with porcine cardiac myosin covered by complete freund's adjuvant (CFA) on day 1 and 8 to establish animal model of experimental autoimmune myocarditis, and then the 50 mice divided into different subgroups: EAM group, CTLA4-Ig group (injected with CTLA4-Ig,1mg/kg), anti-CD40LmAb group (injected with anti-CD40LmAb,1mg/kg), CTLA4-Ig+anti-CD40LmAb group (injected with CTLA4-Ig+anti-CD40L mAb,1mg/kg) and IgG group (injected with mice IgG,1mg/kg); (2) normol control group consisted 10mice, which was immunized with PBS and CFA without porcine cardiac myosin. On day 21 after the first immunization, all mice were sacrificed. HE staining was used to oberve the change of the histopathological characteristics, Myocardical enzymes (Tn-I,CK-MB) were detected to reflect myocardial damage. Immuno-histochemicalstaining and realtime fluorescence quantitative PCR (RQ-PCR) detection were used to position and qualitative analysis the expression of CTLA4 and CD40L. IFN-γand IL-4 mRNA in muscular tissues were detected by RQ-PCR, sera IFN-γand IL-4 were determined by ELISA.Results:1. Animal model The incidenee of experimental autoimmune myocardltis was 100%. Body weight of the mice in EAM group and IgG group grew slower than normal control group, even decreased from the second week to the third week.2. Pathology testing HE staining showed: Myocardial cells in normal control group arranged in uniform. All mice in EAM group and IgG group developed typical severe autoimmune myocarditis, large numbers of inflammatory cells infilatrated all layers of ventricular wall, degeneration and necrosis were also evident. Inflammatory cells significantly decreased after immunologic intervention,there were a few focal inflammatory cells in CTLA4-Ig group and anti-CD40LmAb group, minimal infliammatory cells were observed in CTLA4-Ig+anti-CD40LmAb group. Pathological scores of mice in CTLA4-Ig+anti-CD40LmAb group were significantly lower than CTLA4-Ig group and anti-CD40LmAb group(P<0.05), but had no significant difference between CTLA4-Ig group and anti-CD40LmAb group.3. Myocardical enzymes Sera Tn-I and CK-MB in EAM group and IgG group were markedly higher than those of control group(P<0.01). After immunologic intervention , sera Tn-I and CK-MB significantly decreased(P<0.05), those in CTLA4-Ig+anti-CD40LmAb group were lower than CTLA4-Ig group and anti-CD40LmAb group(P<0.05), but had no significant difference between CTLA4-Ig group and anti-CD40LmAb group(P>0.05).4. The expression of CTLA4 and CD40L in ventricular tissue There was almost no expression of CTLA4 and CD40L in the cardiac myocytes of normal mice, whereas CTLA4 and CD40L were clearly induced on the infiltrating cells in mice of EAM group and IgG group, CTLA4 and CD40L mRNA were also significantly enhanced. After immunologic intervention, the expression of CTLA4 and CD40L significantly decreased(P<0.05). those in CTLA4-Ig+anti-CD40LmAb group were lower than CTLA4-Ig group and anti-CD40LmAb group(P<0.05), but had no significantly difference between CTLA4-Ig group and anti-CD40LmAb group (P>0.05). Dynamic changes of CTLA4 and CD40L showed significantly positive correlation with the changes of myocarditical histoPathological scores.5. The expression of IFN-γand IL-4 mRNA in ventricular tissue The expression of IFN-γmRNA and IL-4 mRNA in cardiac myocytes markedly increased in mice of EAM group and IgG group compared to nomal control group (P<0.01),the Th1 cytokine IFN-γmRNA increased more significant than Th2 cytokine IL-4 mRNA. After immunologic intervention, the expression of Th1 cytokine IFN-γmRNA decreased, whereas Th2 cytokine IL-4 mRNA increased in CTLA4-Ig group and anti-CD40LmAb group,but the expression of IFN-γmRNA and IL-4 mRNA were all depressed in CTLA4-Ig+anti-CD40LmAb group.6. The level of IFN-γand IL-4 in serum The level of sera IFN-γin EAM group and IgG group was markedly higher than that in control group(P<0.01), but there was no significantly difference of sera IL-4. After immunologic intervention, the expression of Th1 cytokine IFN-γdecreased whereas Th2 cytokine IL-4 increased.Conclusions:1. Repetitively immunized with porcine cardiac myosin was capable of inducing experimental autoimmune myocarditis.2. Either Single or combined use of CTLA4-Ig and anti-CD40mAb could remarkably decrease the myocardical inflammation and alleviate autoimmune damage in murine acute myocarditis. Intervention alliance had a better effectiveness than sigle intervention.