Expression Of MHC Class â…¡ Molecule And Changes Of Immune Function In Autoimmune Myocardial Disease

Objective: To explore the expression of CD4~+T cells and MHCclassⅡmolecules, the changes of cytokine levels such as IFN-γ、IL-2、IL-4and IL-10, and changes in myocardial pathology during thedevelopment of experimental autoimmune myocarditis in Balb/c miceinduced by immunization of myosin, thus to provide an experimentalbasis for the prognostic assessment and treatment for myocarditis andidiopathic expansionary cardiomyopathy.Methods:1. Founding mice model of myosin-inducedcardiomyopathy: Inbred strain of Balb/c mice were60male and female,8weeks old, weighing16-20g, divided into2groups,30as theexperimental group,30as the control group, each group is further dividedinto three groups. Experimental groups of mice in the inguinal andaxillary subcutaneous injection of pig cardiac myosin and completeFreund’s adjuvant, a mixture of200ul(containing myosin100ug),immune Balb/c mice,8days and30days after primary immunization inthe same way re-immunization. The control group was injected with amixture of potassium phosphate buffer and CFA(without myosin) in thesame manner.14,21and60days after the first immunization,10mice in the experimental group were randomly selected and10mice from thecontrol group. Removing eyes to get blood, using Elisa to measure theconcentration of serum anti-myosin and the serum of IFN-γ、IL-2、IL-4and IL-10levels. After blood collection, mice were sacrificed and theirhearts were removed immediately fixed in formalin. Slicing afterparaffin-embedded, observe the specimen under the optical microscopeafter routine hematoxylin-eosin staining. Immunohistochemicaldetermination of expression of CD4~+T cells and MHC class Ⅱmolecules.3. Statistical analysis: Analyze data with statistical softwareSPSS17.0. Results were expressed as average±standard deviation,geometric mean expressed the average of antibody titers, single factoranalysis of variance, groups were analyzed with LSD t-test. Correlationanalysis were analyzed with Spearman analysis. P values less than0.05isconsidered to be statistical significance.Results:1.The pathological change of the myocardial tissue:Each period of the control group compared with the same period in theexperimental group, the heart of the naked eye view of no obvious change.Microscopic observation of cardiac muscle cell structure of the controlgroup were normal, the myofibrils arranged in neat, uniform cytoplasmand clear strips, normal stromal cells, no inflammatory cell infiltration, nopathological changes. Experimental group of30mice can be seen varyingdegrees of inflammation, infection rate was100%. Mice were killed by 14days after the first immunization, the heart could be observed ininflammation appear, such as myocardial cell swelling, granulardegeneration, surrounded by inflammatory cells could be seen oozing,mainly lymphocytes, the lesions were focal distribution, inflammatorycell infiltration of the lesion mainly focused on the epicardial and aroundthe small blood vessels.21days after the initial immunization, thesechanges increased, a large number of inflammatory cells could be seenaround the myocardial cells and myocardial interstitial, and myocardialcell swelling, degeneration and necrosis. On the sixtieth day, the numberof inflammatory cell infiltration in the myocardial tissue reduced,necrosis of cardiac myocytes were replaced by fibrous tissue andmyocardial fibrosis.2. Expression of CD4~+T cells and MHC class Ⅱmolecule in the myocardial tissue: There was almost no expression ofCD4~+T cells in the myocardial tissue of the control group of mice.Compared with the control group, the expression of CD4~+T cells in theexperimental group were increased in each period, markedly at14,21days(65.13±4.45vs0；17.20±1.16vs0；5.14±0.35vs0，P<0.01) andon the sixtieth day, the expression of CD4~+T cells has been significantlydecreased, but there was still a small amount of it(2.14±0.85vs0，P<0.05). The expression of CD4~+T cells and the integral of themyocarditis was positively correlated(r=0.91、0.87、0.72，P<0.05). MHCclass Ⅱ molecules had low levels of expression in control group. Compared with control group, the expression of MHC class Ⅱ moleculesincreased, markedly at14,21days(83.25±1.05vs17.08±0.44；70.63±3.55vs16.31±0.62，P<0.01), and a positive correlation was foundbetween the expression of MHC class Ⅱ molecules and the integral ofthe myocarditis(r=0.85、0.82，P<0.05).3. Serum myosin antibody levels:Control group had low antibody titer of serum myosin. Compared withcontrol group,14and21days after the initial immunization, serum ofmice in test group can be detected to a high titer of antibodies, comparingwith the control group, which had a significant difference(1:200vs1:50；1:400vs1:75，P<0.01), but on the sixtieth day, compared with the21days, the antibody titers decreased significantly(1:400vs1:100，P<0.05).4. Changes of cytokine levels: Serum cytokine levels in control grouphad no obvious changes at different times. Compared with control group,the content of the experimental group of Th1and Th2cells secretecytokines were elevated during different periods.14days after the initialimmunization, serum levels of Th1-related cytokines IFN-γ and IL-2were significantly increased, and gradually reached a peak at21days(42.65±2.92vs14.65±2.02，81.04±0.20vs31.15±4.33，55.92±3.08vs15.87±1.64，93.15±3.06vs30.96±3.15，P<0.01). But during therecovery period, which was60days after the initial immunization,Th1-related cytokines decreased significantly and returned to normallevels. However, in acute phase, which were14days and21days after the initial immunization, Th2-related cytokines IL-4and IL-10contentdid not change obviously, but they were significantly increased at60dayscompared with the control group(5.13±0.30vs0.38±0.27，8.46±0.86vs4.09±0.22，P <0.05).Conclusion: Typical autoimmune myocarditis could be induced byimmunization of pig cardiac myosin in Balb/c mice. MHC class Ⅱmolecules had abnormal expression early in the autoimmune response.CD4~+T cells resisted damage also caused itself myocardial injury.Th1/Th2imbalance participated in the development of the disease.