| BackgroundSince the nineties of last century, many researchers found that there were autoantibodies against the second extracellular loop of M2-muscarinic acetylcholine receptor (M2-AA) in the serum of patients with cardiovascular disease in high titer. Further functional studies found that the M2-AA played a role of negative chronotropic effect on the myocardial cells in vitro, which was similar to M2 receptor agonist carbachol, but the stimulation is not easy to desensitization. Therefore, we speculate that maybe the M2-AA is able to interfere with the nomal function of heart according to activating M2 receptor in the long-term, which result in the pathological injuries to the heart.We have immunized rats with the synthesis peptides against the second extracellular loop of the M2 receptor as antigens for long time, then we observed:with the increase of M2-AA titer and extend of interaction time, myocardial remodeling occurred gradually, in the end, the the pathological and functional changes which was similar to of dilated cardiomyopathy appeared. During the immunization time, the degeneration of vacuolar presented in mitochondria of myocardial cells, with the loss of myocardial cells in focus, and filtration of lymphocytes. These results suggested that the M2-AA may be involved in autoimmune-mediated pathogenesis of heart disease.However, the above-mentioned study is not sufficient to conclude that M2-AA can result in cardiac injuries directly. Because the model was active immunization model, in one hand, the else factors except M2-AA can not be excluded in the model, for instance, whether the antigen is involved in the occurance of cardiac injuries; on the other hand, the titer of M2-AA produced by active immunization model highly more than the serum of the clinical patients. Thus, it is difficult to explain the clinical cases by active immunization model. Consequently, establishment of passive immunization model of animals whose concentration of M2-AA similar to the clicical situations is the essential and basic experiment method of the further study about the pathophysiology significance of M2-AA.In the active immunization model above, the infiltration of a large number of lymphocytes in the damaged myocardial tissue was also an important pathological phenomenon, hinting that a certain factors activated the immune system of an organism. As we all know, T lymphocytes are the responsible for the immunologic response, which have multiple biological functions of killing the target cells directly, assisting or inhibiting the antibodies produce by B cells, specific response of antigens and promoting mitogen, and production of cytokines and such on. It is reported that there are M2 receptors in the T lymphocytes of human and murine, and the parasympathetic neurotransmitter ACh plays a role in immune regulation. In view of M2-AA is similar to agonist-like activation to M2 receptors, so we speculated that M2-AA in the circulating blood may also activate M2 receptors of T lymphocytes, thereby affecting the activity of T lymphocytes and immune function. However, whether M2-AA is directly involved in activation of T lymphocytes needs to be further identified. If it is, the forms of participation need to be studied.In the specific immune response of body, CD4+T lymphocytes are the main effective T cells involved in all stages of the immune response, and play a key role in immune regulation. According to the traditional view, CD4+T lymphocytes were divided into type 1 T helper cells (T helper cell 1, Th1), type 2 T helper cells (T helper cell 2, Th2) and regulatory T cells (regulatory T cells, Treg). In recent years, researchers found that there is a new CD4+T lymphocytes intype17 T helper cells (T helper cell 17, Th17), which involved in body's own immune and inflammatory responses. Th17 lymphocytes can secret interleukin-17 (IL-17, namely IL-17A), IL-17F, IL-21, IL-22, IL-6, TNF-a (Tumor Necrosis Factorα,) and so on. These cytokines play the role of collective mobilization, collection and activated neutrophils. Some studies reported that Th17 cells and their characteristic cytokines IL-17 may be an important autoimmune factors in myocardial injury. If we can confirm whether M2-AA directly affect the functional activity of Th17 cells specifically, may be it will provide direct evidence that M2-AA is capable of regulating immune cell function and activity, which supply a new starting point of further study of the physiological significance to M2-AA.Objective(1) The passive immunization rat models were establishment by injection of M2-AA to observe whether long-term presence of M2-AA can lead to changes of cardiac structure and function, as well as in immune function of body, in order to explore whether the M2-AA has a direct role in leading to myocardial injury and regulation of immune function.(2) Isolation and culture of rat T lymphocytes to observe whether the M2-AA could affect the proliferation and secretion of T lymphocytes, in order to investigate whether the M2-AA can activate T-lymphocyte-mediated immune response, and explore other role sites of M2-AA except to the heart. (3) Isolation and culture of human peripheral blood mononuclear cells, the native CD4+T cells were induced by the inducers and activators to differentiate to Th17 lymphocytes, in order to observe the influence of M2-AA on functional activites of Th17 lymphocytes.Methods(1) The rats model of active immunization are established with synthetic antigenic peptide according to 163-184 amino acid sequence of the second extracellular loop of M2 receptor, and SA-ELISA method is used for monitoring the production of M2-AA.(2) Affinity chromatography is used to purify M2-AA IgGs in the serum of the immune rats. SDS-PAGE gel electrophoresis is used for determine the purity of IgGs, and BCA method is used for measuring the content of proteins. ELISA method is used for identifying the purified M2-AA IgGs subsets.(3) Passive immunization rat models of M2-AA were established by the choosing the serum M2-AA negative animal. The MS2000 Biological Signal Recording and Analysis System were used to monitor the cardiac function in a regular time, and myocardial tissue samples were collected and treated to observe under the light and electron microscope.(4) The T lymphocytes from spleen in rats passive immunization M2-AA were isolated. Cell staining by immunofluorescence was used, and CD4+/CD8+T-lymphocyte ratio is determined by flow cytometry.(5) The T lymphocytes from spleen of normal rats were acutely isolated and cultured, then the cell staining by immunofluorescence was used and the T lymphocytes were identified by laser scanning confocal microscope.(6) CCK-8 method was used to detect the activity levels of T-lymphocyte after the accession of M2-AA, in order to determinate the impact of M2-AA on the proliferation function of T-lymphocyte. ELISA kits wer used to detect the levels of IL-4, IL-10, IL-17, IL-21 and IL-22 in the supernatant of cultured T-lymphocyte after intervention by M2-AA.(7) Ficoll-Hypaque density gradient centrifugation was used to isolate and culture the peripheral blood mononuclear cells in human. Th17 cells were promoted to differentiate and maturate by adding inducing agents and stimulants, and cell staining by immunofluorescence (including intracellular cytokine staining) was used, in order to identify them by laser scanning confocal microscope.(8) CCK-8 method was used to detect activity level of Th17 cells after intervention by M2-AA, to observe the impact on the proliferation function of Th17 cells by M2-AA; ELISA kits were used to detect the levels of IL-17, IL-21 and IL-22 in the supernatants of cultured Th17 cell after intervention by M2-AA.Results1. Preparation, purification, qualitation and quantitation of M2-AA1.1 The active immunized rat models were set up successfullyThe results determined by SA-ELISA showed that 2 weeks after immunization, the level of M2-AA in the sera of M2 immunized group has already raised and reached the peak in 8 weeks (OD Values:2.93±0.11 vs.0.21±0.02, P＜0.01). Then to 10 weeks, the levels of M2-AA decreased, but it was still higher than that before initial immunization (P＜0.01) and the control group (P＜0.01). (Fig.1-1). The result suggests that the active immunized rat models were set up successfully.1.2 Purification, quantitative qualitative and detection the subtype of M2-AA IgGsIgGs in the sera of immunized group were purified by affinity chromatography, so the total IgGs including M2-AA were obtained. The specificity of purified IgGs was determined by the SDS-PAGE. The results showed that two straps of 55KD and 25KDa appeared which represented one heavy chain and one light chain, respectively (Fig.l-2A).The concentration of purified IgGs was 2.39 mg/mL, which was determined by BCA kits.The subtype of purified M2-AA IgGs was mainly IgG2a (Fig.l-2B).2. The heart injury appeared in the M2-AA passive immunized rat models2.1 The passive immunized rat models were established successfully4 weeks after passive immunization, the sera level of M2-AA in the immunized group was significantly higher than that in the control group (0.355±0.03 vs.0.103±0.04, P<0.05). Then the sera level of M2-AA persisted at stable level. It was still higher than that in control group in 24 weeks after immunization (0.388±0.04 vs.0.098±0.08, P＜0.01) (Fig.1-3). The result suggests the passive immunized rat models were set up successfully.2.2 The cardiac function decreased in the passive immunized rat16 weeks after passive immunization, the LVEDP in the M2-AA group has been higher than that in the control group (1.53±0.91 vs.1.38±0.61, P<0.05) (Fig.l-4B), while the+dp/dtmax was significantly lower than the control group (302.43±39.1 vs.369.18±30.6, P＜0.05) (Fig.l-4C). In 24 weeks in the M2-AA immune rats, the LVSP was lower than that in the control group (9.06±2.20 vs.13.92±2.81, P<0.05), but the LVEDP was higher than the corresponding control group (2.96±0.52 vs.1.29±0.61, P<0.01),+dp/dtmax decreased (213.06±40.32 vs.360.32±26.81, P<0.01) (Fig.l-4A), and the-dp/dtmax increased(-163.96±28.52 vs.-225.29±12.71, P<0.01) (Fig.l-4B, D), these results indicate decline in cardiac function.2.3 The ratios of heart weight to body weight (HW/BW) decreased in the passive immunized rats16 weeks after passive immunization, the ratio of heart weight to body weight (HW/BW) was significantly lower than that in the control group (2.23±0.10 vs.2.48±0.11, P＜0.05), and it decreased continuously until the experiment was ended up (Fig.1-5C).2.4 Myocardial injury induced by passive immunization rats with M2-AAHeart stereological examination showed 24 weeks after immunization with M2-AA, the heart size became larger and the heart chamber expanded (Fig.l-5A, B). While there were no significant changes in the heart at other time points of immunization.HE staining of myocardial tissue showed:24 weeks after immunization, there were immune inflammatory cells infiltration in the myocardial cells of M2-AA group, and the emergence of infiltration with inflammatory cells, while the control group without such changes in myocardial tissue (Fig.1-6).Electron microscope displayed marked myocardial fibrosis, deposition with collagen in sarcoplasmic reticulum, and swelling and degeneration in mitochondria, while the control group does not appear similar to myocardial change(Fig.l-7).These results suggest that long-standing of M2-AA in vivo can lead to myocardial injury.2.5 The ratios of CD4+/CD8+ T-lymphocytes from spleen cells increased in passive immunization of rats with M2-AABefore 12 weeks after immunization, there was no significant difference in ratios of CD4+/CD8+ T-lymphocytes in the M2-AA immune group and the control group. It was significantly higher in the M2-AA group at 12th week (3.06±0.28 vs.1.92±0.38, P＜0.05), and it continuous increase to 24 weeks with more notable difference (3.47±0.31 vs.1.82±0.33, P＜0.01) (Fig.1-8).This result indicates that long-standing of M2-AA in vivo leads to cellular immune function disturbance in the body.3. M2-AA accelerated proliferation and secretion of T lymphocytes3.1 The activation and identification of mature T lymphocytes The T lymphocytes were round or oval-shaped without ConA; when were stimulated and cultured for 72h by, the cell volume augmented, with the number increased significantly, and the pattern were irregular-shaped, long spindle or triangular, etc(Fig.2-1).The T lymphocytes were identified by immunofluorescence, according to the characteristic surface markers of T lymphocytes. The fluorescently labeled antibodies of CD3-FITC and CD4-PE were used. The result showed most of the cell surface display of green fluorescent-positive, indicating that T lymphocytes; a small number of cell surface was green and red fluorescence double positive, these were the helper T cells (Th cells) (Fig.2-2).3.2 T lymphocytes from rat spleen could be activated by Con AAfter adding ConA to the cultured rat spleen lymphocytes, the cell activity was significantly higher than that of the control group (OD value,0.442±0.05 vs.0.126±0.03, P＜0.01) (Fig.2-3), which indicated that Con A could promote T lymphocytes proliferation.3.3 M2-AA promoted proliferation of T lymphocytes in a dose-dependent mannerO.1μmol/L of the M2-AA elevated the cell activity significantly (OD values,0.561±0.08 vs. 0.439±0.05, P<0.05), the Oxotremorine also showed the similar effect in the same concentration (P＜0.05) (Fig.2-4).Three concentrations of M2-AA (0.01,0.1, 1μmol/L) could promote T lymphocyte proliferation in a concentration-dependent manner (OD values:0.01:0.496±0.06 vs.0.311±0.05, P<0.05; 0.1:0.687±0.08 vs.0.420±0.06, P＜00.01; 1:0.783±0.11 vs.0.461±0.08, P＜0.01). The same concentration of Oxotremorine also promoted T lymphocytes proliferation in a dose-dependent (Fig.2-5).3.4 M2-AA promoted T-lymphocyte proliferation through the M2 receptorThe effect of M2-AA on the proliferation of T lymphocytes could be inhibited by the M2 receptor antagonist Methoctraine (Fig.2-6). In addition, the effect of M2-AA could also be inhibited by M2R peptides (Fig.2-7). The results indicate that the M2-AA promoted T-lymphocyte proliferation through the M2 receptor. 3.5 M2-AA increased the secretion of IL-4 and IL-10 by T-lymphocyte0.1μmol/L and 1μmol/L of the M2-AA were able to increase the secretion of IL-4 (Fig.2-8) and IL-10 (Fig.2-9) by T-lymphocyte, while M2-AA had no effect on the secretion of IL-17, IL-21or IL-22(Tab.1).4. M2-AA promoted the proliferation and secretion of Th17 lymphocytes4.1 The morphology and identification of Th17 lymphocytes The lymphocytes showed a normal round or oval without any stimulant. When added stimulants for 7 days, the cell morphology changed significantly, they were in irregular shapes, spindle-cell type, triangular, etc. Which indicated that the Thl7 lymphocytes have been differentiated (Fig.3-1).As there are no characteristic surface markers of Th17 cells so far, we used the membrane CD3-FIT.C and CD4-APC fluorescence antibody staining, combined with intracellular cytokine IL-17-PE staining methods to identify the Th17 lymphocytes. The surface of Th17 lymphocytes cell were green and blue fluorescent double-positive, moreover, the red fluorescence was detected in intracellular (Fig.3-2).4.2 M2-AA induced a dose-dependent proliferation of Th17 lymphocytesAfter added the inducers and activators, the activities of Th17 lymphocytes increased significantly (0.398±0.12 vs.0.051±0.03, P＜0.01), which suggested that add inducers and activators could promote the mature of Th17 lymphocytes (Fig.3-3).0.1μmol/L of the M2-AA increased the activity of Th17 lymphocytes (0.554±0.09 vs. 0.419±0.05, P<0.05). The same concentration of Oxotremorine also exhibited the same effects (P＜0.05) (Fig.3-4).Three concentrations of M2-AA (0.01,0.1, 1μmol/L) promoted Th17 lymphocytes proliferation in a concentration-dependent manner (0.01:0.443±0.03 vs.0.404±0.03, P<0.05; 0.1: 0.554±0.09 vs.0.424±0.06, P<0.01; 1:0.569±0.07 vs.0.449±0.06, P＜0.01). The same concentration of Oxotremorine also promoted Th17 lymphocytes proliferation by a concentration-dependent manner (Fig.3-5).4.3 M2-AA promoted Th17 lymphocyte proliferation through the M2 receptorThe effect of M2-AA on the proliferation of Th17 lymphocytes could be inhibited by the M2 receptor antagonist Methoctraine (0.449±0.03 vs.0.439±0.05, P>0.05) (Fig.3-6). In addition, the effect of M2-AA could also be inhibited by M2R peptides (0.437±0.04 vs.0.439±0.05, P>0.05) (Fig.3-7). These results indicate that the M2-AA promoted Th17 lymphocytes proliferation through the M2 receptor.4.4 M2-AA promoted Th17 cell secretion of IL-17, IL-21 and IL-220.1μmol/L of the M2-AA increased the secretion of IL-17 (0.049±0.01 vs.0.043±0.01, P＜0.05) and IL-21 (0.048±0.01 vs.0.042±0.01, P＜0.05) by the Thl7 lymphocytes (Fig.3-8,3-9), the effects of 1μmol/L M2-AA were more obvious (P＜0.01). In addition, 1μmol/L of the M2-AA increased the secretion of IL-22 (0.053±0.01 vs.0.044±0.01, P＜0.05) (Fig.3-10). Similarly, 0.1μmol/L and 1μmol/L Oxotremorine also increased the secretion of the three kinds of cytokines.Conclusions(1) The M2-AA can lead to myocardial injury and cellular immune function disturbance in the condition of long-standing and match the concentration of the clinical serum titer.(2) Within a certain range, M2-AA promotes T-lymphocyte proliferation and enhances the secretion of cytokines in a concentration-dependent manner in vitro. In addition to the cardiomyocyte, the T lymphocytes are also the role of M2-AA target.(3) Within a certain range, M2-AA promoted the proliferation of Th17 lymphocytes in dose-dependent manner in vitro, and increased the function of secretion inflammatory cytokines.
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