ABSTRACT
Objective To screen out the cytokines relating to cardiac insufficiency caused by au-toantibodies against the second extracellular loop of the β1-adrenoceptor(β1-AA) using cytokine chip tech-nique, and to analyze the changes in signaling pathways. Methods Blood samples were collected from 67 patients with coronary artery disease(CAD) and 42 healthy subjects. ELISA was performed to detect β1-AA in plasma. BALB/c mice were passively immunized with the monoclonal antibodies against β1-AA (β1-AA mAb). Dynamic changes in mouse cardiac structure and functions were detected by heart ultrasound. Hema-toxylin and eosin (HE) and Masson staining were used to observe morphological changes in heart tissues.Cytokine chip technique was used to screen out the cytokines causing myocardial injury. Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis were used to classify the differentially expressed cytokines. Results Patients with CAD showed increased titer and posi-tive rate of β1-AA as compared with healthy subjects(P<0.001). The mouse model of heart injury was in-duced by β1-AA at the 8th week after immunization. A total of 37 differentially expressed cytokines were found in the model group,of which 11 cytokines were up-regulated and 26 cytokines were down-regulate as compared with those in the mouse control group. The level of CXCL16 was significantly increased in β1-AA-positive mice. GO analysis showed that CXCL16 was mainly involved in life processes including the positive regulation of cell death, migration, locomotion and cellular component movement. KEGG pathway enrich-ment analysis showed CXCL16 was significantly enriched in the pathway of cytokine-cytokine receptor inter-action and chemokine signaling pathway. ELISA showed that compared with β1-AA-negative patients, CXCL16 level was significantly increased in β1-AA-positive patients (P<0.01). A positive correlation was found between β1-AA and CXCL16 (P<0.01,r=0.43). Conclusion CXCL16 may play a critical role in the development of cardiac insufficiency induced by β1-AA.
ABSTRACT
Objective To investigate the effects of β1-adrenergic receptor autoantibodies (β1-AA) on the proliferation of different subtypes of T lymphocytes in patients with heart failure .Methods β1-AA-positive IgG antibodies isolated from patients with heart failure were purified by using affinity chromatog -raphy.CD3 +CD4 +T and CD3 +CD8 +T lymphocytes were sorted by flow cytometry analysis .The prolifera-tion of different subtypes of T lymphocytes was tested by using CCK-8 kit.Tests for lactate dehydrogenase ( LDH) level and cell apoptosis were performed to evaluate T lymphocytes damage .Results The prolifera-tion of activated lymphocytes was inhibited by β1-AA isolated from the serum of patients with heart failure in a concentration dependent manner , but it could be blocked by β1 receptor blocker .The damage of lympho-cytes induced by β1-AA was increased.Moreover, β1-AA promoted the necrosis and apoptosis of CD 3 +CD4 +T and CD3 +CD8 +T lymphocytes and thus inhibited the proliferation of them .Conclusion β1-AA isolated from the serum of patients with heart failure inhibited the proliferation of CD 3 +CD4 +T and CD3 +CD8 +T lymphocytes through increasing the necrosis and apoptosis of them .This study suggests that β1-AA might induce immune disorders in addition to causing pathological changes in heart tissues .
ABSTRACT
Objective To investigate the effects of autoantibodies (β1-AA) against second extra-cellular loop of the β1-adrenergic receptor (β1-AR-ECⅡ) in sera of patients with dilated cardiomyopathy (DCM) on proliferation of rat CD4+T lymphocytes.Methods β1-AA in the sera of patients with DCM was purified by affinity chromatography .CD4+T lymphocytes were isolated by immunomagnetic microbeads from peripheral blood mononuclear cells of rats and its positive rate was detected by flow cytometry .CCK-8 meth-od was used to detect the proliferation of CD 4+T lymphocytes and flow cytometry was performed to measure the ratio of CD4+/CD8+T lymphocyte .Results The purity of isolated rat CD 4+T lymphocytes by immu-nomagnetic microbeads reached 97.7%.The proliferation of CD4+T lymphocytes stimulated by CD3/CD28 was inhibited by β1-AA in a concentration-dependent manner .However , IgG antibodies extracted from sera of healthy controls did not suppress lymphocyte proliferation (P>0.05).The suppression effect of β1-AA was inhibited after binding to antigenic peptides corresponding to β1-AR-ECⅡ and was completely blocked by metoprolol, a specific antagonist of β1-adrenergic receptor (β1-AR).In addition,β1-AA had no effects on the ratio of CD4+/CD8+T lymphocyte .Conclusion β1-AA isolated from DCM patients suppresses the pro-liferation of CD4+T lymphocytes through β1-AR pathway , which indicates that β1-AA can directly reduce the number of T lymphocytes and impair the function of T lymphocytes , resulting in immune system disorders and the development of DCM .
ABSTRACT
Objective To investigate the influence of autoantibodies against the second extracellular loop of β1-adrenoceptor ( β1-AA) on the proliferation ability of LPS-stimulated rat B lymphocytes.Methods Active immunization assay was used to obtain adequate IgGs in which β1-AA was positive; MACS (magnetic activated cell sorting) assay was used for gaining rat splenic B lymphocytes; CCK8 assay was used for detecting the influence of β1-AA to the proliferation abilities of silent and LPS-stimulated rat splenic B lymphocytes.Results β1-AA (0.1 μmol/L pIgGs ) promoted the proliferation of LPS-stimulated rat splenic B lymphocytes(A values:0.739±0.036 vs 0.533±0.032,P＜0.05),and the effect was likely to be concentration-dependent.And the effect could be blocked by β1-AR blocker or β2-AR blocker partially,and could be blocked by β1-AR blocker and β2-AR blocker completely; β1-AA had no proliferation effect on silent rat splenic B lymphocytes.Conclusion β1-AA could promote the proliferation of LPS-stimulated rat splenic B lymphocytes via β1-AR and β2-AR which were on the surface of B lymphocytes.Thus,it could provide new clues for complex pathologic mechanism of cardiovascular patients in which β1-AA is positive.