The Influence Of Autoantibodies Against β₁-Adrenoceptor On Rat B Lymphocytes Proliferation And The Possible Mechanism

BackgroundsCardiovascular disease is one kind of the common diseases that threaten human health, andits mortality rate is always at the top of our resident deaths. Nowadays, cardiovascular diseasehas become one of the China’s major public health problems. The level of diagnosis andtreatment rises continuously, however, the fatality rate of cardiovascular disease is still very highbecause of various cardiovascular causes and complicated pathological mechanisms, whichsuggests that there are still other unknown factors involved in the development and progressionof cardiovascular disease. With the development of nerve-endocrine-immune network theory, therole of immune factors in cardiovascular disease has been an increasing concerm of manyresearchers. Wallukat et al firstly discovered that the autoantibody against β₁-Adrenoceptor（β₁-AR） in the sera of idiopathic dilated cardiomyopathy patients in1987. And β₁-AA was shortfor autoantibody against β₁-Adrenoceptor. After that, β₁-AA was also detected in the sera ofpatients with Chagas’ heart disease, primary electrical cardiac abnormalities and othercardiovascular diseases by many researchers. And the positive rate and titers of β₁-AA weremuch higher than healthy people. Further studies reported thatβ₁-AA recognized and bound toβ₁-AR-ECIIwhich was on the surface of the cardiomyocytes to display agonist-like effects suchas increasing the beating frequency of cultured neonatal rat cardiomyocytes, enhancing thenormal atrial muscle contraction, increasing cAMP content in the cardiomyocytes andintracellular Ca2+. All these results showed β₁-AA may participate in the pathophysiologicalprocess of many cardiovascular diseases. Our group also found that long time existence of β₁-AAincreased the capacity of producing antibody, as well as the changes of cardiac dysfunction andstructure, suggesting that β₁-AA may lead to functional change of immune system. When Blymphocytes are activated by many factors, they proliferate and become plasma cells through aseries of process of differentiation. Finally, plasma cells secrete antibodies. The high counts of Blymphocytes in the sera of patients were closely related to the mortality of cardiovasculardiseases. Therefore, exploring whether β₁-AA promote the proliferation of B lymphocytes willbe helpful for elucidating the pathogenesis of patients with cardiovascular disease whose sera isβ₁-AA-positive. Section OneThe influence of B lymphocytes induced by β₁-AAObjectiveTo explore whether β₁-AA can promote proliferation of rat B lymphocytesMethods1. The preparation of β₁-AAHuman β₁-AR-ECII（197²23, H-W-W-R-A-E-S-D-E-A-R-R-C-Y-N-D-P-K-C-C-D-F-V-T-N-R-A, the homology between human and rats is100%, the purity>95%） was injected torats for acquiring β₁-AA-positive IgGs （pIgGs）, and the pseudo immune group was used to offerβ₁-AA-negative IgGs （nIgGs）. IgGs were purified by Mab Trap Kit. The purity of purified IgGswas detected by SDS-PAGE gel electrophoresis, and protein quantification of IgGs wasdetermined by the BCA Protein Quantitation Kit.2. The acquirement of B lymphocytesRat spleen lymphocytes were isolated by Ficoll density gradient centrifugation, and Blymphocytes were sorted from spleen lymphocytes according to using the method of MagneticActivated Cell Sorting （MACS）; the positivity rate and the viability of B lymphocytes weredetected by flow cytometry; B lymphocytes were cultured in RPMI1640solution in cellculture box where was on the condition of37℃,5%CO2, saturated humidity.3. The detection of proliferative ability of B lymphocytes induced by β₁-AA according toCCK8kitPIgGs and nIgGs were used to resting B lymphocytes and LPS-stimulated B lymphocytesfor48hours, respectively. Then number of B lymphocytes was detected by CCK8kit to explorethe proliferation effects on B lymphocytes induced by β₁-AA.Results1. Acquirement of β₁-AA according to active immunization of rats by using β₁-AR-ECIIantigen peptide1.1The establishment of active immunization modelTwo weeks after rats were immuned by injecting the β₁-AR-ECIIantigen peptides, the levelof β₁-AA detected by ELISA in the sera already increased from0.41±0.06to0.75±0.11（P= 0.006, P<0.01）. And the level of β₁-AA rise rapidly over time and reached a peak at8weeks, Avalue of3.01±0.020, and was significantly higher than the same period of pseudo-immunegroup the corresponding pseudo immunohistochemical （A:0.42±0.10, P=0, P<0.01）. The resultsindicate: β₁-AR-ECIIantigen peptide active immune rat model was successfully established, and8weeks after immunization of rats with abdominal aortic blood taking and leaving serum（Figure1）.1.2Determination of the qualitation and quantitation of β₁-AA proteinSerum total IgGs was purified by affinity chromatography kit. Purity of purified IgGs wasdetected by SDS-PAGE gel electrophoresis. The results showed two bands were displayed afterSDS-PAGE gel electrophoresis, one band was the IgG heavy chain （55KD）,and the other arepresentative of the light chain （25KD）, the two bands were consistent with IgG standardpreparation （Figure2）, showed that the purification effect was ideal. The concentration ofpurified IgGs was detected by BCA Protein Quantitation Kit （Figure3and table1）.2. Isolation of rat B lymphocytes2.1The positivity rate of rat B lymphocytes after MACSRat spleen lymphocytes were isolated by Ficoll density gradient centrifugation. AfterMACS, the positivity rate of B lymphocytes was92.7%（Figure4）. The results indicated that ratB lymphocytes were isolated successfully.2.2The determination of the activity of rat B lymphocytes after MACSThe activity of rat B lymphocytes was detected by Guava PCA instrument, and the cellsurvival rate was94.6%（Figure5）.3. There was no proliferation effect on resting B lymphocytes induced by β₁-AAAfter resting rat B lymphocytes were acted by0.1M pIgGs for24hours, there was nostatistical difference from the change of A value （A:0.320±0.012vs.0.309±0.011, t=0.060,P=0.524, P>0.05, Figure6）. The results indicated that β₁-AA had not affect on the quantitychange of resting B lymphocytes.4. β₁-AA promoted the proliferation of LPS-activated B lymphocytes0.1M pIgGs promoted LPS-stimulated rat B lymphocytes proliferation （A:0.739±0.036vs.0.533±0.032, P<0.05）, however,0.1M β₁-AA negative IgGs （β₁-AA-negative IgGs, nIgGs） When β₁-AA and β₁-AR-ECIIwere used at the same time compared with nIgGs,proliferation effect on rat B lymphocytes disappeared （A:0.552±0.044vs.0.560±0.048, P=0.238,P>0.05, Figure7）; β₁-AR-ECIIitself had not affect on activated B lymphocytes proliferation （A:0.575±0.052vs.0.533±0.032, P=0.078, P>0.05, Figure7）.These results suggested the proliferation of LPS-activated B lymphocytes was induced byβ₁-AA.5. The proliferation effects on LPS stimulated B lymphocytes induced by β₁-AA hadconcentration-dependent trend24hours after different concentrations of β₁-AA （0.001M,0.01M,0.1M,1M pIgGs）acting on B lymphocytes which were pre-stimulated48hours by LPS, compared with nIgGsgroup, A values were increased from0.536±0.040,0.542±0.036,0.538±0.030,0.561±0.051to0.612±0.029,0.654±0.047,0.739±0.036,0.768±0.030, respectively. The result predicted LPSstimulated rat B lymphocyte proliferation induced by β₁-AA was concentration-dependent（Figure8）.Summaryβ₁-AA promoted LPS-activated rat B lymphocytes proliferation, and the effect wasconcentration-dependent; however, β₁-AA did not promote resting B lymphocytes proliferation.Section TwoThe possible mechanism of B lymphocytes proliferation induced byβ₁-AAObjectivesTo explore the possible mechanism of of B lymphocytes proliferation induced by β₁-AA.Methods1. The acquirement of B lymphocytes and CD4⁺T lymphocytesRat spleen lymphocytes were separated by Ficoll density gradient centrifugation. Blymphocytes and CD4⁺T lymphocytes were sorted by MACS; positivity rates and viability of thetwo types of cells were detected by flow cytometry. 2. The detection of β₁-AR on the surface of B lymphocytesβ₁-AR gene expression in B lymphocytes was detected by Real-Time PCR technique, andβ₁-AR protein expression on the surface of B lymphocytes was detected by immunofluorescencemethod.3. CCK8kit for detection of B lymphocyte proliferationβ₁-AR blocker metoprolol, β₂-AR antagonist ICI118551, β₁/β₂-AR blockers Nadolol werepre-incubated to investigate whether β-AR pathways played a role in B lymphocytesproliferation induced by β₁-AA. The culture supernatant of ConA-stimulated CD4⁺Tlymphocytes acting by β₁-AA on resting B lymphocytes for24hours was to explore whetherβ₁-AA affect on B lymphocyte number by acting on CD4⁺T lymphocytes indirectly.Results1. The expression of β₁-AR on the surface of B lymphocytes1.1β₁-AR gene expression in the rat B lymphocytesThe results of the Real Time-PCR according to using the company’s synthetic β₁-AR, β₂-ARshowed there were a small amount of β₁-AR gene expression and a large number of β₂-AR geneexpression in B lymphocytes （Figure9）.1.2β₁-AR protein expression in rat B lymphocytesImmunofluorescence assay was used for the detection of β₁-AR protein expression on thesurface of B lymphocytes. The result predicted that β₁-AR was present on the membrane surfacesof B lymphocytes （Figure10）.2. β₁/β₂-AR partially mediated the proliferation effect of activated B lymphocytes inducedby β₁-AA2.1β₁-AR mediated activated B lymphocytes proliferation effect induced by β₁-AAPre-action of0.01M β₁-AR blocker Metoprolol partially blocked activated B lymphocytesproliferation induced by pIgGs （0.739±0.036vs.0.672±0.028, P=0.008, P<0.01, Figure11）, butstill higher than the PBS solvent control group of0.533+0.032（P=in0, P<0.01, Figure11）;Metoprolol itself had no effect（0.533±0.032vs.0.523±0.036, P=0.695, P>0.05, Figure12）. Theresults suggested that β₁-AA may promote the activated B lymphocytes proliferation partially byβ₁-AR on the surface of B lymphocytes.2.2β₂-AR mediated activated B lymphocytes proliferation effect induced by β₁-AA Pre-action of0.01M β₂-AR antagonist ICI118551made A value from the original0.739±0.036to0.638±0.033（P<0.01）, but still higher than the PBS solvent control group of0.533±0.032（P=0, P<0.01）; ICI118551had no effect itself （0.533±0.032vs.0.511±0.031,P=0.390, P>0.05, Figure12）. The results suggested that β₁-AA partially promoted activated Blymphocytes proliferation by the β₂-AR on the surface of B lymphocytes.2.3β₁/β₂-AR both mediated the proliferation effect on activated B lymphocytes induced byβ₁-AAPre-action of Nadolol as non-specific antagonist of β₁/β₂-AR, made the proliferation effecton activated B lymphocytes induced by β₁-AA disappeard （A:0.528±0.062vs.0.533±0.032,P>0.05）; Nadolol itself had no effect（0.533±0.032vs.0.528±0.061, P=0.855, P>0.05, Figure12）.The results suggested proliferation effect on activated B lymphocytes induced by β₁-AA may bemediated by both of β₁-AR and β₂-AR signal pathway.3. β₁-AA indirectly promoted resting B lymphocytes proliferation possibly through theactivated CD4⁺T lymphocytes3.1The preparation of Rat CD4⁺T lymphocytesRat spleen lymphocytes were isolated by Ficoll density gradient centrifugation. AfterMACS sorting, the positivity rate CD4⁺T lymphocytes was97.7%（Figure13）. The activity ofCD4⁺T lymphocytes was detected by Guava PCA instrument, which was more than90%.3.2The supernatant of activated CD4⁺T lymphocyte acted by β₁-AA promoted resting Blymphocytes proliferationAfter PBS,0.1M pIgGs,0.1M nIgGs were acted on the Con A-stimulated CD4⁺Tlymphocytes respectively for72hours, the three culture supernatant were extracted to apply toresting B lymphocytes for24hours, the results showed that A values were0.460±0.033,0.480±0.054,0.552±0.055（F=6.061, P=0.012, P<0.5）. Compared with PBS group, IgGsnegative group did not promote the proliferation effect of resting B lymphocytes （0.480±0.054vs.0.460+0.033, P=0.483, P>0.05）. Compared with group nIgGs, group pIgGs promoted restingB lymphocytes proliferation （0.55±0.055vs.0.480±0.054, P=0.02, P<0.05）（Figure14）. Theresults indicated that β₁-AA promoted the proliferation of resting B lymphocytes through actingon CD4⁺T lymphocytes. Summaryβ1-AA may promote activated B lymphocytes proliferation through β1/β2-AR on the surfaceof B lymphocytes; β1-AA may also promote the proliferation of resting B lymphocytes throughacting on the activated CD4⁺T lymphocytes.Conclusionβ1-AA promoted the proliferation of activated B lymphocytes through the β1/β2-AR on thesurface of B lymphocytes concentration-dependently, and β1-AA possibly also promoted theproliferation of resting B lymphocytes by acting on the activated CD4⁺T lymphocytes.