| BackgroundThe neuroendocrine-immune network is an important regulatory system in that maintains homeostasis throughout the body though its tight regulation and its interconnectivity between the disparate elements of the immune and neuroendocrine systems. Evidence demonstrates that changes in the neuroendocrine-immune networks that disrupt homeostasis lead to pathological processes and/or to the development of certain diseases.Sympathetic nervous is an important component of the neuroendocrine-immune network, which plays an important role in maintaining the homeostasis of the system. In physiological conditions,catecholamines released by sympathetic nerve terminals may bind to the adrenergic receptors (ARs) which are distributed to the target organ and modulate the functions of the target organ. Several studies have demonstrated that the excessive release of catecholamines closely correlates with the development and occurrence of multiple diseases. However, as the hypersympathetic state in some pathology is not induced by the excessive release of catecholamines, it suggests that other unknown factors may be involved.Since the 1990s, serum autoantibodies directed against the second extracellular loop of theβ1-adrenoceptor (β1-AR-ECII,β1-AA) has been documented in several cardiovascular disease patients such as dilated cardiomyopathy, primary electrical cardiac abnormalities, chagas disease and so on. These antibodies boundβ1-AR-ECII and mediated catecholamine-like actions, which suggested thatβ1-AA may correlate with the the hypersympathetic state through theβ1-AR.In the past ,the research about theβ1-AA is limited to its effect on the cardiovascular system, With the development of the neuroendocrine-immune network, the influence of autoantibodies on the body may be re-examined. It is repoted that theβ-AR agonist isoproterenol (ISO) can modulate the function of immune system. Previously, we documented that long-term active immunization with a synthetic peptide corresponding toβ1-AR-ECII induces cardiac structure and function and altered the CD4+/CD8+ T lymphocyte ratio in the peripheral blood, which indicated the ability ofβ1-AA to perturb immune homeostasis. However, one hand, active immunization means to make the antigenic peptides into rats and the antigenic peptides itself may directly influence the immune system, so we cannot concluded that long-term existence of theβ1-AA will make the change of the immune system. On the other hand, the serum level ofβ1-AA was much higher than the actual level in clinical patients during the long-term active immunization, so we cannot use the results to explain the clinical phenomenon. Therefore,we should establish a much better model to mimic clinical condition.T lymphocytes are one of the most important cell populations in the immune system and their proliferation is the precise to play a variety of biological functions. It has been reported that catecholamines may modulate the target cells throughβ1-AR-cAMP-PKA pathway,β2-AR-cAMP-PKA pathway,β1/β2-AR-PKC pathway and Ca2+ pathway. However, a large number of studies have shown that there is mainlyβ2-ARs subtype on the surface of T lymphocytes in the rat, in the rat lymphocyte surface there is almost noβ1-AR on it. Then, how the antibodies will affect T lymphocytes proliferation in rats? Which pathways by? What role of the Ca2+ ?The key characteristics of the T lymphocyte function depends on the type of cytokines. IL-2 and IFN-γthat are synthesised and secreted by Th1 cells reflect the cellular immune function. IL-4 which are synthesised and secreted by Th2 cells reflect the humoral immune function.So we research the state of immune function through the secretion of cytokines.It is reported that the human T Lymphocytes expressβ1-AR,β2-AR andβ3-AR on their surface. Because of almost noβ1-AR expression on T lymphocyte surface in rats, we cannot conclude that the influence ofβ1-AA to the immune system in clinical patients. Therefore, we use the T lymphocytes from human peripheral blood and observe their change of proliferation and secretory, and further analysis of its pathway.Therefore, the present study was designed to answer the following questions: (1) to establish the passive immunization rat models which was mimic clinical condition, and to explore whether theβ1-AA has a direct role in regulation of immune function during the long-term presence of it. (2) to observe the proliferation and secretory functions affects ofβ1-AA on rat T lymphocytes and explore its pathway, and analysis the differences of pathway between human T lymphocytes and rat T lymphocytes. (3) to observe the effects ofβ1-AA on human peripheral blood T lymphocytes (proliferation and secretory functions) and explore its pathway.Section One long-term presence ofβ1-AA can lead to changes of immune functionObjectiveThe passive immunization rat models were establishment by injection ofβ1-AA to observe whether long-term presence ofβ1-AA can lead to changes of immune function of body.1. Experimental SubjectsMale Wistar rats2. Methods2.1 PeptideA peptide corresponding to amino acids 197–223 of humanβ1-AR-ECII (197aa-223aa,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)) was synthesised by GL Biochem Ltd (Shanghai, China) at a purity of 95%.2.2 Acquirement ofβ1-AA①immunization group: (n=40): inject the mixture of antigen peptides and immunoadjuvant into the dermis of back at the dose of 0.4μg/g, boosting for every 2 weeks. The experiment lasts 8 weeks.②Vehicle group(n=40): inject the mixture of saline and immunoadjuvant into the dermis of back boosting for every 2 weeks. The experiment lasts 8 weeks.Detecting its valency by Streptavidin-enzyme linked immunosorbent assay (SA-ELISA). Extracting and purifyingβ1-AA serum by MAb Trap Kit. The purity of the antibody solution was detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then the protein quantification of the extract was detected with the BCA Kit.2.3 Passive immunizationβ1AAb group: According to the results of the pre-experiment, injected 2μg/g of the purifiedβ1-AA into the normal rats via the tail vein, every 2 weeks to enhance immunity, and the boosting dose should correspond to the level ofβ1-AA in the immune rats, for a whole period of 20 weeks of immunization.Negative sera group: Injected 2μg/g of negative IgG via tail vein, with the same immunization procedures and test methods.2.4 Examination of cardiac function of the ratsAfter establishment of passive immunization model, cardiac function of the rats were examined at 0w,4w,8w,12w,16w,20w. After emergence of the left ventricular waveform, using BL-410 biological signal analysis system to analyze and record the cardiac function parameters, including left ventricular systolic pressure (LVSP), left ventricular diastolic pressure (LVDP), left ventricular maximum rate of pressure rise (+dp/dtmax) and left ventricular maximum rate of pressure decline (-dp/dtmax).2.5 Isolate rat spleen lymphocytesRat spleens were aseptically removed and placed into PBS. Spleens were disassociated in a 200 mesh nylon filters, and the filtrates were collected. Hypotonic Solution was to used to destruct red blood cells, and then obtained spleen lymphocytes.2.6 Cell staining by immunofluorescence was used, and CD4+/CD8+T-lymphocyte ratio is determined by flow cytometryPeripheral blood (1×106/mL) 100μL was added FITC labeled CD4 antibody and PE labeled CD8 antibody, while added FITC or PE labeled IgG1 monoclonal antibody as negative control. Then incubated at room temperature 20min, washed 3 times with PBS; and resuspended cells to measure the T lymphocyte CD4+ / CD8+ by flow cytometry.2.7 Plaque forming cell (PFC) assaysThe primary in vivo antibody response to SRBC was determined using a modified hemolytic plaque assay. Four days before sacrifice, rats were injected intraperitoneally with 1mL of 20% (v/v) sheep red blood cells (SRBC) in saline. On the day of sacrifice, spleens were aseptically removed. Single spleen cell suspensions were prepared and an aliquot of them were assayed for the number of antibody secreting cells in both direct and indirect plaque forming cell (PFC) assays.2.8 HE staining of heart, liver, kidney of passive immunization ratsDeparaffinize with xylene; dehydrated by alcohol, washed briefly in distilled water; stain in Harris hematoxylin solution for 5min, counterstain in eosin-phloxine for 2min; routine dehydration, transparency, mounted with gum, then observed th heart, liver, kidney pathology slice under light microscope.Results1 Preparation ofβ1-AA1.1 The active immunized rat models were set up successfullySA-ELISA results showed that the first 2 weeks after immunization, the level ofβ1-AA in the immunized group has already raised from 0.41±0.05 to 0.73±0.06 (P <0.05); and reached the peak at 8 weeks (3.02±0.09 vs. 0.40±0.02, P<0.01). While the level ofβ1-AA has no significant change in vehicle group (P> 0.05) (Figure. 1-1). The results suggested that the active immunization model was set up successfully.1.2 Purification, quantitative and qualitative ofβ1-AA IgGsIgGs in the sera were purified by affinity chromatography. 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 (Figure.1-2). The concentration of purified IgGs was 8 mg/mL, which was determined by BCA kits.2 The passive immunized rat models were established successfully2.1 The level ofβ1-AA in rats during the process of passive immunization could be maintained.Before passive immunization, the OD value was 0.08±0.030 in rats'sera. 4 weeks after passive immunization, the sera level ofβ1-AA in the immunized group was significantly higher than that in the control group (OD value:0.22±0.029 vs. 0.09±0.04,P<0.01). Then the sera level ofβ1-AA persisted at stable level. It was still higher than that in control group (P <0.01, Figure. 1-3). 2.2 The change of the cardiac function in the passive immunized rat8 weeks after passive immunization, LVSP and +dp / dtmax in immunized group had been higher than that in the control group, while the LVEDP and -dp/dtmax did not change significantly (P>0.05). Passive immunization with the time, the cardiac function declined in immunized group. 20 weeks after passive immunization, LVSP , +dp / dtmax and -dp/dtmax in immunized group declined significantly, However, LVEDP increased significantly (Figure. 1-4A, B, C, D). These results indicate decline in cardiac function.3 long-term presence ofβ1-AA could lead to changes of immune function3.1 The ratios of CD4+/CD8+ T lymphocytes increased in passive immunization of rats withβ1-AA4 weeks after immunization, the ratios of CD4+/CD8+ T lymphocytes had been increased , but there was no significant difference between theβ1-AA immune group and the control group. It was significantly higher in theβ1-AA group at 8th week (2.92±0.39 vs. 2.00±0.45,P<0.05), and it continuous increase to 20 weeks with more notable difference (3.49±0.42 vs. 1.89±0.40,P<0.01) (Figure. 1-5).This result indicates that long-standing ofβ1-AA in vivo leads to cellular immune function disturbance in the body.3.2 the ability of antibody production had been enhanced during long-term presence ofβ1-AAThe number of rat spleen AFC in direct and in indireact responses to immunization with sRBC were conspicuous increased at 8 weeks after passive immunization (P<0.05). Meanwhile, the difference is more obviously untill 20 weeks after passive immunization (P<0.01). (Figure. 1-6A, B)The result showed us that the ability of antibody production had been enhanced during long-term presence ofβ1-AA3.3 Long-term existence ofβ1-AA could result in the mononuclear cell infiltration of heart, liver and kindney by HE staining20 weeks after immunization, HE staining of myocardial tissue showed: there were immune inflammatory cells infiltration in the myocardial cells ofβ1-AA group, while the vehicle group without such changes in myocardial tissue(Figure. 1-7); HE colouration of liver in the rats displayed a large amount of mononuclear cell collected at the edge of the liver (Figure. 1-8); HE coloration of the kidney displayed that a lot of mononuclear cell infiltration could be seen around the renal glomerulus in rats (Figure. 1-9), no obvious changes were observed in the vehicle group. Summary1. Long-term passive immunization with purifiedβ1-AA could establish the animal model which matched with clinical patient .2. Long-term presence ofβ1-AA could cause dysfunction of cellular immunity.3. Long–term presence ofβ1-AA could enhance the ability of antibody production.Section Two Proliferation and Secretion Effects of Autoantibodies Against the Second Extracellular Loop ofβ1-Adrenoceptor on Rat T Lymphocytes throughβ2-ARobjective1.To observe the proliferation and secretion effects ofβ1-AA on rat T lymophoctes, and to explore its pathway.2. To analysis the role of calcium in proliferation effect ofβ1-AA on rat T LymphocytesMaterials and Methods1. Experimental SubjectsMale Wistar rats2. Methods2.1 Isolate CD3+ rat T lymphocytesRat spleens were aseptically removed and placed into PBS. Spleens were disassociated in a 200 mesh nylon filters, and the filtrates were collected. Hypotonic Solution was to used to destruct red blood cells, and then obtained spleen lymphocytes. The CD3+ T lymphocytes were isolated by immunomagnetic mircobeads. The positive rate of the CD3+ T lymphocytes was detected by flow cytometry technology, and the cell activity was measured by Guava PCA CytoSoft6.0.2 software.2.2 Detection expression ofβ1-AR on the rat lymphocytes by immunofluorescenceThe separated T lymphocytes on smear were fixed with 4% paraformaldehyde at room temperature 20min; After washing three times with PBS; 3) the slides were incubated with 10% bovine serum albumin for 2 h at 37°C, the blocking serum was washed, then added specific primary antibody and incubated overnight at 4°C. After washing with PBS three times, the slides were covered with the fluorescein isothiocyanate (FITC)-labelled secondary antibody and incubated 37°C for half an hour in the dark, then removed the second antibody, and added DAPI staining solution at room temperature over 15min. After washing three times with PBS , examined through confocal laser scanning microscopy.2.3 Experimental groups2.3.1 The proliferation effect ofβ1-AA on human T lymphocytes was observed , and the groups were designed as follows(1) Vehicle group: separated rat lymphocytes + saline(2)β1-AA group: separated rat lymphocytes +β1-AA2.3.2 According to the activation characteristics of T lymphocytes, the groups were designed 2 groups(1) Vehicle group: separated rat T lymphocytes + saline(2) ConA group: to stimulate T lymphocytes with 5μg/mL Concanavalin (ConA),. After 72 h, T lymphocytes were transferred into 96-well plates (100μL/well). Next, the cells were subjected to different treatments for 48 h. The groups were designed as follows:2.3.3 In order to observe the proliferation effect ofβ1-AA on ConA activated T lymphocytes, the groups were divided into 5(1) ConA group (saline);(2)β1-AA negative serum IgGs group (0.1μmol / L);(3) Different concentrations ofβ-AR agonist isoproterenol group (ISO, 0.01μmol / L, 0.1μmol / L, 1μmol / L);(4) Different concentrations ofβ1-AA group (0.01μmol / L, 0.1μmol / L, 1μmol / L);(5)β1-AA (0.1μmol / L) +β1-AR antigen peptide group (3μmol / L);2.3.4 To observe the different interventions on the proliferation effect ofβ1-AA, the groups were designed as follows(1)β1-AA (0.1μmol / L) +β1-AR blocker (Atenolol) group (1μmol / L);(2)β1-AA (0.1μmol / L) +β2-AR antagonist (ICI118, 551) group (1μmol / L);(3)β1-AA (0.1μmol / L) +β1/β2-AR blocker (Nadolol) group (1μmol / L);(4)β1-AA (0.1μmol / L) +β-AR blocker (Propranolol) group (1μmol / L);(5)β1-AA (0.1μmol / L) + PKA inhibitor (H-89) (1μmol / L); (6)β1-AA (0.1μmol / L) + PKC inhibitor (Chelerythrine Chloride) group (1μmol / L);(7)β1-AA (0.1μmol / L) + PKA inhibitor (H-89) (1μmol / L) + PKC inhibitor (Chelerythrine Chloride) (1μmol / L);(8)β1-AA (0.1μmol / L) + L-Ca channel blocker verapamil (1μmol / L);(9)β1-AA (0.1μmol / L) + IP3 receptor antagonist heparin (1μmol / L);(10)β1-AA (0.1μmol / L) + Ca2+ release-activated Ca2+ channels (CRAC) blocker SKF96365 (1μmol / L);2.3.5 In order to observe different blockers on T lymphocyte proliferation, the groups were divided into 6 groups(1)β1-AR blocker (Atenolol) group (1μmol / L);(2)β2-AR blocker (ICI118, 551) group (1μmol / L);(3)β1/β2-AR blocker (Nadolol) group (1μmol / L);(4)β-AR blocker (Bupranolol) group (1μmol / L);(5) PKA inhibitor (H-89) group (1μmol / L);(6) PKC inhibitor (Chelerythrine) group (1μmol / L);2.4 CCK-8 method was used to detect the activity levels of rat T lymphocytesCells were cultured in 96-well plates and stimulated by different factors for 48 hours. A volume of 10μL of CCK-8 solution was added to each well, and cells were incubated for 2 hours at 37°C. The amount of formazan dye generated by cellular dehydrogenase activity was measured by absorbance at 450 nm with a microplate fluorometer. Repeat 8 times each group.2.5 ELISA kits were used to detect the levels of cAMP and the level of IL-2,IFN-γ,IL-4Dispense 100μL of standards and specimens into appropiate wells, incubated at 37℃for 60-90min; wash 4 times with washing buffer; dispense Enzyme Conjugate Reagent into each well, incubated at 37℃for 60min; wash 4 times with washing buffer; then add substrate working solution and stop solution respectively to each well. Using a microtiter plate reader, read the optical density at 450nm.2.6 Detection of intracellular free calcium through confocal laser scanning microscopy in rat T lymphocyteIsolated rat T lymphocytes, the cells were loaded the fluorescent indicator Fluo-3/AM (3μmol / L), and incubated for 30min at 37°C in the dark. After washing three times with PBS, analyzed the results with the image analysis software LSM510 and used Fluorescence intensity (F value) to indicate the level of cytosolic calcium.Results1 Rat CD3+ T lymphocytes were separated successfullyThe purity of selected rat CD3+ T lymphocytes by immunomagnetic separation was 92.2% (Figure. 2-1), and the viability was more than 95 %. Cell morphology was uniform under light microscope, suggesting that T lymphocytes successful separation (Figure. 2-2,2-3).2 Expression ofβ1-ARs had not been detected on rat T lymphocytes.To further verify whether the expression of rat T lymphocytesβ1-AR, we used immunofluorescence techniques to identify: DAPI staining of lymphocytes nuclear was blue, the negative control group (Figure. 2-4A) and experimental group (Figure. 2-4B) did not detect green fluorescence (FITC-labelled secondary antibody). The result suggested that there was noβ1- AR expression on rat T lymphocytes.3β1-AA had no effect on rat T lymphocytes without ConA stimulationIn addition to containing a large number of T (90% -95%) in separated lymphocytes, it contained small amounts of other monocytes.We found thatβ1-AA had no proliferation effects on these cells (P> 0.05) (Figure. 2-5).4 ConA can promote the proliferation of rat T lymphocyteAfter adding ConA to the cultured rat T lymphocytes, the cell activity was significantly higher than that of the control group (OD value, 0.38±0.04 vs. 0.18±0.05, P<0.01) (Figure. 2-6).5β1-AA promoted the proliferation of ConA-stimulated T lymphocytes in a dose-dependent mannerDifferent concentrations ofβ1-AA (0.01, 0.1, 1μM) promoted ConA-stimulated T lymphocyte proliferation in a concentration-dependent manner(OD value, 0.50±0.04 vs. 0.37±0.04, P<0.01; 0.61±0.02 vs. 0.38±0.06, P<0.01; 0.68±0.05 vs. 0.42±0.04, P<0.01) (Figure.2-7). However, the IgG antibodies extracted from the serum of the vehicle group rats did not enhance lymphocyte proliferation (P>0.05). The proliferation effect ofβ1-AA was inhibited by the addition of antigenic peptides corresponding toβ1-AR-ECII (Figure. 2-8).6 The possible mechanism of the proliferation effects ofβ1-AA on rat T lymphocytesWe found that the proliferation effect ofβ1-AA on rat T lymphocytes wasn't inhibited by Atenolol, but the effect could be blocked completely by ICI118,551, Nadolol and Bupranolol. Alone, Atenolol, ICI118,551, Nadolol and Bupranolol had no effect on ConA-stimulated T lymphocytes proliferation (P>0.05) (Figure. 2-9A, B). Meanwhile,β1-AA could enhance the level of cAMP, and the elevated cAMP couldn't be inhibited by Atenolol, but could be inhibited by ICI118, 551, Nadolol and Bupranolol (Figure. 2-10). Further research found that PKA inhibitor H-89 or PKC inhibitor chelerythrine couldn't inhibite completely the effect, but H-89 and chelerythrine working together could completely inhibit theβ-AA-induced proliferation of T lymphocytes ( Figure. 2-11).The result suggested thatβ2-AR-cAMP-PKA/β2-AR-PKC pathway may be involved inβ1-AA on human T lymphocytes proliferation. 7 the role of calcium in proliferation effect ofβ1-AA on rat T Lymphocytes 7.1β1-AA could increase intracellular calcium of rat T lymphocytesAfter additonβ1-AA to the ConA stimulated T lymphocytes, and we found that the average fluorescence intensity increased from 200±8.5 increased to 628±25 (P <0.01). However, this change could be inhibited by ICI118,551(P> 0.05) (Figure. 2-12, 2-13).The results suggest thatβ1-AA could increased T lymphocytes'intracellular calcium levels through activation ofβ2-AR.7.2 Different calcium channel blockers could inhibit the proliferation effect ofβ1-AA on rat T Lymphocytes in different degreeIn order to study of the role of calcium in proliferation effect ofβ1-AA on rat T Lymphocytes, we observed different calcium channel blockers (verapamil, heparin and SKF96365) on proliferation effect ofβ1-AA on rat T Lymphocytes, and found that they all could inhibit the proliferation effect in different degree(Figure. 2-14).8β1-AA promoted rat T lymphocyte IL-2, IFN-γand IL-4 secretionDifferent concentrations ofβ1-AA (0.01, 0.1, 1μM) could increase the secretion of IL-2, IFN-γand IL-4 a concentration-dependent manner (Figure. 2-15A,2-16A,2-17A). Further, these cytokines secretion were completely blocked by ICI118, 551, Bupranolol and Nadolol (Figure. 2-15B,2-16B,2-17B).Summary1.β1-AA could promote ConA-stimulated rat T-lymphocyte proliferation in a dose-dependent manner throughβ2-AR-cAMP-PKA/β2-AR-PKC pathways.2.β1-AA could promote ConA-stimulated rat T-lymphocyte proliferation through increasing intracellular calcium level.3.β1-AA could promote ConA-stimulated rat T-lymphocyte IL-2, IFN-γ, IL-4 secretion.Section Three Proliferation and Secretion Effects of Autoantibodies against the Second Extracellular Loop ofβ1-Adrenoceptor on Human T LymphocytesObjectiveTo observe the proliferation and secretion effects ofβ1-AA on human T lymophoctes, and explore its pathway.Materials and Methods1. Experimental Subjectshealth adult peripheral blood2.Methods2.1 Separation and identification of CD3+ T lymphocytes in human peripheral bloodPeripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation, and CD3+ T lymphocytes were separated by immunomagnetic mircobeads.2.2 Experimental groups2.2.1 The proliferation effect ofβ1-AA on human T lymphocytes was observed , and the groups were designed as follows(1) Control group: separated huaman T lymphocytes + saline(2)β1-AA group: separated huaman T lymphocytes +β1-AA2.2.2 According to the activation characteristics of T lymphocytes, the groups were designed 2 groups(1) Control group(2) ConA group: to stimulate T lymphocytes with 5μg/mL of Concanavalin (ConA). After 72 h, T lymphocytes were transferred into 96-well plates (100μL/well). Next, the cells were subjected to different treatments for 48 h. The groups were designed as follows:2.2.3 In order to observe the proliferation effect ofβ1-AA on ConA activated T lymphocytes, the groups were divided into 5(1) ConA group (saline);(2)β1-AA negative serum IgGs group (0.1μmol / L);(3) Different concentrations ofβ-AR agonist isoproterenol group (ISO, 0.01μmol / L, 0.1μmol / L, 1μmol / L);(4) Different concentrations ofβ1-AA group (0.01μmol / L, 0.1μmol / L, 1μmol / L);(5)β1-AA (0.1μmol / L) +β1-AR antigen peptide group (3μmol / L);2.2.4 To observe the different interventions on the proliferation effect ofβ1-AA, the groups were designed as follows(1)β1-AA (0.1μmol / L) +β1-AR blocker (Atenolol) group (1μmol / L);(2)β1-AA (0.1μmol / L) +β2-AR antagonist (ICI118, 551) group (1μmol / L);(3)β1-AA (0.1μmol / L) +β1/β2-AR blocker (Nadolol) group (1μmol / L);(4)β1-AA (0.1μmol / L) +β-AR blocker (Propranolol) group (1μmol / L);(5)β1-AA (0.1μmol / L) + PKA inhibitor (H-89) (1μmol / L);(6)β1-AA (0.1μmol / L) + PKC inhibitor (Chelerythrine Chloride) group (1μmol / L);(7)β1-AA (0.1μmol / L) + PKA inhibitor (H-89) (1μmol / L) + PKC inhibitor (Chelerythrine Chloride) (1μmol / L);2.2.5 In order to observe different blockers on T lymphocyte proliferation, the groups were divided into 6 groups(1)β1-AR blocker (Atenolol) group (1μmol / L);(2)β2-AR antagonist (ICI118, 551) group (1μmol / L);(3)β1/β2-AR blocker (Nadolol) group (1μmol / L);(4)β-AR blocker (Bupranolol) group (1μmol / L);(5) PKA inhibitor (H-89) group (1μmol / L);(6) PKC inhibitor (Chelerythrine) group (1μmol / L);2.3 CCK-8 method was used to detect the activity levels of rat human lymphocytesCCK-8 method was same with the section two. 2.4 ELISA kits were used to detect the levels of cAMP and the level of IL-2,IFN-γ,IL-4The method was same with the section two.Results1β1-AA had no effect on human T lymphocytes without ConA stimulationIn addition to containing a large number of T (90% -95%) in separated lymphocytes, it contained small amounts of other monocytes. In order to detect the role ofβ1-AA on these cells , we added the antibody to the separated cell, and found thatβ1-AA had no proliferation effects on these cells (P> 0.05) (Figure. 3-1).2 ConA can promote the proliferation of human peripheral blood T lymphocyteThe cell activity was significantly higher than that of the control group (OD value, 0.38±0.06 vs. 0.13±0.04, P<0.01) (Figure. 3-2), which indicated that Con A could be successfully activated T lymphocytes3β1-AA could promote ConA stimulated proliferation of human peripheral blood T lymphocytes3.1β1-AA promoted the proliferation of ConA-stimulated T lymphocytes in a dose-dependent mannerDifferent concentrations ofβ1-AA (0.01, 0.1, 1μM) promoted ConA-stimulated T lymphocyte proliferation in a concentration-dependent manner (OD value: 0.01: 0.47±0.05 vs. 0.39±0.04, P <0.05; 0.1: 0.61±0.04 vs. 0.40±0.03, P <0.01; 1: 0.67±0.05 vs. 0.42±0.04, P <0.01) (Figure.3-3). However, the IgG antibodies extracted from the serum of the vehicle group rats did not enhance lymphocyte proliferation. The proliferation effect ofβ1-AA was inhibited by the addition of antigenic peptides corresponding toβ1-AR-ECII (Figure 3-4).These results suggest thatβ1-AA could promote the proliferation of ConA activated human peripheral blood T lymphocyte.3.2 The possible mechanism of the proliferation effects ofβ1-AA on human T lymphocytes3.2.1β1-AA promoted the proliferation of ConA-stimulated T lymphocytes byβ1-AR-cAMP-PKA pathwayTo investigate whether theβ1-AA on the T lymphocyte proliferation was through theβ1-AR-cAMP-PKA pathway, the study conducted the following experiment.T lymphocytes activated byβ1-AA were pretreated with theβ1-AR antagonist Atenolol and theβ-AR antagonist Bupranolol, respectively. The proliferation effect ofβ1-AA on lymphocytes was partially inhibited by Atenolol, but the effect can be blocked completely by non-specific β-AR antagonist Bupranolol (Figure. 3-5A). Alone, the atenolol and Bupranolol had no effect on ConA-stimulated T lymphocytes proliferation (P>0.05) (Figure. 3-5B). At the same time,β1-AA could enhance the level of cAMP (300±7.5pmol/mL vs. ConA group 120±6.8 pmol/mL, P<0.01), and the elevated cAMP couldn't be inhibited by Atenolol, but could be inhibited by Bupranolol (Figure. 3-6). Further research found that PKA inhibitor H-89 couldn't inhibite completely the effect (Figure. 3-7A); the H-89 alone had no effect on the proliferation of T lymphocytes (P> 0.05) (Figure. 3-7B).The results suggest that there may be other pathways involved in proliferation effects ofβ1-AA on human T lymphocytes besidesβ1-AR-cAMP-PKA pathway. 4.2.2β1-AA promoted the proliferation of ConA-stimulated T lymphocytes byβ2-AR-cAMP-PKA pathwayTo further analyze the pathway ofβ1-AA on human peripheral blood T lymphocyte proliferation, T lymphocytes activated byβ1-AA were pretreated with theβ2-AR antagonist ICI118,551 and theβ1/β2-AR antagonist Nadolol, respectively. We found that the proliferation effect ofβ1-AA on lymphocytes was partially blocked by ICI118,551, but the effect can be blocked completely by Nadolol (Figure. 3-8A). These antagonists had no effect on T lymphocyte proliferation (Figure. 3-8B). Further, elevated cAMP levels could't be inhibited completely by ICI118, 551, but could be inhibited completely by Nadolol (P>0.05) (Figure. 3 -9).The results suggest thatβ2-AR-cAMP-PKA pathway may be involved inβ1-AA on human T lymphocytes proliferation. 4.2.3β1/β2-AR-PKC pathway were involved inβ1-AA on human T lymphocytes proliferationIn view of the fact thatβ1-AA on human T lymphocytes proliferation could be partially inhibited by H-89, we selected the PKC inhibitor chelerythrine to treat human T lymphocytes activated byβ1-AA, and we found that the proliferatin effect was also partially inhibited theβ1- AA stimulated the proliferation of T lymphocytes. Further, chelerythrine and H-89 could completely inhibited this effect (Figure. 3-10).Takeing into account that PKC could be actived byβ1/β2-AR, whether PKC originated fromβ1- orβ2-AR activation during the process ofβ1-AA enhancing human T lymphocytes proliferation? We performed the following experiment. At first, we blockedβ2-AR receptor pathway (ICI118, 551), then added PKA inhibitor H-89, and found that the proliferation effect could be partially blocked. Further, we continued to add PKC inhibitor and found the effect could be completely blocked (Figure. 3-11). Secondly, we blockedβ1-AR receptor pathway (Atenolol), and then joined the H-89, also found that the proliferation effect couldn't be completely blocked, while adding chelerythrine, its proliferation could be completely blocked (Figure. 3-12).The results suggest thatβ1/β2-AR-PKC pathway were involved inβ1-AA on human T lymphocytes proliferation.5β1-AA promoted human T lymphocyte IL-2, IFN-γand IL-4 secretionT lymphocyte function depends on the type of cytokine secretion, the study examined the IL-2, IFN-γand IL-4 secretion levels to reflect the body's cellular and humoral immune function. Different concentrations ofβ1-AA (0.01, 0.1, 1μM) could increase the secretion of IL-2, IFN-γand IL-4 a concentration-dependent manner (Figure. 3-13A ,3-14A ,3-15A). However, IgG antibodies purified from the serum of rats in the negative control group did not promote IL-2, IFN-γand IL-4secretion. Additionally, the enhanced IL-2, IFN-γand IL-4 secretion observed followingβ1-AA treatment was inhibited by preincubation with antigenic peptides that correspond toβ1-AR-ECII(Figure. 3-13B ,3-14B ,3-15B). Further, these cytokines secretion were partially blocked by Atenolol and ICI118, 551, and completely blocked by Bupranolol and Nadolol (Figure. 3-13C ,3-14C ,3-15C).Summary1.β1-AA could promote ConA-stimulated human T-lymphocyte proliferation in a dos...
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