β1-Adrenoceptor Autoantibodies from DCM Patients Enhance the Proliferation of T Lymphocytes through the β1-AR/cAMP/PKA and p38 MAPK Pathways

Background Autoantibodies against the second extracellular loop of the β1-adrenergic receptor (β1-AA) not only contribute to increased susceptibility to heart failure, but also play a causative role in myocardial remodeling through their sympathomimetic-like effects that are induced upon binding to the β1-adrenergic receptor. However, their role in the function of T lymphocytes has never been previously investigated. Our present study was designed to determine whether β1-AA isolated from the sera of dilated cardiomyopathy (DCM) patients caused the proliferation of T cells and the secretion of cytokines. Methods Blood samples were collected from 95 DCM patients as well as 95 healthy subjects, and β1-AA was detected using ELISA. The CD3+T lymphocytes were selected separately through flow cytometry and the effect of β1-AA on T lymphocyte proliferation was examined by CCK-8 kits and CFSE assay. Western blotting was used to analyze the expressions of phospho-VASP and phospho-p38 MAPK. Results β1-AA enhanced the proliferation of T lymphocytes. This effect could be blocked by the selective β1-adrenergic receptor antagonist metoprolol, PKA inhibitor H89, and p38 MAPK inhibitor SB203580. Furthermore, the expression of the phosphorylated forms of phospho-VASP and phospho-p38 MAPK were markedly increased in the presence of β1-AA. β1-AA also inhibited the secretion of interferon-γ (IFN-γ) while promoting an increase in interleukin-4 (IL-4) levels. Conclusions These results demonstrate that β1-AA isolated from DCM patients binds to β1-AR on the surface of T cells, causing changes in T-cell proliferation and secretion through the β1-AR/cAMP/PKA and p38 MAPK pathways.


Introduction
Dilated cardiomyopathy (DCM), a heart condition characterized by left ventricular dilation and progressive loss of cardiac function, represents the main cause of severe heart failure in younger adults and thus is a challenge for public health [1]. About one-third of DCM cases are genetic in origin, whereas the etiology of the remaining 70% is poorly understood [2]. Autoimmune responses against various myocardial antigens have been proposed to play an important role in the triggering or progression of DCM [3][4]. However, the mechanisms involved in its pathological process have not been elucidated.
Recent studies have reported that the activation of T lymphocytes and an increase in inflammatory cytokines are involved in chronic heart failure due to DCM [5,6]. A role for altered T cell proliferation was indicated by our previous studies which reported elevated ratios of CD4 + /CD8 + T lymphocytes during heart failure in rats [7]. These researches suggest that some certain elements may contribute to T lymphocyte disorder in the pathogenesis of chronic heart failure.
Evidences suggest that antigens newly exposed to the immune system upon cardiac damage trigger a myocardial autoimmune response, leading to ventricular remodeling and further damage to the myocardium [8]. In the 1990s, investigators reported that the autoimmune antibody (b 1 -AA) against the second extracellular loop (b 1 -AR-EC II amino acid residues 197-223, 100% sequence identity between humans and rats [9]) of the b 1 -adrenergic receptor is present in the sera of patients with cardiovascular diseases [10][11][12][13]. This led to the proposal stating that b 1 -AA acts similarly to a b 1 -adrenergic receptor agonist based on findings of an increased beating rate in neonatal rat cardiomyocytes [14]. We have reported that the long-term presence of b 1 -AA led to myocardial damage with increased ratios of CD4 + /CD8 + T lymphocytes [15]. These clinical and experimental results, taken together with the expression of b 1 -AR on the surface of T lymphocytes [16], strongly suggest that b 1 -AA might mediate T cell abnormalities in chronic heart failure patients. However, it is not known if the b 1 -AA isolated from DCM patients, which mimics the action of catecholamine, could recognize the cognate receptor and interfere with T lymphocytes.
Therefore, the aims of the current study were as follows: (1) to observe the effects of b 1 -AA on the proliferation and secretion of T cells, and (2) to identify the signaling pathways that mediate T cell responses.

Animals
Healthy male 8-week-old Sprague-Dawley rats, with normal blood pressure and heart rate, weighing 200 g to 240 g, were selected for this study. The experimental procedures were conducted in adherence to the ''Guiding Principles in the Use and Care of Animals'' published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996), the Guide for the Care and Use of Laboratory Animals protocol, published by the Ministry of the People's Republic of China (issued on 3 June 2004), and approved by the Institutional Animal Care and Use Committee of Capital Medical University.

Patients and Samples
The study adheres to the principles of the Declaration of Helsinki and Title 45, U.S. Code of Federal Regulations, Part 46, Protection of Human Subjects, Revised 13 November 2001, effective 13 December 2001. Ninety-five chronic heart failure patients were recruited from the Air Force General Hospital of the People's Liberation Army and General Hospital of Tonghua Mining Group Co., LTD, all of which were suffering from dilated cardiomyopathy (DCM) (New York Heart Association functional class II to IV), with a left ventricular diastolic volume .110 ml/m 2 and an ejection fraction ,45% (by echocardiography). DCM was diagnosed when coronary heart diseases were excluded by angiography and exposures to cardiotoxic substances, myocarditis, or other systemic heart diseases were not evident from clinical history. In ventriculography, all patients exhibited a diffuse reduction in wall motion. At the time of sample acquisition, all patients were stable under therapy with diuretics, ACE inhibitors, digitalis, and nitrates. The control group consisted of 95 healthy subjects randomly selected from the same community with normal clinical, ECG, and echocardiography examinations. On the basis of the resulting measurements of b 1 -AA, the DCM patients were divided into a b 1 -AA-positive group (n = 44) and a b 1 -AA-negative group (n = 51). Clinical characteristics are summarized in Table 1 and Table 2. Venous blood samples were collected without an anticoagulant. After centrifugation at 4uC, the serum was immediately separated and stored at 280uC until assay.
The Institutional Committee for the Protection of Human Subjects of Capital Medical University approved this research protocol. All patients were informed of the purpose and protocol of the investigational nature of the study. Both oral informed consent and written consent were obtained.

Peptide Synthesis
The peptide corresponding to the sequence (amino acid residues 197-223) of the second extracellular loop of the human b 1 -AR [15]: T-N-R-C was synthesized using an automated peptide synthesizer by solid-phase methods. Peptide purity was judged by highperformance liquid chromatography (HPLC) using an automated amino-acid analyzer. Peptide preparations were 98% pure as judged by analytical HPLC. This work was performed by a contractor (Qiang Yao, Shanghai Bio Scientific Commercial Development Co. Ltd., China).

Enzyme-linked Immunosorbent Assay (ELISA)
The titer of b 1 -AA was measured by ELISA, and the results are expressed as optical-density (OD) units according to published methods [17]. Briefly, the synthetic peptide described above (5 mg/ml) in 100 mmol/l Na 2 CO 3 (pH 11.0), was coated onto the wells of microtiter plates overnight at 4uC. The wells were then saturated with 0.1% PMT buffer [0.1% (w/v) albumin bovine V, Table 1. Clinical characteristics of patients with chronic heart failure due to DCM (mean 6 SD).  The OD values were measured at 405 nm using a microplate reader (Spectra Max Plus, Molecular Devices, USA). We also calculated antibody titer according to the ratio (P/N) of OD values of positive/negative controls [(specimen OD-blank control OD)/ (negative control OD-blank control OD)] [7]. Control samples were prepared as follows: 95 sera samples from healthy humans with an OD value of less than 2.5 times the background OD were pooled and centrifuged at 1,500 rpm for 10 min, and the supernatants were then divided into small aliquots and stored for subsequent use. Samples positive or negative for b 1 -AA were defined as P/N $2.1 or P/N #1.5, respectively.

Preparation of Immunoglobulin G
Immunoglobulin G fractions (IgG) from the sera of 44 b 1 -AApositive or from 51 b 1 -AA-negative DCM patients were prepared by MabTrap Kit (Amersham Bioscience, Sweden). The concentrations (mg/ml) and specificities of purified IgGs were determined by the Bicinchoninic Acid (BCA) Protein Assay (Pierce, USA) and ELISA, respectively.

Isolation and Culture of CD3 + T Cells
Rats were anesthetized with ether, and a blood sample was taken from the abdominal aorta. Mononuclear cells were prepared from the freshly drawn blood samples by Ficoll-Hypaque (1.077 g/l) density gradients. Whole blood (40 ml) was collected with heparin and centrifuged. After the plasma was discarded, white cells and erythrocytes were taken and suspended in 10 ml of PBS (pH 7.4). These suspensions were added to 5 ml of Ficoll-Hypaque and then centrifuged at 2,000 rpm for 20 min. Mononuclear cells were collected, washed twice with PBS, and centrifuged at 1,500 rpm for 10 min. To eliminate adherent cells (monocytes), cell suspensions were placed into culture flasks with 5 ml of Roswell Park Memorial Institute medium 1640 (RPMI) with gentamicin (100 mg/ml), L-glutamine (2 mmol/l), and 10% fetal calf serum (Invitrogen, USA). Cells were incubated at 37uC in humidified air containing 5% CO 2 for 30 min. Nonadherent cells were collected, washed, and isolated by centrifugation at 1,500 rpm for 10 min and suspended in culture medium. CD3 + T cells were selected from the mononuclear cells using a flow cytometer (BD Biosciences, USA).

Immunofluorescence Staining
CD3 + T cells were gently washed with PBS (pH 7.4) and immediately fixed with 4% paraformaldehyde (w/v) for 20 min. Cells were blocked in PBS containing 5% bovine serum albumin (BSA) (w/v). The cells were then incubated overnight at 4uC with the IgG fractions (25 mg/ml) from b 1 -AA-positive DCM patients at a 1:500 dilution. Following three PBS washes, cells were incubated in donkey anti-human IgG tagged with fluorescein isothiocyanate (FITC) as the secondary antibody for 1 hour in the dark at 37uC. After being rinsed with PBS, cover slips with mounting medium containing 49, 6-diamidino-2-phenylindole (DAPI) stain nuclei were coated. Negative controls were performed by omitting primary antibodies. Images were acquired using a Zeiss 510 Meta Confocal microscope (63 power oil 1.40 NA (Zeiss, Germany), pinhole equals 1.0 Airy Disc) using the Carl Zeiss Imaging software.

Culture of Beating Neonatal Cardiomyocytes
Hearts were removed aseptically from 1 to 2-day-old Sprague-Dawley rats and cultured as described [7]. The number of beats of a selected isolated myocardial cell or a cluster of synchronously contracting cells in each of 10 fields was counted for 15 s each. The IgG fractions from b 1 -AA-positive DCM patients and corresponding receptor agonists were added, and the cells were observed for 5 min after each addition. This procedure was repeated three times in different cultures to yield results representing a total of 30 cells or cell clusters. The basal beating rate was 145615 beats/min. CD3 + T Cell Proliferation Assays 1. CCK-8 assay. CD3 + T cells (5610 5 cells/ml) were cultured for 48 h with or without mitogens in either the presence or absence of b 1 -AA (12.5 mg/ml, 25 mg/ml, and 50 mg/ml), H89 (1 mmol/l), metoprolol (1 mmol/l), isoproterenol (0.1 mmol/l), and SB203580 (1 mmol/l). The mitogens used were 3 mg/ml soluble mouse anti-rat CD3 mAb (Clone 1F4, Biolegend, USA) and 1 mg/ ml soluble mouse anti-rat CD28 mAb (Clone JJ319, Biolegend).
Agonists were added to cell suspensions together with the mitogens while the antagonists were added 1 h before the agonists. After each treatment, 10 ml CCK-8 solution was added to each well, and the cells were incubated for 4 hours at 37uC. The absorbance at 450 nm was measured using a microplate reader with the wavelength correction set to 630 nm.
2. CFSE-labeling of lymphocytes. CFSE (10 mmol/l in DMSO (Invitrogen) was diluted in PBS. CD3 + T lymphocytes were suspended in PBS supplemented with 0.05% BSA and 4 mmol/l CFSE (2610 7 cells/ml) for 10 min at 37uC in a 5% CO 2 atmosphere. Cells were washed, diluted in 0.5 ml culture medium and incubated for 30 min at 37uC in 5% CO 2 to stabilize the CFSE-labeling. The efficiency of labeling untreated cells was .95%.

Analysis of cAMP Production
T lymphocytes were washed twice in Tris buffer containing 120 mmol/l NaCl, 1 mmol/l MgCl 2 , 5 mmol/l KCl, 0.6 mmol/l CaCl 2 , 25 mmol/l Tris (hydroxymethyl-amino-ethane), 5 mmol/l glucose, and 0.1 mmol/l human albumin, adjusted to pH 7.4 with HCl. Cells were suspended in RPMI 1640/FBS medium to a final density of 2610 6 cells/ml. The samples were incubated with 1methyl-3-isobutylxanthine (0.5 mmol/l) for 10 min and then stimulated for 10 min with anti-rat CD3/CD28 mAb in either the absence or presence of the b 1 -AA or agonist, with or without an antagonist. Reactions were terminated by adding 2 N HCl-0.1 mol/l EDTA followed by incubating the samples at 80uC for 10 min. After centrifugation of the precipitated protein, the samples were neutralized with CaCO 3 and cAMP was measured using an enzyme immunoassay (Biotrak, Amersham, UK) as specified by the manufacturer. The cAMP concentrations are expressed as pg/ml.
Analysis of VASP-Ser157 Phosphorylation, p38 MAPK Phosphorylation, Total VASP, and Total p38 MAPK Cells were lysed in PRO-PREP protein extract solution. The sample was centrifuged at 10,000 rpm for 20 min at 4uC. Protein concentration was determined by the BCA assay (Pierce). An equal volume of 26 SDS sample buffer (0.1 mol/ l Tris-Cl, 20% glycerol, 4% SDS, and 0.01% bromophenol blue) was added to an aliquot of the supernatant fraction from the lysates, and the mixture was boiled for 5 min. Aliquots of 30 mg of protein were subjected to 10% SDS-polyacrylamide gel electrophoresis for 1 h 30 min at 110 V. The separated proteins were transferred to PVDF membranes for 2 h at 20 mA with a SD Semi-dry Transfer Cell (Bio-Rad). The membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) for 2 h at room temperature. The membranes were then incubated with rabbit polyclonal anti-rat phospho-VASP (Ser 157), anti-rat VASP, anti-rat phosphor-p38 MAPK, and anti-rat p38 MAPK all diluted at 1:1000 in 5% nonfat milk in TBS-T overnight at 4uC, and then the bound antibody was detected using horseradish peroxidase-conjugated anti-rabbit IgG. The membranes were washed and proteins detected using a Western Blotting Luminol Reagent system and autoradiography.

Analysis of Cytokine Production
IFN-c and IL-4 levels in culture supernatants were analyzed using a commercial ELISA kit (R&D Systems, USA), according to the manufacturer's protocol. The absorbance at 450 nm was measured using a microplate reader with the wavelength correction set to 630 nm.

Statistical Analysis
Values are expressed as means 6 standard deviation (SD). Statistical analysis was performed with SPSS 13.0 software. The student t-test was used to compare two independent sample means, and one-way ANOVA was used to compare the means of more than two samples. A value of p,0.05 was considered statistically significant.

Serum Levels of b 1 -AA were Markedly Increased in DCM Patients Compared with Healthy Subjects
We tested for the presence of autoantibodies directed against the second extracellular loop of b 1 -AR by ELISA in sera collected from 95 patients with chronic heart failure due to DCM and from 95 control subjects. Subjects' clinical data are summarized in Table 1 and Table 2. There was no difference in age or gender distribution. Compared with healthy individuals, serum titers of b 1 -AA were markedly increased in DCM patients (0.55360.028 vs. 0.16760.0102, p,0.01) (Fig. 1A). As illustrated in Fig. 1B, only 8 of 95 healthy subjects were b 1 -AA positive (8.42%), whereas 44 of 95 DCM patients had a P/N value greater than 2.1 (46.3% positive). These data demonstrate that serum levels of b 1 -AA were markedly increased in DCM patients with heart failure when compared with healthy subjects. b 1 -AA Bound to b 1 -ARs on the Surface of CD3 + T Cells To determine the purity of the CD3 + T cells preparation, multicolor flow cytometry was used. As illustrated in Fig. 2A, after sorting, the CD3 + T cells represented 92.2% of the cell population. We next employed immunofluorescence staining to determine whether the IgG fraction isolated from the b 1 -AA-positive sera of DCM patients could bind to b 1 -ARs. We found that b 1 -AA (25 mg/ml) showed that the b 1 -AR staining was localized mainly to the membrane, while DAPI staining was only observed in the nucleus (Fig. 2C). Therefore, we concluded that the IgG fraction isolated from b 1 -AA-positive sera of DCM patients exhibited a pattern of b 1 -AR specific binding virtually identical to commercially available b 1 -AR-specific antibodies.  We found that b 1 -AA enhanced T cell proliferation in a concentration-dependent manner (Fig. 4A). Therefore, we chose 25 mg/ml for further study because it is comparable to the concentration in the sera of heart failure patients [18][19]. Freshly isolated CD3 + T cells were stimulated with anti-CD3/CD28 mAb in either the presence or absence of b 1 -AA for 48 h. As summarized in Fig. 4, the presence of b 1 -AA increased CD3 + T cell proliferation (0.12760.028 vs. 0.074560.016, p,0.05) (Fig. 4B). However, administration of b 1 -AA-negative IgG purified from 51 DCM patients did not detectably affect proliferation (0.08460.0059 vs. 0.074560.016, p.0.05) (Fig. 4B). Similar results were obtained when we used the CFSE assay (Figs. 4C and 4D). Taken together, the results presented in Fig. 4 demonstrate that b 1 -AA present in DCM patients induced CD3 + T cells proliferation. b 1 -AA Enhanced T Lymphocyte Proliferation through the b 1 -AR/cAMP/PKA Pathway The most common signaling mechanism initiated by b 1 -AR stimulation is the b 1 -AR/cAMP/PKA pathway [18,20]. To determine whether b 1 -AA-stimulated T cell proliferation resulted from the triggering of this pathway, the selective b 1 -AR antagonist metoprolol (1 mmol/l) and the PKA inhibitor H89 (1 mmol/l) were used to block the pathway before b 1 -AA administration, and then the activity of PKA was determined. The results demonstrated that T cell proliferation mediated by b 1 -AA was partially inhibited by metoprolol (0.09460.0044 vs.  (Fig. 5A). Additionally, we determined the accumulation of intracellular cAMP in T lymphocytes stimulated with b 1 -AA (25 mg/ml). As summarized in Fig. 5B, basal levels of cAMP (11969.63 pg/ml) were detected in T lymphocytes stimulated with anti-CD3/CD28 mAb alone. b 1 -AA significantly enhanced accumulation of intracellular cAMP levels (294619.4 pg/ml, p,0.01), whereas metoprolol (1 mmol/l) antagonized the b 1 -AAinduced accumulation of cAMP (Fig. 5B). In the immunoblot analysis, b 1 -AA stimulated VASP phosphorylation, which was inhibited by metoprolol and H89. However, it had no effect on total VASP (Fig. 5C, 5D). Collectively, these results suggest that the b 1 -AR/cAMP/PKA pathway was involved in T cell proliferation stimulated by b 1 -AA.

Involvement of p38 MAPK in b 1 -AA-mediated T Lymphocyte Proliferation
In immune cells, p38 MAPK plays a role in regulating the production of mature T cells [21][22]. To investigate the role of activation of p38 MAPK in b 1 -AA-stimulated T cell proliferation, the selective p38 MAPK antagonist SB203580 (1 mmol/l) was used to block the pathway before stimulation with b 1 -AA (25 mg/ml), and then the activity of p38 MAPK was determined. As depicted in Fig. 6A (Fig. 6A). In immunoblot analysis, b 1 -AA treatment of anti-CD3/CD28-mAb-activated T cells resulted in increased p38 MAPK phosphorylation, while total p38 MAPK remained unchanged (Fig. 6B, 6C). These data demonstrate a role for p38 MAPK activation in T cell proliferation mediated by b 1 -AA. Taken together, both the b 1 -AR/cAMP/PKA pathway and p38 MAPK activation were involved in the production of mature T cells.  (Fig. 7A). The effect was completely blocked by the addition of the selective b 1 -AR antagonist metoprolol (1 mmol/l) (p.0.05) (Fig. 7A). We next examined the effects of b 1 -AA on the production of IL-4 in T cells. The results suggest that b 1 -AA promoted the secretion of IL-4 (959.37661.79 pg/ml vs. 413.19632.495 pg/ml, p,0.01) (Fig. 7B), while the increase in IL-4 was antagonized by metoprolol (1 mmol/l) (p.0.05). Collectively, these results suggest that b 1 -AA regulated the secretion of T cells.

Discussion
The concentration of circulating autoantibodies directed against the second extracellular loop of b 1 -AR is known to be increased in patients with heart failure [17] when compared with healthy subjects. Our present results confirm these findings, and we report here that autoantibodies directed against the second extracellular loop of b 1 -AR were present in 46.3% of sera collected from 95 patients with heart failure due to DCM, which was significantly higher than in the samples from 95 healthy subjects. Moreover, we noted that their overall prevalence was 8.42% in healthy subjects, which is clearly more than has been reported to date [19,20,23]. We believe that these differences are essentially due to different methods used to detect the autoantibodies as well as the discriminant criteria. In the present study, the P/N ratio was used to represent the positive rate of b 1 -AA detection, which can effectively reduce the risk for false negative results [6]. Furthermore, during the myocardial remodeling process in rats, the generation of b 1 -AA showed a characteristic self-growth and timecourse decline, and the existence of b 1 -AA in rats lasted for a short period, with the titers gradually tapering after about two to three months [2]. Additionally, in concert with our animal experiments, it was reported, clinically, that autoantibodies against the b 1 -AR existing in the sera of DCM patients also present a time-course decrease and cardiac autoantibodies in patients with DCM become undetectable with disease progression [24][25]. Therefore, the presence of b 1 -AA in the sera of DCM patients showed a timecourse decrease, and the different pathological processes of heart failure patients could lead to the changes of incidence of b 1 -AA. Moreover, using the immunofluorescence and radioligand-binding techniques, we could demonstrate that b 1 -AA isolated from DCM patients recognized b 1 -ARs expressed either on CD3 + T cells or on H9c2 rat cardiomyoblast cells transiently transfected with b 1 -AR (Fig. S1C, Fig. 2), whereas after b 2 -ARs expressed on CD3 + T cells were blocked by the specific b 2 -AR antagonist ICI118551, CD3 + T cells can still be stained (Fig. S1B). Additionally, in order to enhance specificity, the monoclonal antibody, which was obtained by immunizing Balb/C mice with free peptide H26R corresponding to the second extracellular loop of the human b 1 -AR, and had the same biological effect with b 1 -AAIgGs isolated from heart failure patients (Fig. S3), was used to serve as positive control (Fig.  S1A). These experiments revealed that b 1 -AA isolated from DCM patients can bind to b 1 -ARs on CD3 + T cells, and did not crossreact with the very closely related b 2 -AR expressed on CD3 + T cells. All of these antibodies were directed against the second extracellular domain, which is known to affect ligand binding [26] and may induce immune responses [11]. They all increased the beat frequency and cAMP level of cultured cardiomyocytes (Fig.  S4), in the same way as the effect of IgG fractions isolated from individual b 1 -AA-positive sera of DCM patient (Fig. S5). Our results indicated that b 1 -AA may be one of the abnormal immune phenomena in heart failure and suggests their involvement in the pathophysiology of essential heart failure.
Recent clinical reports showed that the selective b 1 -AR antagonist, metoprolol, decreased the frequency and the geometric mean titer of b 1 -AA [23,27]. Moreover, Wallukat [28] demonstrated that the b 1 -adrenergic receptor antagonists were able to block the effect of the antibodies and displace the anti-b 1adrenergic receptor antibodies from their binding sites on the receptor, leading to a decrease of b 1 -AA titers in patients with  heart failure. Therefore, the DCM patients involved in the current study were required to stop treatment with b 1 -AR antagonists for one week.
T lymphocytes recognize antigens, help or suppress B cells to produce antibodies, and secrete cytokines [29]. The activation and proliferation of T lymphocytes are required for these functions. Therefore, the state of immune function is usually reflected by the proliferation of T lymphocytes. In the present study, in order to exclude the interference of other cells, FACS was used to separate CD3 + T lymphocytes to specifically determine how they are affected by b 1 -AA. We found that b 1 -AA isolated from DCM patients had no effect on the numbers of resting CD3 + T cells (Fig.  S6A), but it can enhance the proliferation of anti-rat CD3/CD28 mAbs-induced CD3 + T cells. Therefore, in this study, the CD3 + T cells were stimulated with anti-rat CD3/CD28 mAbs first, and then the effect of b 1 -AA on activated CD3 + T cells was analyzed. Furthermore, we have isolated CD3 + T cells from b 1 -AA-positive,negative DCM patients and healthy control subjects. Additionally, the CD3 + T cells were stimulated with anti-human CD3/CD28 mAbs first, and then the role of b 1 -AA in the proliferation of activated CD3 + T cells was observed. We found that b 1 -AA from DCM patients significantly enhanced the proliferation of CD3 + T cells isolated from b 1 -AA-positive and -negative DCM patients as well as healthy control subjects (Fig. S6B, C, D).
According to published paper [14], the patients with DCM also develop functionally active antibodies against the first extracellular loop of the b 1 -AR (b 1 -AR-EC I ). Therefore, to detect whether there were anti-b 1 -AR-EC I -antibodies in DCM patients involved in this study, we added peptide corresponding to the sequence of the 1st (b 1 -AR-EC I ) as negative control, and found that the supernatant produced by incubating b 1 -AA-positive IgG with b 1 -AR-EC I still promoted CD3 + T lymphocytes proliferation (Fig. S7A). However, the supernatant produced by incubating b 1 -AA-positive IgG with peptide corresponding to the sequence of the 2nd (b 1 -AR-EC II ) had no effect on CD3 + T lymphocytes (Fig. S7B). These results suggest that b 1 -AA-positive IgG isolated from DCM patients promoted the proliferation of CD3 + T lymphocytes by binding to b 1 -AR-EC II .
The most common signaling mechanism initiated by b 1 -AR stimulation is the b 1 -AR/cAMP/PKA pathway [18,20]. In order to explore whether b 1 -AA promoted T lymphocyte proliferation through this pathway, the b 1 -AR selective antagonist metoprolol was added to T lymphocytes before b 1 -AA administration. As a result, the proliferative effect could be blocked, suggesting that b 1 -ARs on the surface of CD3 + T cells could be activated by the b 1 -AA from DCM patients. Furthermore, we also found that each b 1 -AA sample increased mitogen-stimulated cAMP production in a receptor-mediated fashion. Previous studies have demonstrated the PKA-dependent effects in immune cells by either assessing agonist-stimulated PKA activity through in vitro assays or demonstrating the actions of pharmacologic PKA inhibitors and activators [30][31]. Here we used a more direct approach in which we analyzed PKA activity by assaying for the phosphorylation of VASP at Ser157, which is mediated directly and selectively by PKA [32][33]. We found that b 1 -AR activation by b 1 -AA rapidly led to VASP phosphorylation  at Ser157. The selective b 1 -AR antagonist metoprolol decreased the level of VASP phosphorylation stimulated by b 1 -AA. In addition, inhibition of PKA by compound H89 abrogated b 1 -AAinduced phosphorylation of VASP at Ser157. However, b 1 -AA had no effect on total VASP. Taken together, all of these results strongly implicate the b 1 -AR/cAMP/PKA pathway as the principal signaling system modulating the b 1 -AA-induced phosphorylation of VASP at Ser157.
In T cells, one physiological effect of p38 MAPK activity is the regulation of cell growth and cell death, which are especially important in the thymus during T cell development [34]. Dysregulation of p38 MAPK can result in negative selectioninduced cell death and the subsequent absence of T cell populations in the peripheral immune system [35]. Our present results suggest that the proliferation of T cells induced by b 1 -AA was partially blocked by the p38 MAPK-selective inhibitor SB203580. Moreover, immunoblot assays revealed an apparent increase in phosphorylation of p38 MAPK following treatments with b 1 -AA, in the absence of an effect on total p38 MAPK levels. These results indicated that activation of p38 MAPK was correlated with the production of mature T cells. Furthermore, when H89 and SB203580 were used together before stimulation with b 1 -AA, the proliferation of T cells was also partially inhibited. These results suggest that both b 1 -AR/cAMP/PKA and p38 MAPK pathways contributed to b 1 -AA-mediated proliferation of T cells. However, other pathways may also participate in this process. The study by Antonio et al. [18] showed that the effect of b 1 -AA on cardiomyocytes could be blocked by tyrosine kinase inhibitor PP2. Other studies have also reported that the role of b 1 -AR could be mediated by PI3-kinase, PKC, or PKA in the trigger phase of ischemic preconditioning [36]. Based on the studies mentioned, further researches are necessary to investigate the other possible pathways stimulated by b 1 -AA.
The main form of T cell activation is to secrete cytokines. Therefore, in the current study, the levels of IFN-c and IL-4, the characteristic cytokines secreted by T cells, were chosen to detect the effect of b 1 -AA on T lymphocytes secretion. IFN-c is a major proinflammatory effector and regulatory cytokine produced by activated T lymphocytes, which can inhibit humoral immunity by suppressing the production of Th2 cells, but promotes cellmediated immunity [37]. Our results suggest that both b 1 -AApositive and -negative IgGs might inhibit the secretion of IFN-c, though the effect of b 1 -AA-positive IgGs was more pronounced than that of b 1 -AA-negative IgGs. However, the IgGs isolated from healthy subjects did not enhance IFN-c secretion (Fig. S8A). Moreover, in order to analyze whether the reduction of IFN-c production caused by the b 1 -AA-negative IgG preparation of DCM patients might have an effect of anti-b 1 -AR-EC I -antibodies, we added peptide corresponding to the sequence of the 1st extracellular loop of the receptor (b 1 -AR-EC I ) as a negative control. We found unexpectedly that the supernatant produced by incubating b 1 -AA-negative IgGs with b 1 -AR-EC I also decreased IFN-c production, however, compared with b 1 -AA-negative IgGs, the inhibition of IFN-c level was alleviated (Fig. S9). These results suggest that there may be anti-b 1 -AR-EC I -antibodies in b 1 -AAnegative DCM patients, which inhibited IFN-c production. Besides that, heart failure itself may be a risk factor in inhibiting cell-mediated immunity, and the effect may be magnified due to the presence of b 1 -AA. However, the mechanism for the b 1 -AAmediated decrease in IFN-c production is unknown, and further investigations are needed to explain this phenomenon.
IL-4 is the characteristic cytokine secreted by Th2 cells, which decreases the production of Th1 cells and promotes humoral immunity [38][39]. It has been reported that the b-AR agonist isoproterenol may activate Th2 cells and promote IL-4 production mainly via binding to b 2 -ARs expressed on T lymphocytes [40]. In the current study, we found that b 1 -AA from DCM patients can enhance IL-4 release through combining with b 1 -ARs also expressed on T lymphocytes, but the IgGs isolated from healthy subjects did not enhance IL-4 secretion (Fig. S8B). These results suggest that although b 1 -AA mediates b 1 -AR agonist-like actions; it is different from the b 1 -AR agonist isoproterenol and might activate Th2 cells through the b 1 -AR pathway. Additionally, b 1 -AA serves as a kind of antibody that is produced by B cells and Th2 cells, and the present study shows that b 1 -AA may enhance humoral immunity, while inhibiting cell-mediated immunity. This suggests that there is positive feedback between b 1 -AA and Th2 cells. However, further studies are required to support this conclusion.
We observed here the direct effect of IgG fractions from b 1 -AApositive sera on CD3 + T lymphocytes isolated from patients with heart failure due to DCM, and demonstrated that T lymphocyte, in addition to cardiomyocyte, may also be one of the important targets of b 1 -AA isolated from DCM patients. The vicious circle between the immune system and b 1 -AA may exacerbate autoantibody-positive diseases.
Nonetheless, our work leaves some unanswered questions and paths for future work. The b 1 -AA used in this study was not specific for the second extracellular loop of b 1 -AR, and some nonspecific IgGs were involved. Additionally, because of clinical and ethical reasons, DCM patients involved in the current study only stopped b-blocker therapy for one week, which can not completely preclude effects of b-blocker on b 1 -AA synthesis. Therefore, further studies using monoclonal antibodies specific for the second extracellular loop of b 1 -AR would be carried out to yield more conclusive results. Moreover, whether there are any differences in T lymphocytes in b 1 -AA positive and negative patients has not been elucidated. In addition, in the present study, ELISA and beating rate of isolated neonatal cardiomyocytes were employed to detect the titer and function of b 1 -AA in the patients with DCM. However, recent research reported that a novel molecular and/or fluorescence-based diagnostic method of detecting b 1 -AA in patients with heart failure was proved to be fast and highly sensitive [41]. Therefore, further investigations using new diagnostic methods would be conducted to provide functional and conclusive diagnostic data.  Figure S6 The effects of b 1 -AA on CD3 + T cells proliferation. A. b 1 -AA had no effect on resting rat CD3 + T cells. B, C, D. b 1 -AA enhanced the proliferation of activated CD3 + T cells isolated from b 1 -AA-positive/2negative DCM patients and healthy subjects, respectively. **p,0.01 versus vehicle group; ## p,0.01 versus negative IgG group. n = 6 per group. Data are presented as means 6 SD of 3 independent experiments. (TIF) Figure S7 The proliferation of CD3 + T lymphocytes induced by b 1 -AA was blocked by b 1 -AR-EC II . The supernatant produced by incubating b 1 -AA with b 1 -AR-EC I promoted CD3 + T lymphocytes proliferation. The proliferation of CD3 + T lymphocytes induced by b 1 -AA was blocked by b 1 -AR-EC II . **p , 0.01, *p , 0.05 versus. vehicle group. n = 6 per group. Data are presented as means 6 SD of 3 independent experiments. (TIF) Figure S8 The IgGs isolated from healthy subjects revealed no effect on the levels of IFN-c and IL-4 in CD3 + T cells. n = 6 per group. Data are presented as means 6 SD of 3 independent experiments. (TIF) Figure S9 The reduce of IFN-c induced by b 1 -AAnegative IgG was partially blocked by b 1 -AR-EC I . b 1 -AA-negative IgG (25 mg/ml) and the peptide corresponding to the sequence of the first extracellular loop of the human b 1 -AR (b 1 -AR-EC I , 1 mmol/l) co-incubated for 1 h at 37uC, supernatants were then collected to treat CD3 + T lymphocytes, and finally IFNc level was analyzed by ELISA. **p , 0.01, *p , 0.05 versus. vehicle group; DD p,0.01 versus negative IgG group. n = 6 per group. Data are presented as means 6 SD of 3 independent experiments. (TIF)