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Dopamine receptor DR2 expression in B cells is negatively correlated with disease activity in rheumatoid arthritis patients
Immunobiology 220 (2015) 323–330
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Dopamine receptor DR2 expression in B cells is negatively correlated with disease activity in rheumatoid arthritis patients L. Wei a,1 , C. Zhang b,1 , H.Y. Chen a , Z.J. Zhang a , Z.F. Ji a , T. Yue c , X.M. Dai a , Q. Zhu c , L.L. Ma a , D.Y. He c , L.D. Jiang a,∗ a
Department of Rheumatology, Zhongshan Hospital, Fudan University, Shanghai, China Department of Orthopedics, Zhongshan Hospital of Fudan University, Shanghai, China c Department of Rheumatology, Shanghai Guanghua Hospital of Integrated Chinese & Western Medicine, Shanghai, China b
a r t i c l e
i n f o
Article history: Received 16 September 2014 Received in revised form 19 October 2014 Accepted 20 October 2014 Available online 5 November 2014 Keywords: B cells Disease activity Dopamine receptor Neuro-immune interaction Rheumatoid arthritis
a b s t r a c t Objective: Dopamine receptor (DR) signaling is involved in the pathogenesis of autoimmune diseases. We aimed to measure the expression levels of DR1-5 on B cells from patients with rheumatoid arthritis (RA) and to analyze the relationship between DRs and clinical manifestations, inflammatory biomarkers, functional status and disease activity. Methods: A total of 29 patients with RA, 12 healthy donors and 12 patients with osteoarthritis (OA) were recruited in this study. Flow cytometry was used to measure the levels of DR1-5 expressed on B cells. The relationships between B cell DR expressions and clinical features in RA patients were analyzed using the Spearman correlation test. Results: The expression levels of B cell DR1-5 in both the RA and OA groups were lower than those in healthy controls. After 3 months of medication, all five receptors were elevated in RA patients, with DR2 and DR3 being significantly increased from the baseline. DR2 expression on B cells was negatively correlated with inflammatory biomarkers and disease activity. Conclusion: RA patients had lower expression level of DR2 on B cells compared to the healthy controls, and the level of DR2 negatively correlated with the disease activity. DR2 and DR3 might be novel predictors of patient responses to disease modifying antirheumatic drug therapy. © 2014 Elsevier GmbH. All rights reserved.
Introduction
Abbreviations: RA, Rheumatoid arthritis; DR, dopamine receptor; OA, osteoarthritis; cAMP, cyclic adenosine monophosphate; SLE, systemic lupus erythematous; DMARD, disease modifying antirheumatic drug; TNF-␣, tumor necrosis factor-␣; TJC, tender joint count; SJC, swollen joint count; HAQ, health assessment questionnaire; PGA, patient global assessment; EGA, evaluator global assessment; VAS, visual analog score; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; anti-CCP, anti-cyclic citrullinated protein; RF, rheumatoid factor; DAS28, 28-joint disease activity score; SDAI, simplified disease activity index; CDAI, clinical disease activity index; PBMCs, peripheral blood mononuclear cells; APC, allophycocyanin; SD, standard deviation; IQR, inter-quartile range; MTX, methotrexate; HCQ, hydroxychloroquine; SSZ, sulfasalazine; LEF, leflunomide; ACPA, anti-citrullinated protein antibody; DC, dendritic cell; NK cell, natural killer cell; aa, amino acid. ∗ Corresponding author at: Department of Rheumatology, Zhongshan Hospital, Fudan University, No. 180, Road Fenglin, Shanghai 200032, PR China. Tel.: +86 021 64041990 2940. E-mail address: [email protected] (L.D. Jiang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.imbio.2014.10.016 0171-2985/© 2014 Elsevier GmbH. All rights reserved.
Rheumatoid arthritis (RA) is a heterogeneous immune disorder characterized by chronic erosive inflammatory synovitis with the production of multiple autoantibodies, which indicate the abrogation of self-tolerance. Genetic and environmental factors impinge upon all stages of RA pathogenesis. To date, RA pathogenesis has been explained at multiple levels; however, the exact pathogenic mechanism involved remains unclear. The neuro-immune interaction, a holistic theory, allows us to gain a better understanding of the development and progression of autoimmune diseases. Recently, neurotransmitters have been extensively studied, especially monoamines in the central and peripheral immune systems. Dopamine, a catecholamine, exerts its function by targeting specific DR to modify the activation (Mikulak et al. 2014), proliferation (Saha et al. 2001), differentiation (Nakano et al. 2009a,b), chemotaxis, homing (Watanabe et al. 2006) and apoptosis of immune cells (Oberbeck et al. 2006). The five DRs, termed DR1, DR2, DR3, DR4, and DR5, are G-protein coupled receptors that can be further categorized into two classes based on their ability to
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modulate cyclic adenosine monophosphate (cAMP) production, the D1-like (DR1 and DR5) and D2-like (DR2, DR3, and DR4) receptors. D1-like receptors activate the G␣s/olf G protein subunit, resulting in the activation of adenylyl cyclase and the upregulation of cAMP. D2-like receptors activate the G␣i/o G protein subunit, leading to the down-regulation of endogenous cAMP by inhibiting adenylyl cyclase. Thus, D1- and D2-like receptors often play antagonistic roles. However, sometimes they can operate in a synergistic way (Paul et al. 1992). Many lines of evidence support the reciprocal effect of dopamine on autoimmune diseases. There are extensive interactions between sympathetic innervations and lymphoid organs (Bellinger et al. 2008), as well as the musculoskeletal system (including the synovial cavity) (Jänig and Green 2014). Interestingly, immune cells also can synthesize, transport and release dopamine (Nakano et al. 2009a,b). In immunopathologies, alterations in serum dopamine levels have been noted. These pathophysiological findings provide a basis for its function in the immune system. Additionally, dopaminergic therapies, using either dopamine itself or DR agonists/antagonists in patients with Parkinson’s disease or multiple sclerosis, have shown effects on the T cell proteome (Alberio et al. 2012) and polarization (Ferreira et al. 2014). Expressions of the DRs by T cells are also related to the severity of schizophrenia (BritoMelo et al. 2012). These studies have demonstrated the role of dopamine in orchestrating the immune response in the development of neuropsychiatric disorders. Moreover, many investigations have been conducted to assess the distribution and modulation of the dopaminergic system in autoimmune diseases, including systemic lupus erythematosus (SLE) (Jafari et al. 2013), primary Sjögren syndrome (Wester et al. 1991), RA (Capellino et al. 2014; Nakano et al. 2011; Nakashioya et al. 2011) and inflammatory bowel disease (Magro et al. 2002). Measurement of DRs by flow cytometry revealed that all five dopamine receptors are expressed in immune cells, including a low expression level in T cells and high expression level in B cells (McKenna et al. 2002). However, the exact phenotypic distribution of different subtypes of these receptors is unknown in B cells in immnopathologies. Here, we generated a comprehensive profile of these five DRs on B cells in patients with RA and analyzed the correlation between DR expression levels and disease activity, functional status, and serum biomarkers. In this study of the dopamine system, we shed some new light on the pathogenesis of RA and identify new potential therapeutic targets.
Materials and methods Study population Healthy blood donors, patients with RA and patients with osteoarthritis (OA) satisfied the classical diagnostic criteria (Arnett et al. 1988; Altman et al. 1986) were recruited from the Department of Rheumatology, Zhongshan Hospital from July 12, 2013 to May 31, 2014. Inclusion criteria for the RA group encompassed patients with active disease (the 28-joint disease activity score [DAS28] according to the CRP formula > 3.2) and disease modifying anti-rheumatic drug (DMARD) naive or no DMARDs use within the previous 3 months. Patients who had received anti-tumor necrosis factor␣ (TNF-␣) or glucocorticoid therapy were excluded. The other exclusion criteria for both the RA and OA groups were infectious or inflammatory diseases, endocrine disorders, any past or current psychiatric or neurological diseases, pregnant or planning to be pregnant, lactation, liver or kidney dysfunction, cardiovascular disease, cancer, any drug history that would affect the sympathomimetric or sympatholytic system, and recent severe stress events. All subjects were provided with written informed consent. This study
was conducted according to the Declaration of Helsinki and was approved by the ethics committee of Zhongshan Hospital.
Assessment and follow-up Complete medical histories, physical examinations, and radiological and laboratory tests were conducted for all individuals. Tender joint count (TJC), swollen joint count (SJC), health assessment questionnaire (HAQ), patient global assessment (PGA; visual analog score from 0 to 100 mm), evaluator global assessment (EGA; visual analog score from 0 to 100 mm), routine blood tests, liver and renal function, erythrocyte sedimentation rate (ESR; mm/h) and Creactive protein (CRP; mg/dl) were recorded. For all patients, the assessments were performed by one rheumatologist. ELISA tests for anti-cyclic citrullinated protein (anti-CCP; mg/L) antibody and rheumatoid factor (RF; IU/ml) were also performed. A DAS28 (CRP formula), simplified disease activity index (SDAI) (Smolen et al. 2003) and clinical disease activity index (CDAI) (Greenberg et al. 2009) were all calculated.
Antibodies and reagents Rabbit anti-human polyclonal antibodies (lgG) to DR1 (corresponding to amino acids [aa] 9–21 of human DR1), DR3 (corresponding to aa 2–10 of human DR3), DR4 (corresponding to aa 176–185 of human DR4), and DR5 (corresponding to aa 2–10 of human DR5) were purchased from Calbiochem (Merck Millipore, Germany) and anti-DR2 (raised against 11 aa near the N-terminus ligand binding domain of rat DR2 and cross-reacts with human DR2) was from LifeSpan BioSciences (Seattle, WA, USA). Allophycocyanin (APC)-conjugated CD19 and its isotype control were obtained from Becton Dickinson (San Jose, CA, USA). Histopaque1077 and goat anti-rabbit CF405M-conjugated antibody were from Sigma–Aldrich (St. Louis, MO, USA). Normal rabbit IgG antibody as an isotype control was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Peripheral blood mononuclear cells (PBMCs) isolation Peripheral overnight fasting blood (10 ml) from the cubital vein was preserved in tubes (10 ml, BD Biosciences) containing ethylene diamine tetraacetic acid (EDTA). PBMCs were separated using Ficoll–Hypaque density centrifugation. Cells were washed three times in phosphate buffered saline and prepared at a final concentration of 1 × 106 /ml.
Dopamine receptor detection B cells were labeled with APC-conjugated CD19 mouse antihuman monoclonal antibody. Cells were incubated with DR polyclonal (DR1-5) antibodies (1:100 dilution) at 4 ◦ C for 30 min and were then washed twice with staining buffer and incubated with secondary CF405M-conjugated goat anti-rabbit antibody (1:100 dilution) at 4 ◦ C for 30 min. Samples with ‘no primary antibody’ (NPA) or normal rabbit sera (Rab) control (1:100) were used as two negative controls. Stained cells were analyzed using a Conto II flow cytometer (BD Biosciences). Data analysis was performed with Diva software (BD Biosciences) and FlowJo V.7.6.4 (Treestar Inc., Ashland, OR, USA). A minimum of 1,000,000 cells were analyzed from each sample. The results were finally expressed as the percentage of positive cells (%). BDR (%) was the percentage of CD19+ DR+ cells among CD19+ cells.
L. Wei et al. / Immunobiology 220 (2015) 323–330 Table 1 The general baseline characteristics of RA patients. Baseline characteristics
RA group (n = 29)
Male/female Age (years), mean (SD) Disease duration (month), median (IQR) TJC, mean (SD) SJC, mean (SD) ESR (mm/h), mean (SD) CRP (mg/L), median (IQR) Anti-CCP antibody, median (IQR) RF (IU/ml), median (IQR) Pain (VAS, 1–100 mm), median (IQR) PGA (VAS, 1–100 mm), median (IQR) EGA (VAS, 1–100 mm), median (IQR) HAQ, median (IQR) DAS28, mean (SD) 3.2 ≤ DAS28 < 5.3, n (%)t 5.3 ≤ DAS28, n (%) CDAI, mean (SD) SDAI, mean (SD)
5/24 53.69 (12.05) 42 (6–81) 17.56 (7.92) 16.26 (8.02) 69.44 (33.66) 30.90 (16.00–58.92) 236.29 (73.50–501.00) 118.00 (34.90–446.00) 70.00 (60–85) 70.00 (50–80) 70.00 (55–85) 1.00 (0.2–2.00) 6.38 (1.41) 7 (24.14) 22 (75.86) 46.06 (19.19) 50.53 (21.17)
TJC, tender joint count; SJC, swollen joint count; ESR, erythrocyte Sedimentation Rate; CRP, C-reactive Protein; anti-CCP antibody, anti-cyclic citrullinated protein antibody; RF, rheumatoid factor; HAQ, Health Assessment Questionnaire Disability Index; PGA, patient global assessment; EGA, evaluator global assessment; VAS, visual analog score; DAS28, 28-joint disease activity score; CDAI, clinical disease activity index; SDAI, simplified disease activity index; SD, standard deviation; IQR, inter quartile range. Data are presented as means ± SD or means (IQR) as appropriate.
Statistical analysis Continuous data were expressed as means ± standard deviation (SD) or medians (inter-quartile range, IQR) according to the data distribution. Statistical analyses of nonparametric data were performed using the Mann–Whitney U-test. The pre- and posttreatment expression levels of B cell DRs were compared using the Wilcoxon rank test. The relationship between the DR expression levels and the disease activity score, inflammatory markers, autoantibodies, and functional status were analyzed using Spearman’s correlation test. A P-value < 0.05 was considered to indicate a statistically significant difference. Results Baseline characteristics There were 29 eligible RA patients included in this study. Patients’ demographic and clinical data are shown in Table 1. Five patients were male and eight patients were treatment-naive. The mean age of RA patients was 53.80 ± 11.58 years old and the median disease duration was 42 (6–81) months. All patients had moderate to severe disease activity according the DAS28 score with a mean value of 6.38 ± 1.41, and a majority of these patients (75.86%) had severe disease activity. Only three patients had radiological bone erosions. As controls, 12 OA patients and 12 healthy donors were also recruited. The mean ages of these patients were 56.75 ± 9.19 and 24.58 ± 2.07 years old, with the sex ratio (male/female) of 1:1 and 1:2, respectively. The ages of the OA and RA groups were not significantly different (P = 0.368); however, both groups were significantly older than the healthy controls (P < 0.001). B cell DR1-5 expression levels in RA patients The expression levels of DR1-5 on B cells were investigated by flow cytometry (Figs. 1 and 2A and B). In RA patients, a lower percentage of DR1+ cells (2.88%, 1.49–4.89%) were detected compared to healthy controls (4.45%, 3.04–9.90%; P = 0.068), which was significantly higher than that detected in the OA group (1.56%,
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0.77–3.47%; P = 0.007). B cell DR1 expression in the RA group was slightly higher than that in the OA group, but this difference did not reach our threshold for statistical significance (P = 0.150). B cell DR2 expression levels were 6.94% (2.52–15.40%) in the RA group and 3.58% (2.58–8.13%) in the OA group, which were both markedly lower than that in the healthy controls (28.1%, 14.38–42.35%; P = 0.003 or P = 0.001, respectively). No difference was observed between the RA and OA groups. In RA patients, the expression levels of DR3 (2.90%, 0.98–8.98%), DR4 (2.24%, 0.79–3.68%), and DR5 (2.39%, 1.36–10.27%) showed no differences with the OA group (DR3, 2.48% (IQR: 1.11–4.45%); DR4, 1.84% (IQR: 0.68–4.30%); DR5, 1.90%, (IQR: 0.98–5.95%)) or the healthy control groups (DR3, 3.19% (IQR: 2.05–8.14%); DR4, 3.99% (IQR: 2.56–6.66%); DR5, 2.89%, (IQR: 2.57–7.27%)). However, trends for higher expression levels of DR15 in RA patients compared to OA patients, and lower expression levels of DR1-5 in RA patients compared to healthy controls were observed. Dopamine receptor expression levels varied greatly in the three groups, with the variation for DR2 expression being the most striking (Fig. 2B). When the RA patients were divided into two groups based on the DAS28 score, 7 were in the moderate group (3.2 < DAS28 ≤ 5.3) and 22 were in the severe group (DAS28 > 5.3). DR2 expression in the severe group was much lower than that in the moderate group (5.98%, IQR: 2.08–13.70% vs. 15.00%, IQR: 10.14–27.08%; P = 0.047). The expression of other DRs showed no significant differences between these two groups. Five DRs were further divided into D1-like (DR1 and DR5) and D2-like (DR2, DR3, and DR4) receptors. D1-like receptor expression was 5.32% (4.10–15.79%) in the RA group, which showed no difference with the healthy controls (8.02%, 5.66–19.86%) or the OA group (3.78%, 2.06–8.93%). The frequency of D2-like receptor expression was 13.50% (6.94–38.20%) in the RA group, which had a low expression level compared to the healthy controls (38.02%, 22.97–48.30%; P = 0.021) and showed no difference with the OA group (10.61%, 6.14–14.68%; P = 0.214). The relationship between B cell DR1-5 expression levels and clinical features A Spearman correlation analysis was performed to investigate the relationships between DRs and clinical manifestations, inflammatory markers, disease activity, and functional capabilities (Table 2). DR2 was found to be negatively correlated with ESR (r = −0.623, P < 0.001; Fig. 3A), CRP (r = −0.556, P = 0.002; Fig. 3B), DAS28 (r = −0.471, P = 0.01; Fig. 3C), SDAI (r = 0.441, P = 0.017, Fig. 3E) and EGA (r = −0.459, P = 0.012). Other DRs, including DR1, DR3, and DR5, were negatively associated with RF (r = −0.396, −0.427, −0.492, respectively; P-values were all <0.05). Besides, DR1 expression was correlated with CRP (r = −0.451, P = 0.014) and DR4 was negatively correlated with HAQ (r = −0.387, P = 0.038). D1-like receptors were also associated with RF (r = −0.504, P = 0.007). A correlation could be observed between RF and D2-like receptors (r = −0.499, P = 0.008). Additionally, D2-like receptors also correlated negatively with ESR (r = −0.431, P = 0.019), CRP (r = −0.522, P = 0.004), EGA (r = −0.387, P = 0.038), and DAS28 (r = −0.384, P = 0.04). Comparison of the baseline and post-treatment levels of DRs In our cohort, 14 of 29 RA patients who had good intention to treat were administered DMARDs and were followed up every month at our clinic. DMARDs included methotrexate (MTX, nine patients), TNF-␣ blockers (five patients), hydroxychloroquine (HCQ, four patients), low dose glucocorticoid (four patients), penicillamine (three patients), Tripterygium wilfordii (two patients), sulfasalazine (SSZ, four patients), iguratimod (one patient), and
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Fig. 1. Expression of DRs by B cells in RA patients compared with healthy controls and OA patients. (A–E) Representative dot plots of the expression of DR1-5 by B cells in RA patient using flow cytometric analysis. (F) Comparison of the DR1-5 expressions by B cells in RA patients (n = 29) with that in OA patients (n = 12) and healthy controls (n = 12) (Mann–Whitney test). Data were expressed as median (IQR). Asterisks represent statistically significant differences (P < 0.05).
leflunomide (LEF, one patient). After 3 months of therapy, peripheral blood was collected and laboratory tests, disease activity, and functional status were evaluated by the same rheumatologist. The ESR and CRP of the patients dropped significantly to 27.5 ± 19.74 mm/h and 3.82 mg/L, respectively. The tender and swollen joints were also improved. The mean TJC was 3.57 ± 4.97 and SJC was 2.93 ± 5.66. The DAS28 score was 3.11 ± 1.33. Out of the 14 patients, 2 remained severe disease activity (DAS28 > 5.3) and 3 who showed moderate activity. The rest of the 9 patients
achieved low disease activity or remission (DAS28 < 3.2). Only one patient had elevated liver enzyme levels at her 3-month follow-up visit. Dopamine receptor expression levels were measured again to compare the post-treatment with the baseline levels (Table 3). After treatment, B cell DR2 expression levels were significantly higher, increasing from 6.76% (2.44–15.85%) at baseline to 20.5% (15.70–43.35%) post-treatment (P < 0.05; Fig. 2B). DR3 expression also increased from baseline (2.41%, 0.54–6.14%) to the posttreatment level (6.51%, 3.81–14.95%; P < 0.05).
Table 2 Spearman correlation analyses of the expression of DRs by B cells and clinical features.
TJC SJC ESR CRP CCP RF HAQ PAIN PGA EGA DAS28 CDAI SDAI
BDR1
BDR2
BDR3
BDR4
BDR5
−0.011 0.179 −0.131 −0.451* 0.031 −0.396* −0.274 −0.063 −0.126 −0.210 −0.275 −0.146 −0.207
−0.075 −0.043 −0.623** −0.556** −0.313 −0.306 −0.341 −0.188 0.146 −0.459* −0.471* −0.358 −0.441*
−0.1 0.026 −0.044 −0.306 −0.256 −0.427* −0.224 0.163 0.095 −0.115 −0.156 −0.028 −0.125
−0.140 0.028 −0.186 −0.152 −0.269 −0.374 −0.387* 0.101 0.018 −0.091 −0.098 −0.03 −0.071
0.129 0.286 −0.239 −0.048 −0.153 −0.492* −0.340 0.075 0.038 −0.094 0.057 0.086 0.06
All correlations were assessed using Spearman’s rank order correlation coefficient (r). * P-value < 0.05. ** P-value < 0.01. See abbreviations in Table 1.
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Fig. 2. Expression of DR2 by B cells in RA patients compared to healthy controls and OA patients. (A) Representative flow cytometric analysis of DR2 expression by CD19+ B cells. Data at baseline from healthy controls (n = 12) and patients with OA (n = 12) or RA (n = 29) are shown. (B) A comparison of DR2 expression in B cells in healthy controls, OA patients, and RA patients (Mann–Whitney test). (C) A comparison of the pre- and post-treatment levels of DR2 expressed by B cells in RA patients (n = 14; Wilcoxon rank test). Lines within the dot plot represent medians. Two-tailed P-values < 0.05 were considered to indicate statistically significant differences.
Discussion The regulatory role of the dopaminergic system in the immune response has been reviewed in detail elsewhere (Sarkar et al. 2010; Basu and Dasgupta 2000; Pacheco et al. 2014). Alterations in DR expression levels have been detected in autoimmune diseases. Jafari et al. found that SLE patients showed lower expression levels of the DR gene DR2 and higher expression of DR4 by realtime PCR in PBMC, and further attributed altered DR2 and DR4 gene expression levels to T cells rather than B cells (Jafari et al. 2013). In rheumatoid arthritis, dopamine released by dendritic cells (DC) can contribute to DC–T cell interactions, which affect T cell
Table 3 A comparison of DR2 expression by B cells of RA patients at the baseline and after 3 months of DMARDs therapy. Receptor
Pre-treatment (%)
Post-treatment (%)
Ba BDR1 BDR2 BDR3 BDR4 BDR5
9.00 (5.64–16.76) 3.04 (1.34–5.16) 6.76 (2.44–15.85) 2.41 (0.54–6.14) 1.51 (0.50–3.95) 2.63 (0.48–5.43)
9.43 (4.63–10.88) 6.24 (3.45–19.20) 20.5b (15.70–43.35) 6.51b (3.81–14.95) 3.63 (1.01–12.95) 4.67 (2.67–16.15)
The Wilcoxon rank test was used to compare the baseline pre-treatment with the post-treatment levels of CD19+ DR2+ B cells in RA patients (n = 14). a Percentage of CD19+ B cells among lymphocytes. b Two-tailed P-value < 0.05.
differentiation (Nakano et al. 2009a,b). The roles of the dopaminergic system were further investigated using DR agonists/antagonists in animal models. After treatment with the D1-like receptor antagonist SCH23390, mice with collagen-induced arthritis exhibited lower arthritis scores and less severe joint destruction than control mice (Nakashioya et al. 2011). The therapeutic effect of SCH23390 was also shown in another study in which the authors found that in the human RA/SCID mouse chimera model, SCH23390 suppressed cartilage destruction by inhibiting the IL-6–IL-17 axis, while the D2like receptor antagonist haloperidol worked in the opposite way (Nakano et al. 2011). Synovial fibroblasts, which are one of the most important participants in the pathogenesis of RA, have been shown to express all five DRs by immunohistochemistry. IL-6 and IL-8 production by synovial fibroblasts can be inhibited by dopamine via the D1-like receptor (Capellino et al. 2014). Tanaka and colleagues studied the regulation of DR functions in osteoclastogenesis (Hanami et al. 2013). The functions of the DR depend upon the experiment conditions, specific DR subtype, cell type and cell status. Pharmacological or physiological doses of dopamine can affect experimental results obtained either in vitro or in vivo. The five dopamine receptor subtypes, even those among the same class (D1- or D2-like), can vary in their downstream signaling pathways and biological effects. DR1 is low expressed in immune cells has not been thoroughly investigated. Dopamine mediates IL-10 and TNF-␣ release by activating DR2 and DR3, respectively (Besser et al. 2005). Stimulation of DR3 also increases IFN-␥ secretion in T cell blasts (Ilani et al. 2004).
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Fig. 3. The relationship between DR2 expressed by CD19+ B cells and clinical data in RA patients (n = 29). (A) ESR (mm/H). (B) CRP (mg/L). (C) DAS28 score. (D) CDAI score. (E) SDAI score. Data from 29 patients with RA at baseline were compared by Spearman correlation analysis. Two-tailed P-values < 0.05 were considered to indicate statistically significant differences.
By targeting DR4, dopamine can induce T cell quiescence (Sarkar et al. 2006). Dopamine regulates NK cell cytotoxicity through the cAMP–PKA–CREB signaling pathway (Zhao et al. 2013). By binding to DR5, dopamine exerts an inhibitory function to ‘switch-off’ activated natural killer (NK) cells (Mikulak et al. 2014). Central memory T cells (TCM ) and effector memory T cells (TEM ) express more D2than D1-like receptors. By contrast, naive T cells express higher level of D1-like receptor (Kustrimovic et al. 2014), which indicates that the cell type and activation state play key roles in establishing the DR repertoire. Unfortunately, most studies that focused on dopamine and DR functions in immune cells did not include B cells. Only one study found that dopamine could induce the apoptosis of B cells, which occurred independently of the DRs (Meredith et al. 2006). B cells are responsible for producing RF and anti-CCP autoantibody, which are serological hallmarks of RA. The appearance of RF and anti-citrullinated protein antibody (ACPA) can precede the onset of clinical symptoms of RA by 10 years, indicating that humoral autoimmunity is an early critical event in the pathogenesis of RA. However, B cells can influence the immune response in ways that extend beyond autoantibody production. As effective antigen presenting cells, B cells present self-antigen to CD4+ T cells and activate them through the expression of co-stimulatory molecules. Different B cell subsets can also release a variety of cytokines (e.g., IL-2, IL-4, IL-6, IL-10, and IFN-␥) in a non-classical antibodyindependent manner (Bao and Cao 2014). Thus, they have been recently established to be endowed with great potential to regulate the immune system, and can serve pathogenic or protective roles by producing cytokines that amplify or suppress inflammation. Additionally, they are involved in the formation of ectopic lymphoid structures, especially in the synovial tissue of RA patients. Ectopic lymphoid organogenesis promotes T cell activation and aggravates synovial hyperplasia and angiogenesis, leading to
cartilage and bone erosion (Silverman and Carson 2003). During the past decades, a close relationship has been established between B cells and the skeletal system in animal models and experimental studies have advanced our understanding of RA and B cells. Indeed, B cell development can be blocked by bone cell inactivation. In turn, abnormalities in B cells can affect bone mass (Manilay and Zouali 2014). However, the exact molecular mechanism whereby B cells are closely related to the bone and joint remains unclear. Not only are their functions complicated, their classifications vary based on different B cell surface markers and functions. Unlike T cells, which are extensively studied in immune disorders, B cells still need to be explored because of their diversified functions and complex classifications. Our study profiled the DR1-5, at the protein level by flow cytometry. Pathological status was one factor associated with dopamine receptor expression. Furthermore, expression levels of all DRs in the RA and OA groups were lower than those in healthy controls, which indicated that, similar to the central nervous system, age may act as a confounding factor in immune cell DR expression. However, between the two disease entities, B cell levels of DR expression showed a higher tendency in RA patients than in OA patients. Capellino et al. reported that mRNA levels of DR genes were markedly elevated in synovial fibroblasts of RA patients compared to OA patients and could represent potential markers of inflammation (Capellino et al. 2014). We found that B cell D2 expression level was lower in RA patients than in healthy controls. DR2 was also negatively correlated with acute phase reactive protein, ESR, and CRP, as well as the disease activity scores DAS28 and SDAI. Additionally, either D1- or D2-like receptors were correlated with RF. After DMARDs therapy, the levels of DR2 and DR3 on B cells increased, which were accompanied by reduced acute phase reactants and disease activity. Although the underlying mechanism of dopaminergic signaling in B cells remains enigmatic, it might
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be a significant regulator in a direct or indirect way that influences the immune response. B-cell depletion therapy (Chen and Cohen 2012) and bromocriptine (McMurray 2001), a DR2 receptor agonist, have been found to be effective in the treatment of RA, supporting this hypothesis and also highlighting a possible role of a dopamine receptor as a therapeutic target. We propose that B cell dopamine receptors could be involved in the production of autoantibodies, such as RF, and B cell-producing cytokines, thereby affecting Th1/Th2/Th17 cell polarization and regulating bone metabolism. However, testing these theories will require additional evidence from basic and clinical studies. To our knowledge, the present study is the first to report B cell DR expression levels in RA patients. We used quantitative and effective ways to measure DR1-5 expression at the protein level and we monitored the changes after DMARDs therapy. However, some flaws and limitations exist in this study. Our sample size was limited. Therefore, its implication needs further large scale study. As the age of healthy controls was much younger than that of RA patients, this represents a confounding factor that was not excluded. Moreover, most of the DMARDs used in our therapy act on T cells rather than B cells. Additional in-depth studies will be needed to investigate the B cell dopaminergic system in RA and the effects of the therapeutic targeting of these receptors on B cells. Conflict of interest The authors declare that they have no conflict of interests. Disclosure None. Acknowledgements This research was the major program funded by of Shanghai Committee of Science and Technology (grant no. 12JC1402500, The mechanism of dopaminergic system modulation on T cell polarization). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.imbio. 2014.10.016. References Alberio, T., Pippione, A.C., Comi, C., Olgiati, S., Cecconi, D., Zibetti, M., Lopiano, L., Fasano, M., 2012. Dopaminergic therapies modulate the T-CELL proteome of patients with Parkinson’s disease. IUBMB Life 64, 846–852. Altman, R., Asch, E., Bloch, D., Bole, G., Borenstein, D., Brandt, K., Christy, W., Cooke, T.D., Greenwald, R., Hochberg, M., 1986. Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association. Arthritis Rheum. 29, 1039–1049. Arnett, F.C., Edworthy, S.M., Bloch, D.A., McShane, D.J., Fries, J.F., Cooper, N.S., 1988. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 31, 315–324. Bao, Y., Cao, X., 2014. The immune potential and immunopathology of cytokine-producing B cell subsets: a comprehensive review. J. Autoimmun., http://dx.doi.org/10.1016/j.jaut.2014.04.001 [Epub ahead of print]. Basu, S., Dasgupta, P.S., 2000. Dopamine, a neurotransmitter, influences the immune system. J. Neuroimmunol. 102, 113–124. Bellinger, D.L., Millar, B.A., Perez, S., Carter, J., Wood, C., ThyagaRajan, S., Molinaro, C., Lubahn, C., Lorton, D., 2008. Sympathetic modulation of immunity: relevance to disease. Cell Immunol. 252, 27–56. Besser, M.J., Ganor, Y., Levite, M., 2005. Dopamine by itself activates either D2, D3 or D1/D5 dopaminergic receptors in normal human T-cells and triggers the selective secretion of either IL-10, TNFalpha or both. J. Neuroimmunol. 169, 161–171. Brito-Melo, G.E., Nicolato, R., de Oliveira, A.C., Menezes, G.B., Lélis, F.J., Avelar, R.S., Sá, J., Bauer, M.E., Souza, B.R., Teixeira, A.L., Reis, H.J., 2012. Increase in
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Immunobiology journal homepage: www.elsevier.com/locate/imbio
Dopamine receptor DR2 expression in B cells is negatively correlated with disease activity in rheumatoid arthritis patients L. Wei a,1 , C. Zhang b,1 , H.Y. Chen a , Z.J. Zhang a , Z.F. Ji a , T. Yue c , X.M. Dai a , Q. Zhu c , L.L. Ma a , D.Y. He c , L.D. Jiang a,∗ a
Department of Rheumatology, Zhongshan Hospital, Fudan University, Shanghai, China Department of Orthopedics, Zhongshan Hospital of Fudan University, Shanghai, China c Department of Rheumatology, Shanghai Guanghua Hospital of Integrated Chinese & Western Medicine, Shanghai, China b
a r t i c l e
i n f o
Article history: Received 16 September 2014 Received in revised form 19 October 2014 Accepted 20 October 2014 Available online 5 November 2014 Keywords: B cells Disease activity Dopamine receptor Neuro-immune interaction Rheumatoid arthritis
a b s t r a c t Objective: Dopamine receptor (DR) signaling is involved in the pathogenesis of autoimmune diseases. We aimed to measure the expression levels of DR1-5 on B cells from patients with rheumatoid arthritis (RA) and to analyze the relationship between DRs and clinical manifestations, inflammatory biomarkers, functional status and disease activity. Methods: A total of 29 patients with RA, 12 healthy donors and 12 patients with osteoarthritis (OA) were recruited in this study. Flow cytometry was used to measure the levels of DR1-5 expressed on B cells. The relationships between B cell DR expressions and clinical features in RA patients were analyzed using the Spearman correlation test. Results: The expression levels of B cell DR1-5 in both the RA and OA groups were lower than those in healthy controls. After 3 months of medication, all five receptors were elevated in RA patients, with DR2 and DR3 being significantly increased from the baseline. DR2 expression on B cells was negatively correlated with inflammatory biomarkers and disease activity. Conclusion: RA patients had lower expression level of DR2 on B cells compared to the healthy controls, and the level of DR2 negatively correlated with the disease activity. DR2 and DR3 might be novel predictors of patient responses to disease modifying antirheumatic drug therapy. © 2014 Elsevier GmbH. All rights reserved.
Introduction
Abbreviations: RA, Rheumatoid arthritis; DR, dopamine receptor; OA, osteoarthritis; cAMP, cyclic adenosine monophosphate; SLE, systemic lupus erythematous; DMARD, disease modifying antirheumatic drug; TNF-␣, tumor necrosis factor-␣; TJC, tender joint count; SJC, swollen joint count; HAQ, health assessment questionnaire; PGA, patient global assessment; EGA, evaluator global assessment; VAS, visual analog score; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; anti-CCP, anti-cyclic citrullinated protein; RF, rheumatoid factor; DAS28, 28-joint disease activity score; SDAI, simplified disease activity index; CDAI, clinical disease activity index; PBMCs, peripheral blood mononuclear cells; APC, allophycocyanin; SD, standard deviation; IQR, inter-quartile range; MTX, methotrexate; HCQ, hydroxychloroquine; SSZ, sulfasalazine; LEF, leflunomide; ACPA, anti-citrullinated protein antibody; DC, dendritic cell; NK cell, natural killer cell; aa, amino acid. ∗ Corresponding author at: Department of Rheumatology, Zhongshan Hospital, Fudan University, No. 180, Road Fenglin, Shanghai 200032, PR China. Tel.: +86 021 64041990 2940. E-mail address: [email protected] (L.D. Jiang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.imbio.2014.10.016 0171-2985/© 2014 Elsevier GmbH. All rights reserved.
Rheumatoid arthritis (RA) is a heterogeneous immune disorder characterized by chronic erosive inflammatory synovitis with the production of multiple autoantibodies, which indicate the abrogation of self-tolerance. Genetic and environmental factors impinge upon all stages of RA pathogenesis. To date, RA pathogenesis has been explained at multiple levels; however, the exact pathogenic mechanism involved remains unclear. The neuro-immune interaction, a holistic theory, allows us to gain a better understanding of the development and progression of autoimmune diseases. Recently, neurotransmitters have been extensively studied, especially monoamines in the central and peripheral immune systems. Dopamine, a catecholamine, exerts its function by targeting specific DR to modify the activation (Mikulak et al. 2014), proliferation (Saha et al. 2001), differentiation (Nakano et al. 2009a,b), chemotaxis, homing (Watanabe et al. 2006) and apoptosis of immune cells (Oberbeck et al. 2006). The five DRs, termed DR1, DR2, DR3, DR4, and DR5, are G-protein coupled receptors that can be further categorized into two classes based on their ability to
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modulate cyclic adenosine monophosphate (cAMP) production, the D1-like (DR1 and DR5) and D2-like (DR2, DR3, and DR4) receptors. D1-like receptors activate the G␣s/olf G protein subunit, resulting in the activation of adenylyl cyclase and the upregulation of cAMP. D2-like receptors activate the G␣i/o G protein subunit, leading to the down-regulation of endogenous cAMP by inhibiting adenylyl cyclase. Thus, D1- and D2-like receptors often play antagonistic roles. However, sometimes they can operate in a synergistic way (Paul et al. 1992). Many lines of evidence support the reciprocal effect of dopamine on autoimmune diseases. There are extensive interactions between sympathetic innervations and lymphoid organs (Bellinger et al. 2008), as well as the musculoskeletal system (including the synovial cavity) (Jänig and Green 2014). Interestingly, immune cells also can synthesize, transport and release dopamine (Nakano et al. 2009a,b). In immunopathologies, alterations in serum dopamine levels have been noted. These pathophysiological findings provide a basis for its function in the immune system. Additionally, dopaminergic therapies, using either dopamine itself or DR agonists/antagonists in patients with Parkinson’s disease or multiple sclerosis, have shown effects on the T cell proteome (Alberio et al. 2012) and polarization (Ferreira et al. 2014). Expressions of the DRs by T cells are also related to the severity of schizophrenia (BritoMelo et al. 2012). These studies have demonstrated the role of dopamine in orchestrating the immune response in the development of neuropsychiatric disorders. Moreover, many investigations have been conducted to assess the distribution and modulation of the dopaminergic system in autoimmune diseases, including systemic lupus erythematosus (SLE) (Jafari et al. 2013), primary Sjögren syndrome (Wester et al. 1991), RA (Capellino et al. 2014; Nakano et al. 2011; Nakashioya et al. 2011) and inflammatory bowel disease (Magro et al. 2002). Measurement of DRs by flow cytometry revealed that all five dopamine receptors are expressed in immune cells, including a low expression level in T cells and high expression level in B cells (McKenna et al. 2002). However, the exact phenotypic distribution of different subtypes of these receptors is unknown in B cells in immnopathologies. Here, we generated a comprehensive profile of these five DRs on B cells in patients with RA and analyzed the correlation between DR expression levels and disease activity, functional status, and serum biomarkers. In this study of the dopamine system, we shed some new light on the pathogenesis of RA and identify new potential therapeutic targets.
Materials and methods Study population Healthy blood donors, patients with RA and patients with osteoarthritis (OA) satisfied the classical diagnostic criteria (Arnett et al. 1988; Altman et al. 1986) were recruited from the Department of Rheumatology, Zhongshan Hospital from July 12, 2013 to May 31, 2014. Inclusion criteria for the RA group encompassed patients with active disease (the 28-joint disease activity score [DAS28] according to the CRP formula > 3.2) and disease modifying anti-rheumatic drug (DMARD) naive or no DMARDs use within the previous 3 months. Patients who had received anti-tumor necrosis factor␣ (TNF-␣) or glucocorticoid therapy were excluded. The other exclusion criteria for both the RA and OA groups were infectious or inflammatory diseases, endocrine disorders, any past or current psychiatric or neurological diseases, pregnant or planning to be pregnant, lactation, liver or kidney dysfunction, cardiovascular disease, cancer, any drug history that would affect the sympathomimetric or sympatholytic system, and recent severe stress events. All subjects were provided with written informed consent. This study
was conducted according to the Declaration of Helsinki and was approved by the ethics committee of Zhongshan Hospital.
Assessment and follow-up Complete medical histories, physical examinations, and radiological and laboratory tests were conducted for all individuals. Tender joint count (TJC), swollen joint count (SJC), health assessment questionnaire (HAQ), patient global assessment (PGA; visual analog score from 0 to 100 mm), evaluator global assessment (EGA; visual analog score from 0 to 100 mm), routine blood tests, liver and renal function, erythrocyte sedimentation rate (ESR; mm/h) and Creactive protein (CRP; mg/dl) were recorded. For all patients, the assessments were performed by one rheumatologist. ELISA tests for anti-cyclic citrullinated protein (anti-CCP; mg/L) antibody and rheumatoid factor (RF; IU/ml) were also performed. A DAS28 (CRP formula), simplified disease activity index (SDAI) (Smolen et al. 2003) and clinical disease activity index (CDAI) (Greenberg et al. 2009) were all calculated.
Antibodies and reagents Rabbit anti-human polyclonal antibodies (lgG) to DR1 (corresponding to amino acids [aa] 9–21 of human DR1), DR3 (corresponding to aa 2–10 of human DR3), DR4 (corresponding to aa 176–185 of human DR4), and DR5 (corresponding to aa 2–10 of human DR5) were purchased from Calbiochem (Merck Millipore, Germany) and anti-DR2 (raised against 11 aa near the N-terminus ligand binding domain of rat DR2 and cross-reacts with human DR2) was from LifeSpan BioSciences (Seattle, WA, USA). Allophycocyanin (APC)-conjugated CD19 and its isotype control were obtained from Becton Dickinson (San Jose, CA, USA). Histopaque1077 and goat anti-rabbit CF405M-conjugated antibody were from Sigma–Aldrich (St. Louis, MO, USA). Normal rabbit IgG antibody as an isotype control was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Peripheral blood mononuclear cells (PBMCs) isolation Peripheral overnight fasting blood (10 ml) from the cubital vein was preserved in tubes (10 ml, BD Biosciences) containing ethylene diamine tetraacetic acid (EDTA). PBMCs were separated using Ficoll–Hypaque density centrifugation. Cells were washed three times in phosphate buffered saline and prepared at a final concentration of 1 × 106 /ml.
Dopamine receptor detection B cells were labeled with APC-conjugated CD19 mouse antihuman monoclonal antibody. Cells were incubated with DR polyclonal (DR1-5) antibodies (1:100 dilution) at 4 ◦ C for 30 min and were then washed twice with staining buffer and incubated with secondary CF405M-conjugated goat anti-rabbit antibody (1:100 dilution) at 4 ◦ C for 30 min. Samples with ‘no primary antibody’ (NPA) or normal rabbit sera (Rab) control (1:100) were used as two negative controls. Stained cells were analyzed using a Conto II flow cytometer (BD Biosciences). Data analysis was performed with Diva software (BD Biosciences) and FlowJo V.7.6.4 (Treestar Inc., Ashland, OR, USA). A minimum of 1,000,000 cells were analyzed from each sample. The results were finally expressed as the percentage of positive cells (%). BDR (%) was the percentage of CD19+ DR+ cells among CD19+ cells.
L. Wei et al. / Immunobiology 220 (2015) 323–330 Table 1 The general baseline characteristics of RA patients. Baseline characteristics
RA group (n = 29)
Male/female Age (years), mean (SD) Disease duration (month), median (IQR) TJC, mean (SD) SJC, mean (SD) ESR (mm/h), mean (SD) CRP (mg/L), median (IQR) Anti-CCP antibody, median (IQR) RF (IU/ml), median (IQR) Pain (VAS, 1–100 mm), median (IQR) PGA (VAS, 1–100 mm), median (IQR) EGA (VAS, 1–100 mm), median (IQR) HAQ, median (IQR) DAS28, mean (SD) 3.2 ≤ DAS28 < 5.3, n (%)t 5.3 ≤ DAS28, n (%) CDAI, mean (SD) SDAI, mean (SD)
5/24 53.69 (12.05) 42 (6–81) 17.56 (7.92) 16.26 (8.02) 69.44 (33.66) 30.90 (16.00–58.92) 236.29 (73.50–501.00) 118.00 (34.90–446.00) 70.00 (60–85) 70.00 (50–80) 70.00 (55–85) 1.00 (0.2–2.00) 6.38 (1.41) 7 (24.14) 22 (75.86) 46.06 (19.19) 50.53 (21.17)
TJC, tender joint count; SJC, swollen joint count; ESR, erythrocyte Sedimentation Rate; CRP, C-reactive Protein; anti-CCP antibody, anti-cyclic citrullinated protein antibody; RF, rheumatoid factor; HAQ, Health Assessment Questionnaire Disability Index; PGA, patient global assessment; EGA, evaluator global assessment; VAS, visual analog score; DAS28, 28-joint disease activity score; CDAI, clinical disease activity index; SDAI, simplified disease activity index; SD, standard deviation; IQR, inter quartile range. Data are presented as means ± SD or means (IQR) as appropriate.
Statistical analysis Continuous data were expressed as means ± standard deviation (SD) or medians (inter-quartile range, IQR) according to the data distribution. Statistical analyses of nonparametric data were performed using the Mann–Whitney U-test. The pre- and posttreatment expression levels of B cell DRs were compared using the Wilcoxon rank test. The relationship between the DR expression levels and the disease activity score, inflammatory markers, autoantibodies, and functional status were analyzed using Spearman’s correlation test. A P-value < 0.05 was considered to indicate a statistically significant difference. Results Baseline characteristics There were 29 eligible RA patients included in this study. Patients’ demographic and clinical data are shown in Table 1. Five patients were male and eight patients were treatment-naive. The mean age of RA patients was 53.80 ± 11.58 years old and the median disease duration was 42 (6–81) months. All patients had moderate to severe disease activity according the DAS28 score with a mean value of 6.38 ± 1.41, and a majority of these patients (75.86%) had severe disease activity. Only three patients had radiological bone erosions. As controls, 12 OA patients and 12 healthy donors were also recruited. The mean ages of these patients were 56.75 ± 9.19 and 24.58 ± 2.07 years old, with the sex ratio (male/female) of 1:1 and 1:2, respectively. The ages of the OA and RA groups were not significantly different (P = 0.368); however, both groups were significantly older than the healthy controls (P < 0.001). B cell DR1-5 expression levels in RA patients The expression levels of DR1-5 on B cells were investigated by flow cytometry (Figs. 1 and 2A and B). In RA patients, a lower percentage of DR1+ cells (2.88%, 1.49–4.89%) were detected compared to healthy controls (4.45%, 3.04–9.90%; P = 0.068), which was significantly higher than that detected in the OA group (1.56%,
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0.77–3.47%; P = 0.007). B cell DR1 expression in the RA group was slightly higher than that in the OA group, but this difference did not reach our threshold for statistical significance (P = 0.150). B cell DR2 expression levels were 6.94% (2.52–15.40%) in the RA group and 3.58% (2.58–8.13%) in the OA group, which were both markedly lower than that in the healthy controls (28.1%, 14.38–42.35%; P = 0.003 or P = 0.001, respectively). No difference was observed between the RA and OA groups. In RA patients, the expression levels of DR3 (2.90%, 0.98–8.98%), DR4 (2.24%, 0.79–3.68%), and DR5 (2.39%, 1.36–10.27%) showed no differences with the OA group (DR3, 2.48% (IQR: 1.11–4.45%); DR4, 1.84% (IQR: 0.68–4.30%); DR5, 1.90%, (IQR: 0.98–5.95%)) or the healthy control groups (DR3, 3.19% (IQR: 2.05–8.14%); DR4, 3.99% (IQR: 2.56–6.66%); DR5, 2.89%, (IQR: 2.57–7.27%)). However, trends for higher expression levels of DR15 in RA patients compared to OA patients, and lower expression levels of DR1-5 in RA patients compared to healthy controls were observed. Dopamine receptor expression levels varied greatly in the three groups, with the variation for DR2 expression being the most striking (Fig. 2B). When the RA patients were divided into two groups based on the DAS28 score, 7 were in the moderate group (3.2 < DAS28 ≤ 5.3) and 22 were in the severe group (DAS28 > 5.3). DR2 expression in the severe group was much lower than that in the moderate group (5.98%, IQR: 2.08–13.70% vs. 15.00%, IQR: 10.14–27.08%; P = 0.047). The expression of other DRs showed no significant differences between these two groups. Five DRs were further divided into D1-like (DR1 and DR5) and D2-like (DR2, DR3, and DR4) receptors. D1-like receptor expression was 5.32% (4.10–15.79%) in the RA group, which showed no difference with the healthy controls (8.02%, 5.66–19.86%) or the OA group (3.78%, 2.06–8.93%). The frequency of D2-like receptor expression was 13.50% (6.94–38.20%) in the RA group, which had a low expression level compared to the healthy controls (38.02%, 22.97–48.30%; P = 0.021) and showed no difference with the OA group (10.61%, 6.14–14.68%; P = 0.214). The relationship between B cell DR1-5 expression levels and clinical features A Spearman correlation analysis was performed to investigate the relationships between DRs and clinical manifestations, inflammatory markers, disease activity, and functional capabilities (Table 2). DR2 was found to be negatively correlated with ESR (r = −0.623, P < 0.001; Fig. 3A), CRP (r = −0.556, P = 0.002; Fig. 3B), DAS28 (r = −0.471, P = 0.01; Fig. 3C), SDAI (r = 0.441, P = 0.017, Fig. 3E) and EGA (r = −0.459, P = 0.012). Other DRs, including DR1, DR3, and DR5, were negatively associated with RF (r = −0.396, −0.427, −0.492, respectively; P-values were all <0.05). Besides, DR1 expression was correlated with CRP (r = −0.451, P = 0.014) and DR4 was negatively correlated with HAQ (r = −0.387, P = 0.038). D1-like receptors were also associated with RF (r = −0.504, P = 0.007). A correlation could be observed between RF and D2-like receptors (r = −0.499, P = 0.008). Additionally, D2-like receptors also correlated negatively with ESR (r = −0.431, P = 0.019), CRP (r = −0.522, P = 0.004), EGA (r = −0.387, P = 0.038), and DAS28 (r = −0.384, P = 0.04). Comparison of the baseline and post-treatment levels of DRs In our cohort, 14 of 29 RA patients who had good intention to treat were administered DMARDs and were followed up every month at our clinic. DMARDs included methotrexate (MTX, nine patients), TNF-␣ blockers (five patients), hydroxychloroquine (HCQ, four patients), low dose glucocorticoid (four patients), penicillamine (three patients), Tripterygium wilfordii (two patients), sulfasalazine (SSZ, four patients), iguratimod (one patient), and
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Fig. 1. Expression of DRs by B cells in RA patients compared with healthy controls and OA patients. (A–E) Representative dot plots of the expression of DR1-5 by B cells in RA patient using flow cytometric analysis. (F) Comparison of the DR1-5 expressions by B cells in RA patients (n = 29) with that in OA patients (n = 12) and healthy controls (n = 12) (Mann–Whitney test). Data were expressed as median (IQR). Asterisks represent statistically significant differences (P < 0.05).
leflunomide (LEF, one patient). After 3 months of therapy, peripheral blood was collected and laboratory tests, disease activity, and functional status were evaluated by the same rheumatologist. The ESR and CRP of the patients dropped significantly to 27.5 ± 19.74 mm/h and 3.82 mg/L, respectively. The tender and swollen joints were also improved. The mean TJC was 3.57 ± 4.97 and SJC was 2.93 ± 5.66. The DAS28 score was 3.11 ± 1.33. Out of the 14 patients, 2 remained severe disease activity (DAS28 > 5.3) and 3 who showed moderate activity. The rest of the 9 patients
achieved low disease activity or remission (DAS28 < 3.2). Only one patient had elevated liver enzyme levels at her 3-month follow-up visit. Dopamine receptor expression levels were measured again to compare the post-treatment with the baseline levels (Table 3). After treatment, B cell DR2 expression levels were significantly higher, increasing from 6.76% (2.44–15.85%) at baseline to 20.5% (15.70–43.35%) post-treatment (P < 0.05; Fig. 2B). DR3 expression also increased from baseline (2.41%, 0.54–6.14%) to the posttreatment level (6.51%, 3.81–14.95%; P < 0.05).
Table 2 Spearman correlation analyses of the expression of DRs by B cells and clinical features.
TJC SJC ESR CRP CCP RF HAQ PAIN PGA EGA DAS28 CDAI SDAI
BDR1
BDR2
BDR3
BDR4
BDR5
−0.011 0.179 −0.131 −0.451* 0.031 −0.396* −0.274 −0.063 −0.126 −0.210 −0.275 −0.146 −0.207
−0.075 −0.043 −0.623** −0.556** −0.313 −0.306 −0.341 −0.188 0.146 −0.459* −0.471* −0.358 −0.441*
−0.1 0.026 −0.044 −0.306 −0.256 −0.427* −0.224 0.163 0.095 −0.115 −0.156 −0.028 −0.125
−0.140 0.028 −0.186 −0.152 −0.269 −0.374 −0.387* 0.101 0.018 −0.091 −0.098 −0.03 −0.071
0.129 0.286 −0.239 −0.048 −0.153 −0.492* −0.340 0.075 0.038 −0.094 0.057 0.086 0.06
All correlations were assessed using Spearman’s rank order correlation coefficient (r). * P-value < 0.05. ** P-value < 0.01. See abbreviations in Table 1.
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Fig. 2. Expression of DR2 by B cells in RA patients compared to healthy controls and OA patients. (A) Representative flow cytometric analysis of DR2 expression by CD19+ B cells. Data at baseline from healthy controls (n = 12) and patients with OA (n = 12) or RA (n = 29) are shown. (B) A comparison of DR2 expression in B cells in healthy controls, OA patients, and RA patients (Mann–Whitney test). (C) A comparison of the pre- and post-treatment levels of DR2 expressed by B cells in RA patients (n = 14; Wilcoxon rank test). Lines within the dot plot represent medians. Two-tailed P-values < 0.05 were considered to indicate statistically significant differences.
Discussion The regulatory role of the dopaminergic system in the immune response has been reviewed in detail elsewhere (Sarkar et al. 2010; Basu and Dasgupta 2000; Pacheco et al. 2014). Alterations in DR expression levels have been detected in autoimmune diseases. Jafari et al. found that SLE patients showed lower expression levels of the DR gene DR2 and higher expression of DR4 by realtime PCR in PBMC, and further attributed altered DR2 and DR4 gene expression levels to T cells rather than B cells (Jafari et al. 2013). In rheumatoid arthritis, dopamine released by dendritic cells (DC) can contribute to DC–T cell interactions, which affect T cell
Table 3 A comparison of DR2 expression by B cells of RA patients at the baseline and after 3 months of DMARDs therapy. Receptor
Pre-treatment (%)
Post-treatment (%)
Ba BDR1 BDR2 BDR3 BDR4 BDR5
9.00 (5.64–16.76) 3.04 (1.34–5.16) 6.76 (2.44–15.85) 2.41 (0.54–6.14) 1.51 (0.50–3.95) 2.63 (0.48–5.43)
9.43 (4.63–10.88) 6.24 (3.45–19.20) 20.5b (15.70–43.35) 6.51b (3.81–14.95) 3.63 (1.01–12.95) 4.67 (2.67–16.15)
The Wilcoxon rank test was used to compare the baseline pre-treatment with the post-treatment levels of CD19+ DR2+ B cells in RA patients (n = 14). a Percentage of CD19+ B cells among lymphocytes. b Two-tailed P-value < 0.05.
differentiation (Nakano et al. 2009a,b). The roles of the dopaminergic system were further investigated using DR agonists/antagonists in animal models. After treatment with the D1-like receptor antagonist SCH23390, mice with collagen-induced arthritis exhibited lower arthritis scores and less severe joint destruction than control mice (Nakashioya et al. 2011). The therapeutic effect of SCH23390 was also shown in another study in which the authors found that in the human RA/SCID mouse chimera model, SCH23390 suppressed cartilage destruction by inhibiting the IL-6–IL-17 axis, while the D2like receptor antagonist haloperidol worked in the opposite way (Nakano et al. 2011). Synovial fibroblasts, which are one of the most important participants in the pathogenesis of RA, have been shown to express all five DRs by immunohistochemistry. IL-6 and IL-8 production by synovial fibroblasts can be inhibited by dopamine via the D1-like receptor (Capellino et al. 2014). Tanaka and colleagues studied the regulation of DR functions in osteoclastogenesis (Hanami et al. 2013). The functions of the DR depend upon the experiment conditions, specific DR subtype, cell type and cell status. Pharmacological or physiological doses of dopamine can affect experimental results obtained either in vitro or in vivo. The five dopamine receptor subtypes, even those among the same class (D1- or D2-like), can vary in their downstream signaling pathways and biological effects. DR1 is low expressed in immune cells has not been thoroughly investigated. Dopamine mediates IL-10 and TNF-␣ release by activating DR2 and DR3, respectively (Besser et al. 2005). Stimulation of DR3 also increases IFN-␥ secretion in T cell blasts (Ilani et al. 2004).
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Fig. 3. The relationship between DR2 expressed by CD19+ B cells and clinical data in RA patients (n = 29). (A) ESR (mm/H). (B) CRP (mg/L). (C) DAS28 score. (D) CDAI score. (E) SDAI score. Data from 29 patients with RA at baseline were compared by Spearman correlation analysis. Two-tailed P-values < 0.05 were considered to indicate statistically significant differences.
By targeting DR4, dopamine can induce T cell quiescence (Sarkar et al. 2006). Dopamine regulates NK cell cytotoxicity through the cAMP–PKA–CREB signaling pathway (Zhao et al. 2013). By binding to DR5, dopamine exerts an inhibitory function to ‘switch-off’ activated natural killer (NK) cells (Mikulak et al. 2014). Central memory T cells (TCM ) and effector memory T cells (TEM ) express more D2than D1-like receptors. By contrast, naive T cells express higher level of D1-like receptor (Kustrimovic et al. 2014), which indicates that the cell type and activation state play key roles in establishing the DR repertoire. Unfortunately, most studies that focused on dopamine and DR functions in immune cells did not include B cells. Only one study found that dopamine could induce the apoptosis of B cells, which occurred independently of the DRs (Meredith et al. 2006). B cells are responsible for producing RF and anti-CCP autoantibody, which are serological hallmarks of RA. The appearance of RF and anti-citrullinated protein antibody (ACPA) can precede the onset of clinical symptoms of RA by 10 years, indicating that humoral autoimmunity is an early critical event in the pathogenesis of RA. However, B cells can influence the immune response in ways that extend beyond autoantibody production. As effective antigen presenting cells, B cells present self-antigen to CD4+ T cells and activate them through the expression of co-stimulatory molecules. Different B cell subsets can also release a variety of cytokines (e.g., IL-2, IL-4, IL-6, IL-10, and IFN-␥) in a non-classical antibodyindependent manner (Bao and Cao 2014). Thus, they have been recently established to be endowed with great potential to regulate the immune system, and can serve pathogenic or protective roles by producing cytokines that amplify or suppress inflammation. Additionally, they are involved in the formation of ectopic lymphoid structures, especially in the synovial tissue of RA patients. Ectopic lymphoid organogenesis promotes T cell activation and aggravates synovial hyperplasia and angiogenesis, leading to
cartilage and bone erosion (Silverman and Carson 2003). During the past decades, a close relationship has been established between B cells and the skeletal system in animal models and experimental studies have advanced our understanding of RA and B cells. Indeed, B cell development can be blocked by bone cell inactivation. In turn, abnormalities in B cells can affect bone mass (Manilay and Zouali 2014). However, the exact molecular mechanism whereby B cells are closely related to the bone and joint remains unclear. Not only are their functions complicated, their classifications vary based on different B cell surface markers and functions. Unlike T cells, which are extensively studied in immune disorders, B cells still need to be explored because of their diversified functions and complex classifications. Our study profiled the DR1-5, at the protein level by flow cytometry. Pathological status was one factor associated with dopamine receptor expression. Furthermore, expression levels of all DRs in the RA and OA groups were lower than those in healthy controls, which indicated that, similar to the central nervous system, age may act as a confounding factor in immune cell DR expression. However, between the two disease entities, B cell levels of DR expression showed a higher tendency in RA patients than in OA patients. Capellino et al. reported that mRNA levels of DR genes were markedly elevated in synovial fibroblasts of RA patients compared to OA patients and could represent potential markers of inflammation (Capellino et al. 2014). We found that B cell D2 expression level was lower in RA patients than in healthy controls. DR2 was also negatively correlated with acute phase reactive protein, ESR, and CRP, as well as the disease activity scores DAS28 and SDAI. Additionally, either D1- or D2-like receptors were correlated with RF. After DMARDs therapy, the levels of DR2 and DR3 on B cells increased, which were accompanied by reduced acute phase reactants and disease activity. Although the underlying mechanism of dopaminergic signaling in B cells remains enigmatic, it might
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be a significant regulator in a direct or indirect way that influences the immune response. B-cell depletion therapy (Chen and Cohen 2012) and bromocriptine (McMurray 2001), a DR2 receptor agonist, have been found to be effective in the treatment of RA, supporting this hypothesis and also highlighting a possible role of a dopamine receptor as a therapeutic target. We propose that B cell dopamine receptors could be involved in the production of autoantibodies, such as RF, and B cell-producing cytokines, thereby affecting Th1/Th2/Th17 cell polarization and regulating bone metabolism. However, testing these theories will require additional evidence from basic and clinical studies. To our knowledge, the present study is the first to report B cell DR expression levels in RA patients. We used quantitative and effective ways to measure DR1-5 expression at the protein level and we monitored the changes after DMARDs therapy. However, some flaws and limitations exist in this study. Our sample size was limited. Therefore, its implication needs further large scale study. As the age of healthy controls was much younger than that of RA patients, this represents a confounding factor that was not excluded. Moreover, most of the DMARDs used in our therapy act on T cells rather than B cells. Additional in-depth studies will be needed to investigate the B cell dopaminergic system in RA and the effects of the therapeutic targeting of these receptors on B cells. Conflict of interest The authors declare that they have no conflict of interests. Disclosure None. Acknowledgements This research was the major program funded by of Shanghai Committee of Science and Technology (grant no. 12JC1402500, The mechanism of dopaminergic system modulation on T cell polarization). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.imbio. 2014.10.016. References Alberio, T., Pippione, A.C., Comi, C., Olgiati, S., Cecconi, D., Zibetti, M., Lopiano, L., Fasano, M., 2012. Dopaminergic therapies modulate the T-CELL proteome of patients with Parkinson’s disease. IUBMB Life 64, 846–852. Altman, R., Asch, E., Bloch, D., Bole, G., Borenstein, D., Brandt, K., Christy, W., Cooke, T.D., Greenwald, R., Hochberg, M., 1986. Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association. Arthritis Rheum. 29, 1039–1049. Arnett, F.C., Edworthy, S.M., Bloch, D.A., McShane, D.J., Fries, J.F., Cooper, N.S., 1988. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 31, 315–324. Bao, Y., Cao, X., 2014. The immune potential and immunopathology of cytokine-producing B cell subsets: a comprehensive review. J. Autoimmun., http://dx.doi.org/10.1016/j.jaut.2014.04.001 [Epub ahead of print]. Basu, S., Dasgupta, P.S., 2000. Dopamine, a neurotransmitter, influences the immune system. J. Neuroimmunol. 102, 113–124. Bellinger, D.L., Millar, B.A., Perez, S., Carter, J., Wood, C., ThyagaRajan, S., Molinaro, C., Lubahn, C., Lorton, D., 2008. Sympathetic modulation of immunity: relevance to disease. Cell Immunol. 252, 27–56. Besser, M.J., Ganor, Y., Levite, M., 2005. Dopamine by itself activates either D2, D3 or D1/D5 dopaminergic receptors in normal human T-cells and triggers the selective secretion of either IL-10, TNFalpha or both. J. Neuroimmunol. 169, 161–171. Brito-Melo, G.E., Nicolato, R., de Oliveira, A.C., Menezes, G.B., Lélis, F.J., Avelar, R.S., Sá, J., Bauer, M.E., Souza, B.R., Teixeira, A.L., Reis, H.J., 2012. Increase in
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