Acta Pharmacologica Sinica 2017: 1466–1474 © 2017 CPS and SIMM All rights reserved 1671-4083/17 www.nature.com/aps Original Article The immunoglobulin D Fc receptor expressed on fibroblast-like synoviocytes from patients with rheumatoid arthritis contributes to the cell activation Yu-jing WU#, Wen-sheng CHEN#, Heng-shi CHEN, Xing DAI, Jin DONG, Ying WANG, Ling-ling ZHANG, Yan CHANG, Qiong HUANG, Xiao-yi JIA, Wei WEI* Institute of Clinical Pharmacology, Anhui Medical University, Key Laboratory of Anti-inflammatory and Immune Medicine, Ministry of Education, Anhui Collaborative Innovation Center of Anti-inflammatory and Immune Medicine, Hefei 230032, China Abstract Immunoglobulin IgD might play an important role in autoimmune diseases, but the function of IgD has remained elusive, despite multiple attempts to define its biological function. Fibroblast-like synoviocytes (FLSs) are specialized cells of the synovium that play a key role in the pathogenesis of rheumatoid arthritis (RA). In this study we explored the possible roles of excessive IgD expression on the function of FLSs from RA patients (RA-FLSs). We showed that IgD Fc receptor (IgDR) was constitutively expressed on FLSs, and was significantly elevated in RA-FLSs compared with FLSs prepared from synovial tissues of healthy controls (HC-FLSs). Furthermore, IgDR was mainly detected on the cell surface and in the cytoplasm. We further detected the intrinsic binding affinity of IgD to IgDR on HC-FLSs with an equilibrium dissociation constant (KD) of 0.067 nmol/L. Incubation of RA-FLSs with IgD (1–10 μg/mL) for 48 h dose- dependently promoted the expression of IgDR, and stimulated the production of inflammatory cytokines and chemokines, such as IL-1β, IL-6, monocyte chemotactic protein (MCP)-1, TNF-α and receptor activator of nuclear factor-κB ligand (RANKL), thus potentially contributing to IgD-IgDR crosslinking. Moreover, incubation with IgD (0.1–10 μg/mL) for 48 h dose-dependently enhanced viability for both HC-FLSs and RA-FLSs. Our results demonstrate that IgDR is expressed on RA-FLSs and contributes to the activation of FLSs, and suggest that IgD-IgDR is a potential novel immunotherapeutic target for the management of RA. Keywords: rheumatoid arthritis; immunoglobulin D; immunoglobulin D Fc receptor; fibroblast-like synoviocytes; fluorescence based receptor binding assay; inflammatory cytokines Acta Pharmacologica Sinica (2017) 38: 1466–1474; doi: 10.1038/aps.2017.105; published online 3 Aug 2017 Introduction might play an important role in autoimmune diseases, and The immunoglobulin IgD can be expressed as membrane the function of IgD has remained elusive since its discovery, IgD (mIgD) representing one of two B cell receptors with an despite multiple attempts to define its biological function. attached signaling machinery or can be secreted as an antibody Many lymphoid and non-lymphoid cells express surface (sIgD)[1]. IgD has been found to be increased in several dis- membrane receptors, and many studies have focused on specific eases, such as IgD myeloma, tuberculosis, chronic obstructive structures located in the constant region of immunoglobulin pulmonary diseases, Hodgkin’s disease, hyper IgE and hyper molecules (FcR)[6], such as immunoglobulin A (IgA), IgE, IgG, IgD syndromes[2]. Furthermore, sIgD levels are increased and IgM. Surprisingly, an Fc receptor specific for IgD (FcδR or in autoimmune disorders such as rheumatoid arthritis (RA) IgDR) has been suggested to exist on T cells[7]; however, early and systemic lupus erythematosus (SLE)[1-4]. However, high attempts to detect its expression have produced variable and expression of sIgD is associated with increased protein-like conflicting results[8]. Through quantitative immunofluorescence sediments and cell necrosis in the kidney, spleen and liver in techniques, binding of IgD to T cells and basophilic cell lines has transgenic mice[5]. Recent studies have suggested that IgD been demonstrated, and an increase in δ+ T cells in vivo has also been observed, thus suggesting that a putative IgDR on T cells may play a role regulating IgD[2, 4, 9]. Recently, our group has # These authors contributed equally to this work. reported further evidence of the existence of an IgDR, demon- *To whom correspondence should be addressed. E-mail [email protected] strating that human IgD antibody induces cell proliferation in Received 2016-12-30 Accepted 2017-04-04 the human Burkitt lymphoma B cell line, Daudi, potentially www.chinaphar.com Wu YJ et al 1467 by activating the putative IgDR and consequently initiating was carried out in accordance with the Declaration of Helsinki a tyrosine phosphorylation signaling cascade that accelerates (2008) and was approved by the Ethics Committee of Anhui [10] the G1/S transition . Furthermore, we have reported that Medical University. Written informed consent was obtained in RA patients, the levels of sIgD, mIgD and IgDR are higher from each participant before the start of the study. than those in healthy controls, and after treatment with IgD antibodies, the expression of IgDR on peripheral T and B cells Flow cytometry analysis significantly increases, thereby possibly contributing to RA Expression of IgDR was detected by treatment of FLSs with pathogenesis[11]. biotinylated IgD. For detecting IgD binding receptors, a total RA is a chronic inflammatory autoimmune disease charac- of 1×106 cells were washed twice with PBS and incubated with terized by synovial hyperplasia and chronic inflammation. 1 µg of biotinylated IgD for 30 min at room temperature (RT). A marked increase in macrophage-like and fibroblast-like After washing, APC-Cy7 conjugated streptavidin was added, synoviocytes (FLSs) in the synovial membrane and the accom- and cells were incubated for an additional 30 min on ice and panying inflammatory infiltration of other immune cells such washed again. Cells stained with APC-Cy7 conjugated strep- as synovial macrophages result in bone erosion and joints tavidin alone were used as an isotype control. For assessment destruction. Accumulating evidence indicates that activated of IgD, a total of 1×106 cells were washed twice with PBS and FLSs in RA patients contribute to joint destruction, and the incubated with PE conjugated anti-IgD antibodies for 30 min possible roles of excessive IgD expression on the function of at RT, and the IgG1 isotype control was also matched. To FLSs from RA patients (RA-FLSs) are discussed in this work. determine the ability of IgD to induce the expression of IgDR, The expression of IgDR has been described on CD4+ and CD8+ FLS were stimulated with IgD for 48 h or were left unstimu- T cells in mice and humans[1, 3, 11]. However, the role of IgDR lated. After incubation, expression of IgDR was detected with on cells of the synovial infiltrate has not yet been studied. biotinylated IgD followed by APC-Cy7 conjugated streptavi- Therefore, we investigated the expression of IgDR in RA-FLSs din. Data were acquired on a FC 500 flow cytometer (Beck- and explored the effects of IgD. We identified IgD-binding man Coulter Ltd, CA, USA ) and analyzed with CXP analysis IgDR as a ~70 kDa protein complex that was detected on FLSs software (Beckman Coulter Ltd, CA, USA). by using flow cytometry and confocal laser scanning micros- copy. Studies of the binding properties indicated that human Confocal microscopy analysis IgD bound to IgDR with an equilibrium dissociation constant FLSs were plated on coverslips and fixed with 4% paraformal- (KD) of 0.067 nmol/L. Furthermore, we found that IgD sig- dehyde for 20 min at RT. Then, cells were permeabilized with nificantly promoted the viability of FLSs, induced cytokines/ 0.2% Triton X-100 for 10 min at RT and washed with PBS three chemokine secretion and increased the expression of IgDR in times. After being blocked with 1% BSA in PBS for 1 h at RT, FLSs, thus potentially contributing to RA pathogenesis. biotinylated IgD was added, and cells were incubated over- night at 4 °C. After being washed with PBS three times, cells Materials and methods were stained with streptavidin labeled APC-Cy7 secondary Reagents antibody for 2 h at 37 °C. Cells were then washed with PBS Human IgD was purchased from Abcam (Cambridge, MA, buffer three times. When the coverslips were dry, a mounting USA). Biotinylated IgD was prepared in our laboratory with solution containing DAPI (Vector Laboratories, Inc, Burlin- a protein biotinylation kit from Pierce Biotechnology (Rock- game, CA, USA) was applied, and images were taken using a ford, IL, USA), according to the manufacturer’s instructions[12]. SP8 confocal laser scanning microscope (Leica, Wetzlar, Ger- APC-Cy7 conjugated streptavidin, PE conjugated anti-IgD many). The expression of IgDR was quantitatively analyzed antibody and IgG1 isotype control were purchased from BD using ImageJ software. Pharmingen (San Diego, CA, USA). Fluorescence based receptor binding assay and Scatchard analysis Preparation and culture of FLSs Protocols were used to evaluate the intrinstic binding affinity Human synovial membranes were obtained from patients of fluorescence labeled IgD antibody to IgD on IgDR in FLSs. with RA undergoing arthroscopy. The diagnosis of RA con- Binding of IgD for detection by flow cytometry was done as formed to the American College of Rheumatology revised described below. Various concentrations of IgD (0.003, 0.01, criteria from 1987[13]. Normal knee tissues were obtained from 0.03, 0.1, 0.3, 1, 3, 10 μg/mL) were used. Maximal binding of donors undergoing traumatic surgery. Synovial tissues were IgD was observed on FLS in 6-well microtiter plates at 1×106 minced and digested with type IA collagenase for 3 h, filtered, cells/well, incubated at 37 °C for 2 h in fresh medium (with extensively washed and then cultured in DMEM contain- 0.1% BSA), then cells were washed three times with PBS at RT, ing 20% fetal bovine serum (FBS, Sigma-aldich, MO, USA) at then with buffer appropriate to the assay.