Chemerin activates fibroblast-like synoviocytes in patients with rheumatoid arthritis
© Kaneko et al.; licensee BioMed Central Ltd. 2011
Received: 9 March 2011
Accepted: 29 September 2011
Published: 29 September 2011
Chemerin is a chemotactic agonist identified as a ligand for ChemR23 that is expressed on macrophages and dendritic cells (DCs). In this study, we analyzed the expression of chemerin and ChemR23 in the synovium of rheumatoid arthritis (RA) patients and the stimulatory effects of chemerin on fibroblast-like synoviocytes (FLSs) from RA patients.
Chemerin and ChemR23 expression in the RA synovium was ascertained by immunohistochemistry and Western blot analysis. Chemerin expression on cultured FLSs was analyzed by ELISA. ChemR23 expression on FLSs was determined by immunocytochemistry and Western blot analysis. Cytokine production from FLSs was measured by ELISA. FLS cell motility was evaluated by utilizing a scrape motility assay. We also examined the stimulating effect of chemerin on the phosphorylation of mitogen-activated protein kinase (MAPK), p44/42 mitogen-activated protein kinase (ERK1/2), p38MAPK, c-Jun N-terminal kinase (JNK)1/2 and Akt, as well as on the degradation of regulator of NF-κB (IκBα) in FLSs, by Western blot analysis.
Chemerin was expressed on endothelial cells and synovial lining and sublining cells. ChemR23 was expressed on macrophages, immature DCs and FLSs and a few mature DCs in the RA synovium. Chemerin and ChemR23 were highly expressed in the RA synovium compared with osteoarthritis. Chemerin and ChemR23 were expressed on unstimulated FLSs. TNF-α and IFN-γ upregulated chemerin production. Chemerin enhanced the production of IL-6, chemokine (C-C motif) ligand 2 and matrix metalloproteinase 3 by FLSs, as well as increasing FLS motility. The stimulatory effects of chemerin on FLSs were mediated by activation of ERK1/2, p38MAPK and Akt, but not by JNK1/2. Degradation of IκB in FLSs was not promoted by chemerin stimulation. Inhibition of the ERK1/2, p38MAPK and Akt signaling pathways significantly suppressed chemerin-induced IL-6 production. Moreover, blockade of the p38MAPK and Akt pathways, but not the ERK1/2 pathway, inhibited chemerin-enhanced cell motility.
The interaction of chemerin and ChemR23 may play an important role in the pathogenesis of RA through the activation of FLSs.
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial hyperplasia, joint destruction and infiltration of the synovium by immunocytes, including lymphocytes, macrophages and dendritic cells (DCs) [1–3]. In addition to these cells, fibroblast-like synoviocytes (FLSs) play a major role in the pathogenesis of RA [4, 5] in that they produce a variety of cytokines, chemokines and matrix-degrading enzymes that mediate interaction with neighboring inflammatory and endothelial cells and are responsible for the progressive inflammation in the joints and destruction of the articular cartilage and bone [6–8].
Chemerin is the ligand protein for ChemR23, a G protein-coupled receptor expressed on macrophages, DCs and natural killer cells (NK cells) [9–11]. Chemerin is characterized as a strong chemoattractant factor for ChemR23-expressing cells and acts at subnanomolar concentrations [9, 12]. Chemerin is synthesized as an inactive precursor protein, prochemerin, which binds ChemR23 with low affinity [9, 13]. It can be rapidly converted into a full ChemR23 agonist by the proteolytic removal of the last six or seven amino acids by neutrophil-derived proteases (elastase and cathepsin G), mast cell products (tryptase), proteases of the coagulation cascade [14, 15] and certain bacterial proteases  at the inflammatory site.
Investigators in recent studies have reported that the expression of chemerin correlates with ChemR23-positive cell recruitment in human skin inflammatory diseases, such as systemic lupus erythematosus, oral lichen planus and psoriasis [10, 12, 17]. Moreover, it has been reported that chemerin and ChemR23 are expressed by human articular chondrocytes  and endothelial cells [19, 20]. The interaction of chemerin with ChemR23 is assumed to play an important role not only in the migration of macrophages and DCs to the sites of inflammation but also in the mediating inflammatory signaling to articular chondrocytes and endothelial cells. However, there is little information on the expression and function of chemerin in the RA synovium.
In the present study, we analyzed the expression of chemerin and ChemR23 in the RA synovium and evaluated the function of chemerin in cultured FLSs isolated from the synovium of RA patients to explore a possible role of chemerin and ChemR23 interaction in the pathogenesis of RA.
Materials and methods
Synovial tissue samples were obtained from nine RA patients who fulfilled the American College of Rheumatology diagnostic criteria for RA  and from four patients with osteoarthritis (OA) who underwent total knee joint replacement surgery. Signed consent forms were obtained before the operations. All patients with RA and OA were female. In RA patients, the mean age (± SEM) was 71.0 ± 3.7 years, mean disease duration (± SEM) was 10.75 ± 3.5 years, and their mean C-reactive protein concentration (± SEM) was 0.33 ± 0.17 mg/dl. Among the patients with RA, seven patients were seropositive for rheumatoid factor and five were positive for anticitrullinated protein antibodies. Two of the patients with RA were receiving prednisolone, one was being treated with bucillamine (an analogue of D-penicillamine), one was receiving methotrexate monotherapy, one was being treated with methotrexate and etanercept and three were was taking tocilizumab. Three patients were receiving no medication at the surgery. The experimental protocol was approved by the Ethics Committee of Tokyo Medical and Dental University.
Immunohistochemical analysis was conducted on OCT compound-embedded sections of frozen synovial tissues. Briefly, 8-μm-thick cryostat sections were fixed in cold acetone (stored at -20°C) for three minutes. Endogenous peroxidase activity was blocked by incubation in 2% H2O2 in methanol for 15 minutes, and then nonspecific binding was blocked with 10% normal goat serum in PBS for 40 minutes. Serial sections were then incubated for two hours at 4°C with 1 μg/ml rabbit anti-chemerin pAb (affinity-purified from rabbit serum immunized with glutathione S-transferase-chemerin (E21-S157) fusion protein; provided by KAN Research Institute, Kobe, Japan), 1 μg/ml rabbit anti-ChemR23 pAb (Acris Antibodies, Herford, Germany) or normal rabbit immunoglobulin G (IgG) (Sigma-Aldrich, St Louis, MO, USA) as an isotype control. Antibody binding was detected using the EnVision+ Kit (DakoCytomation, Carpinteria, CA, USA).
For immunofluorescence double-staining with CD68, CD1a, DC-LAMP or vimentin, and ChemR23, nonspecific binding was blocked with 1% blocking reagent (Roche, Manheim, Germany) in PBS, and then the sections were incubated overnight at 4°C with rabbit anti-ChemR23 pAb or normal rabbit IgG (control) at 1 μg/ml. The samples were then incubated with Alexa Fluor 568-conjugated goat anti-rabbit IgG (4 μg/ml; Invitrogen, Carlsbad, CA, USA) for one hour at room temperature. Next the sections were incubated overnight with 2.35 μg/ml mouse anti-CD68 mAb (KP1; DakoCytomation), 10 μg/ml mouse anti-CD1a mAb (BL6; Immunotech, Marseille, France), 10 μg/ml mouse anti-DC-LAMP mAb (104.G4; Immunotech) or 1 μg/ml mouse anti-vimentin mAb (V9; DakoCytomation) at 4°C. Subsequently, the samples were incubated with 4 μg/ml Alexa Fluor 488-conjugated goat anti-mouse IgG2a (Invitrogen) or anti-mouse IgG1 (Invitrogen) for one hour at room temperature. The slides were examined using a Biozero Fluorescence Microscope (Keyence, Tokyo, Japan).
For whole-mount staining, human synovial tissues from RA patients were washed vigorously in ice-cold PBS, then fixed and permeabilized with a BD Cytofix/Cytoperm Kit (BD Biosciences, San Jose, CA, USA). The whole-mount specimens of synovial tissues were then stained with rabbit anti-ChemR23 pAb or rabbit IgG as an isotype control, followed by secondary antibodies conjugated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (BD Biosciences). The specimens were further stained with Alexa Fluor 633-labeled phalloidin (BD Biosciences). The specimens were embedded in a 30% solution of glycerol in PBS and analyzed with a DM IRE2 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany).
Synovial tissues from RA patients were minced and incubated with 0.5 mg/ml collagenase (Sigma-Aldrich) for one hour at 37°C, then pressed through a metal screen to obtain single-cell suspensions. The harvested cells were plated in cell culture plates and incubated with DMEM (Sigma-Aldrich) supplemented with 10% FCS (Sigma-Aldrich). Adherent cells were maintained in the medium as FLSs and used after five passages  in the experiments that followed.
For immunofluorescence double-staining of cultured FLSs with ChemR23 and vimentin, FLSs were incubated on eight-well chamber slides (4 × 104 cells/well) (IWAKI, Tokyo, Japan) at 37°C overnight. The adherent cells were fixed in cold acetone for three minutes. Nonspecific binding was blocked with 10% normal goat serum in PBS, then the cells were incubated overnight at 4°C with rabbit anti-ChemR23 pAb or normal rabbit IgG at 1 μg/ml. Next the cells were incubated with Alexa Fluor 568-conjugated goat anti-rabbit IgG (4 μg/ml) for one hour at room temperature. After that step, the cells were incubated overnight with 1 μg/ml mouse anti-vimentin mAb at 4°C, followed by incubation with 1 μg/ml Alexa Fluor 488-conjugated goat anti-mouse IgG1 for one hour at room temperature. The slides were examined using a Biozero Fluorescence Microscope (Keyence).
ELISA for chemerin and inflammatory mediators produced by cultured fibroblast-like synoviocytes
FLSs were cultured overnight in 96-well plates (2 × 104 cells/well) in DMEM with 10% FCS and then incubated with or without recombinant human TNF-α (0.1, 1 or 10 ng/ml) or IFN-γ (1, 10 or 100 ng/ml) (both from R&D Systems, Minneapolis, MN, USA) at 37°C for 48 hours. The concentration of chemerin in culture supernatant was measured with an ELISA kit (R&D Systems) according to the instructions provided by the manufacturer.
To determine the effects of chemerin on the production of IL-6, chemokine (C-C motif) ligand 2 (CCL2) and matrix metalloproteinase 3 (MMP-3) by FLSs, the cells were cultured separately overnight in 96-well plates (4 × 104 cells/well), then incubated with or without recombinant bioactive human chemerin (2, 10 or 50 nM; R&D Systems) in FCS-free DMEM at 37°C for 24 and 48 hours. The protein levels of IL-6, CCL2 and MMP-3 in the supernatant were assessed using ELISA kits (R&D Systems) according to the manufacturer's instructions. To block the signaling pathway through p44/42 mitogen-activated protein kinase (ERK1/2), p38 mitogen-activated protein kinase (p38MAPK) or Akt, FLSs (4 × 104 cells/well) were pretreated with specific inhibitors, including a mitogen-activated protein kinase kinase (MEK) inhibitor (10 μM PD98059; Calbiochem, Merck KgaA, Germany), a p38MAPK inhibitor (10 μM SB203580; Calbiochem) or phosphoinositide 3-kinase (PI3K) inhibitor (10 μM LY294002; Cell Signaling Technology, Danvers, MA, USA) for 30 minutes before stimulation with 50 nM chemerin. To confirm the lack of a significant amount of endotoxin in the stimulation procedure, FLSs were incubated with 50 μg/ml polymyxin B in addition to 50 nM chemerin.
Scrape motility assay
RA FLSs were plated at a density of 1 × 105 cells/ml in 12-well plates (22.1-mm diameter) in DMEM with 10% FCS. After overnight incubation, the tip of a plastic pipette was drawn across the center of the well to produce a scraped area . The culture wells were washed twice with PBS, and free cells were removed. Then the cells were pretreated with or without pertussis toxin (PTX) (0.5 μg/ml), PD98059 (10 μM), SB203580 (10 μM) or LY294002 (10 μM) for 30 minutes, followed by incubation with 2 or 10 nM of chemerin and CCL2 in FCS-free medium. Immediately after scraping (zero hours) as well as after 24-hour incubation, the process of two-dimensional FLS migration into the cell-free area at the center of the well was photographed with a Nikon TE2000U inverted microscope (Nikon Instruments, Tokyo, Japan) and printed. Cells that migrated into the scraped area after 24 hours were counted by an observer blinded to the study design. The fold increase in the cells was calculated (number of cells with chemerin with or without signal inhibitor per number of cells without treatment).
Western blot analysis
Synovial tissues from patients with RA and OA were lysed with radioimmunoprecipitation assay (RIPA) buffer (Millipore, Temecula, CA, USA) containing protease inhibitor (Roche) and phosphatase inhibitor cocktail (Sigma-Aldrich) for 30 minutes at 4°C. RA FLSs (3 × 105 cells/well) were cultured overnight in 60-mm dishes in DMEM with 10% FCS, then the medium was replaced with FCS-free DMEM. This step was followed by incubation with or without recombinant human TNF-α (10 ng/ml), IFN-γ (100 ng/ml), transforming growth factor-β1 (TGF-β1; 1 ng/ml), IL-1β (5 ng/ml) or IL-6 (20 ng/ml) (all from R&D Systems) at 37°C for 24 hours. After incubation, cells were collected and lysed with the RIPA buffer containing protease inhibitor and phosphatase inhibitor cocktail for 30 minutes at 4°C.
A total of 20 μg of protein were boiled in the presence of SDS sample buffer and separated on a 10% SDS-polyacrylamide gel (ATTO, Tokyo, Japan). Proteins were then electrotransferred onto a polyvinylidene fluoride microporous membrane (Millipore) in a semidry system. The membrane was blocked with 5% skim milk for one hour at room temperature, then the immunoblots were incubated overnight with rabbit anti-chemerin pAb (1:1, 000 dilution; Phoenix Pharmaceuticals, Burlingame, CA, USA) or rabbit anti-ChemR23 pAb (1:500 dilution; Abcam, Cambridge, UK) in Can Get Signal Immunoreaction Enhancer Solution (Toyobo Co, Ltd, Tokyo, Japan) at 4°C. Peroxidase-conjugated goat anti-rabbit IgG pAb (GE Healthcare, Birmingham, UK) was used as the secondary antibody.
To detect phosphorylated and total ERK1/2, p38MAPK, c-Jun N-terminal kinase (JNK)1/2 or Akt, cells were incubated in a medium supplemented with 10 nM human recombinant chemerin prior to lysis for 5, 15 or 30 minutes. A total of 7.5 μg of protein was boiled in the presence of SDS sample buffer and separated on 10% SDS-polyacrylamide gel. After blotting, the membranes were blocked with Block Ace blocking solution (Snow Brand Milk Products Co, Ltd, Tokyo, Japan) for one hour at room temperature, then the immunoblots were incubated overnight with rabbit anti-phospho-ERK1/2 pAb (1:1, 000 dilution), rabbit anti-pan-ERK1/2 pAb (1:1, 000 dilution), rabbit anti-phospho-p38MAPK pAb (1:1, 000 dilution), mouse anti-pan-p38MAPK mAb (1:1, 000 dilution) (all from Cell Signaling Technology), mouse anti-phospho-JNK mAb (1:300 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-pan-JNK pAb (1:1, 000 dilution), rabbit anti-phospho-Akt pAb (1:2, 000 dilution) or rabbit anti-pan-Akt pAb (1:1, 000 dilution) (all from Cell Signaling Technology) in Tris-buffered saline (TBS) containing 0.1% Tween 20 at 4°C. To detect the expression of the regulator of NF-κB, (IκBα), and β-actin, the immunoblots were incubated overnight with rabbit anti-IκBα pAb (1:1, 000 dilution; Santa Cruz Biotechnology) at 4°C or with mouse anti-β-actin mAb (1:5, 000 dilution; Sigma-Aldrich) for one hour at room temperature in TBS containing 0.1% Tween 20. Peroxidase-conjugated goat anti-rabbit IgG pAb (GE Healthcare), rabbit anti-mouse IgG pAb (SouthernBiotech, Birmingham, AL, USA), goat anti-mouse IgG pAb (Promega, Madison, WI, USA) or donkey anti-rabbit IgG pAb (GE Healthcare) was used as the secondary Ab. ECL Plus detection reagent and the ImageQuant LAS 4000 Mini Biomolecular Imager (both from GE Healthcare) were used in conjunction with MultiGauge software (Fujifilm, Tokyo, Japan) to detect and quantitate the bands.
Data are presented as means ± SEM. Student's t-tests were applied to compare two groups, and one-way analysis of variance and Dunnett's multiple comparison tests were used to compare three or more groups. P < 0.05 was considered statistically significant.
Expression of chemerin and ChemR23 in the rheumatoid arthritis synovium
Expression of chemerin and ChemR23 in cultured rheumatoid arthritis fibroblast-like synoviocytes
ChemR23 expression on FLSs was determined by immunocytochemical analysis and Western blot analysis. Double-staining revealed that cultured FLSs expressed both ChemR23 and vimentin (Figures 4C through 4E). The use of a specific ChemR23 Ab also showed its expression in RA FLSs on Western blots (Figure 4F). Stimulation with TNF-α, IFN-γ, TGF-β, IL-1β and IL-6 did not show any effect on the expression of ChemR23 in RA FLSs.
Chemerin enhances IL-6, CCL2 and MMP-3 production by fibroblast-like synoviocytes
Chemerin enhances cell motility of rheumatoid arthritis fibroblast-like synoviocytes
Chemerin induces activation of ERK1/2, p38MAPK and Akt of fibroblast-like synoviocytes
IκBα is one of the regulatory proteins of NF-κB, a transcriptional factor which induces various proinflammatory cytokines, including TNF-α and IL-6 [29, 30]. When the cells were stimulated for NF-κB activation, IκBα was degraded and the amount of the protein was decreased . Therefore, we analyzed the effect of chemerin on the degradation of IκBα in FLSs to clarify whether NF-κB is involved in chemerin-induced FLS activation. Figures 7F and 7L indicate that chemerin does not induce IκBα degradation in FLSs.
Involvement of ERK1/2, p38MAPK and Akt pathways in chemerin-induced IL-6 production and cell motility of fibroblast-like synoviocytes
In this study, we have shown that chemerin and ChemR23 are highly expressed in RA synovium. Chemerin enhanced the production of IL-6, CCL2 and MMP-3 from RA FLSs and promoted cell motility. These results suggest that chemerin activates FLSs in the RA synovium and is probably involved in the pathogenesis of RA.
Our results show upregulation of chemerin and accumulation of ChemR23+ cells in RA synovium. We found that chemerin was expressed on synovial lining and sublining cells and on endothelial cells. Moreover, our study shows that FLSs isolated from RA synovium also produce chemerin in vitro and that this production is upregulated by stimulation with TNF-α and IFN-γ, which characterize the inflammatory environment in the RA synovium. In this regard, it was reported that the concentration of chemerin in the synovial fluid was much higher in RA than that in OA (358 ng/ml vs < 1 ng/ml) . ChemR23 was expressed on most macrophages, CD1a+ immature DCs and FLSs in RA synovium. A few DC-LAMP+ mature DCs also expressed ChemR23. It was previously reported that ChemR23 was expressed on immature DCs derived from peripheral blood and that the expression was downregulated by maturation induced by lipopolysaccharide or CD40L .
The unique pathway of activation of the chemerin precursor, prochemerin, has been well-investigated [13–15, 35, 36]. Prochemerin is considered to be activated through C-terminal six- or seven-amino acid processing by protease and to be produced by neutrophils and mast cells at inflammatory sites. Neutrophils and mast cells are known to release protease-rich granules and to secrete immune mediators to activate themselves and other immune cells, triggering positive regulatory feedback that leads to acute or chronic RA inflammation [37–39]. Considered together, our data suggest that chemerin is produced at high levels by FLSs in RA synovium and that extracellular protease is produced by preexistent neutrophils and mast cells converted from inactive prochemerin into bioactive chemerin, which have powerful chemoattractant properties for migration of macrophages and DCs into the RA synovium.
The present results reveal that the expression of ChemR23 on RA FLSs and chemerin itself activate FLSs to enhance the production of IL-6 and CCL2. IL-6 is considered to have pleiotropic functions, including the regulation of maturation and activation of T and B cells, macrophages, osteoclasts, chondrocytes and endothelial cells in RA [40, 41]. Clinically, tocilizumab, a humanized mAb specific for IL-6R, has outstanding anti-inflammatory effects, including suppression of disease activity and erosive progression in patients with RA that is resistant to traditional disease-modifying antirheumatic drugs . CCL2 is known as a prototype chemokine that attracts monocytes, T cells, NK cells and basophils into the RA synovium . Therefore, our results indicate that chemerin may be involved in the enhancement of local proinflammatory cytokine and chemokine production by RA FLSs, leading to persistent amplification of inflammation in the RA synovium, possibly in an autocrine or paracrine manner.
The present results indicate that chemerin enhances the cell motility of RA FLSs, whereas CCL2 does not promote FLS migration by this examination procedure. These data suggest that the effect of chemerin for FLS mobility is not affected by inducing the production of CCL2. In the RA joints, the pannus tissue migrates and invades the cartilage and bone, which contribute to damaging these structures . FLSs are the predominant cell type in pannus tissue, especially at the pannus-cartilage junction . FLSs retrieved from synovial tissues directly cause cartilage degradation when cocultured with macrophages in vitro , suggesting that FLS migration and invasion play a central role in pannus tissue-related cartilage degradation in RA. Moreover, our results show that chemerin enhances MMP-3 production from RA FLSs, which is a proteolytic enzyme with cartilage degradation properties. Collectively, our results suggest that chemerin plays an important role in cartilage destruction through FLS activation.
The present results show that chemerin enhances the activation of ERK1/2, p38MAPK and Akt, but not of JNK1/2 and NF-κB, in FLSs. Moreover, pretreatment with a specific inhibitor of MEK, p38MAPK, and PI3K suppressed chemerin-induced IL-6 production, and p38 MAPK and PI3 kinase inhibitor reduced RA FLS cell motility. These results suggest the involvement of both the MAPK (including MEK-ERK1/2 and p38MAPK) and PI3K-Akt pathways in chemerin-induced IL-6 production by RA FLSs. The p38MAPK and PI3K-Akt pathways are also involved in cell motility induced by chemerin. Chemerin activated macrophage adhesion to fibronectin by activation of p38MAPK and PI3K-Akt signaling pathway . These results suggest that chemerin activates macrophages as well as FLSs in RA synovium.
Our results identify the important role of chemerin in the activation of FLSs in RA synovium, suggesting that chemerin and ChemR23 interaction might play a role in the pathogenesis of RA.
chemokine (C-C motif) ligand 2
Dulbecco's modified Eagle's medium
enzyme-linked immunosorbent assay
p44/42 mitogen-activated protein kinase
fetal calf serum
c-Jun N-terminal kinase
mitogen-activated protein kinase
mitogen-activated protein kinase kinase
nuclear factor κB
- NK cells:
natural killer cells
transforming growth factor β1
tumor necrosis factor α.
We thank Aya Sato for excellent technical support and Timothy Sullivan and Thomas J Schall (ChemoCentryx, Inc, Mountain View, CA, USA) for critical advice. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Health, Labor and Welfare and the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and by the Japanese Ministry of Education, Global Center of Excellence (GCOE) Program, the International Research Center for Molecular Science in Tooth and Bone Diseases, and the Mitsui Life Social Welfare Foundation.
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