A role of monocyte chemoattractant protein-4 (MCP-4)/CCL13 from chondrocytes in rheumatoid arthritis Takuji Iwamoto 1,2 , Hiroshi Okamoto 1 , Shu Kobayashi 1,2 , Katsunori Ikari 1 , Yoshiaki Toyama 2 , Taisuke Tomatsu 1 , Naoyuki Kamatani 1 and Shigeki Momohara 1 1 Institute of Rheumatology, Tokyo Women’s Medical University, Japan 2 Department of Orthopedic Surgery, School of Medicine, Keio University, Tokyo, Japan Rheumatoid arthritis (RA) is a chronic, symmetric poly- articular joint disease that primarily affects the small joints of the hands and feet [1]. It is characterized by infiltration of inflammatory cells such as monocytes and T-lymphocytes into the joints, leading to synovial proliferation and progressive destruction of cartilage and bone [2]. Although the basic mechanisms of RA are widely accepted, the pathogenesis of the disease is not fully understood. Chemokines in humans comprise more than 50 small (8–10 kDa) heparin-binding proteins that were origi- nally identified by their chemotactic activity on bone marrow-derived cells [3,4]. They are classified into four families on the basis of the location of cysteine residues. The four chemokine groups are CC, C, CXC, and CX3C, and their receptors are consequently classi- fied as CCR, CR, CXCR, and CX3CR. Chemokines and chemokine receptors have been shown to be involved in a variety of inflammatory diseases by recruiting leukocytes to the inflammatory site [5]. It is well known that synovial tissue and synovial fluid from RA patients contain increased concentrations of sev- eral chemokines, such as interleukin (IL)-8) ⁄ CXCL8, interferon-c (IFN-c)-inducible protein-10 ⁄ CXCL10, monokine induced by interferon-c ⁄ CXCL9, stromal cell-derived factor-1 ⁄ CXCL12, monocyte chemotactic protein (MCP)-1 ⁄ CCL2, macrophage inflammatory protein-1a ⁄ CCL3, and fractalkine ⁄ CXC3CL1 [6]. Keywords chondrocytes; extracellular signal-regulated kinase (ERK); monocyte chemoattractant protein-4 (MCP-4) ⁄ CCL13; rheumatoid arthritis Correspondence H. Okamoto, Institute of Rheumatology, Tokyo Women’s Medical University, 10-22 Kawada-cho, Shinjuku, Tokyo 162-0054, Japan Fax: +81 3 5269 1726 Tel: +81 3 5269 1725 E-mail: hokamoto@ior.twmu.ac.jp (Received 14 April 2007, revised 6 July 2007, accepted 26 July 2007) doi:10.1111/j.1742-4658.2007.06013.x We studied the role of monocyte chemoattractant (MCP)-4 ⁄ CCL13 in the pathogenesis of rheumatoid arthritis (RA). MCP-4 was highly expressed in cartilage from RA patients. Interferon-c significantly stimulated MCP-4 ⁄ CCL13 production in human chondrocytes, and this effect was enhanced in combination with interleukin-1b or tumor necrosis factor-a. MCP-4 ⁄ CCL13 induces the phosphorylation of extracellular signal-regulated kinase in fibroblast-like synoviocytes and activates cell proliferation, and PD98059 completely inhibits these effects. These data suggest that interferon-c in combination with interleukin-1b ⁄ tumor necrosis factor-a activates the pro- duction of MCP-4 ⁄ CCL13 from chondrocytes in RA joints, and that secreted MCP-4 ⁄ CCL13 enhances fibroblast-like synoviocyte proliferation by activating the extracellular signal-regulated kinase mitogen-activated protein kinase cascade. Abbreviations DAB, 3¢3-diaminobenzidine tetrahydrochloride; ERK, extracellular signal-regulated kinase; FLS, fibroblast-like synoviocyte; IFN-c, interferon-c; IL, interleukin; MCP, monocyte chemoattractant protein; OA, osteoarthritis; RA, rheumatoid arthritis; SNP, single-nucleotide polymorphism; TNF-a, tumor necrosis factor-a; XTT, sodium 3¢-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate. 4904 FEBS Journal 274 (2007) 4904–4912 ª 2007 The Authors Journal compilation ª 2007 FEBS These chemokines are implicated in RA pathogenesis via the recruitment and retention of leukocytes into the joints. In addition to functioning in cell traffic, several chemokines are reported to enhance the prol- iferation of fibroblast-like synoviocytes (FLSs) and upregulate gelatinase and collagenase production by FLSs [7]. Thus, chemokines are key molecules in RA pathogenesis and are potential therapeutic targets for RA [8]. Although macrophages and FLSs are considered to be the most potent producers of chemokines in the synovial compartment, chondrocytes also have the ability to produce chemokines [8–10]. In our previous report, we found that mRNA expression of MCP- 4 ⁄ CCL13 was significantly higher in cartilage from RA patients than from osteoarthritis (OA) patients or nor- mal controls, and the concentration of MCP-4 ⁄ CCL13 protein in synovial fluid was also significantly higher in RA patients than in OA patients [11]. MCP-4 ⁄ CCL13 is a recently identified CC chemo- kine from a human cDNA library that directs the migration of eosinophils, monocytes and T-lympho- cytes through several chemokine receptors, including CCR-2 and CCR-3 [12,13]. The role of MCP-4 ⁄ CCL13 in disease is less well defined, but recent studies suggest that it is involved in inflammatory cell recruit- ment in allergic disorders such as asthma and atopic dermatitis [14–17]. In the present study, we further determined the role of MCP-4 ⁄ CCL13 in RA pathogenesis. We investi- gated the role of several stimuli on the expression of MCP-4 ⁄ CCL13 by human chondrocytes, and the sig- nal transduction pathways controlling FLS prolifera- tion by MCP-4 ⁄ CCL13. In addition, we conducted a case-control study using single-nucleotide polymor- phisms (SNPs) to determine whether MCP-4 ⁄ CCL13 could be a genetic risk factor for RA. Results Production of MCP-4 ⁄ CCL13 by human chondrocytes To identify the stimulatory signals that activate the production of MCP-4 ⁄ CCL13 from human chondro- cytes, we investigated the effect of several cytokines reported to have roles in RA pathogenesis. Human chondrocytes from RA or OA patients were cultured in the presence of IL-1b, tumor necrosis factor-a (TNF-a) or IFN-c, and various combinations of these three cytokines. Chondrocytes from both RA and OA patients gave similar results, and we present the data obtained with RA-derived chondrocytes. MCP- 4 ⁄ CCL13 protein concentrations in culture superna- tants were evaluated by ELISA. IFN-c significantly stimulated MCP-4 ⁄ CCL13 production in a dose-depen- dent manner, whereas IL-1b and TNF-a had no signif- icant effect (Fig. 1A). Interestingly, stimulation of MCP-4 ⁄ CCL13 production by IFN-c was significantly and remarkably enhanced when IFN-c was combined with IL-1b or TNF-a (Fig. 1B). To determine whether these observed effects occur at the transcriptional level, quantitative real-time PCR analysis was performed on IL-1b, TNF-a, and IFN-c. Consistent with the ELISA data, IFN-c significantly stimulated mRNA expression of MCP-4 ⁄ CCL13 in MCP-4 concentration (pg/mL) MCP-4 concentration (pg/mL) 0 100 200 300 IFN-γ γ 0 1 10 1000100 400 A B 0 10010 0 10010 IL-1 β TNF- α (ng/mL) * * 0 100 200 300 IFN- γ (ng/mL) 400 500 600 IL-1 β (ng/mL) TNF- α (ng/mL) 10 10 10 10 10 100 100 10 100 10 # # 1000 Fig. 1. MCP-4 ⁄ CCL13 protein production by human chondrocytes from RA and OA patients. Human chondrocytes were cultured in the presence of (A) IL-1b (0–100 ngÆmL )1 ; open bars), TNF-a (0–100 ngÆmL )1 ; solid bars) and IFN-c (0–1000 ngÆmL )1 ; shaded bars) for 48 h, or (B) IFN-c (shaded bars), IFN-c + IL-1b (open bars), and IFN-c + TNF-a (solid bars) for 48 h. MCP-4 ⁄ CCL13 protein concentrations in these cell culture supernatants were evaluated by ELISA. Bars show the mean and SD of four separate experiments. Statistical evaluation was performed using one-way ANOVA followed by Tukey’s method for multiple comparisons. *P<0.01 compared with vehicle-treated control. # P<0.01 compared with the sample cultured with IFN-c alone. T. Iwamoto et al. Role of MCP-4 ⁄ CCL13 in rheumatoid arthritis FEBS Journal 274 (2007) 4904–4912 ª 2007 The Authors Journal compilation ª 2007 FEBS 4905 a dose-dependent manner. IL-1b or TNF-a also enhanced the mRNA expression of MCP-4 ⁄ CCL13 induced by IFN-c. Chondrocytes from both RA and OA patients gave similar results, and we present the data obtained with RA-derived chondrocytes. These results suggest that these stimulatory effects occur at the transcriptional level (Fig. 2A,B). Effect of MCP-4 ⁄ CCL13 on extracellular signal-regulated kinase (ERK) phosphorylation Western blot analysis was performed to investigate whether MCP-4⁄ CCL13 could activate ERK, which is widely known to play a major role in cell prolifera- tion [20]. The FLSs from both RA and OA patients were similar, and we present the data from the RA-derived FLSs. As expected, phosphorylation of ERK was induced by stimulation with MCP-4 ⁄ CCL13 in RA FLSs. This activation peaked at about 10–20 min, and returned to basal levels within 60 min (Fig. 3A). Incubation with 100 lm PD98059, a spe- cific ERK activation inhibitor, was sufficient to abol- ish ERK activation by MCP-4 ⁄ CCL13 in RA FLSs (Fig. 3B). To confirm the involvement of ERK phosphorylation in the proliferative effect on RA FLSs of MCP-4 ⁄ CCL13, an XTT {sodium 3¢-[1-(phenylaminocarbonyl)- 3,4-tetrazolium]-bis-(4-methoxy-6-nitro) benzene sulfo- nic acid hydrate} cell proliferation assay was performed, using PD98059 to antagonize the phosphorylation of ERK. As representative results of western blot ana- lyses were obtained at a concentration of 100 ngÆmL )1 , an XTT cell proliferation assay was performed with the same concentrations. MCP-4 ⁄ CCL13 enhanced the proliferation of FLSs in a dose-dependent manner, and PD98059 completely inhibited the stimulatory effect of MCP-4 ⁄ CCL13 on FLS proliferation. PD98059 did not inhibit basal proliferation of RA FLSs (Fig. 4A). The FLSs from both RA and OA patients were similar, and we present the data from the RA-derived FLSs. Association study using SNPs of MCP-4 ⁄ CCL13 As we found that MCP-4 ⁄ CCL13 was one of the key molecules in the pathogenesis of RA, we studied the association of the MCP-4 ⁄ CCL13 gene with RA sus- ceptibility. We selected two SNPs in MCP-4 ⁄ CCL13 (T887C and rs159313), for the following reasons. Two single-nucleotide T–to–C polymorphisms (T896C and T887C) were reported in the MCP-4 ⁄ CCL13 core pro- moter region [22]. They are located 896 and 887 bp before the transcription initiation site, and were reported to have direct effects on the transcript level of the gene [22]. After preliminary analysis, we selected T887C for a larger-scale study, because T896C and T887C were in complete linkage disequilibrium (D¢ ¼ 1.0, r 2 ¼ 1.0). On the basis of information from the National Center of Biotechnology Information data- base, three SNPs (rs3136677, rs159313 and rs2072069) were found in MCP-4 ⁄ CCL13. Among them, we selected SNP rs159313 for the study, because one SNP (rs3136677) was nonpolymorphic in Japanese popula- tions, and other two SNPs (rs159313 and rs2072069) were in complete linkage disequilibrium (D¢ ¼ 1.0, r 2 ¼ 1.0) according to the International HapMap pro- ject (public release 19) [23]. 08412 IFN-γ IFN-γ 1000 ng/mL IFN-γ 100 ng/ml IFN-γ 10 ng/ml 1 ng/ml IFN-γ 0.1 ng/ml IFN-γ 1000 IFN-γ 100 ng/mL IFN-γ 10 ng/mL 1 ng/mL IFN-γ 0.1 ng/mL -delta delta Ct-delta delta Ct Time (hours) * * * * 2 4 6 8 A B IFN-γ 10 ng/mL + Il -1β 10 ng/mL IFN-γ + IL-1β IFN-γ 10 ng/mL Il -1β 10 ng/mL + TNF-α 10 ng/mL IL-1β + TNF-α TNF-α 10 ng/mLTNF-α Il -1β 10 ng/mLIL-1β IFN-γ 10 ng/mL + TNF-α 10 ng/mL IFN-γ + TNF-α 2 4 0 6 8 08412 Time (hours) # # Fig. 2. MCP-4 ⁄ CCL13 mRNA expression by human chondrocytes from RA and OA patients. Total RNA from chondrocytes stimulated with different cytokines for 4–12 h was harvested and transcribed to cDNA by reverse transcription. cDNA was used for TaqMan quantitative real-time PCR: (A) with IFN-c (0–1000 ngÆmL )1 ), and (B) with IL-1b, TNF-a, IFN-c, IL-1b + TNF-a, IFN-c + IL-1b, and IFN-c + TNF-a. The figure shows expression of MCP-4 ⁄ CCL13 mRNA relative to time-matched vehicle-treated controls using the comparative threshold cycle (Ct) method. Data are mean ± SD of four separate experiments. Statistical evaluation was performed using one-way ANOVA followed by Tukey’s method for multiple comparisons. *P<0.01 compared with vehicle-treated control. # P<0.01 compared with the sample cultured with IFN-c alone. Role of MCP-4 ⁄ CCL13 in rheumatoid arthritis T. Iwamoto et al. 4906 FEBS Journal 274 (2007) 4904–4912 ª 2007 The Authors Journal compilation ª 2007 FEBS According to the genotyping results, these two SNPs were present in Hardy–Weinberg equilibrium in both cases and controls. No statistically significant differ- ences in genotype or allele frequencies were observed between cases and controls. We failed to find signifi- cant differences even when RA patients were stratified according to the rheumatoid factor status (Table 1). These data indicate that the MCP-4 ⁄ CCL13 gene may not be responsible for the onset of RA. Discussion MCP-1 ⁄ CCL2, MCP-2 ⁄ CCL8, MCP-3 ⁄ CCL7 and MCP-4 ⁄ CCL13 constitute a subfamily of CC chemo- kines that share structural and functional features. MCP-1 ⁄ CCL2 was the first to be identified [24], and MCP-4 ⁄ CCL13 is the most recently identified chemo- kine, and is a potent chemoattractant for eosinophils, monocytes and T-lymphocytes [12,13]. MCP-4⁄ CCL13 expression is upregulated at sites of inflammation in a number of different diseases, including asthma [14–16], atherosclerosis [25], acute renal inflammation [26], and atopic dermatitis [17]. MCP-4 ⁄ CCL13 was also highly expressed in articular cartilage from patients with RA [11]. In the present study we demonstrated MCP- 4 ⁄ CCL13 protein production by human chondrocytes, and showed for the first time that MCP-4 ⁄ CCL13 from chondrocytes is actively involved in RA patho- genesis. We also demonstrated that IFN-c was the main stimulus for MCP-4 ⁄ CCL13 production, and that TNF-a and IL-1b enhanced the stimulatory effect of IFN-c. According to the analysis of the human MCP- 4 ⁄ CCL13 gene in dermal fibroblasts, the core promoter region contained IFN-c-response elements as well as nuclear factor-jB-like consensus sequences [27]. Fur- thermore, MCP-4 ⁄ CCL13 mRNA expression was reported to be upregulated by stimulation with TNF-a and IFN-c in dermal fibroblasts [27]. Similar results were obtained in human airway epithelial cell lines after stimulation with the cytokine TNF-a alone or in combination with IFN-c [28]. In contrast, our experi- ments indicated that IFN-c was the main stimulus for MCP-4 ⁄ CCL13 production by human chondrocytes. We observed no significant stimulation of induction of MCP-4 ⁄ CCL13 mRNA expression by TNF-a alone in human chondrocytes, whereas TNF-a greatly enhanced the expression in combination with IFN-c. IFN-c is produced by T-cells and by natural killer cells infiltrating the inflamed synovium, and is secreted into the joint space, although its role in the progres- sion of articular injury remains controversial [29]. To date, divergent in vitro effects of IFN- c have been reported in the literature. IFN-c induces the produc- tion of nitric oxide, IL-6 and prostaglandin E 2 by human chondrocytes [30]. In contrast, IFN-c inhibits TNF-a- and IL-1b-induced collagenase and stromely- sin production by chondrocytes, as well as TNF-a- and IL-1b-stimulated proteoglycan degradation [31,32]. Furthermore, the effects of IFN-c in the treatment of pERK A B 1/2 ERK 1/2 MCP-4 (min) 025 2010 30 60 120 Relative Intensity 1.00 1.03 16.55 47.73 46.16 23.29 1.04 1.05 Relative Intensity 1.00 0.96 0.94 0.95 0.98 0.97 0.99 1.01 PD98059 IL-1β pERK 1/2 ERK 1/2 MCP-4 +++ 10μ Μ 100μ Μ Relative Intensity 1.00 40.01 9.20 0.96 27.88 Relative Intensity 1.00 0.96 0.98 1.01 1.03 Fig. 3. Induction of ERK phosphorylation by MCP-4 ⁄ CCL13 in RA FLSs. FLSs were cul- tured overnight in serum-free DMEM. (A) FLSs were incubated in the presence of MCP-4 ⁄ CCL13 (100 ngÆmL )1 ) for an addi- tional 0–120 min. (B) FLSs were incubated in the presence of MCP-4 ⁄ CCL13 (100 ngÆmL )1 ) for 20 min with or without PD98059 (10–100 lgÆmL )1 ). As a positive control, FLSs were incubated with IL-1b (5 ngÆmL )1 ) for 20 min. Cell lysates were examined for ERK activation by western blotting with phospho-p44 ⁄ 42 MAP kinase mouse monoclonal antibody (pERK1 ⁄ 2). Total p44 ⁄ 42 MAP kinase antibody was used to verify equal protein loading. The result is one representative example from three independent experiments. T. Iwamoto et al. Role of MCP-4 ⁄ CCL13 in rheumatoid arthritis FEBS Journal 274 (2007) 4904–4912 ª 2007 The Authors Journal compilation ª 2007 FEBS 4907 RA are unclear. A statistically significant improvement was observed among the RA patients treated with recombinant IFN-c in one double-blind study of 91 patients [33], whereas current evidence shows that anti- IFN-c therapy is significantly superior to placebo in 30 patients with RA [34]. It is widely accepted that TNF-a is the key molecule in RA pathogenesis, as demonstrated by the clinical benefit of TNF-a-neutra- lizing therapy [35]. Although approximately 40% of patients show dramatic responses, the remainder show some evidence of persistent synovitis or minimal clini- cal benefit [1]. The results of the present study suggest that IFN-c may contribute to the progression of joint inflammation, in part by modulating MCP-4 ⁄ CCL13 production by human chondrocytes. We have also demonstrated the ability of MCP- 4 ⁄ CCL13 to phosphorylate ERK mitogen-activated protein (MAP) kinase, and have shown that induction of FLS proliferation by MCP-4 ⁄ CCL13 is dependent on the phosphorylation of ERK MAP kinase. It is widely accepted that the progressive destruction of articular cartilage is reliant on the evolution of hyper- plastic synovial tissue, and that hyperplasia of FLSs is dependent on dysregulated proliferation and apoptosis [1,36]. Key regulators of this proliferation include the recently recognized macrophage migration inhibitory factor and proinflammatory cytokines such as TNF-a and IL-1b through the nuclear factor- jB and ⁄ or MAP kinase signal transduction pathways [37–40]. To date, several chemokines, including MCP-1, stromal cell- derived factorF-1a, IFN-c-inducible protein, monokine induced by IFN-c and MCP-4 ⁄ CCL13 are also known to enhance FLS proliferation, although the signal transduction pathways underlying the proliferation remain unclear [7,11]. We hypothesized that ERK acti- vation might be involved in the proliferative effect of MCP-4 ⁄ CCL13, as the ERK cascade has been reported to be a central pathway that transmits signals from many extracellular agents to regulate cellular pro- cesses such as proliferation, differentiation and cell cycle progression in various cells [22,41]. As expected, the MCP-4 ⁄ CCL13–ERK cascade was indeed involved in the proliferation of synovial cells, as shown here. In addition, ERK is reported to have a role in the expres- sion of matrix metalloproteinases (MMPs), such as MMP-1, MMP-2, and MMP-9, and contributes to the degradation of extracellular matrix for the invasion of melanoma cells [42]. Several lines of evidence have shown that MMPs are involved in the joint degrada- tion process in RA. Thus, MCP-4 ⁄ CCL13 might have roles not only in the proliferation of synovial cells but also in the invasion of synovial cells, resulting in pan- nus formation and destruction of joints in RA. Taken together with these results, MCP-4⁄ CCL13 secreted from chondrocytes in the joints plays an important role in the development of aggressive synovial tissues in RA, as illustrated in Fig. 4B. There are numerous reports showing the importance of synovial cells in RA pathogenesis. Our data support the notion that chon- drocytes are also actively involved in RA pathogenesis. In conclusion, we have shown that MCP-4 ⁄ CCL13 is produced by human chondrocytes from RA patients Absorbance 0.8 0.9 1.0 1.1 MCP-4 (ng/mL) PD98059 * ** * 100 + 0 100 + 10010 + ** 1.2 A B Chondrocytes MCP-4 Proliferation IL-1β, TNF-α IFN-γ Th1 cells Synovial Cells IL-18 Fig. 4. (A) Effects of inhibition of ERK phosphorylation on RA FLS proliferation. FLSs were treated with MCP-4 ⁄ CCL13 (0– 100 ngÆmL )1 ) with and without the addition of MAP kinase kinase inhibitor PD98059 (100 lgÆmL )1 ) for 48 h. MCP-4 ⁄ CCL13 signifi- cantly increased RA FLS proliferation, and inhibition of ERK phos- phorylation by PD98059 significantly inhibited FLS proliferation. PD98059 did not inhibit basal proliferation of RA FLSs. Bars show the mean and SD of three indepemdent experiments. Statistical evaluation was performed using one-way ANOVA followed by Tukey’s method for multiple comparisons.*P<0.05; **P<0.01. (B) Schematic representation of the role of MCP-4 ⁄ CCL13 in RA. A vicious circle is formed between chondrocytes and synovium in the affected joint. IFN-c is produced by Th1 (T helper 1) cells infiltrating the synovium and activates the expression of MCP-4 ⁄ CCL13. Then, MCP-4 ⁄ CCL13 stimulates the proliferation of synovial cells, which produce inflammatory cytokines (IL-b, TNF-a). Together, these cyto- kines, with IFN-c, further enhance the production of MCP-4 ⁄ CCL13 by chondrocytes. Role of MCP-4 ⁄ CCL13 in rheumatoid arthritis T. Iwamoto et al. 4908 FEBS Journal 274 (2007) 4904–4912 ª 2007 The Authors Journal compilation ª 2007 FEBS stimulated by IFN-c and TNF-a ⁄ IL-1b. In addition, MCP-4 ⁄ CCL13 has significant effects on FLS prolifer- ation that are dependent on the activation of ERK MAP kinase. These data suggest that MCP-4 ⁄ CCL13 is a significant contributor to synovial hyperplasia in RA, and that MCP-4 ⁄ CCL13 may serve as a new tar- get for anti-RA therapy. Experimental procedures Preparation of articular cartilage and synovial tissue Human articular cartilage and synovial tissue were obtained from OA and RA patients (n ¼ 5 in each group) who were undergoing total knee replacement at Tokyo Women’s Medical University, Tokyo, Japan. OA was diagnosed by physical examination along with radiographic findings, and RA patients met the 1987 disease criteria of the American College of Rheumatology [18]. All samples were obtained with informed consent. All experiments were approved by the Ethical Committee of Tokyo Women’s Medical University. Isolation and culture of chondrocytes and FLSs Tissue was obtained under aseptic conditions and was finely minced. Chondrocytes were isolated by sequential enzy- matic digestion at 37 °C: 5 mgÆmL )1 pronase (Kaken Phar- maceutical Co., Ltd, Tokyo, Japan) for 1 h, followed by 2mgÆmL )1 collagenase (Sigma Chemical Co., St Louis, MO, USA) for 6 h at 37 °C in DMEM (Nikken Bio Medi- cal Laboratory, Kyoto, Japan) with antibiotics (100 unitsÆ mL )1 penicillin, 100 lgÆmL )1 streptomycin; Gibco BRL, Grand Island, NY, USA). FLSs were also isolated by diges- tion with 1 mgÆmL )1 collagenase for 3 h at 37 °Cin DMEM. The digested tissue was briefly subjected to centri- fugation at 1500 g at 37 °C for 15 min using an MX-100 centrifuge (TOMY Seiko, Tokyo, Japan) with TMP-11 angle-type rotor, and the resulting pellet was washed three times in NaCl ⁄ P i . The isolated cells were seeded at high density in tissue culture flasks and cultured in DMEM sup- plemented with 10% heat-inactivated fetal bovine serum (Tissue Culture Biologicals, Tulare, CA, USA) at 37 °Cin a humidified atmosphere of 5% CO 2 ⁄ 95% air. The culture medium was changed every 3–5 days, and nonadherent lymphoid cells were removed. At confluence, chondrocytes and FLSs were detached and passaged once, and then seeded at high density and allowed to grow in DMEM supplemented as above. Chondrocytes were used between passages 1 and 3, and FLSs were used between passages 5 and 8 for the following experiments. In some cases, carti- lage tissue slices were obtained for immunohistochemical analysis. Effect of cytokines on MCP-4 production by human chondrocytes Human chondrocytes from four RA patients and three OA patient were cultured in DMEM supplemented with 10% fetal bovine serum in 12-well culture plates. At confluence, the culture medium was replaced with serum-free DMEM. After 24 h, chondrocytes were incubated for an additional 48 h in the absence or presence of recombinant human IL-1b (0–100 ngÆmL )1 ; R&D Systems), recombinant human TNF-a (0–100 ngÆmL )1 ; R&D Systems), recombinant human IFN-c (0–1000 ngÆmL )1 ; R&D Systems) and combi- nations of these cytokines. The culture supernatant was collected and stored at ) 80 °C. MCP-4 concentrations in these supernatants were evaluated as described above. Experiments were performed three times with each of the four independent cultures. Table 1. Summary of the association of MCP-4 in rheumatoid arthritis cases and controls. The major allele was always referred to as allele 1 and the minor allele as allele 2. SNP, single-nucleotide polymorphism; RF, rheumatoid factor; MAF, minor allele frequency; OR, odds ratio; 95% CI, confidence interval. SNP Genotype Cases Controls Allele 1 versus allele 2 a 1 ⁄ 11⁄ 22⁄ 2 Total MAF 1 ⁄ 11⁄ 22⁄ 2 Total MAF v 2 OR (95% CI) P rs159313 total 400 533 189 1122 0.41 152 227 75 454 0.42 0.23 0.96 (0.82–1.13) 0.63 RF + 350 467 167 984 0.41 0.17 0.97 (0.82–1.14) 0.68 RF - 50 66 22 138 0.4 0.24 0.93 (0.70–1.24) 0.62 T-887C total 924 193 9 1126 0.094 368 75 4 447 0.093 0.006 1.01 (0.77–1.34) 0.94 RF + 810 168 9 987 0.094 0.014 1.02 (0.77–1.35) 0.91 RF – 114 25 0 139 0.09 0.022 0.96 (0.58–1.56) 0.88 a Distribution of the frequency of allele 1 versus allele 2 in the cases compared with the controls. T. Iwamoto et al. Role of MCP-4 ⁄ CCL13 in rheumatoid arthritis FEBS Journal 274 (2007) 4904–4912 ª 2007 The Authors Journal compilation ª 2007 FEBS 4909 Quantitative real-time PCR Total RNA was harvested from chondrocytes stimulated with cytokines for 4–12 h using the RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen, Chats- worth, CA, USA). cDNA was synthesized from 0.3 lgof total RNA in a 20 lL reaction using TaqMan Reverse Tran- scription Reagents (Applied Biosystems, Tokyo, Japan). TaqMan quantitative real-time PCR was performed using the ABI Prism 7900HT sequence detection system and Taq- Man PCR Master Mix according to the manufacturer’s pro- tocol (Applied Biosystems). Primers and probes for human MCP-4 ⁄ CCL13 and human glyceraldehyde-3-phosphate dehydrogenase were purchased from Applied Biosystems. RNA samples lacking reverse transcriptase were used with each real-time PCR experiment to verify the absence of genomic DNA. The incubation was initiated at 50 °C for 2 min, and this was followed by 95 °C for 10 min, and 40 cycles at 95 °C for 15 s and 65 °C for 1 min. Samples were compared using the comparative threshold cycle (Ct) method to determine MCP-4 mRNA expression relative to the time-matched vehicle-treated control. The parameter Ct is the PCR cycle number at which the fluorescence generated by cleavage of the probe reaches a fixed threshold above baseline. For each sample, the MCP-4 ⁄ CCL13 Ct value was normalized using DCt ¼ MCP-4 ⁄ CCL13 Ct ) glyceralde- hyde-3-phosphate dehydrogenase Ct. To determine relative expression levels, the following formula was used: DDCt ¼ sample DCt ) time-matched control DCt, and the value used to plot relative MCP-4 ⁄ CCL13 expression of each sample was calculated using the expression 2 –DDCt . Western blot analysis The phosphorylation of p44 ⁄ 42 MAP kinase, or ERK, was assessed by western blotting. In brief, FLSs were cultured in DMEM supplemented with 10% fetal bovine serum on a 10 cm culture dish. At 80% confluence, the culture medium was replaced with serum-free DMEM. After 24 h, FLSs were incubated in the presence of recombinant human MCP-4 (rHuMCP-4, 100 ngÆmL )1 ; R&D Systems) for an additional 0–120 min. In addition, FLSs were incubated in the presence of recombinant human MCP-4 (100 ngÆmL )1 ) for 20 min with or without a specific inhibitor of MAP kinase kinase, PD98059 (Calbiochem, San Diego, CA, USA; 10–100 lgÆmL )1 ). As a positive control, FLSs were incu- bated with recombinant human IL-1b (5 ngÆmL )1 ) for 20 min. Cells were lysed with Cell Lysis Buffer (Cell Signal- ing Technology, Beverly, MA, USA). After incubation on ice for 10 min, the protein concentration was determined, and the lysates were stored at ) 80 °C. Equal amounts of cellular proteins were separated by SDS ⁄ PAGE and trans- ferred to Immune-Blot poly(vinylidene difluoride) mem- brane (Bio-Rad, Hercules, CA, USA). Immunoblotting was performed using phospho-p44 ⁄ 42 MAP kinase mouse monoclonal antibody (Cell Signaling Technology; diluted 1 : 5000) and p44 ⁄ 42 MAP kinase antibody (Cell Signaling Technology; diluted 1 : 1000) to verify equal protein loading. Cell proliferation assay FLSs were seeded at a density of 1 · 10 3 cells per well in 96-well microtiter plates in 100 lL of serum-free DMEM per well, and were treated with recombinant human MCP- 4 (0–100 ngÆmL )1 ) for 48 h. The activation of ERK was antagonized with PD98059 (100 lgÆmL )1 ). Cell prolifera- tion was evaluated by measuring the number of viable cells using the XTT assay by using the XTT Cell Proli- feration Kit II (Roche Applied Science, Mannheim, Germany) [19]. Formazan product in the supernatant was measured in terms of absorbance values at 490 nm by using an ELISA plate reader. The absorbance values obtained from culture medium without cells were sub- tracted from the values obtained with cells. Experiments were performed six times with each of the three indepen- dent cultures. Genetic association study using SNPs The study was part of an RA cohort project (IORRA: Institute of Rheumatology RA cohort), and was approved by Tokyo Women’s Medical University Genome Ethics Committee [20]. Out of the registered RA patients, DNA samples were obtained from 1284. Informed written consent was obtained from every subject. Of these, 1128 samples were randomly selected for this study. Eighty-eight per cent of them were rheumatoid factor positive. They were mostly females (82.6%), and the mean age of the patients was 57.6 years (range: 19–85 years). Four hundred and fifty-five population-based control DNA samples were obtained from the Pharma SNP consortium (http://www.jpma.or.jp/ psc/index.html). All control subjects were matched for sex, ethnic origin, and geographical area. SNP genotyping was performed using the TaqMan fluoro- genic 5¢-nuclease assay (Applied Biosystems) according to the manufacturer’s instructions, as described previously [21]. Statistical methods Data are presented as the mean ± standard deviation (SD). Statistical comparisons were performed using either the Mann–Whitney U-test or one-way ANOVA followed by Tukey’s method for multiple comparisons, as appropri- ate. Hardy–Weinberg equilibrium and associations between RA and each of the SNPs were estimated by the chi-square test. Statistical significance was established at the P<0.05 level. All analyses were carried out using the r software package, version 2.0.1 (http://www.r-project.org/). Role of MCP-4 ⁄ CCL13 in rheumatoid arthritis T. Iwamoto et al. 4910 FEBS Journal 274 (2007) 4904–4912 ª 2007 The Authors Journal compilation ª 2007 FEBS Acknowledgements This work was supported, in part, by grants-in-aid from the Ministry of Education, Culture, Sports, Sci- ence and Technology of Japan. The expert technical help of Yukiko Katagiri is gratefully acknowledged. References 1 Firestein GS (2003) Evolving concepts of rheumatoid arthritis. Nature 423, 356–361. 2 Feldmann M, Brennan FM & Maini RN (1996) Rheumatoid arthritis. Cell 85, 307–310. 3 Charo IF & Ransohoff RM (2006) The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med 354, 610–621. 4 Sallusto F, Mackay CR & Lanzavecchia A (2001) The role of chemokine receptors in primary, effector, and memory immune responses. Annu Rev Immunol 18, 593–620. 5 Luster AD (1998) Chemokines ) chemotactic cytokines that mediate inflammation. N Engl J Med 338, 436–445. 6 Koch AE (2005) Chemokines and their receptors in rheumatoid arthritis: future targets? Arthritis Rheum 52, 710–721. 7 Garcia-Vicuna R, Gomez-Gaviro MV, Dominguez-Luis MJ, Pec MK, Gonzalez-Alvaro I, Alvaro-Gracia JM & Diaz-Gonzalez F (2004) CC and CXC chemokine receptors mediate migration, proliferation, and matrix metalloproteinase production by fibroblast-like synoviocytes from rheumatoid arthritis patients. Arthritis Rheum 50, 3866–3877. 8 Haringman JJ, Ludikhuize J & Tak PP (2004) Chemokines in joint disease: the key to inflammation? Ann Rheum Dis 63 , 1186–1194. 9 Pulsatelli L, Dolzani P, Piacentini A, Silvestri T, Ruggeri R, Gualtieri G, Meliconi R & Facchini A (1999) Chemokine production by human chondrocytes. J Rheumatol 26, 1992–2001. 10 Villiger PM, Terkeltaub R & Lotz M (1992) Monocyte chemoattractant protein-1 (MCP-1) expression in human articular cartilage. Induction by peptide regula- tory factors and differential effects of dexamethasone and retinoic acid. J Clin Invest 90, 488–496. 11 Iwamoto T, Okamoto H, Iikuni N, Takeuchi M, Toyama Y, Tomatsu T, Kamatani N & Momohara S (2006) Monocyte chemoattractant protein-4 (MCP- 4) ⁄ CCL13 is highly expressed in cartilage from patients with rheumatoid arthritis. Rheumatology (Oxford) 45, 421–424. 12 Garcia-Zepeda EA, Combadiere C, Rothenberg ME, Sarafi MN, Lavigne F, Hamid Q, Murphy PM & Luster AD (1996) Human monocyte chemoattractant protein (MCP)-4 is a novel CC chemokine with activities on monocytes, eosinophils, and basophils induced in aller- gic and nonallergic inflammation that signals through the CC chemokine receptors (CCR)-2 and -3. J Immunol 157, 5613–5626. 13 Uguccioni M, Loetscher P, Forssmann U, Dewald B, Li H, Lima SH, Li Y, Kreider B, Garotta G, Thelen M et al. (1996) Monocyte chemotactic protein 4 (MCP-4), a novel structural and functional analogue of MCP-3 and eotaxin. J Exp Med 183, 2379–2384. 14 Lamkhioued B, Garcia-Zepeda EA, Abi-Younes S, Nakamura H, Jedrzkiewicz S, Wagner L, Renzi PM, Allakhverdi Z, Lilly C, Hamid Q et al. (2000) Monocyte chemoattractant protein (MCP)-4 expression in the air- ways of patients with asthma. Induction in epithelial cells and mononuclear cells by proinflammatory cyto- kines. Am J Respir Crit Care Med 162 (2 Part 1), 723– 732. 15 Kalayci O, Sonna LA, Woodruff PG, Camargo CA Jr, Luster AD & Lilly CM (2004) Monocyte chemotactic protein-4 (MCP-4; CCL-13): a biomarker of asthma. J Asthma 41, 27–33. 16 Taha RA, Minshall EM, Miotto D, Shimbara A, Luster A, Hogg JC & Hamid QA (1999) Eotaxin and mono- cyte chemotactic protein-4 mRNA expression in small airways of asthmatic and nonasthmatic individuals. J Allergy Clin Immunol 103 (3 Part 1), 476–483. 17 Taha RA, Minshall EM, Leung DY, Boguniewicz M, Luster A, Muro S, Toda M & Hamid QA (2000) Evidence for increased expression of eotaxin and monocyte chemotactic protein-4 in atopic dermatitis. J Allergy Clin Immunol 105 , 1002–1007. 18 Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS et al. (1998) The American Rheuma- tism Association 1987 revised criteria for the classifica- tion of rheumatoid arthritis. Arthritis Rheum 31, 315–324. 19 Okamoto H, Cujec TP, Okamoto M, Peterlin BM, Baba M & Okamoto T (2000) Inhibition of the RNA-depen- dent transactivation and replication of human immuno- deficiency virus type 1 by a fluoroquinoline derivative K-37. Virology 272, 402–408. 20 Matsuda Y, Singh G, Yamanaka H, Tanaka E, Urano W, Taniguchi A, Saito T, Hara M, Tomatsu T & Kamatani N (2003) Validation of a Japanese version of the Stanford Health Assessment Questionnaire in 3,763 patients with rheumatoid arthritis. Arthritis Rheum 49, 784–788. 21 Ikari K, Kuwahara M, Nakamura T, Momohara S, Hara M, Yamanaka H, Tomatsu T & Kamatani N (2005) Association between PADI4 and rheumatoid arthritis: a replication study. Arthritis Rheum 52, 3054–3057. 22 Kalayci O, Birben E, Wu L, Oguma T, Storm Van’s Gravesande K, Subramaniam V, Sheldon HK, T. Iwamoto et al. Role of MCP-4 ⁄ CCL13 in rheumatoid arthritis FEBS Journal 274 (2007) 4904–4912 ª 2007 The Authors Journal compilation ª 2007 FEBS 4911 Silverman ES & Lilly CM (2003) Monocyte chemoattractant protein-4 core promoter genetic variants: influence on YY-1 affinity and plasma levels. Am J Respir Cell Mol Biol 29, 750–756. 23 Altshuler D, Brooks LD, Chakravarti A, Collins FS, Daly MJ & Donnelly PA (2005) Haplotype map of the human genome. Nature 437, 1299–1320. 24 Furutani Y, Nomura H, Notake M, Oyamada Y, Fukui T, Yamada M, Larsen CG, Oppenheim JJ & Matsushima K (1989) Cloning and sequencing of the cDNA for human monocyte chemotactic and activating factor (MCAF). Biochem Biophys Res Commun 159, 249–255. 25 Berkhout TA, Sarau HM, Moores K, White JR, Elshourbagy N, Appelbaum E, Reape RJ, Brawner M, Makwana J, Foley JJ et al. (1997) Cloning, in vitro expression, and functional characterization of a novel human CC chemokine of the monocyte chemotactic protein (MCP) family (MCP-4) that binds and signals through the CC chemokine receptor 2B. J Biol Chem 272, 16404–16413. 26 Chakravorty SJ, Howie AJ, Girdlestone J, Gentle D & Savage CO (2001) Potential role for monocyte chemo- tactic protein-4 (MCP-4) in monocyte ⁄ macrophage recruitment in acute renal inflammation. J Pathol 194, 239–246. 27 Hein H, Schluter C, Kulke R, Christophers E, Schroder JM & Bartels J (1999) Genomic organization, sequence analysis and transcriptional regulation of the human MCP-4 chemokine gene (SCYA13) in dermal fibro- blasts: a comparison to other eosinophilic beta-chemo- kines. Biochem Biophys Res Commun 255, 470–476. 28 Stellato C, Collins P, Ponath PD, Soler D, Newman W, La Rosa G, Li H, White J, Schwiebert LM, Bickel C et al. (1997) Production of the novel C-C chemokine MCP-4 by airway cells and comparison of its biological activity to other C-C chemokines. J Clin Invest 99, 926– 936. 29 Feldmann M, Brennan FM & Maini RN (1996) Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 14, 397–440. 30 Henrotin YE, Zheng SX, Labasse AH, Deby GP, Criel- aard JM & Reginster JY (2000) Modulation of human chondrocyte metabolism by recombinant human inter- feron. Osteoarthritis Cartilage 8, 474–482. 31 Bunning RA & Russell RG (1989) The effect of tumor necrosis factor alpha and gamma-interferon on the resorption of human articular cartilage and on the pro- duction of prostaglandin E and of caseinase activity by human articular chondrocytes. Arthritis Rheum 32, 780–784. 32 Andrews HJ, Bunning RA, Plumpton TA, Clark IM, Russell RG & Cawston TE (1990) Inhibition of interleu- kin-1-induced collagenase production in human articular chondrocytes in vitro by recombinant human interferon- gamma. Arthritis Rheum 33, 1733–1738. 33 Lemmel EM, Brackertz D, Franke M, Gaus W, Hartl PW, Machalke K, Mielke H, Obert HJ, Peter HH, Sieper J et al. (1988) Results of a multicenter placebo- controlled double-blind randomized phase III clinical study of treatment of rheumatoid arthritis with recom- binant interferon-gamma. Rheumatol Int 8, 87–93. 34 Sigidin YA, Loukina GV, Skurkovich B & Skurkovich S (2001) Randomized, double-blind trial of anti-inter- feron-gamma antibodies in rheumatoid arthritis. Scand J Rheumatol 30, 203–207. 35 Lipsky PE, van der Heijde DM, St Clair EW, Furst DE, Breedveld FC, Kalden JR, Smolen JS, Weisman M, Emery P, Feldmann M et al. (2000) Infliximab and methotrexate in the treatment of rheumatoid arthritis. Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant Therapy Study Group. N Engl J Med 343, 1594–1602. 36 Qu Z, Garcia CH, O’Rourke LM, Planck SR, Kohli M & Rosenbaum JT (1994) Local proliferation of fibro- blast-like synoviocytes contributes to synovial hyperpla- sia. Results of proliferating cell nuclear antigen ⁄ cyclin, c-myc, and nucleolar organizer region staining. Arthritis Rheum 37, 212–220. 37 Lacey D, Sampey A, Mitchell R, Bucala R, Santos L, Leech M & Morand E (2003) Control of fibroblast-like synoviocyte proliferation by macrophage migration inhibitory factor. Arthritis Rheum 48, 103–109. 38 Inoue H, Takamori M, Nagata N, Nishikawa T, Oda H, Yamamoto S & Koshihara Y (2001) An investiga- tion of cell proliferation and soluble mediators induced by interleukin 1beta in human synovial fibroblasts: com- parative response in osteoarthritis and rheumatoid arthritis. Inflamm Res 50, 65–72. 39 Schett G, Tohidast-Akrad M, Smolen JS, Schmid BJ, Steiner CW, Bitzan P, Zenz P, Redlich K, Xu Q & Steiner G (2000) Activation, differential localization, and regulation of the stress-activated protein kinases, extracellular signal-regulated kinase, c-JUN N-terminal kinase, and p38 mitogen-activated protein kinase, in synovial tissue and cells in rheumatoid arthritis. Arthritis Rheum 43, 2501–2512. 40 Youn J, Kim HY, Park JH, Hwang SH, Lee SY, Cho CS & Lee SK (2002) Regulation of TNF-alpha-medi- ated hyperplasia through TNF receptors, TRAFs, and NF-kappaB in synoviocytes obtained from patients with rheumatoid arthritis. Immunol Lett 83, 85–93. 41 Rubinfeld H & Seger R (2005) The ERK cascade: a prototype of MAPK signaling. Mol Biotechnol 31, 151–174. 42 Smalley KS (2003) A pivotal role for ERK in the onco- genic behaviour of malignant melanoma? Int J Cancer 104, 527–532. Role of MCP-4 ⁄ CCL13 in rheumatoid arthritis T. Iwamoto et al. 4912 FEBS Journal 274 (2007) 4904–4912 ª 2007 The Authors Journal compilation ª 2007 FEBS . Yamanaka H, Tanaka E, Urano W, Taniguchi A, Saito T, Hara M, Tomatsu T & Kamatani N (2003) Validation of a Japanese version of the Stanford Health Assessment. Questionnaire in 3,763 patients with rheumatoid arthritis. Arthritis Rheum 49, 784–788. 21 Ikari K, Kuwahara M, Nakamura T, Momohara S, Hara M, Yamanaka H, Tomatsu