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Rheumatology Advance Access originally published online on November 22, 2005
Rheumatology 2006 45(4):421-424; doi:10.1093/rheumatology/kei209
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© The Author 2005. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Monocyte chemoattractant protein-4 (MCP-4)/CCL13 is highly expressed in cartilage from patients with rheumatoid arthritis

T. Iwamoto1,2, H. Okamoto1, N. Iikuni1, M. Takeuchi3, Y. Toyama2, T. Tomatsu1, N. Kamatani1 and S. Momohara1

1 Institute of Rheumatology, Tokyo Women's Medical University, 2 Department of Orthopaedic Surgery, School of Medicine, Keio University and 3 Institute for Drug Discovery, Yamanouchi Pharmaceutical Co., Ltd, Tberaki, Japan.

Correspondence to: H. Okamoto, Institute of Rheumatology, Tokyo Women's Medical University, 10-22 Kawada-cho, Shinjuku, Tokyo 162-0054, Japan. E-mail: hokamoto{at}ior.twmu.ac.jp


   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. To study the role of monocyte chemoattractant protein-4 (MCP-4)/CCL13 in the pathogenesis of rheumatoid arthritis (RA), we analysed the expression of MCP-4/CCL13 in chondrocytes, synovial fluid and serum from patients with RA and investigated the effect of MCP-4/CCL13 on the proliferation of synovial cells.

Methods. Human articular cartilage specimens were obtained from joints from RA and osteoarthritis (OA) patients and normal joints (controls). Transcript levels of MCP-4 in cartilage were determined by real-time polymerase chain reaction. Protein levels were measured by enzyme-linked immunoassay. Cultured fibroblast-like synoviocytes (FLS) were treated with various concentrations of recombinant MCP-4/CCL13 protein, and cell proliferation was evaluated with a viability assay.

Results. The gene expression of MCP-4 was significantly higher in cartilage from RA patients than in that from OA patients (P = 0.00902) and in normal cartilage (P = 0.00902). The concentration of MCP-4/CCL13 protein in serum from RA patients (mean 94.7 ± 37.6 pg/ml) was significantly higher than in serum from OA patients (mean 49.2 ± 31.2 pg/ml, P = 0.0051) and controls (mean 32.6 ± 23.9 pg/ml, P = 0.0001). The concentration of MCP-4/CCL13 protein in synovial fluid from RA patients (mean 247.2 ± 161.2 pg/ml) was also significantly higher than in that from OA patients (mean 29.6 ± 50.5 pg/ml, P = 0.000019). Moreover, MCP-4/CCL13 enhanced the proliferation of FLS in a dose-dependent manner.

Conclusions. MCP-4/CCL13 is highly expressed in RA joints at the mRNA and protein levels. Our results suggest that MCP-4/CCL13 is secreted from chondrocytes and activates the proliferation of rheumatoid synovial cells, thereby leading to joint destruction in RA.

KEY WORDS: Rheumatoid arthritis, Monocyte chemoattractant protein-4, Chemokines


   Introduction
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 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is a chronic disease characterized by infiltration of inflammatory cells such as monocytes and T lymphocytes into the joints, leading to synovial inflammation and progressive destruction of cartilage. Although the basic mechanisms of RA are widely accepted, the pathogenesis of the disease is not fully understood. The CC chemokines, such as monocyte chemoattractant protein-1 (MCP-1)/CCL2, macrophage inflammatory protein-1{alpha} (MIP-1{alpha})/CCL3, MIP-1ß/CCL4, and RANTES/CCL5, are considered to be implicated in RA pathogenesis via the recruitment and retention of monocytes and T lymphocytes into the joints [1]. In addition to functions in cell traffic regulation, several chemokines are reported to enhance the migration and proliferation of fibroblast-like synoviocytes (FLS) and up-regulate gelatinase and collagenase production by FLS [2]. Thus, chemokines are key molecules in the pathogenesis of RA and are potential therapeutic targets for RA.

We compared the patterns of gene expression in cartilage from RA patients, patients with osteoarthritis (OA) and normal joints using DNA microarray analysis and found that MCP-4/CCL13 was highly expressed in RA cartilage. To understand the role of cartilage MCP-4/CCL13 expression in RA, we investigated the expression of MCP-4/CCL13 in cartilage, synovial fluid and serum. We also investigated the expression of MCP-4/CCL13 receptors, CCR3 and CCR11 in synovial tissues from RA patients, and studied the proliferative effects of MCP-4/CCL13 on synovial cells to understand the role of MCP-4/CCL13 in the pathogenesis of RA.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Preparation of articular cartilage, synovial fluid and serum
Human articular cartilage specimens were obtained from OA and RA patients (n = 5 in each group) who were undergoing total knee replacement at Tokyo Women's Medical University, Tokyo, Japan. To serve as a control, normal human articular cartilage was obtained from three patients without a history of joint disease who were undergoing joint surgery after femoral neck fracture and whose cartilage was free of lesions. OA was diagnosed by physical examination along with radiographic findings, and RA patients had disease that met the 1987 criteria of the American College of Rheumatology [3]. Serum samples were obtained from RA patients (n = 23), OA patients (n = 8) and patients without a history of joint disease whose joints were considered to be normal (n = 10). Synovial fluid was obtained from OA and RA patients (n = 24 in each group). All samples were obtained with informed consent from these patients. All the experiments were approved by the Ethical Committee of Tokyo Women's Medical University.

Real-time PCR
Cartilage was dissected, and total RNA was directly isolated from cartilage with Isogen (Nippon Gene, Tokyo, Japan) and an RNeasy Mini Kit (Qiagen, Chatsworth, CA, USA). Complementary DNA was synthesized from 1 µg of total RNA, using Superscript II and random hexamers (Invitrogen, San Diego, CA, USA). Real-time polymerase chain reaction (PCR) was performed using the ABI Prism 7900HT sequence detection system and SYBR Green PCR Master Mix in accordance with the protocol suggested by the manufacturer (PE Applied Biosystems, Foster City, CA, USA). The primer sets used were as follows: MCP-4/CCL13, sense 5'-CAAACTGGGCAAGGAGATCTG-3', antisense 5'- GGCCCAGGTGTTTCATATAATTCT-3' (PCR product 61 bp); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), sense 5'-GGGAAGGTGAAGGTCGGA-3', antisense 5'-GCAGCCCTGGTGACCAG-3' (PCR product 62 bp). PCR conditions were as follows: 10 min at 95°C, followed by 40 cycles each consisting of 15 s at 95°C and 1 min at 59°C. Dissociation curve analyses were performed to verify that there was neither unspecific amplification nor formation of primer dimers. Values were calculated based on standard curves generated for each gene. Normalization of samples was determined by dividing the number of copies of MCP-4/CCL13 transcript by the number of copies of GAPDH transcript and dividing this number by the minimum data in normal samples, which was defined as 1.

Measurement of MCP-4/CCL13 levels
Serum and synovial fluid samples were stored at –20°C until use. MCP-4/CCL13 concentrations in these samples were evaluated by enzyme-linked immunosorbent assay (ELISA) with the Quantikine human MCP-4 immunoassay according to the instructions of the manufacturer (R&D Systems, Minneapolis, MN, USA). The sensitivity of the ELISA kit was 1.92 pg/ml for MCP-4. Results are presented as mean ± S.E.M.

Immunohistochemistry
Sections (4 µm thick) of acetone-fixed, frozen synovial tissues were used for immunohistochemical staining with the ChemMate Envision kit (Dako Cytomation, Kyoto, Japan). Briefly, sections were incubated with anti-human CCR3 monoclonal antibody (Sigma, St Louis, MO, USA; diluted 1:200) or CCR11 monoclonal antibody (Calbiochem, San Diego, CA USA; diluted 1:200) in a humid chamber for 60 min at 37°C. An isotype control was used. After washing, sections were then incubated with five drops of horseradish peroxidase-conjugated dextran polymer reagent (ChemMate Envision kit K5007) in a humid chamber for 30 min at 37°C. 3'3-Diaminobenzidine tetrahydrochloride (DAB) was used as the chromogen (Liquid DAB K3466; Dako Cytomation). Stained tissues were examined by light microscopy.

Cell proliferation assay
To study the biological effects of MCP-4/CCL13 on the proliferation of synovial cells, cultured synovial fibroblasts were treated with various concentrations of recombinant MCP-4 protein for 96 h, and cell proliferation was evaluated by measuring the number of viable cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [4]. As a positive control, FLS were incubated with IL-1ß (5 ng/ml). Experiments were performed six times and the average was obtained.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Expression of MCP-4/CCL13 in chondrocytes
Our preliminary study using DNA microarray analysis revealed a high level of expression of MCP-4/CCL13 in RA cartilage (relative intensity in normal, OA, and RA cartilage was 5.2, 13.5 and 124.5, respectively). To confirm this evidence, we performed real-time PCR of the MCP-4 gene in cartilage from RA, OA and normal joints. Gene expression of MCP-4/CCL13 was significantly higher in cartilage from RA patients than in that from OA patients (P = 0.00902) and normal cartilage (P = 0.00902) (Fig. 1A). There was no significant difference between OA patients and controls.


Figure 1
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  		FIG. 1. (A) Comparative expression of the gene for monocyte chemoattractant protein-4 (MCP-4/CCL13) in RA, OA and normal cartilage. (B) Serum levels of MCP-4/CCL13 in RA, OA and normal subjects. (C) Synovial fluid levels of MCP-4/CCL13 in RA and OA. Bars show the mean. Statistical comparisons were carried out using the non-parametric Mann–Whitney U-test.

 
Levels of MCP-4/CCL13 in synovial fluid and serum
We next analysed the concentrations of MCP-4/CCL13 protein in serum obtained from the three groups, and in synovial fluid obtained from RA patients and OA patients by using ELISA. Concentrations of MCP-4/CCL13 protein in serum from RA patients (mean 94.7 ± 37.6 pg/ml) were significantly higher than those in serum from OA patients (mean 49.2 ± 31.2 pg/ml, P = 0.00512) and controls (mean 32.6 ± 23.9 pg/ml, P = 0.000105) (Fig. 1B and C). Moreover, the concentration of MCP-4/CCL13 protein in synovial fluid from RA patients (mean 247.2 ±161.2 pg/ml) was also significantly higher than that in fluid from OA patients (mean 29.6 ± 50.5 pg/ml). Our data demonstrate that MCP-4/CCL13 is highly expressed in RA joints, not only at the mRNA level, but also at the protein level.

Expression of MCP-4 receptors in synovial tissues
MCP-4/CCL13 is highly expressed in the RA joints and several chemokines are reported to enhance the proliferation of FLS [2]. Therefore, we hypothesized that MCP-4/CCL13 in joints might play important roles in the pathogenesis of RA. To prove this hypothesis, we confirmed the expression of the MCP-4/CCL13 receptors CCR3 and CCR11 in synovial tissues from RA patients (Fig. 2A) [5]. Therefore, MCP-4/CCL13 expressed in chondrocytes in RA joints could bind to its receptor on synovial cells. Next, we analysed the expression of CCR3 and CCR11 in chondrocytes from RA patients. As shown in Fig. 2B, CCR3 and CCR11 were also expressed in chondrocytes.


Figure 2
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  		FIG. 2. (A) Immunohistochemical studies with anti-CCR3 antibody and anti-CCR11 antibody, showing significant expression of CCR3 and CCR11 in synovial tissues (x400). An isotype control was used (x400). (B) Immunohistochemical studies with anti-CCR3 antibody and anti-CCR11 antibody, showing significant expression of CCR3 and CCR11 in articular chondrocytes (x400). An isotype control was used (x400). (C) Enhancement of synovial cell proliferation by MCP-4/CCL13. Data are mean ± S.D. *P<0.05; **P<0.01 vs experiments without MCP-4/CCL13 and IL-1 (–), by paired t-test.

 
Enhancement of proliferation of fibroblast-like synoviocytes by MCP-4/CCL13
It has been reported that some chemokines, such as MCP-1, IP-10 and SDF-1{alpha} can induce the proliferation of synovial cells [2]. We hypothesized that MCP-4/CCL13 could enhance the proliferation of synovial cells, as fierce proliferation of synovial cells is one of the pathological features of RA. To this end, we studied the biological effects of MCP-4/CCL13 on the proliferation of FLS. We observed that MCP-4/CCL13 enhanced the proliferation of FLS in a dose-dependent manner (Fig. 2C).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
MCPs constitute a subfamily of CC chemokines that share structural and functional features, and they are considered to be major chemoattractants for monocytes, eosinophils, basophils and lymphocytes in chronic inflammatory diseases. Among them, MCP-4/CCL13 is the most recently identified chemokine from a human heart cDNA library [5]. There are some reports showing that MCP-4/CCL13 is up-regulated at sites of inflammation and that MCP-4/CCL13 may play a major role in the pathophysiological mechanisms of allergic disorders such as asthma and atopic dermatitis, which are considered to be Th2-dominant diseases [6, 7]. To our knowledge, there has been no report showing a relationship between MCP-4/CCL13 and RA; therefore, this is the first report to describe the expression of MCP-4/CCL13 in RA patients. Here, we have shown that proliferation of rheumatoid synovial cells might be activated by MCP-4/CCL13 secreted from chondrocytes in the affected joints following the development of aggressive synovial tissue. The MCP-4/CCL13 receptors CCR3 and CCR11 were expressed not only on synovial tissue but also chondrocytes from RA patients, suggesting that there might be autocrine/paracrine signalling in chondrocytes in RA. Therefore, MCP-4/CCL13 could play important roles in joint destruction in RA.

These results indicate that MCP-4/CCL13 plays an important role in the development of RA, and MCP-4/CCL13 may serve as a new target for the development of anti-RA therapy.
Figure 3


   Acknowledgments
 
This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

The authors have declared no conflicts of interest.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Patel DD, Zachariah JP, Whichard LP. CXCR3 and CCR5 ligands in rheumatoid arthritis synovium. Clin Immunol 2001;98:39–45.[CrossRef][ISI][Medline]
  2. García-Vicuña R, Gómez-Gaviro MV, Domínguez-Luis MJ et al. CC and CXC chemokine receptors mediate migration, proliferation, and matrix metalloproteinase production by fibroblast-like synoviocytes from rheumatoid arthritis patients. Arthritis Rheum 2004;50:3866–77.[CrossRef][Medline]
  3. Arnett FC, Edworthy SM, Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24.[ISI][Medline]
  4. Okamoto H, Cujec TP, Okamoto M, Peterlin BM, Baba M, Okamoto T. Inhibition of the RNA-dependent transactivation and replication of human immunodeficiency virus type 1 by a fluoroquinoline derivative K-37. Virology 2000;272:402–8.[CrossRef][ISI][Medline]
  5. Garcia-Zepeda EA, Combardiere C, Rothenberg ME et al. Human monocyte chemoattractant protein (MCP)-4 is a novel CC chemokine with activities on monocytes, eosinophils, and basophils induced in allergic and nonallergic inflammation that signals through the CC chemokine receptors (CCR)-2 and -3. J Immunol 1996;157:5613–26.[Abstract]
  6. Lamkhioued B, Garcia-Zepeda EA, Abi-Younes S et al. Monocyte chemoattractant protein (MCP)-4 expression in the airway of patients with asthma. Am J Respir Crit Care Med 2000;162:723–32.[Abstract/Free Full Text]
  7. Taha RA, Minshall EM, Leung DY et al. Evidence for increased expression of eotaxin and monocyte chemotactic protein-4 in atopic dermatitis. J Allergy Clin Immunol 2000;105:1002–7.[CrossRef][ISI][Medline]
Submitted 24 July 2005; revised version accepted 19 October 2005.
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