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. 2001 Jul;125(1):155–161. doi: 10.1046/j.1365-2249.2001.01542.x

Selective recruitment of CCR6-expressing cells by increased production of MIP-3α in rheumatoid arthritis

T Matsui *, T Akahoshi *, R Namai *, A Hashimoto *, Y Kurihara *, M Rana *, A Nishimura *, H Endo *, H Kitasato †, S Kawai ‡, K Takagishi §, H Kondo *
PMCID: PMC1906097  PMID: 11472439

Abstract

Infiltration of various types of leucocytes has been shown to play a crucial role in the pathogenesis of rheumatoid arthritis (RA). Macrophage inflammatory protein-3α (MIP-3α) is a recently identified chemokine which is a selective chemoattractant for leucocytes such as memory T cells, naïve B cells and immature dendritic cells. In this study, we investigated the expression of MIP-3α and its specific receptor CCR6 in the inflamed joints of patients with RA. Increased amounts of MIP-3α were found by ELISA in synovial fluids (SF) of patients with RA. MIP-3α was apparently detected in all synovial tissue specimens of RA patients (n = 6), but it could not be detected in that of osteoarthritis (OA) patients (n = 4). Expression of MIP-3α was detected especially in the sublining layer, and infiltrating mononuclear cells in RA synovial tissue. Gene expression of MIP-3α was also found in six out of 11 RA-synovial fluid cells by RT-PCR. Cultured synovial fibroblasts derived from either RA or OA patients were capable of producing MIP-3α in response to IL-1β and TNFα in vitro. Furthermore, expression of CCR6 was found in infiltrating mononuclear cells in the cellular clusters and around the vessels of RA synovial tissue. These findings indicate that increased production of MIP-3α may contribute to the selective recruitment of CCR6-expressing cells in RA.

Keywords: MIP3K, CCR6, chemokines, dendritic cells

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease characterized by destruction of the inflamed joints. Infiltration of various types of leucocytes is involved in the joint inflammation, and leucocyte accumulation has been shown to play a crucial role in the pathogenesis of RA [1].

Recently, several chemokines and their specific receptors have been identified. Chemokines are a family of structurally-related, low molecular weight proteins that primarily promote leucocyte recruitment and activation [2]. Accumulating evidence indicates that chemokines are involved in a variety of immunological and inflammatory responses mediated by their diverse ability to promote leucocyte migration and activation, to modulate haematopoiesis and angiogenesis, and to inhibit HIV infection [3–5]. Chemokines can be divided into four subfamilies (CXC, CC, CX3C and C) based on the position and structure of the first two cysteine amino acid residues [2]. Expression of several chemokines and their receptors has been found in RA, suggesting the possible contribution of these chemokines to leucocyte accumulation and leucocyte-mediated inflammatory responses [6–9].

The CC chemokines include MCP-1, MIP-1α, MIP-1β, RANTES and Eotaxin, and these chemokines are predominantly chemotactic for monocytes, lymphocytes, basophils and eosinophils. Recently, a novel member of the CC subfamily was identified and named MIP-3α, or LARC (liver and activation-regulated chemokine) [10]. MIP-3α shows some differences in its amino acid sequence and gene location compared with other CC chemokines. A specific receptor for MIP-3α has been identified and named CCR6 [11,12]. MIP-3α has a chemoattractant effect on CCR6-expressing leucocytes such as memory T cells, naïve B cells and immature dendritic cells [13–15]. It is well known that rheumatoid synovial tissues contain abundant memory T cells as well as B cells and dendritic cells [1,16,17]. However, the mechanisms whereby these leucocytes are recruited into inflamed joints are not well known. Of particular interest is the recruitment and functional maturation of dendritic cells in RA [18,19]. Mature dendritic cells are professional antigen-presenting cells and potent initiators of the immune response by activating naïve T cells. Dendritic cells are derived from the bone marrow, with their precursors being recruited into tissues from the blood and subsequently undergoing maturation [20]. Because of the pathological significance of dendritic cells for immunologically-mediated inflammation in autoimmune diseases, evaluation of the mechanisms regulating the recruitment and maturation of these cells in RA seems to be important [18,19].

Therefore, we investigated the expression of MIP-3α and its specific receptor CCR6 in the inflamed joints of patients with RA. We also investigated the functional role of proinflammatory cytokines in the production of MIP-3α by cultured synovial cells.

Materials and methods

Reagents

Recombinant human interleukin-1β (IL-1β) and tumour necrosis factor (TNF)-α were obtained from Genzyme (Cambridge, MA). Recombinant human MIP-3α and transforming growth factor-β1 (TGF-β1) were purchased from Pepro Tech (London, UK). Mouse antibodies directed against MIP-3α and CCR6 were obtained from R & D Systems (Minneapolis, MN). Gout anti-MIP-3α antibody was purchased from Genzyme-Techne (Minneapolis, MN). Biotin-SP-conjugated F(ab′)2 fragment goat anti-mouse IgG was obtained from Jackson ImmunoResearch Lab. (West Grove, PA). A DIG-High Prime DNA Labelling and Detection Kit was obtained from Roche Diagnostics GmbH (Mannheim, Germany) and a Vectorstain ABS kit was purchased from Vector Laboratories Inc. (Burlingame, CA). 3,3′-diaminobenziidine (DAB) was purchased from DOJINDO (Kumamoto, Japan).

Patients

After informed consent had been obtained, synovial tissue was obtained during total knee joint replacement in six patients with RA and four patients with OA. The RA patients were classified according to the 1987 revised criteria of the American College of Rheumatology. All patients had active disease and were receiving non-steroidal anti-inflammatory drugs. Among the RA patients, four were also taking low-dose steroids and two were taking D-penicillamine.

Synovial fluid was collected by routine knee joint aspiration from 20 patients with RA and 18 patients with OA. Infiltrating cells and SF were separated from these SF samples by centrifugation and were stored at −70°C until use.

Cell culture

Human synovial tissue specimens were excised and digested with collagenase (0·2 mg/ml) at 37°C for 1 h. Adherent synoviocytes were maintained in Dulbecco's modified Eagle's medium (gibco BRL, Grand Island, NY) supplemented with 10% fetal calf serum (JRH Bioscience, Lenexa, KS), 10 mm HEPES, 100 units/ml penicillin and 100 µg/ml streptomycin, at 37°C in humidified 5% CO2. Synovial fibroblasts from passages 5–8 were incubated in culture dishes (Sumitomo Bakelite Co., Ltd, Tokyo, Japan) for several days. After confluence was reached, the cultures were stimulated with various cytokines for the indicated periods, and the cells were harvested and stored at −70°C until use.

Determination of MIP-3α by specific ELISA

MIP-3α was determined by a sandwich ELISA using gout anti-human MIP-3α antibody, monoclonal mouse anti-human MIP-3α antibody and biotin-SP-conjugated F(ab′)2 fragment gout anti-mouse IgG.

Polymerase chain reaction and Southern hybridization

Total RNA was extracted from the cells isolated from SF or from cultured synovial fibroblasts by the acid guanidium–phenol–chloroform method. RNA yields were determined spectrophotometrically by measurement of the absorbance at 260 nm. Expression of the MIP-3α and CCR6 genes was assessed by the reverse-transcriptase polymerase chain reaction (RT-PCR) followed by Southern blot hybridization. In brief, 2 µg RNA was reverse-transcribed into single-stranded cDNA by incubation for 1 h at 42°C with 40 µl of a reverse transcription reaction mixture containing random hexadeoxynucleotide primer and Rous-associated virus 2 reverse transcriptase (Takara, Kyoto, Japan). PCR was performed in a thermal cycler with 25 µl reaction mixture containing 5 µl cDNA solution, 2·5 units Taq DNA polymerase, and 0·5 µm each of the sense and anti-sense primers. Amplification was achieved by 30 cycles of denaturing at 95°C for 1 min, annealing at 52°C (for MIP-3α) or 56°C (for CCR6) for 30 s, and extension at 72°C for 30 s.

The primers for MIP-3α were 5′-TTGGATCCTGCTGCTAC TCCACCTCTG-3′ and 5′-TTCTCGAGTATATTTCACCCAAG TCTGTTTT-3′ [21], and the PCR product obtained with these primers was 210 base pairs (bp) in size. The primers for CCR6 were 5′-TTGGATCCGTGGGGGCTGTCAGTCATCAT-3′ and 5′-TTCTCGAGCTGCCCAATAAAAGCGTAGA-3′ [14], and the PCR product was 451 bp in size. Beta 2-microglobulin (β2m) was used as the internal control, with primers of 5′-TTCTG GCCTGGAGGGCATCC-3′ and 5′-ATCTTCAAACCTCCATGA TG-3′ [22] yielding an expected PCR product of 340 bp.

PCR products were subjected to electrophoresis on 2% agarose gel, transferred to nylon membranes and hybridized with dioxigenin (DIG) end-labelled oligonucleotide probes. The following probes were used in this study: 5′-GATGTCACAGCCT TCATTGG-3′ for MIP-3α, 5′-AAGCCAGAAACACAAGCACC AC-3′ for CCR6 and 5′-ACACGGCAGGCATACTCATC-3′ for β2m. Three-primed end-labelling of the probes and detection after hybridization were performed using a DIG-High Prime DNA Labelling and Detection Kit. The membrane was subsequently exposed to Fuji RX-U film.

In situ hybridization

In situ hybridization was performed as described previously [23]. In brief, mRNA for MIP-3α and CCR6 was amplified by RT-PCR using two sets of specific primers (MIP-3α: 5′-TTGGATCCTGC TGCTACTCCACCTCTG-3′ and 5′-TTCTCGAGTATATTTCA CCCAAGTCTGTTTT-3′; CCR6: 5′-TTGGATCCGTGGGGGCT GTCAGTCATCAT-3′ and 5′-TTCTCGAGCTGCCCAATAAAA GCGTAGAGC-3′). PCR products were cloned using cloning vector pBluescript (Stratagene, La Jolla, CA), and the cDNAs of the MIP-3α and CCR6 clones were identified by direct sequencing. Subsequently, sense and antisense DIG-labelled riboprobes for MIP-3α and CCR6 were generated.

Cryosections of synovium were mounted on silane-coated glass slides and fixed with 4% (w/v) paraformaldehyde. The sections were treated with 10 µg/ml proteinase K, and hybridized with the labelled riboprobes in hybridization solution (Novagen, Madison, WI) for 18 h at 50°C in moistened plastic boxes. After hybridization, the sections were treated with 20 µg/ml RNase and washed extensively. Then, probe binding was visualized with alkaline-phosphate conjugated anti-dioxigenin antibody in 5-bromo-4-chloro-3 indolyl-phosphate and 4-nitroblue tetrazolium chloride solution (Roche Diagnostics GmbH, Mannheim, Germany). The slides were subsequently counterstained with haematoxylin before examination.

Immunohistochemistry

Immunoperoxidase staining was carried out using a Vectorstain ABC kit according to the manufacturer's protocol. Sections prepared from frozen samples were incubated in methanol containing 3% (v/v) H2O2 for 20 min to enhance endogenous peroxidase activity. Then, the sections were pre-incubated with 0·3% (v/v) bovine serum albumin (Vector Laboratories, Inc., Burlingame, CA) in PBS for 20 min and subsequently incubated with diluted goat serum for 30 min, followed by additional incubation with antibodies against human MIP-3α or CCR6 in a humidified chamber for 1 h. Purified murine IgG (DAKO, Carpinteria, CA) was used as the control. Next, the sections were washed with PBS and treated with the biotinylated secondary antibodies for 30 min. After washing again with PBS, colour was developed by treatment with DAB and the sections were counterstained with haematoxylin.

Results

Detection of MIP-3α in SF

Immunoreactive MIP-3α was determined in SF from 20 patients with RA and 18 patients with OA by specific ELISA. As shown in Fig. 1, increased levels of MIP-3α were found in SF from RA patients. The mean concentration of MIP-3α in these 20 SF samples was 12·2 ± 12·1 ng/ml. Although one sample from OA patients contained an increased level of MIP-3α, the mean concentration of 18 SF samples from OA patients was relatively low (3·2 ± 5·0 ng/ml). The mean levels of MIP-3α in the RA and OA groups differed significantly (P = 0·0027).

Fig. 1.

Fig. 1

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MIP-3α in synovial fluids. Concentration of MIP-3α in SF of RA patients (n = 20) and OA patients (n = 18) was determined by ELISA. Bars show the mean ±s.d. The mean concentration of MIP-3α in RA-SF is significantly higher than that of OA-SF (P = 0·0027).

Immunohistological analysis of MIP-3α in RA

Expression of MIP-3α was investigated immunohistochemically using rabbit anti-human MIP-3α antibody and synovial tissue specimens obtained from six patients with RA and four patients with OA. As shown in Fig. 2A, MIP-3α was detected in synovial tissue specimens of RA patients but could not be detected in those of OA patients (Fig. 2B). MIP-3α expression was especially noted in the sublining tissue at sites of enhanced synovial proliferation (Fig. 2C). Infiltrating mononuclear cells also expressed MIP-3α protein (data not shown).

Fig. 2.

Fig. 2

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Expression of MIP-3α and CCR6 in RA. Expression of MIP-3α and CCR6 was determined by immunostaining and in situ hybridization of frozen synovial tissue from RA and OA patients, as described in Materials and methods. (a) Immunostaining of MIP-3α in RA synovial tissue (original magnification, ×100). (b) Immunostaining of MIP-3α in OA synovial tissue (original magnification, ×100). (c) Higher magnification of Fig. 2a (original magnification, ×200). (d) In situ hybridization of MIP-3α mRNA in RA synovial tissue (original magnification, ×200). (e) Immunostaining of CCR6-expressing cells in RA synovial tissue (original magnification, ×200). (f) In situ hybridization of CCR6 mRNA in RA synovial tissue (original magnification: ×200). Results were reproducible in the synovial tissue specimens obtained from six RA patients and four OA patients.

Gene expression of MIP-3α in RA

In order to assess MIP-3α gene expression in RA synovial tissue, we performed in situ hybridization using DIG-labelled riboprobes. In the RA synovial tissue specimens, expression of MIP-3α transcripts was detected by the anti-sense riboprobe. MIP-3α mRNA was expressed by the synovial lining cells and the cells in the sublining region, consisting of infiltrating cells and synovial fibroblasts (Fig. 2D). No signals were observed when the sense riboprobe was used on the same tissue specimens (data not shown).

CCR6-immunopositive cells in RA

Because of the enhanced expression of MIP-3α in RA synovial tissue, we investigated the expression of CCR6, which is the specific and selective receptor for MIP-3α. Immunostaining of RA synovial tissue specimens for CCR6 was performed using a monoclonal anti-human CCR6 antibody. As shown in Fig. 2E, CCR6 was clearly detected in the infiltrating mononuclear cells in the cellular clusters. Positive staining of CCR6 was not found in OA synovial tissue (data not shown).

Gene expression of CCR6 in RA

Expression of CCR6 mRNA was also evaluated by in situ hybridization. Figure 2F demonstrates the expression of CCR6 transcripts in RA synovial tissue using the anti-sense riboprobe. CCR6 gene expression was prominent in the infiltrating mononuclear cells around the vessels. Positive signals for CCR6 mRNA were also observed in endothelial cells. Essentially no hybridization was seen when the sense riboprobe was used (data not shown).

Expression of MIP-3α in SF cells

In order to evaluate MIP-3α expression by infiltrating cells in RA, expression of the MIP-3α gene by SF cells was investigated after these cells were freshly isolated from 11 RA patients. Expression of MIP-3α was determined by RT-PCR followed by Southern blot hybridization, and the gene was detected in six of 11 samples (Fig. 3). In contrast, MIP-3α transcripts were not detected by the same RT-PCR method in peripheral blood leucocytes from 10 RA patients and seven healthy volunteers (data not shown). These findings suggested that cells infiltrating in SF may be one of the sources of MIP-3α in RA.

Fig. 3.

Fig. 3

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Expression of MIP-3α by infiltrating cells from RA-SF. Gene expression of MIP-3α and the internal control (β2-microglobulin) was determined in SF cells from 11 RA patients by RT-PCR and Southern hybridization.

MIP-3α production by synovial fibroblasts

MIP-3α staining of synovial fibroblasts in RA synovial tissue indicated that these fibroblasts were a possible source of MIP-3α production. Therefore, we investigated MIP-3α production by synovial fibroblasts in vitro. Cultured synovial fibroblasts obtained from RA patients were stimulated with various cytokines for 1 h, and MIP-3α gene expression was evaluated by RT-PCR followed by Southern blot hybridization. As shown in Fig. 4a, IL-1β and TNF-α, both at 1 ng/ml, significantly enhanced MIP-3α gene expression by synovial fibroblasts. TGF-β has been shown to play a role in a number of inhibitory effects on immune responses. Therefore, we investigated the effects of TGF-β on MIP-3α expression. TGF-β failed to decrease basal expression as well as IL-1β-induced expression of MIP-3α (Fig. 4a,b). RT-PCR analysis also demonstrated that simultaneous stimulation of synovial fibroblasts with IL-1β and TNFα might not exert synergistic effects. Synovial fibroblasts derived from RA and OA patients were subsequently incubated with IL-1β at 1 ng/ml for the indicated periods, and the time course of MIP-3α gene expression was evaluated. It was found that IL-1β rapidly induced MIP-3α expression of both synovial fibroblasts as early as 30 min after the stimulation (Fig. 4c).

Fig. 4.

Fig. 4

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Induction of MIP-3. gene expression in synovial fibroblasts. (a) RA synovial fibroblasts were incubated in the presence or absence of cytokines such as IL-1β (1 ng/ml), TNF-α (1 ng/ml) and TGF-β (1 ng/ml) for 1 h. (b) RA synovial fibroblasts were incubated for 1 h with IL-1β (1 ng/ml), or IL-1β (1 ng/ml) and TNF-α (1 ng/ml), or IL-1β (1 ng/ml) and TGFβ (1 ng/ml). (c) Synovial fibroblasts derived from RA patient (RA SyF) and OA patient (OA SyF) were incubated with IL-1β (1 ng/ml) for the indicated periods. Gene expression of MIP-3α and β2-m was determined by RT-PCR and Southern hybridization.

In order to investigate MIP-3α production, synovial fibroblasts were incubated with IL-1β for 24 h, and MIP-3α in the medium was determined by specific ELISA. IL-1β significantly stimulated MIP-3α production from synovial fibroblasts in a concentration-dependent manner (Fig. 5).

Fig. 5.

Fig. 5

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MIP-3α production of cultured synovial fibroblasts by IL-1β. Synovial fibroblasts were stimulated with various concentrations of IL-1β for 24 h and MIP-3α in the culture supernatant fluid was determined by specific ELISA. Data represent the mean ±s.d. of triplicate determinations.

Discussion

In the present study, we obtained evidence of increased production of MIP-3α and of the accumulation of CCR6-expressing mononuclear cells in RA. The MIP-3α-producing cells in RA joints appeared to be infiltrating mononuclear cells as well as synovial fibroblasts. In addition, cultured synovial fibroblasts were shown to produce MIP-3α in response to stimulation with proinflammatory cytokines, IL-1β and TNF-α.

The infiltration of leucocytes into inflamed joints is a characteristic and pathological feature of RA [1]. Recent progress in chemokine research has revealed the mechanisms involved in the selective recruitment of leucocyte subsets by subset-selective expression of chemokine receptors. Type 1 helper T cells (Th1) expressing CCR5 predominantly accumulate in the synovial tissue and synovial fluid of RA patients as a result of increased production of the ligands MIP-1α MIP-1β and RANTES [24]. Also, the migration of macrophages into rheumatoid synovial tissue might be caused mainly by increased production of the CC chemokine MCP-1 [8].

In the present study, we demonstrated that production of MIP-3α was enhanced and that CCR6-positive cells accumulated in the inflamed joints of RA patients. CCR6 and MIP-3α are atypical among promiscuous chemokine receptors and their ligands. MIP-3α is the sole chemokine for CCR6, even though the antibiotic peptide, β-defensin, has recently been identified as a another ligand for CCR6 [25]. Thus, these findings indicated that selective recruitment of CCR6-expressing cells might be caused by local production of MIP-3α.

Expression of CCR6 has previously been identified in resting memory T lymphocytes, immature dendritic cells and B lymphocytes [12–14]. Infiltration of memory T lymphocytes and dendritic cells appears to be essential for the initiation and perpetuation of antigen-specific immune responses. Dendritic cells are derived from multi-potential bone marrow precursors and are the most potent antigen-presenting cells [20]. These cells migrate from the circulation to sites of inflammation where they present antigens to T lymphocytes. Dendritic cells have been shown to undergo phenotypic and functional maturation during the process of migration, and there is evidence that both migration and maturation of dendritic cells are primarily promoted by various tissue cytokines and chemokines [15,20]. Immature dendritic cells selectively express CCR6 and could potentially be attracted to inflamed tissues by the sole ligand of CCR6, MIP-3α [26]. Expression of CCR6 on dendritic cells is subsequently down-regulated during their activation and differentiation [27]. It has been demonstrated that synovial tissue and synovial fluid contain CD4+ T lymphocytes and dendritic cells. Also, histopathological studies of rheumatoid synovial tissue have shown that dendritic cells are clustered with T lymphocytes close to the vessels [18]. Such clustering of dendritic cells may indicate that they are involved in antigen presentation to self-reactive T lymphocytes. Dendritic cells isolated from rheumatoid synovial tissue show an enhanced ability to stimulate the autologous mixed lymphocyte reaction, and express HLA-DR and HLA-DQ molecules as well as co-stimulatory molecules (CD80 and CD86) on the cell surface [28–30]. In the present study, we demonstrated CCR6 expression of infiltrating mononuclear cells in rheumatoid synovial tissue, but expression of CCR6 in sub-populations of mononuclear cells was not well elucidated; our next study will be aimed at investigating this expression.

Rheumatoid synovium contains a number of cytokines and chemokines [1]. Expression of MIP-3β, another chemokine influencing mature dendritic cells [31], has also been found in RA (T. Matsui: unpublished data). Thus, CCR6-expressing immature dendritic cells may undergo differentiation in the RA synovium in response to the increased local production of cytokines. Investigation of the precise influence of MIP-3α on the migration and function of CCR6-expressing leucocytes, such as dendritic cells, T cells and B cells, may help to elucidate the pathological significance of these types of cells in RA.

We also demonstrated that synovial fibroblasts are one of the cellular sources of MIP-3α in RA. Synovial fibroblasts have been shown to produce various chemokines, including IL-8, MCP-1, MIP-1α, MIP-1β and RANTES, in response to stimulation by IL-1 and TNF [8,9,32]. Expression of MIP-3α was also enhanced in cultured synovial fibroblasts. Induction of MIP-3α gene expression was found as early as 30 min after IL-1 stimulation. Thus, MIP-3α seems to be an inflammatory molecule that can be rapidly induced in synovial fibroblasts by proinflammatory cytokines.

Investigation of cytokines and chemokines has elucidated the pathophysiological role of these factors in RA, and has contributed to the development of new therapies targeting these molecules. Further evaluation of MIP-3α and CCR6 may help to determine the precise mechanisms of cellular recruitment in RA.

Acknowledgments

The authors thank Dr Yuichi Sato (Kitasato University) for critical comment on immunostaining. This study was supported by grants from the Ministry of Education and Culture of Japan (10670423).

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