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Rheumatology Advance Access published online on October 4, 2008
Rheumatology, doi:10.1093/rheumatology/ken383
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Functional expression of the chemokine receptor CCR7 on fibroblast-like synoviocytes

H. Brühl1, M. Mack2, M. Niedermeier2, D. Lochbaum1, J. Schölmerich1 and R. H. Straub1

1Department of Internal Medicine I and 2Department of Internal Medicine II, University Hospital, Regensburg, Germany

Correspondence to: Correspondence to: H. Brühl, Department of Internal Medicine I, University Hospital, 93042 Regensburg, Germany. E-mail: hilke.bruehl{at}klinik.uni-regensburg.de


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Acknowledgements
 References
 
Objectives. We have characterized the expression and the function of the chemokine receptor CCR7 on fibroblast-like synoviocytes (FLS) of patients with RA and OA and on dermal fibroblasts.

Methods. FLS were obtained after enzymatic digestion of synovial tissue (ST) of patients with RA and OA undergoing knee replacement surgery and taken into culture for chemokine receptor analysis by RT–PCR, flow cytometry and functional tests. Immunofluorescence for CCR7, fibroblast and T-cell markers was performed on ST of RA and OA patients. To study the response of FLS to CCR7 ligands and other chemokines, migration assays were performed in modified Boyden chambers. After stimulation of FLS with CCR7 ligands, the secretion of VEGF was evaluated by ELISA and Luminex.

Results. CCR7 is expressed on FLS of patients with RA and OA, but not on dermal fibroblasts. FLS migrated in response to the CCR7 ligands, CCL19 and CCL21. Stimulation of FLS with CCL19 resulted in a significantly increased secretion of VEGF of RA- and OA-FLS.

Conclusion. Apart from the migration of FLS in response to CCL19 and CCL21, it was shown that activation of the CCR7 receptor on FLS results in an enhanced VEGF secretion. A considerable expression of CCR7 ligands in proximity to perivascular infiltrates has previously been described in inflamed synovial tissue of RA patients. Stimulation of FLS via CCR7 could thereby contribute to angiogenesis in the synovial tissue.

KEY WORDS: Chemotactic factors, Cytokines and inflammation, Fibroblast, Inflammation, CCR7, Angiogenesis


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Fibroblast-like synoviocytes (FLS) constitutively express the chemokine receptors CCR2, CCR5, CXCR3 and CXCR4. Stimulation of FLS with the corresponding chemokine ligands has been shown to enhance migration, collagenase and gelatinase activity [1]. In addition, stimulation with CCL2, CCL5 and CXCL12 augment IL-6 and IL-8 production by FLS [2]. Here we investigate the expression of the chemokine receptor CCR7 on FLS of patients with OA and RA. The CCR7 ligand CCL19 is expressed both on stromal and on vascular and lymphatic endothelial cells in the synovial tissue of RA patients [3]. The other CCR7 ligand, CCL21, has also been detected in the proximity of perivascular infiltrates and on vascular endothelial cells in RA synovium, where together with CCL19 it was associated with lymphoid follicle formation [4]. In secondary lymphoid organs, CCL19 and CCL20 are constitutively expressed by stromal cells within the T-cell zones. While CCL21 is secreted by endothelial cells of high endothelial venules and lymphatic vessels themselves and is then presented on the luminal surface, CCL19 is known to be transcytosed from the basolateral region to the luminal surface of the endothelium [5]. Activation of FLS by hypoxia or with cytokines (e.g. IL-1β, TNF-{alpha}, IL-18) has been shown to stimulate the secretion of VEGF [6–8]. Here, we characterize the expression of CCR7 on FLS of RA and OA patients and analyse the response of FLS to CCL19 and CCL21 with respect to migration. Further, we study a hitherto unknown function of CCR7 on FLS by analysing the secretion of VEGF after activation with CCL19.


   Methods
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 Methods
Results
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Synovial tissue preparation and cell culture of synovial cells
Approval of the local ethic committee and written informed consent was obtained of all patients according to the Declaration of Helsinki. Synovial tissue from knee joint replacement surgery of patients with OA and RA (according to ACR criteria, Arthritis Rheum 1988;31:315–24) was enzymatically digested for 1–2 h at 37°C (Dispase Grad II, Boehringer Mannheim, Germany). The cells were seeded in RPMI 1640 (Sigma, Steinheim, Germany), 10% FCS (PAN Biotech, Aidenbach, Germany), 1% penicillin/streptomycin (Life Technologies, Inc., Paisley, UK) and cultured for 2–3 weeks (passages 3–5). Dermal fibroblast lines were prepared from normal skin of spare tissue from surgery or purchased from Cambrex, Walkersville, MD, USA. Dermal fibroblasts were cultured in DMEM (low glucose 1% L-glutamine, 1% P/S, 10% FCS). The RA-FLS cell line MH7A [9] was kindly provided by the Riken Cell Bank, Japan. CCL19, CCL21 and CXCL12 were purchased from R&D Systems, MN, USA and TNF-{alpha} from Peprotech, Offenbach, Germany.

FACS analysis
FLS were detached by brief incubation with 2 mM EDTA in PBS followed by washing with RPMI, FCS 10%. 100 000 cells were stained at 4°C by incubation with anti-CCR7 mab (clone 150503, R&D Systems) or an irrelevant IgG2a isotype control (M9144, Sigma) at a concentration of 10 µg/ml for 1 h. After two washing steps, cells were incubated with a PE-conjugated rabbit-anti-mouse F(ab)2 fragment (R0439, DakoCytomation, Hamburg, Germany) for 45 min. After washing, cells were analysed immediately by flow cytometry (Becton-Dickinson, Heidelberg, Germany). Calculations were performed with Cell Quest analysis software.

Migration assay
Migration assays were performed in modified 48-well Boyden chambers. A polycarbonated filter (8 µm pore size, polyverylpyrrolidone-free, Gerbu Biotechnik, Gaiberg, Germany) divided the chamber into an upper and a lower compartment. Twenty thousand FLS/well in 50 µl RPMI (1% BSA) were seeded into the wells of the upper compartment of the Boyden chamber. Each chemokine concentration tested was given into the lower well in replicates of at least 4 in RPMI with 25 µg/ml fibronectin (Roche, Mannheim, Germany) or RPMI with fibronectin alone. Where indicated, FLS were pre-incubated with blocking anti-CCR7 antibodies (clone 150 503, R&D Systems) at 25 µg/ml or an isotype control before migration. The Boyden chamber was incubated at 37°C in 5% CO2 atmosphere for 10 h. The filter was removed from the chamber and the non-migrated cells on the upper side of the filter were scrapped off. The migrated cells on the lower side of the filter were fixed and stained with Hemacolor staining kit (Merck, Darmstadt, Germany) and counted in two microscopic high-power fields of view at a 400-fold magnification.

ELISA and luminex
Quantification of VEGF in the supernatant was performed by ELISA (DuoSet, R&D Systems), according the manufacturer's protocol (Emax, MWG Biotech, Software: SOFTmax, Ebersberg, Germany) or with Fluorokine MAP Multiplex systems from R&D Systems according to the manufacturer's protocol by Luminex (Luminex 100, Software: IS2.2). Stimulation experiments were performed in duplicates or triplicates.

Statistical analyses
All statistical analyses were performed using Student's unpaired t-tests. Results are expressed as the mean ± S.D.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary data
 Acknowledgements
 References
 
Expression of CCR7 on the cell surface of FLS
Cell surface expression of CCR7 was studied on FLS of patients with OA and RA. FLS in passages 3 and 4 were used for expression and functional analysis. Both FLS of patients with OA and RA displayed a strong expression of CCR7 on the cell surface, whereas only a marginal expression was detected on dermal fibroblasts (Fig. 1A and B). Further, we tested an immortalized FLS-cell line (MH7A) from a patient with RA [9] for CCR7 expression. MH7A cells homogeneously expressed CCR7 (Fig. 1A). Chemokine receptor expression was confirmed by quantitative RT–PCR for three randomly selected patients with RA and OA each (supplementary figure 1, available as supplementary data at Rheumatology Online). Double staining with antibodies against CCR7 and a fibroblast marker (prolyl-4 hydroxylase) clearly confirmed CCR7 expression on FLS in the synovial tissue of RA and OA patients (N = 3, each) by IF (supplementary figure 2, available as supplementary data at Rheumatology Online). In addition, CCR7 expression was found on a subpopulation of T cells double-stained for CCR7 and CD3 (supplementary figure 2, available as supplementary data at Rheumatology Online).


Figure 1
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  		FIG. 1. FLS of OA and RA patients express CCR7 and respond to CCR7 stimulation with migration. (A) FACS analysis reveals surface expression of CCR7 (bold line) on FLS from synovial tissue of patients with RA (upper left) and OA (upper right). CCR7 is also expressed on MH7A cells, an FLS cell line from RA synovial tissue (lower left), whereas on dermal fibroblasts no significant surface CCR7 was detectable (lower right). Staining with an unspecific isotype control is given in grey for each cell type. (B) FACS analysis for CCR7 on FLS of 10 RA and 10 OA patients and on six dermal fibroblast cell lines of normal donors. CCR7 surface expression is given as MFI (mean fluorescence intensity of CCR7/isotype control IgG2a). CCR7 expression on FLS of single patients are represented by dots; the mean values are depicted by bars. (C and D). Migration of FLS in response to chemokines was performed in modified 48-well Boyden chambers. FLS exhibited a significant concentration-dependent migration to both CCR7 ligands compared with basal migration with medium alone. Cells were counted on the bottom side of the membrane after 10 h of migration. The migration index of FLS is given on the y-axis. Baseline migration with medium was set to 1. Experiments with FLS of three patients (two OA/one RA for CCL19 and three OA for CCL21) were summarized. The mean migration index and S.D. are given in the bar graph. (E) Chemotaxis of FLS in response to CCL19 was tested with the MH7A cell line. The migration index was significantly decreased by incubating the cells in presence of an equal concentration of CCL19 in the upper compartment. For CXCL12 or CCL21 no significant difference was seen. The migration assay was performed in replicates of four and repeated once with analogous results. The mean and S.D. of one experiment are given in the bar graph. (F) Migration in response to CCL19 was significantly inhibited by blocking the chemokine receptor CCR7 on FLS. FLS were either pre-incubated with a blocking anti-CCR7 antibody (dark bars) or with an isotype control (shaded bars) before migration in response to CCL19 at indicated concentrations in the lower compartment. The experiment was performed with FLS of three different patients with OA and the mean migration indices and S.D.s are summarized in the bar graph.

 
FLS migrate in response to CCR7 ligands
To test the response of FLS to CCL19 and CCL21, migration assays with FLS of RA and OA patients were performed in modified Boyden chambers. FLS of both OA and RA patients exhibited a concentration-dependent migration to CCL19 and CCL21 (Fig. 1C and D). A significant migration was detected in response to 400–1600 ng/ml for CCL19 and 100–1600 ng/ml CCL21 after 10 h (Fig. 1C and D). To evaluate if the cells display a directed migration in response to CCR7 and CXCR4 ligands (1500 ng/ml), the migration of MH7A FLS was studied in the presence of chemokines in the lower compartment alone, compared with chemokines in both the lower and upper compartments. Migration in response to CCL19 was gradient dependent, indicating a chemotactic response (Fig. 1E). For CCL21 and CXCL12, the migration index was not significantly reduced when chemokines were added to the upper compartment, indicating chemokinesis but not chemotaxis (Fig. 1E). The migration of FLS in response to CCL19 was CCR7 dependent, since pre-incubation of cells for 1 h with a blocking anti-CCR7 antibody resulted in a reduced migration index for CCL19 at the concentration of both 500 and 1500 ng/ml (Fig. 1F).

Stimulation with CCR7 ligands results in a significant secretion of VEGF by FLS of RA and OA patients
Activation of FLS, e.g. by IL-1β, TNF-{alpha} or hypoxia, results in an increased secretion of VEGF. Therefore, we tested whether FLS respond to CCR7 ligands with an increased VEGF secretion. Differences in VEGF secretion between basal secretion and CCL19 stimulation were already seen after 24 h, yet because VEGF secretion was low and variable after 24 h in some cell lines we routinely performed stimulation experiments after an incubation of 72 h. After 72 h of stimulation with CCL19, we observed a significant secretion of VEGF into the supernatant of FLS (Fig 2A–C). CCL19 stimulation of FLS resulted in a concentration-dependent release of VEGF (Fig. 2A). At high concentrations, CCL19 was as effective as TNF-{alpha} in stimulating the secretion of VEGF. TNF-{alpha} is one of the most potent stimuli of FLS (Fig. 2A and C). In contrast, stimulation of FLS with CCL21, CXCL12 and CCL5 did not induce a significant VEGF secretion (Fig. 2C). The highly significant increase of VEGF in response to CCL19 at a concentration of 500 ng/ml was further confirmed with FLS of both OA patients (n = 10) and RA patients (n = 5). FLS of RA and OA patients were equally responsive to stimulation with CCL19 (Fig. 2B). Five skin fibroblast lines were tested for VEGF secretion after stimulation with CCL19 (1.5 µg/ml) for 72 h. Stimulation with CCL19 did not result in a significant release of VEGF (80 pg/ml with medium compared with 93 pg/ml with CCL19, P = 0.44).


Figure 2
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  		FIG. 2. FLS respond to CCR7 ligands with an increased VEGF release. (A) Stimulation of FLS of two OA patients and two RA patients with CCL19 induced a concentration-dependent release of VEGF. FLS were stimulated with CCL19 or TNF-{alpha} at the indicated concentrations for 72 h. CCL19-induced VEGF release is given as percentage of basal VEGF secretion. Error bars indicate the S.D. (B) FLS of 5 patients with RA (right) and 10 patients with OA (left) were stimulated with 500 ng/ml CCL19 resulting in a significant increase of VEGF secretion after 72 h compared with baseline secretion (P < 0.01 for RA-FLS and P < 0.001 for OA-FLS). The VEGF secretion after stimulation is given as percentage of baseline secretion (100%) on the y-axis. The dots indicate the response of individual OA- and RA-FLS. The mean VEGF release and the S.D. are shown. FLS of RA patients and OA patients were equally responsive to CCL19 with respect to VEGF secretion. (C) Release of VEGF into the supernatant after stimulation of RA-FLS with CCL19, TNF-{alpha}, CCL21, CXCL12 and CCL5. Data of individual stimulation experiments are given as dots, the mean VEGF release and S.D. are depicted for each chemokine or TNF-{alpha}. TNF-{alpha} (n = 8), CCL19 (n = 12), CCL21 (n = 11), CXCL12 (n = 11) and CCL5 (n = 7).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary data
 Acknowledgements
 References
 
Here we report the functional expression of CCR7 on FLS of patients with RA and OA, but not on dermal fibroblasts. CCR7 expression on cells with mesenchymal origin has previously been shown on breast tumour cells and on melanoma cells, where it has been associated with metastatic spreading and lymph node invasion [10, 11]. Recently, it was reported that lung fibroblasts of patients with idiopathic pulmonary fibrosis/usual interstitial pneumonia but not normal lung fibroblasts express CCR7 and exposure to the CCR7 ligand CCL21 resulted in a significant migratory and proliferative responses [12]. Injection of idiopathic pulmonary fibroblasts but not normal fibroblasts into C.B-17SCID/bg-mice induced patchy interstitial lung infiltrates, which could be inhibited by immunoneutralization of both human CCR7 and CCL21 [13].

CCL21 has preferentially been detected in perivascular areas within the inflamed synovial tissues of RA patients [4]. In addition to perivascular expression, CCL19 has also been detected in stromal areas within the synovial tissue [3]. We found that stimulation of FLS with CCR7 ligands resulted in significantly enhanced migration. In addition, stimulation with CCL19 resulted in an increased secretion of VEGF. VEGF is considered one of the key factors in angiogenesis, one of the hallmarks of synovial inflammation. Blockade of VEGF by an anti-VEGF peptide or soluble VEGF receptor-1 has been shown to inhibit murine CIA [14, 15]. Likewise, VEGF-B knockout mice display a reduced pathology in murine CIA and antigen-induced arthritis [16]. FLS might contribute to angiogenesis in the synovial tissue by the secretion of VEGF in response to CCL19. CCR7 and its ligands have previously been discussed as possible targets to inhibit the immigration of CCR7-positive dendritic cells and T cells into the site of inflammation. The enhanced VEGF secretion after stimulation of FLS with CCL19 might present an additional reason to evaluate the feasibility of an anti-CCR7-based strategy in RA.

Formula


   Supplementary data
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
Acknowledgements
 References
 
Supplementary data are available at Rheumatology Online.


   Acknowledgements
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Acknowledgements
References
 
We thank Sigrid Karrer (Department of Dermatology) and Claus Hellerbrand (Department of Internal Medicine I), University of Regensburg, for providing dermal fibroblasts. The MH7A cell line was generated by K. Miyazawa and provided by the Riken Cell Bank. Financial support was given by the Deutsche Forschungsgemeinschaft BR2139/2-6.

Disclosure statement: The authors have declared no conflicts of interest.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Acknowledgements
 References
 

  1. Garcia-Vicuna R, Gomez-Gaviro MV, Dominguez-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][Web of Science][Medline]
  2. Nanki T, Nagasaka K, Hayashida K, Saita Y, Miyasaka N. Chemokines regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis. J Immunol (2001) 167:5381–5.[Abstract/Free Full Text]
  3. Burman A, Haworth O, Hardie DL, et al. A chemokine-dependent stromal induction mechanism for aberrant lymphocyte accumulation and compromised lymphatic return in rheumatoid arthritis. J Immunol (2005) 174:1693–700.[Abstract/Free Full Text]
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  6. Cha HS, Bae EK, Koh JH, et al. Tumor necrosis factor-alpha induces vascular endothelial growth factor-C expression in rheumatoid synoviocytes. J Rheumatol (2007) 34:16–9.[Abstract/Free Full Text]
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  8. Amin MA, Mansfield PJ, Pakozdi A, et al. Interleukin-18 induces angiogenic factors in rheumatoid arthritis synovial tissue fibroblasts via distinct signaling pathways. Arthritis Rheum (2007) 56:1787–97.[CrossRef][Web of Science][Medline]
  9. Miyazawa K, Mori A, Okudaira H. Establishment and characterization of a novel human rheumatoid fibroblast-like synoviocyte line, MH7A, immortalized with SV40 T antigen. J Biochem (1998) 124:1153–62.[Abstract/Free Full Text]
  10. Muller A, Homey B, Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis. Nature (2001) 410:50–6.[CrossRef][Web of Science][Medline]
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  14. Yoo SA, Bae DG, Ryoo JW, et al. Arginine-rich anti-vascular endothelial growth factor (anti-VEGF) hexapeptide inhibits collagen-induced arthritis and VEGF-stimulated productions of TNF-alpha and IL-6 by human monocytes. J Immunol (2005) 174:5846–55.[Abstract/Free Full Text]
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Submitted 11 January 2008; revised version accepted 2 September 2008.
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