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Chemerin induces CCL2 and TLR4 in synovial fibroblasts of patients with rheumatoid arthritis and osteoarthritis
Experimental and Molecular Pathology 92 (2012) 90–96
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Chemerin induces CCL2 and TLR4 in synovial fibroblasts of patients with rheumatoid arthritis and osteoarthritis Kristina Eisinger a, Sabrina Bauer a, Andreas Schäffler a, Roland Walter a, Elena Neumann b, Christa Buechler a, Ulf Müller-Ladner b, Klaus W. Frommer b,⁎ a b
Department of Internal Medicine I, Regensburg University Hospital, D-93042 Regensburg, Germany Department of Internal Medicine and Rheumatology, Justus-Liebig-University Gießen, Kerckhoff-Klinik, D-61231 Bad Nauheim, Germany
a r t i c l e
i n f o
Article history: Received 11 October 2011 Available online 19 October 2011 Keywords: Chemerin Synovium Fibroblast Arthritis
a b s t r a c t Introduction: Chemerin stimulates migration of leukocytes to sites of inflammation and also increases inflammatory signaling in chondrocytes suggesting a function of chemerin in joint inflammation. Synovial fibroblasts (SF) are critically involved in synovitis and subsequent cartilage destruction. Here, we analyzed whether synovial fibroblasts express chemerin and its receptor CMKLR1. Further, the role of chemerin in synovial fibroblast chemotaxis, proliferation, insulin response and release of inflammatory proteins was studied. Methods: Synovial tissue sections were labeled with chemerin antibody and chemerin was measured in synovial fluid by ELISA. Chemerin mRNA and protein as well as CMKLR1 expression were determined in SFs from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Effects of chemerin on cytokines, chemokines and matrix metalloproteinases (MMP), and on proliferation, migration and insulin signaling were analyzed appropriately. Results: SFs expressed CMKLR1 and chemerin mRNA, and chemerin protein was found in cell supernatants of synovial fibroblasts. Immunohistochemistry detected chemerin in synovial tissue predominantly localized within the lining layer. Chemerin was present in synovial fluids of RA, OA and psoriatic arthritis patients in similar concentrations. Chemerin neither increased IL-6 levels nor MMP-2 or −9 activity in SFs. Also, it did not act as a chemoattractant for these cells. With respect to intracellular signaling, neither basal nor insulinmediated phosphorylation of Akt was affected. However, chemerin significantly increased TLR4 mRNA and synthesis of CCL2 in SFs while CCL4 and −5 were not altered. Cell proliferation of SFs, however, was modestly reduced by chemerin. Conclusions: These data show that human SFs express both chemerin and its receptor. As chemerin enhanced expression of TLR4 and induced release of CCL2 in SFs, a role of this protein in innate immune systemassociated joint inflammation is proposed. © 2011 Elsevier Inc. All rights reserved.
Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial hyperplasia and progressive joint destruction. Early after disease onset, RA synovial fibroblasts (SFs) become activated and enhance local inflammation by the release of proinflammatory factors. Cytokines and chemokines secreted by RASFs promote the influx of immune cells into the hyperplastic synovium further contributing to synovitis (Neumann et al., 2010). Chemerin, also known as tazarotene-induced gene 2 (TIG2), is a chemotactic protein which has been attributed to the attraction of immune cells including macrophages and dendritic cells (Meder et al., 2003; Yoshimura and Oppenheim, 2008). Its chemotactic activity is mediated
⁎ Corresponding author. Fax: + 49 6036 9962809. E-mail address: [email protected] (K.W. Frommer). 0014-4800/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2011.10.006
by binding of chemerin to the G-protein coupled receptor CMKLR1 (Meder et al., 2003). More recent studies demonstrate that articular chondrocytes express CMKLR1 and even synthesize chemerin, whose expression is induced by IL1-β, at least in murine chondrocytes (Berg et al., 2010; Conde et al., 2011). Chemerin increases production of TNF, IL1-β, IL-6, MMP-1 and MMP-8 in human articular chondrocytes. Higher doses of this chemokine also elevate MMP-2, MMP-3, MMP-13, and IL-8 synthesis. These findings suggest a function of chemerin in joint inflammation and cartilage destruction (Berg et al., 2010). Chemerin seems to be predominantly released by adipocytes and its production and serum levels are increased in obesity (Bauer et al., 2011; Goralski et al., 2007; Sell et al., 2009). Chemerin synthesis in adipocytes is upregulated by proinflammatory cytokines and lipopolysaccharide, and TNF increases circulating chemerin in mice (Cawthorn and Sethi, 2008; Kralisch et al., 2009; Parlee et al., 2010). Systemic chemerin is higher in obese and type 2 diabetic patients, and may contribute to
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insulin resistance by impairing insulin signaling in adipocytes and skeletal muscle cells (Bauer et al., 2011; Ernst and Sinal, 2010; Lehrke et al., 2009; Sell et al., 2009; Weigert et al., 2010a). Chemerin positively correlates with circulating levels of inflammatory cytokines in obesity indicating that increased chemerin levels are associated with inflammation (Ernst and Sinal, 2010; Lehrke et al., 2009; Weigert et al., 2010a). Of note, high levels of chemerin have also been detected in inflamed tissues and body fluids such as psoriatic skin, ascites and synovial joint fluid of patients with rheumatoid arthritis (Albanesi et al., 2009; Wittamer et al., 2003). In obesity, proteins released by adipose tissue contribute to systemic inflammation. Levels of several adipokines such as adiponectin were found to be increased in serum and synovial fluid of patients with arthritis independent of their body mass index. Findings indicate that these adipokines may contribute to inflammation and cartilage destruction (Ferraccioli and Gremese, 2011) as has been shown in vitro for the adipokine adiponectin (Frommer et al., 2010). Murine 3T3-L1 fibroblasts express CMKLR1 whereas chemerin is neither found in the cell lysate nor in the supernatants (Bauer et al., 2011). This suggests that fibroblasts are capable of responding to chemerin. Here, we analyzed the effects of chemerin on SFs from RA and osteoarthritis (OA) patients in order to elucidate the potential role of chemerin in the pathophysiology of these diseases.
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Laboratories). After color development was stopped, slides were in part counter-stained with hematoxylin and then mounted immediately (Aquatex; Merck). For verifying the specificity of antibody binding, negative and isotype controls were also included. Isolation of synovial fibroblasts Synovial tissue samples were obtained from synovial biopsy specimens from RA and OA patients who were undergoing joint surgery. All specimens were obtained with the approval of the Ethics Committee of the Justus-Liebig-University of Gießen. All patients gave informed consent and fulfilled the criteria of the American College of Rheumatology. Following enzymatic digestion, primary synovial fibroblasts were isolated and cultured in supplemented DMEM as described previously (Neumann et al., 2002a, 2002b). Cell culture
Materials and Methods
Primary synovial fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM; PAA Laboratories) containing 10% heatinactivated fetal bovine serum (FBS; Sigma), 12.5 mM HEPES (PAA Laboratories), and 100 units/ml of penicillin/streptomycin (PAA Laboratories), and cultured for a maximum of 8 passages at 37 °C in 10% CO2. For harvesting or subculturing, cells were detached using trypsin–EDTA (PAA Laboratories).
Evaluation of gene expression by real-time RT-PCR
Stimulation of RASFs and OASFs
RNA was isolated with the RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse transcribed into cDNA using a kit from Promega (Mannheim, Germany). Real-time RT-PCR was performed using LightCycler FastStart DNA Master SYBR Green I (Roche, Mannheim, Germany) as recently described (Bauer et al., 2010, 2011). Oligonucleotides were synthesized by Metabion (Planegg-Martinsried, Germany).The primers for human chemerin were chemerin uni (5′-GGT CCA CTG CCC CAT AGA G-3′) and chemerin rev (5′-TTA TCA TGG CTG GGG ATA GAA-3′). The primers for CMKLR1 were CMKLR1 uni (5′-ACC TGC ATG GGA AAA TAT CCT-3′) and CMKLR1 rev (5′-GAG GTT GAG TGT GTG GTA GGG-3′). Primers used to amplify chemokine mRNAs have been recently described (Neumeier et al., 2011). TLR4 was amplified with TLR4 uni (5′-TTC CGC TTC CTG GTC TTA TC-3′) and TLR4 rev (5′-TCA GAG GTC CAT CAA ACA TCA C-3′). For normalization, expression of 18S rRNA was used as published previously (Bauer et al., 2010).
RASFs and OASFs (n =3, each) were grown to 70–80% confluency and stimulated with 360 ng/ml and in some experiments with 1 μg/ml of human recombinant chemerin (Glu21-Ser157, R&D Systems, Wiesbaden-Nordenstadt, Germany) for 24 h unless noted otherwise. The concentration of 360 ng/ml was chosen based on the chemerin level detected by Wittamer et al. (Wittamer et al., 2003) in synovial fluid of arthritis patients. Further, this concentration is within the range of systemic chemerin levels (Sell et al., 2010; Weigert et al., 2010a, 2010b). Insulin (Sigma, Deisenhofen, Germany) stimulation was performed for 20 min. Unstimulated RASFs and OASFs were used as negative controls. Supernatants were collected and frozen at −20 °C until further evaluation.
SDS-PAGE and immunoblotting Proteins (5 μg per lane) were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Bio-Rad, Germany). GAPDH antibody and antibodies against the phosphorylated form of Akt (Ser473) were from New England Biolabs GmbH (Frankfurt, Germany). Incubation with antibodies was performed in 1.5% BSA in TBS, 0.1% Tween. Detection of the immune complexes was performed with the ECL Western blotting detection system (Amersham Pharmacia, Deisenhofen, Germany). Immunohistochemistry Synovial tissue samples from RA and OA patients (n = 3) were embedded into TissueTek® medium and frozen in liquid nitrogen. 5 μm thick cryosections were cut and fixed in acetone. 4% goat serum was used for blocking non-specific primary antibody binding. For immunohistochemical detection, an anti-human chemerin antibody generated in rabbit (H-002-53; Phoenix Pharmaceuticals) was used at a dilution of 1:100 in combination with the Histofine® Simple-Stain MAX/PO rabbit-specific detection system (Nichirei Biosciences) and 3-amino-9-ethylcarbazole (AEC) as a substrate (Vector
Immunoassays The cytokine, chemokine, and chemerin levels in cell culture supernatants were measured using commercially available enzyme-linked immunosorbent assays (ELISAs). Chemerin in synovial fluids was quantified using an ELISA from BioVendor (Heidelberg, Germany). Chemerin in supernatants of fibroblasts was determined using an ELISA from R&D Systems. Further, all other ELISAs used were from R&D Systems. Two-chamber migration assay A two-chamber migration system was used for analyzing whether chemerin acts as a chemoattractant on RASFs. A 24-well plate with 12 Transwell® permeable supports (Corning), in short inserts, was set up according to the manufacturer's instructions. For analyzing RASF migration, inserts with 8 μm pores were used. RASFs used in the migration assay were detached using Accutase (PAA Laboratories). The inserts were filled with 1 ×105 RASFs resuspended in DMEM containing 0.5% heat-inactivated FCS. The bottom wells were filled with DMEM additionally containing 360 ng/ml or 2000 ng/ml chemerin. Gradient-free wells containing 0.5% FBS DMEM in inserts and bottom wells formed the baseline. 0% FBS DMEM served as a negative control (chemorepellent), 10% FBS DMEM as a positive control (i.e. chemoattractant). Each set-up was performed in duplicate wells (n= 2). Migration time was 15 h. At the end of the migration period, cells that did not pass the
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membrane were aspirated, while cells that passed the membrane and dropped into the bottom well were transferred into collection tubes. Additionally, any cells that passed the membrane but may still have been attached to the lower side of it were detached using trypsinEDTA and transferred into the collection tubes. Finally, all cells that actively migrated through the membrane were counted. Cell Titer Blue Cell Viability Assay (CTB Assay) For quantification of cell proliferation, a CTB assay (Promega; Mannheim, Germany) measuring the relative number of viable cells was performed according to the manufacturer's instructions. Cell proliferation was quantified for synovial fibroblasts which had been incubated for 24 h with recombinant chemerin at different concentrations (200 ng/ml, 360 ng/ml and 500 ng/ml). Direct effects of chemerin on cell viability were excluded by a lactate dehydrogenase assay. Zymography Gelatine gels for zymography were obtained from Bio-Rad (Munich, Germany). Assays were performed as recommended by the manufacturer. MMP activity was determined in cell supernatants using 15 μl samples.
informed consent and fulfilled the criteria of the American College of Rheumatology. Statistical analysis Data are shown as mean values ± standard deviation. Data in Fig. 2E are presented as box plots indicating median, lower and upper quartiles and range of values. Statistical differences were analyzed by the two-tailed Mann-Whitney U test or Student's t-test. A value of P b 0.05 was regarded statistically significant. P values of statistically significant differences are indicated as numbers above the bars connecting the values to be compared. Results Expression of chemerin in synovial tissue Immunohistochemical staining showed expression of chemerin in synovial tissue from RA (Fig. 1A–C) and OA (Fig. 1D) patients, which confirms findings recently published by Kaneko et al. (2011). Expression of chemerin was most prominent within the lining layer (Fig. 1A), which consists of synovial fibroblasts and macrophages. Vascular walls also showed increased expression of chemerin (Fig. 1B and C). Chemerin expression appeared to be weaker in RA compared to OA synovial tissue (Fig. 1A and D), which was also found by Kaneko et al.
Synovial fluid Expression of CMKLR1 and chemerin in synovial fibroblasts Synovial fluid was obtained from 17 RA patients, 5 OA patients and 14 patients with psoriatic arthritis (PsA) who were undergoing joint surgery. All specimens were obtained with the approval of the Ethics Committee of the Justus-Liebig-University of Gießen. All patients gave
To find out whether SFs of patients with rheumatoid arthritis (RASFs) and osteoarthritis (OASFs) express chemerin and the chemerin receptor CMKLR1, real-time RT-PCR was performed using RNA isolated
Fig. 1. Expression of chemerin in synovial tissue of patients with rheumatoid arthritis (RA) or osteoarthritis (OA). Immunostaining of chemerin in synovial tissue with (B) or without hematoxylin counterstaining (A, C, D). Expression of chemerin in RA synovial tissue, most prominently within the lining layer (A) and perivascularly (B and C). Expression of chemerin in OA synovial tissue (D). Arrows indicate areas of increased expression.
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Chemerin in synovial fluid Chemerin activity has been found increased in synovial fluid of RA compared to OA patients (Wittamer et al., 2003). However, total chemerin levels measured by ELISA were similarly concentrated in synovial fluid of RA, OA and PsA patients (Fig. 2D). Chemerin is not a chemoattractant for RA fibroblasts Chemerin is known to be a chemoattractant for certain immune cells. We therefore investigated whether it also stimulates migration of RASFs. In this experiment, neither 360 ng/ml of chemerin nor an unphysiologically high level of 2 μg/ml affected migration of RASFs (Fig. 3A). Chemerin reduces proliferation of synovial fibroblasts
Fig. 2. Expression of chemerin and its receptor CMKLR1 in synovial fibroblasts; concentrations of chemerin in synovial fluid. (A) CMKLR1 mRNA in synovial fibroblasts (SFs) of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). (B) Chemerin mRNA in RASFs and OASFs. (C) Chemerin in supernatants of RASFs and OASFs. (D) Chemerin in synovial fluid of patients with RA, OA or psoriatic arthritis (PsA).
from cells obtained of 3 different donors for each disease. Chemerin and CMKLR1 mRNA were found to be expressed in all cell populations. Levels were similar in RASFs and OASFs (Fig. 2A and B). Chemerin was quantified in the cell culture supernatants by ELISA and levels were 250 to 300 pg/mg cellular protein (Fig. 2C).
The effect of 200, 360 and 500 ng/ml chemerin on proliferation of RASFs and OASFs was analyzed. As described previously (Mohr et al., 1975), basal proliferation of RASFs was increased compared to OASFs (data not shown). Only the highest concentration of chemerin (500 ng/ml) reduced proliferation of OASFs and RASFs significantly while only a trend was observed for the other concentrations tested. The effect was similar in all of the SFs; data are summarized for all cell populations (n = 6) in Fig. 3B. To exclude that chemerin affects cell viability, lactate dehydrogenase was measured in the cell supernatants but was not increased by any of the chemerin concentrations used (data not shown). Chemerin does not increase MMP-2 and MMP-9 activity In chondrocytes, chemerin upregulates MMP-2 protein expression (Berg et al., 2010). Also, higher gelanolytic MMP-2 and MMP-9 activity was found in endothelial cells treated with chemerin (Berg et al., 2010; Kaur et al., 2010). In our experiments, zymography revealed weak MMP-2 and strong MMP-9 activity in supernatants of OASFs and RASFs. Dose–response curves were performed using 0, 200, 360, 500,
Fig. 3. Chemerin reduces synovial fibroblast proliferation but does not chemoattract them. (A) The number of migrated cells was determined after using chemerin (360 ng/ml, 2 μg/ml), 0% FBS (negative control) or 10% FBS (positive control) as chemoattractants or chemorepellent, respectively. (B) Viable cells were detected by a CTB assay after 24 h cultivation in the presence of different concentrations of chemerin. (C) MMP-2 and MMP-9 activity was determined by zymography in supernatants of SFs cultivated in the presence of increasing concentrations of chemerin for 24 h. (D) MMP-2 and MMP-9 activity was determined by zymography in supernatants of RASFs of 3 different donors incubated with 360 ng/ml or 1 μg/ml chemerin for 24 h.
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750 and 1000 ng/ml chemerin but MMP activities were not altered. Representative results using one RASF cell population are shown in Fig. 3C. Furthermore, the effect of 360 and 1000 ng/ml chemerin on RASF cells of 3 different donors are shown in Fig. 3D. Chemerin does not alter basal nor insulin-stimulated phosphorylated Akt Chemerin was shown to increase basal phosphorylation of Akt in chondrocytes (Berg et al., 2010) while insulin-stimulated phosphorylation of Akt was impaired in adipocytes and skeletal muscle cells (Ernst and Sinal, 2010; Sell et al., 2009). Incubation of RASFs and OASFs with 360 ng/ml chemerin for 24 h did not increase pAkt (data not shown). Further, insulin-stimulated (0.2 or 2 μM) phosphorylation of this kinase was not impaired in RASFs and OASFs by preincubation with 360 ng/ml chemerin for 24 h (Fig. 4A).
0, 200, 360, 500, 750 and 1000 ng/ml chemerin were also performed but CCL4 and CCL5 were not changed by any of the concentrations used (data not shown). In order to find out whether increased CCL2 protein in the supernatants of the SFs may be partly related to enhanced transcription, real-time RT-PCR was performed. However, mRNA expression of all analyzed chemokines remained unchanged upon incubation with 360 ng/ml chemerin (Fig. 4C and data not shown). Chemerin upregulates TLR4 mRNA in synovial fibroblasts TLR4 is highly expressed in RASF (Ospelt et al., 2008) and contributes to synovial inflammation. Analysis of TLR4 expression revealed that chemerin similarly upregulated TLR4 mRNA in OASFs and RASFs. Combined data are shown in Fig. 4D.
Chemerin upregulates CCL2 in synovial fibroblasts
Discussion
In chondrocytes, chemerin induces the release of proinflammatory cytokines and chemokines (Berg et al., 2010). In SFs, however, IL-6 was not significantly increased by 360 or 1000 ng/ml chemerin after 24 h. Kaneko et al., 2011 found a significant increase of IL-6 but only after 48 h of stimulation with a supraphysiological concentration of chemerin (950 ng/ml). The basal level of IL-8 (CXCL8) secretion of these cells is quite low and was not induced by chemerin either (data not shown). Analysis of monocyte chemoattractant protein (MCP)-1/ CCL2, macrophage inflammatory protein 1β (MIP1β, CCL4) and regulated upon activation normal T cell expressed and secreted (RANTES, CCL5) levels in OASFs and RASFs incubated with 360 ng/ml chemerin for 24 h revealed that only CCL2 was significantly and similarly increased in these cells (Fig. 4B). Similarly, (Kaneko et al., 2011) found a significant increase after incubation with 50 nM (=950 ng/ml) chemerin for 24 h but not with much lower concentrations of chemerin. Basal levels of CCL2, CCL4 and CCL5 in the supernatants of RASFs and OASFs were similar (Fig. 4B and data not shown). Macrophage inflammatory protein 1α (MIP1α, CCL3) levels were below the detection limit of the ELISA (i.e. 2 pg/ml). Dose–response curve analyses using
Synovial fluid of patients with RA contains adipokines such as visfatin, adiponectin and leptin, and their levels are usually higher in RA than in OA (Ibrahim et al., 2008; Schaffler et al., 2003; Sellam and Berenbaum, 2010; Senolt et al., 2010). According to our results, chemerin levels, however, are similar in synovial fluid of RA and OA patients as well as PsA patients. On the other hand, Wittamer et al. (Huss et al., 2010) described increased chemerin activity in synovial fluid of RA patients compared to no activity in synovial fluid of OA patients (Wittamer et al., 2003). In the current study, total levels of chemerin protein were measured. Hence, these data cannot directly be compared. Chemerin is activated by proteases. This proteolytic activation may be increased in highly inflammatory conditions suggesting that chemerin activity may indeed be higher in RA compared to OA (Guillabert et al., 2008; Wittamer et al., 2005). Chemerin is mainly produced by adipocytes but a recent study identified chemerin mRNA and protein in chondrocytes indicating that these cells may also contribute to chemerin levels in synovial fluid (Berg et al., 2010). Here, it is shown that RASFs and OASFs also express chemerin mRNA and release chemerin protein. Further, these cells
Fig. 4. Chemerin induces CCL2 protein and TLR4 mRNA in synovial fibroblasts but does not affect basal or insulin-mediated phosphorylation of Akt. (A) Phosphorylated Akt in RASFs and OASFs preincubated with PBS or 360 ng/ml chemerin for 24 h and subsequently stimulated with 0, 0.2 or 2 μM insulin for 20 min. (B) CCL2 in the supernatants of OASFs and RASFs incubated with 360 ng/ml chemerin for 24 h. (C) CCL2 mRNA in OASFs and RASFs incubated with 360 ng/ml chemerin for 24 h. (D) TLR4 mRNA in OASFs and RASFs incubated with 360 ng/ml chemerin for 24 h.
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express the chemerin receptor CMKLR1 suggesting that chemerin may also affect fibroblast function. Chemerin attracts leukocytes (Yoshimura and Oppenheim, 2008) but migration of RASFs is not stimulated by chemerin. Chemerin-mediated induction of proinflammatory and proangiogenic signalling pathways as well as chemerin-induced release of cytokines, chemokines and MMPs have been described (Berg et al., 2010; Bozaoglu et al., 2010). In RASFs and OASFs, however, chemerin does not upregulate IL-6 and IL-8 synthesis nor MMP-2 and MMP-9 activity. Phosphorylation of Akt at Ser 473 is increased by chemerin in chondrocytes (Berg et al., 2010) but is unchanged in RASFs and OASFs with and without chemerin stimulation. Further, insulin-mediated phosphorylation of this kinase is not affected by chemerin in SFs. While the chemokines IL-8, CCL4 and CCL5 are not increased by chemerin stimulation of SFs, CCL2 release is significantly elevated. Interestingly, the higher release of CCL2 is not accompanied by an induction of CCL2 mRNA. CCL2 is increased upon activation of NFκB, which also mediates a higher release of cytokines like IL-6 (Luo et al., 2008). Absence of chemerin-mediated induction of IL-6 in the SFs suggests that NFκB is not activated and indicates that the higher release of CCL2 depends on posttranscriptional mechanisms. CCL2 attracts immune cells like memory T lymphocytes and natural killer cells, which are major contributors to the pathogenesis of OA and RA (Carr et al., 1994; Ezawa et al., 1997; Huss et al., 2010). In addition, CCL2 mediates angiogenesis via VEGF (Hong et al., 2005). Chemerin therefore seems to contribute similarly to inflammation in OA and RA (Neumann et al., 2010). TLR4 is expressed by SFs and its level was shown to be higher in RASFs compared to OASFs (Ospelt et al., 2008). This difference could not be identified in the SFs analysed herein most likely due to the relatively small number of donors. TLR4 expression is similarly increased by chemerin in OASFs and RASFs. The activation of TLR4 promotes osteoclastogenic activity by inducing receptor activator of nuclear factor ligand (RANKL) thereby promoting bone destruction (Neumann et al., 2010; van den Berg et al., 2007). Inhibition of TLR4 ameliorates the severity of collagen-induced arthritis and spontaneous arthritis caused by interleukin-1 receptor antagonist (IL-1Ra) gene deficiency and lowers IL-1 expression in arthritic joints (Abdollahi-Roodsaz et al., 2007). These findings indicate that higher TLR4 levels in SFs may contribute to increased inflammation and bone destruction. Chemerin modestly reduces proliferation of OASFs and RASFs while having no effect on cell viability. This effect only becomes significant when using relatively high concentrations of chemerin suggesting that this potentially protective function of chemerin, which may antagonize synovial hyperplasia and even inflammation (Karouzakis et al., 2006), may only be relevant in vivo under specific conditions. In summary, the current study shows that chemerin increases specifically CCL2 and TLR4 in RASFs and OASFs while reducing SF proliferation. Therefore, this adipokine may play a small but distinct role in the pathophysiology of arthritis. Authors' contribution KE and KWF performed analyses, participated in the study design and the drafting of the manuscript. SB and RW performed some of the analyses. AS, EN, CB and UML participated in the design, interpretation of the results, coordination of the study and revision of the manuscript. CB participated in the design, interpretation of the results, coordination of the study, and the drafting of the manuscript. All the authors gave their final approval of the manuscript version to be published. Conflict of interest statement The authors have no conflict of interest related to this work.
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Acknowledgment The technical assistance of Yvonne Hader Rosel Engel and Sina Köppert is greatly appreciated. This work was in part supported by the German Research Foundation (BU 1141/7-1, NE1174/3-1 and NE1174/8-1). References Abdollahi-Roodsaz, S., et al., 2007. Inhibition of Toll-like receptor 4 breaks the inflammatory loop in autoimmune destructive arthritis. Arthritis and Rheumatism 56, 2957–2967. Albanesi, C., et al., 2009. Chemerin expression marks early psoriatic skin lesions and correlates with plasmacytoid dendritic cell recruitment. The Journal of Experimental Medicine 206, 249–258. Bauer, S., et al., 2010. Low-abundant adiponectin receptors in visceral adipose tissue of humans and rats are further reduced in diabetic animals. Archives of Medical Research 41, 75–82. Bauer, S., et al., 2011. Sterol regulatory element-binding protein 2 (SREBP2) activation after excess triglyceride storage induces chemerin in hypertrophic adipocytes. Endocrinology 152, 26–35. Berg, V., et al., 2010. Human articular chondrocytes express ChemR23 and chemerin; ChemR23 promotes inflammatory signalling upon binding the ligand chemerin (21–157). Arthritis Research & Therapy 12, R228. Bozaoglu, K., et al., 2010. Chemerin, a novel adipokine in the regulation of angiogenesis. Journal of Clinical Endocrinology and Metabolism 95, 2476–2485. Carr, M.W., et al., 1994. Monocyte chemoattractant protein 1 acts as a T-lymphocyte chemoattractant. Proceedings of the National Academy of Sciences of the United States of America 91, 3652–3656. Cawthorn, W.P., Sethi, J.K., 2008. TNF-alpha and adipocyte biology. FEBS Letters 582, 117–131. Conde, J., et al., 2011. Expanding the adipokine network in cartilage: identification and regulation of novel factors in human and murine chondrocytes. Annals of the Rheumatic Diseases 70, 551–559. Ernst, M.C., Sinal, C.J., 2010. Chemerin: at the crossroads of inflammation and obesity. Trends in Endocrinology and Metabolism 21, 660–667. Ezawa, K., et al., 1997. Comparative analysis of CD45RA- and CD45RO-positive CD4+T cells in peripheral blood, synovial fluid, and synovial tissue in patients with rheumatoid arthritis and osteoarthritis. Acta Medica Okayama 51, 25–31. Ferraccioli, G., Gremese, E., 2011. Adiposity, joint and systemic inflammation: the additional risk of having a metabolic syndrome in rheumatoid arthritis. Swiss Medical Weekly 141, w13211. Frommer, K.W., et al., 2010. Adiponectin-mediated changes in effector cells involved in the pathophysiology of rheumatoid arthritis. Arthritis and Rheumatism 62, 2886–2899. Goralski, K.B., et al., 2007. Chemerin, a novel adipokine that regulates adipogenesis and adipocyte metabolism. Journal of Biological Chemistry 282, 28175–28188. Guillabert, A., et al., 2008. Role of neutrophil proteinase 3 and mast cell chymase in chemerin proteolytic regulation. Journal of Leukocyte Biology 84, 1530–1538. Hong, K.H., et al., 2005. Monocyte chemoattractant protein-1-induced angiogenesis is mediated by vascular endothelial growth factor-A. Blood 105, 1405–1407. Huss, R.S., et al., 2010. Synovial tissue-infiltrating natural killer cells in osteoarthritis and periprosthetic inflammation. Arthritis and Rheumatism 62, 3799–3805. Ibrahim, S.M., et al., 2008. Plasma and synovial fluid adipocytokines in patients with rheumatoid arthritis and osteoarthritis. The Egyptian Journal of Immunology 15, 159–170. Kaneko, K., et al., 2011. Chemerin activates fibroblast-like synoviocytes in patients with rheumatoid arthritis. Arthritis Research & Therapy 13, R158 [Epub ahead of print]. Karouzakis, E., et al., 2006. Molecular and cellular basis of rheumatoid joint destruction. Immunology Letters 106, 8–13. Kaur, J., et al., 2010. Identification of chemerin receptor (ChemR23) in human endothelial cells: chemerin-induced endothelial angiogenesis. Biochemical and Biophysical Research Communications 391, 1762–1768. Kralisch, S., et al., 2009. Interleukin-1beta induces the novel adipokine chemerin in adipocytes in vitro. Regulatory Peptides 154, 102–106. Lehrke, M., et al., 2009. Chemerin is associated with markers of inflammation and components of the metabolic syndrome but does not predict coronary atherosclerosis. European Journal of Endocrinology 161, 339–344. Luo, X., et al., 2008. Extracellular heat shock protein 70 inhibits tumour necrosis factoralpha induced proinflammatory mediator production in fibroblast-like synoviocytes. Arthritis Research & Therapy 10, R41. Meder, W., et al., 2003. Characterization of human circulating TIG2 as a ligand for the orphan receptor ChemR23. FEBS Letters 555, 495–499. Mohr, W., et al., 1975. Proliferation of synovial lining cells and fibroblasts. Annals of the Rheumatic Diseases 34, 219–224. Neumann, E., et al., 2002a. Inhibition of cartilage destruction by double gene transfer of IL-1Ra and IL-10 involves the activin pathway. Gene Therapy 9, 1508–1519. Neumann, E., et al., 2002b. Identification of differentially expressed genes in rheumatoid arthritis by a combination of complementary DNA array and RNA arbitrarily primed-polymerase chain reaction. Arthritis and Rheumatism 46, 52–63. Neumann, E., et al., 2010. Rheumatoid arthritis progression mediated by activated synovial fibroblasts. Trends in Molecular Medicine 16, 458–468.
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Neumeier, M., et al., 2011. Adiponectin stimulates release of CCL2, -3, -4 and − 5 while the surface abundance of CCR2 and − 5 is simultaneously reduced in primary human monocytes. Cytokine. Ospelt, C., et al., 2008. Overexpression of toll-like receptors 3 and 4 in synovial tissue from patients with early rheumatoid arthritis: toll-like receptor expression in early and longstanding arthritis. Arthritis and Rheumatism 58, 3684–3692. Parlee, S.D., et al., 2010. Serum chemerin levels vary with time of day and are modified by obesity and tumor necrosis factor-{alpha}. Endocrinology 151, 2590–2602. Schaffler, A., et al., 2003. Adipocytokines in synovial fluid. JAMA: The Journal of the American Medical Association 290, 1709–1710. Sell, H., et al., 2009. Chemerin is a novel adipocyte-derived factor inducing insulin resistance in primary human skeletal muscle cells. Diabetes 58, 2731–2740. Sell, H., et al., 2010. Chemerin correlates with markers for fatty liver in morbidly obese patients and strongly decreases after weight loss induced by bariatric surgery. Journal of Clinical Endocrinology and Metabolism 95, 2892–2896. Sellam, J., Berenbaum, F., 2010. The role of synovitis in pathophysiology and clinical symptoms of osteoarthritis. Nature Reviews. Rheumatology 6, 625–635.
Senolt, L., et al., 2010. Vaspin and omentin: new adipokines differentially regulated at the site of inflammation in rheumatoid arthritis. Annals of the Rheumatic Diseases 69, 1410–1411. van den Berg, W.B., et al., 2007. Amplifying elements of arthritis and joint destruction. Annals of the Rheumatic Diseases 66 (Suppl. 3), iii45–iii48. Weigert, J., et al., 2010a. Systemic chemerin is related to inflammation rather than obesity in type 2 diabetes. Clinical Endocrinology 72, 342–348. Weigert, J., et al., 2010b. Circulating levels of chemerin and adiponectin are higher in ulcerative colitis and chemerin is elevated in Crohn's disease. Inflammatory Bowel Diseases 16, 630–637. Wittamer, V., et al., 2003. Specific recruitment of antigen-presenting cells by chemerin, a novel processed ligand from human inflammatory fluids. The Journal of Experimental Medicine 198, 977–985. Wittamer, V., et al., 2005. Neutrophil-mediated maturation of chemerin: a link between innate and adaptive immunity. Journal of Immunology 175, 487–493. Yoshimura, T., Oppenheim, J.J., 2008. Chemerin reveals its chimeric nature. The Journal of Experimental Medicine 205, 2187–2190.
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Chemerin induces CCL2 and TLR4 in synovial fibroblasts of patients with rheumatoid arthritis and osteoarthritis Kristina Eisinger a, Sabrina Bauer a, Andreas Schäffler a, Roland Walter a, Elena Neumann b, Christa Buechler a, Ulf Müller-Ladner b, Klaus W. Frommer b,⁎ a b
Department of Internal Medicine I, Regensburg University Hospital, D-93042 Regensburg, Germany Department of Internal Medicine and Rheumatology, Justus-Liebig-University Gießen, Kerckhoff-Klinik, D-61231 Bad Nauheim, Germany
a r t i c l e
i n f o
Article history: Received 11 October 2011 Available online 19 October 2011 Keywords: Chemerin Synovium Fibroblast Arthritis
a b s t r a c t Introduction: Chemerin stimulates migration of leukocytes to sites of inflammation and also increases inflammatory signaling in chondrocytes suggesting a function of chemerin in joint inflammation. Synovial fibroblasts (SF) are critically involved in synovitis and subsequent cartilage destruction. Here, we analyzed whether synovial fibroblasts express chemerin and its receptor CMKLR1. Further, the role of chemerin in synovial fibroblast chemotaxis, proliferation, insulin response and release of inflammatory proteins was studied. Methods: Synovial tissue sections were labeled with chemerin antibody and chemerin was measured in synovial fluid by ELISA. Chemerin mRNA and protein as well as CMKLR1 expression were determined in SFs from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Effects of chemerin on cytokines, chemokines and matrix metalloproteinases (MMP), and on proliferation, migration and insulin signaling were analyzed appropriately. Results: SFs expressed CMKLR1 and chemerin mRNA, and chemerin protein was found in cell supernatants of synovial fibroblasts. Immunohistochemistry detected chemerin in synovial tissue predominantly localized within the lining layer. Chemerin was present in synovial fluids of RA, OA and psoriatic arthritis patients in similar concentrations. Chemerin neither increased IL-6 levels nor MMP-2 or −9 activity in SFs. Also, it did not act as a chemoattractant for these cells. With respect to intracellular signaling, neither basal nor insulinmediated phosphorylation of Akt was affected. However, chemerin significantly increased TLR4 mRNA and synthesis of CCL2 in SFs while CCL4 and −5 were not altered. Cell proliferation of SFs, however, was modestly reduced by chemerin. Conclusions: These data show that human SFs express both chemerin and its receptor. As chemerin enhanced expression of TLR4 and induced release of CCL2 in SFs, a role of this protein in innate immune systemassociated joint inflammation is proposed. © 2011 Elsevier Inc. All rights reserved.
Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial hyperplasia and progressive joint destruction. Early after disease onset, RA synovial fibroblasts (SFs) become activated and enhance local inflammation by the release of proinflammatory factors. Cytokines and chemokines secreted by RASFs promote the influx of immune cells into the hyperplastic synovium further contributing to synovitis (Neumann et al., 2010). Chemerin, also known as tazarotene-induced gene 2 (TIG2), is a chemotactic protein which has been attributed to the attraction of immune cells including macrophages and dendritic cells (Meder et al., 2003; Yoshimura and Oppenheim, 2008). Its chemotactic activity is mediated
⁎ Corresponding author. Fax: + 49 6036 9962809. E-mail address: [email protected] (K.W. Frommer). 0014-4800/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2011.10.006
by binding of chemerin to the G-protein coupled receptor CMKLR1 (Meder et al., 2003). More recent studies demonstrate that articular chondrocytes express CMKLR1 and even synthesize chemerin, whose expression is induced by IL1-β, at least in murine chondrocytes (Berg et al., 2010; Conde et al., 2011). Chemerin increases production of TNF, IL1-β, IL-6, MMP-1 and MMP-8 in human articular chondrocytes. Higher doses of this chemokine also elevate MMP-2, MMP-3, MMP-13, and IL-8 synthesis. These findings suggest a function of chemerin in joint inflammation and cartilage destruction (Berg et al., 2010). Chemerin seems to be predominantly released by adipocytes and its production and serum levels are increased in obesity (Bauer et al., 2011; Goralski et al., 2007; Sell et al., 2009). Chemerin synthesis in adipocytes is upregulated by proinflammatory cytokines and lipopolysaccharide, and TNF increases circulating chemerin in mice (Cawthorn and Sethi, 2008; Kralisch et al., 2009; Parlee et al., 2010). Systemic chemerin is higher in obese and type 2 diabetic patients, and may contribute to
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insulin resistance by impairing insulin signaling in adipocytes and skeletal muscle cells (Bauer et al., 2011; Ernst and Sinal, 2010; Lehrke et al., 2009; Sell et al., 2009; Weigert et al., 2010a). Chemerin positively correlates with circulating levels of inflammatory cytokines in obesity indicating that increased chemerin levels are associated with inflammation (Ernst and Sinal, 2010; Lehrke et al., 2009; Weigert et al., 2010a). Of note, high levels of chemerin have also been detected in inflamed tissues and body fluids such as psoriatic skin, ascites and synovial joint fluid of patients with rheumatoid arthritis (Albanesi et al., 2009; Wittamer et al., 2003). In obesity, proteins released by adipose tissue contribute to systemic inflammation. Levels of several adipokines such as adiponectin were found to be increased in serum and synovial fluid of patients with arthritis independent of their body mass index. Findings indicate that these adipokines may contribute to inflammation and cartilage destruction (Ferraccioli and Gremese, 2011) as has been shown in vitro for the adipokine adiponectin (Frommer et al., 2010). Murine 3T3-L1 fibroblasts express CMKLR1 whereas chemerin is neither found in the cell lysate nor in the supernatants (Bauer et al., 2011). This suggests that fibroblasts are capable of responding to chemerin. Here, we analyzed the effects of chemerin on SFs from RA and osteoarthritis (OA) patients in order to elucidate the potential role of chemerin in the pathophysiology of these diseases.
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Laboratories). After color development was stopped, slides were in part counter-stained with hematoxylin and then mounted immediately (Aquatex; Merck). For verifying the specificity of antibody binding, negative and isotype controls were also included. Isolation of synovial fibroblasts Synovial tissue samples were obtained from synovial biopsy specimens from RA and OA patients who were undergoing joint surgery. All specimens were obtained with the approval of the Ethics Committee of the Justus-Liebig-University of Gießen. All patients gave informed consent and fulfilled the criteria of the American College of Rheumatology. Following enzymatic digestion, primary synovial fibroblasts were isolated and cultured in supplemented DMEM as described previously (Neumann et al., 2002a, 2002b). Cell culture
Materials and Methods
Primary synovial fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM; PAA Laboratories) containing 10% heatinactivated fetal bovine serum (FBS; Sigma), 12.5 mM HEPES (PAA Laboratories), and 100 units/ml of penicillin/streptomycin (PAA Laboratories), and cultured for a maximum of 8 passages at 37 °C in 10% CO2. For harvesting or subculturing, cells were detached using trypsin–EDTA (PAA Laboratories).
Evaluation of gene expression by real-time RT-PCR
Stimulation of RASFs and OASFs
RNA was isolated with the RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse transcribed into cDNA using a kit from Promega (Mannheim, Germany). Real-time RT-PCR was performed using LightCycler FastStart DNA Master SYBR Green I (Roche, Mannheim, Germany) as recently described (Bauer et al., 2010, 2011). Oligonucleotides were synthesized by Metabion (Planegg-Martinsried, Germany).The primers for human chemerin were chemerin uni (5′-GGT CCA CTG CCC CAT AGA G-3′) and chemerin rev (5′-TTA TCA TGG CTG GGG ATA GAA-3′). The primers for CMKLR1 were CMKLR1 uni (5′-ACC TGC ATG GGA AAA TAT CCT-3′) and CMKLR1 rev (5′-GAG GTT GAG TGT GTG GTA GGG-3′). Primers used to amplify chemokine mRNAs have been recently described (Neumeier et al., 2011). TLR4 was amplified with TLR4 uni (5′-TTC CGC TTC CTG GTC TTA TC-3′) and TLR4 rev (5′-TCA GAG GTC CAT CAA ACA TCA C-3′). For normalization, expression of 18S rRNA was used as published previously (Bauer et al., 2010).
RASFs and OASFs (n =3, each) were grown to 70–80% confluency and stimulated with 360 ng/ml and in some experiments with 1 μg/ml of human recombinant chemerin (Glu21-Ser157, R&D Systems, Wiesbaden-Nordenstadt, Germany) for 24 h unless noted otherwise. The concentration of 360 ng/ml was chosen based on the chemerin level detected by Wittamer et al. (Wittamer et al., 2003) in synovial fluid of arthritis patients. Further, this concentration is within the range of systemic chemerin levels (Sell et al., 2010; Weigert et al., 2010a, 2010b). Insulin (Sigma, Deisenhofen, Germany) stimulation was performed for 20 min. Unstimulated RASFs and OASFs were used as negative controls. Supernatants were collected and frozen at −20 °C until further evaluation.
SDS-PAGE and immunoblotting Proteins (5 μg per lane) were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Bio-Rad, Germany). GAPDH antibody and antibodies against the phosphorylated form of Akt (Ser473) were from New England Biolabs GmbH (Frankfurt, Germany). Incubation with antibodies was performed in 1.5% BSA in TBS, 0.1% Tween. Detection of the immune complexes was performed with the ECL Western blotting detection system (Amersham Pharmacia, Deisenhofen, Germany). Immunohistochemistry Synovial tissue samples from RA and OA patients (n = 3) were embedded into TissueTek® medium and frozen in liquid nitrogen. 5 μm thick cryosections were cut and fixed in acetone. 4% goat serum was used for blocking non-specific primary antibody binding. For immunohistochemical detection, an anti-human chemerin antibody generated in rabbit (H-002-53; Phoenix Pharmaceuticals) was used at a dilution of 1:100 in combination with the Histofine® Simple-Stain MAX/PO rabbit-specific detection system (Nichirei Biosciences) and 3-amino-9-ethylcarbazole (AEC) as a substrate (Vector
Immunoassays The cytokine, chemokine, and chemerin levels in cell culture supernatants were measured using commercially available enzyme-linked immunosorbent assays (ELISAs). Chemerin in synovial fluids was quantified using an ELISA from BioVendor (Heidelberg, Germany). Chemerin in supernatants of fibroblasts was determined using an ELISA from R&D Systems. Further, all other ELISAs used were from R&D Systems. Two-chamber migration assay A two-chamber migration system was used for analyzing whether chemerin acts as a chemoattractant on RASFs. A 24-well plate with 12 Transwell® permeable supports (Corning), in short inserts, was set up according to the manufacturer's instructions. For analyzing RASF migration, inserts with 8 μm pores were used. RASFs used in the migration assay were detached using Accutase (PAA Laboratories). The inserts were filled with 1 ×105 RASFs resuspended in DMEM containing 0.5% heat-inactivated FCS. The bottom wells were filled with DMEM additionally containing 360 ng/ml or 2000 ng/ml chemerin. Gradient-free wells containing 0.5% FBS DMEM in inserts and bottom wells formed the baseline. 0% FBS DMEM served as a negative control (chemorepellent), 10% FBS DMEM as a positive control (i.e. chemoattractant). Each set-up was performed in duplicate wells (n= 2). Migration time was 15 h. At the end of the migration period, cells that did not pass the
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membrane were aspirated, while cells that passed the membrane and dropped into the bottom well were transferred into collection tubes. Additionally, any cells that passed the membrane but may still have been attached to the lower side of it were detached using trypsinEDTA and transferred into the collection tubes. Finally, all cells that actively migrated through the membrane were counted. Cell Titer Blue Cell Viability Assay (CTB Assay) For quantification of cell proliferation, a CTB assay (Promega; Mannheim, Germany) measuring the relative number of viable cells was performed according to the manufacturer's instructions. Cell proliferation was quantified for synovial fibroblasts which had been incubated for 24 h with recombinant chemerin at different concentrations (200 ng/ml, 360 ng/ml and 500 ng/ml). Direct effects of chemerin on cell viability were excluded by a lactate dehydrogenase assay. Zymography Gelatine gels for zymography were obtained from Bio-Rad (Munich, Germany). Assays were performed as recommended by the manufacturer. MMP activity was determined in cell supernatants using 15 μl samples.
informed consent and fulfilled the criteria of the American College of Rheumatology. Statistical analysis Data are shown as mean values ± standard deviation. Data in Fig. 2E are presented as box plots indicating median, lower and upper quartiles and range of values. Statistical differences were analyzed by the two-tailed Mann-Whitney U test or Student's t-test. A value of P b 0.05 was regarded statistically significant. P values of statistically significant differences are indicated as numbers above the bars connecting the values to be compared. Results Expression of chemerin in synovial tissue Immunohistochemical staining showed expression of chemerin in synovial tissue from RA (Fig. 1A–C) and OA (Fig. 1D) patients, which confirms findings recently published by Kaneko et al. (2011). Expression of chemerin was most prominent within the lining layer (Fig. 1A), which consists of synovial fibroblasts and macrophages. Vascular walls also showed increased expression of chemerin (Fig. 1B and C). Chemerin expression appeared to be weaker in RA compared to OA synovial tissue (Fig. 1A and D), which was also found by Kaneko et al.
Synovial fluid Expression of CMKLR1 and chemerin in synovial fibroblasts Synovial fluid was obtained from 17 RA patients, 5 OA patients and 14 patients with psoriatic arthritis (PsA) who were undergoing joint surgery. All specimens were obtained with the approval of the Ethics Committee of the Justus-Liebig-University of Gießen. All patients gave
To find out whether SFs of patients with rheumatoid arthritis (RASFs) and osteoarthritis (OASFs) express chemerin and the chemerin receptor CMKLR1, real-time RT-PCR was performed using RNA isolated
Fig. 1. Expression of chemerin in synovial tissue of patients with rheumatoid arthritis (RA) or osteoarthritis (OA). Immunostaining of chemerin in synovial tissue with (B) or without hematoxylin counterstaining (A, C, D). Expression of chemerin in RA synovial tissue, most prominently within the lining layer (A) and perivascularly (B and C). Expression of chemerin in OA synovial tissue (D). Arrows indicate areas of increased expression.
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Chemerin in synovial fluid Chemerin activity has been found increased in synovial fluid of RA compared to OA patients (Wittamer et al., 2003). However, total chemerin levels measured by ELISA were similarly concentrated in synovial fluid of RA, OA and PsA patients (Fig. 2D). Chemerin is not a chemoattractant for RA fibroblasts Chemerin is known to be a chemoattractant for certain immune cells. We therefore investigated whether it also stimulates migration of RASFs. In this experiment, neither 360 ng/ml of chemerin nor an unphysiologically high level of 2 μg/ml affected migration of RASFs (Fig. 3A). Chemerin reduces proliferation of synovial fibroblasts
Fig. 2. Expression of chemerin and its receptor CMKLR1 in synovial fibroblasts; concentrations of chemerin in synovial fluid. (A) CMKLR1 mRNA in synovial fibroblasts (SFs) of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). (B) Chemerin mRNA in RASFs and OASFs. (C) Chemerin in supernatants of RASFs and OASFs. (D) Chemerin in synovial fluid of patients with RA, OA or psoriatic arthritis (PsA).
from cells obtained of 3 different donors for each disease. Chemerin and CMKLR1 mRNA were found to be expressed in all cell populations. Levels were similar in RASFs and OASFs (Fig. 2A and B). Chemerin was quantified in the cell culture supernatants by ELISA and levels were 250 to 300 pg/mg cellular protein (Fig. 2C).
The effect of 200, 360 and 500 ng/ml chemerin on proliferation of RASFs and OASFs was analyzed. As described previously (Mohr et al., 1975), basal proliferation of RASFs was increased compared to OASFs (data not shown). Only the highest concentration of chemerin (500 ng/ml) reduced proliferation of OASFs and RASFs significantly while only a trend was observed for the other concentrations tested. The effect was similar in all of the SFs; data are summarized for all cell populations (n = 6) in Fig. 3B. To exclude that chemerin affects cell viability, lactate dehydrogenase was measured in the cell supernatants but was not increased by any of the chemerin concentrations used (data not shown). Chemerin does not increase MMP-2 and MMP-9 activity In chondrocytes, chemerin upregulates MMP-2 protein expression (Berg et al., 2010). Also, higher gelanolytic MMP-2 and MMP-9 activity was found in endothelial cells treated with chemerin (Berg et al., 2010; Kaur et al., 2010). In our experiments, zymography revealed weak MMP-2 and strong MMP-9 activity in supernatants of OASFs and RASFs. Dose–response curves were performed using 0, 200, 360, 500,
Fig. 3. Chemerin reduces synovial fibroblast proliferation but does not chemoattract them. (A) The number of migrated cells was determined after using chemerin (360 ng/ml, 2 μg/ml), 0% FBS (negative control) or 10% FBS (positive control) as chemoattractants or chemorepellent, respectively. (B) Viable cells were detected by a CTB assay after 24 h cultivation in the presence of different concentrations of chemerin. (C) MMP-2 and MMP-9 activity was determined by zymography in supernatants of SFs cultivated in the presence of increasing concentrations of chemerin for 24 h. (D) MMP-2 and MMP-9 activity was determined by zymography in supernatants of RASFs of 3 different donors incubated with 360 ng/ml or 1 μg/ml chemerin for 24 h.
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750 and 1000 ng/ml chemerin but MMP activities were not altered. Representative results using one RASF cell population are shown in Fig. 3C. Furthermore, the effect of 360 and 1000 ng/ml chemerin on RASF cells of 3 different donors are shown in Fig. 3D. Chemerin does not alter basal nor insulin-stimulated phosphorylated Akt Chemerin was shown to increase basal phosphorylation of Akt in chondrocytes (Berg et al., 2010) while insulin-stimulated phosphorylation of Akt was impaired in adipocytes and skeletal muscle cells (Ernst and Sinal, 2010; Sell et al., 2009). Incubation of RASFs and OASFs with 360 ng/ml chemerin for 24 h did not increase pAkt (data not shown). Further, insulin-stimulated (0.2 or 2 μM) phosphorylation of this kinase was not impaired in RASFs and OASFs by preincubation with 360 ng/ml chemerin for 24 h (Fig. 4A).
0, 200, 360, 500, 750 and 1000 ng/ml chemerin were also performed but CCL4 and CCL5 were not changed by any of the concentrations used (data not shown). In order to find out whether increased CCL2 protein in the supernatants of the SFs may be partly related to enhanced transcription, real-time RT-PCR was performed. However, mRNA expression of all analyzed chemokines remained unchanged upon incubation with 360 ng/ml chemerin (Fig. 4C and data not shown). Chemerin upregulates TLR4 mRNA in synovial fibroblasts TLR4 is highly expressed in RASF (Ospelt et al., 2008) and contributes to synovial inflammation. Analysis of TLR4 expression revealed that chemerin similarly upregulated TLR4 mRNA in OASFs and RASFs. Combined data are shown in Fig. 4D.
Chemerin upregulates CCL2 in synovial fibroblasts
Discussion
In chondrocytes, chemerin induces the release of proinflammatory cytokines and chemokines (Berg et al., 2010). In SFs, however, IL-6 was not significantly increased by 360 or 1000 ng/ml chemerin after 24 h. Kaneko et al., 2011 found a significant increase of IL-6 but only after 48 h of stimulation with a supraphysiological concentration of chemerin (950 ng/ml). The basal level of IL-8 (CXCL8) secretion of these cells is quite low and was not induced by chemerin either (data not shown). Analysis of monocyte chemoattractant protein (MCP)-1/ CCL2, macrophage inflammatory protein 1β (MIP1β, CCL4) and regulated upon activation normal T cell expressed and secreted (RANTES, CCL5) levels in OASFs and RASFs incubated with 360 ng/ml chemerin for 24 h revealed that only CCL2 was significantly and similarly increased in these cells (Fig. 4B). Similarly, (Kaneko et al., 2011) found a significant increase after incubation with 50 nM (=950 ng/ml) chemerin for 24 h but not with much lower concentrations of chemerin. Basal levels of CCL2, CCL4 and CCL5 in the supernatants of RASFs and OASFs were similar (Fig. 4B and data not shown). Macrophage inflammatory protein 1α (MIP1α, CCL3) levels were below the detection limit of the ELISA (i.e. 2 pg/ml). Dose–response curve analyses using
Synovial fluid of patients with RA contains adipokines such as visfatin, adiponectin and leptin, and their levels are usually higher in RA than in OA (Ibrahim et al., 2008; Schaffler et al., 2003; Sellam and Berenbaum, 2010; Senolt et al., 2010). According to our results, chemerin levels, however, are similar in synovial fluid of RA and OA patients as well as PsA patients. On the other hand, Wittamer et al. (Huss et al., 2010) described increased chemerin activity in synovial fluid of RA patients compared to no activity in synovial fluid of OA patients (Wittamer et al., 2003). In the current study, total levels of chemerin protein were measured. Hence, these data cannot directly be compared. Chemerin is activated by proteases. This proteolytic activation may be increased in highly inflammatory conditions suggesting that chemerin activity may indeed be higher in RA compared to OA (Guillabert et al., 2008; Wittamer et al., 2005). Chemerin is mainly produced by adipocytes but a recent study identified chemerin mRNA and protein in chondrocytes indicating that these cells may also contribute to chemerin levels in synovial fluid (Berg et al., 2010). Here, it is shown that RASFs and OASFs also express chemerin mRNA and release chemerin protein. Further, these cells
Fig. 4. Chemerin induces CCL2 protein and TLR4 mRNA in synovial fibroblasts but does not affect basal or insulin-mediated phosphorylation of Akt. (A) Phosphorylated Akt in RASFs and OASFs preincubated with PBS or 360 ng/ml chemerin for 24 h and subsequently stimulated with 0, 0.2 or 2 μM insulin for 20 min. (B) CCL2 in the supernatants of OASFs and RASFs incubated with 360 ng/ml chemerin for 24 h. (C) CCL2 mRNA in OASFs and RASFs incubated with 360 ng/ml chemerin for 24 h. (D) TLR4 mRNA in OASFs and RASFs incubated with 360 ng/ml chemerin for 24 h.
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express the chemerin receptor CMKLR1 suggesting that chemerin may also affect fibroblast function. Chemerin attracts leukocytes (Yoshimura and Oppenheim, 2008) but migration of RASFs is not stimulated by chemerin. Chemerin-mediated induction of proinflammatory and proangiogenic signalling pathways as well as chemerin-induced release of cytokines, chemokines and MMPs have been described (Berg et al., 2010; Bozaoglu et al., 2010). In RASFs and OASFs, however, chemerin does not upregulate IL-6 and IL-8 synthesis nor MMP-2 and MMP-9 activity. Phosphorylation of Akt at Ser 473 is increased by chemerin in chondrocytes (Berg et al., 2010) but is unchanged in RASFs and OASFs with and without chemerin stimulation. Further, insulin-mediated phosphorylation of this kinase is not affected by chemerin in SFs. While the chemokines IL-8, CCL4 and CCL5 are not increased by chemerin stimulation of SFs, CCL2 release is significantly elevated. Interestingly, the higher release of CCL2 is not accompanied by an induction of CCL2 mRNA. CCL2 is increased upon activation of NFκB, which also mediates a higher release of cytokines like IL-6 (Luo et al., 2008). Absence of chemerin-mediated induction of IL-6 in the SFs suggests that NFκB is not activated and indicates that the higher release of CCL2 depends on posttranscriptional mechanisms. CCL2 attracts immune cells like memory T lymphocytes and natural killer cells, which are major contributors to the pathogenesis of OA and RA (Carr et al., 1994; Ezawa et al., 1997; Huss et al., 2010). In addition, CCL2 mediates angiogenesis via VEGF (Hong et al., 2005). Chemerin therefore seems to contribute similarly to inflammation in OA and RA (Neumann et al., 2010). TLR4 is expressed by SFs and its level was shown to be higher in RASFs compared to OASFs (Ospelt et al., 2008). This difference could not be identified in the SFs analysed herein most likely due to the relatively small number of donors. TLR4 expression is similarly increased by chemerin in OASFs and RASFs. The activation of TLR4 promotes osteoclastogenic activity by inducing receptor activator of nuclear factor ligand (RANKL) thereby promoting bone destruction (Neumann et al., 2010; van den Berg et al., 2007). Inhibition of TLR4 ameliorates the severity of collagen-induced arthritis and spontaneous arthritis caused by interleukin-1 receptor antagonist (IL-1Ra) gene deficiency and lowers IL-1 expression in arthritic joints (Abdollahi-Roodsaz et al., 2007). These findings indicate that higher TLR4 levels in SFs may contribute to increased inflammation and bone destruction. Chemerin modestly reduces proliferation of OASFs and RASFs while having no effect on cell viability. This effect only becomes significant when using relatively high concentrations of chemerin suggesting that this potentially protective function of chemerin, which may antagonize synovial hyperplasia and even inflammation (Karouzakis et al., 2006), may only be relevant in vivo under specific conditions. In summary, the current study shows that chemerin increases specifically CCL2 and TLR4 in RASFs and OASFs while reducing SF proliferation. Therefore, this adipokine may play a small but distinct role in the pathophysiology of arthritis. Authors' contribution KE and KWF performed analyses, participated in the study design and the drafting of the manuscript. SB and RW performed some of the analyses. AS, EN, CB and UML participated in the design, interpretation of the results, coordination of the study and revision of the manuscript. CB participated in the design, interpretation of the results, coordination of the study, and the drafting of the manuscript. All the authors gave their final approval of the manuscript version to be published. Conflict of interest statement The authors have no conflict of interest related to this work.
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Acknowledgment The technical assistance of Yvonne Hader Rosel Engel and Sina Köppert is greatly appreciated. This work was in part supported by the German Research Foundation (BU 1141/7-1, NE1174/3-1 and NE1174/8-1). References Abdollahi-Roodsaz, S., et al., 2007. Inhibition of Toll-like receptor 4 breaks the inflammatory loop in autoimmune destructive arthritis. Arthritis and Rheumatism 56, 2957–2967. Albanesi, C., et al., 2009. Chemerin expression marks early psoriatic skin lesions and correlates with plasmacytoid dendritic cell recruitment. The Journal of Experimental Medicine 206, 249–258. Bauer, S., et al., 2010. Low-abundant adiponectin receptors in visceral adipose tissue of humans and rats are further reduced in diabetic animals. Archives of Medical Research 41, 75–82. Bauer, S., et al., 2011. Sterol regulatory element-binding protein 2 (SREBP2) activation after excess triglyceride storage induces chemerin in hypertrophic adipocytes. Endocrinology 152, 26–35. Berg, V., et al., 2010. Human articular chondrocytes express ChemR23 and chemerin; ChemR23 promotes inflammatory signalling upon binding the ligand chemerin (21–157). Arthritis Research & Therapy 12, R228. Bozaoglu, K., et al., 2010. Chemerin, a novel adipokine in the regulation of angiogenesis. Journal of Clinical Endocrinology and Metabolism 95, 2476–2485. Carr, M.W., et al., 1994. Monocyte chemoattractant protein 1 acts as a T-lymphocyte chemoattractant. Proceedings of the National Academy of Sciences of the United States of America 91, 3652–3656. Cawthorn, W.P., Sethi, J.K., 2008. TNF-alpha and adipocyte biology. FEBS Letters 582, 117–131. Conde, J., et al., 2011. Expanding the adipokine network in cartilage: identification and regulation of novel factors in human and murine chondrocytes. Annals of the Rheumatic Diseases 70, 551–559. Ernst, M.C., Sinal, C.J., 2010. Chemerin: at the crossroads of inflammation and obesity. Trends in Endocrinology and Metabolism 21, 660–667. Ezawa, K., et al., 1997. Comparative analysis of CD45RA- and CD45RO-positive CD4+T cells in peripheral blood, synovial fluid, and synovial tissue in patients with rheumatoid arthritis and osteoarthritis. Acta Medica Okayama 51, 25–31. Ferraccioli, G., Gremese, E., 2011. Adiposity, joint and systemic inflammation: the additional risk of having a metabolic syndrome in rheumatoid arthritis. Swiss Medical Weekly 141, w13211. Frommer, K.W., et al., 2010. Adiponectin-mediated changes in effector cells involved in the pathophysiology of rheumatoid arthritis. Arthritis and Rheumatism 62, 2886–2899. Goralski, K.B., et al., 2007. Chemerin, a novel adipokine that regulates adipogenesis and adipocyte metabolism. Journal of Biological Chemistry 282, 28175–28188. Guillabert, A., et al., 2008. Role of neutrophil proteinase 3 and mast cell chymase in chemerin proteolytic regulation. Journal of Leukocyte Biology 84, 1530–1538. Hong, K.H., et al., 2005. Monocyte chemoattractant protein-1-induced angiogenesis is mediated by vascular endothelial growth factor-A. Blood 105, 1405–1407. Huss, R.S., et al., 2010. Synovial tissue-infiltrating natural killer cells in osteoarthritis and periprosthetic inflammation. Arthritis and Rheumatism 62, 3799–3805. Ibrahim, S.M., et al., 2008. Plasma and synovial fluid adipocytokines in patients with rheumatoid arthritis and osteoarthritis. The Egyptian Journal of Immunology 15, 159–170. Kaneko, K., et al., 2011. Chemerin activates fibroblast-like synoviocytes in patients with rheumatoid arthritis. Arthritis Research & Therapy 13, R158 [Epub ahead of print]. Karouzakis, E., et al., 2006. Molecular and cellular basis of rheumatoid joint destruction. Immunology Letters 106, 8–13. Kaur, J., et al., 2010. Identification of chemerin receptor (ChemR23) in human endothelial cells: chemerin-induced endothelial angiogenesis. Biochemical and Biophysical Research Communications 391, 1762–1768. Kralisch, S., et al., 2009. Interleukin-1beta induces the novel adipokine chemerin in adipocytes in vitro. Regulatory Peptides 154, 102–106. Lehrke, M., et al., 2009. Chemerin is associated with markers of inflammation and components of the metabolic syndrome but does not predict coronary atherosclerosis. European Journal of Endocrinology 161, 339–344. Luo, X., et al., 2008. Extracellular heat shock protein 70 inhibits tumour necrosis factoralpha induced proinflammatory mediator production in fibroblast-like synoviocytes. Arthritis Research & Therapy 10, R41. Meder, W., et al., 2003. Characterization of human circulating TIG2 as a ligand for the orphan receptor ChemR23. FEBS Letters 555, 495–499. Mohr, W., et al., 1975. Proliferation of synovial lining cells and fibroblasts. Annals of the Rheumatic Diseases 34, 219–224. Neumann, E., et al., 2002a. Inhibition of cartilage destruction by double gene transfer of IL-1Ra and IL-10 involves the activin pathway. Gene Therapy 9, 1508–1519. Neumann, E., et al., 2002b. Identification of differentially expressed genes in rheumatoid arthritis by a combination of complementary DNA array and RNA arbitrarily primed-polymerase chain reaction. Arthritis and Rheumatism 46, 52–63. Neumann, E., et al., 2010. Rheumatoid arthritis progression mediated by activated synovial fibroblasts. Trends in Molecular Medicine 16, 458–468.
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K. Eisinger et al. / Experimental and Molecular Pathology 92 (2012) 90–96
Neumeier, M., et al., 2011. Adiponectin stimulates release of CCL2, -3, -4 and − 5 while the surface abundance of CCR2 and − 5 is simultaneously reduced in primary human monocytes. Cytokine. Ospelt, C., et al., 2008. Overexpression of toll-like receptors 3 and 4 in synovial tissue from patients with early rheumatoid arthritis: toll-like receptor expression in early and longstanding arthritis. Arthritis and Rheumatism 58, 3684–3692. Parlee, S.D., et al., 2010. Serum chemerin levels vary with time of day and are modified by obesity and tumor necrosis factor-{alpha}. Endocrinology 151, 2590–2602. Schaffler, A., et al., 2003. Adipocytokines in synovial fluid. JAMA: The Journal of the American Medical Association 290, 1709–1710. Sell, H., et al., 2009. Chemerin is a novel adipocyte-derived factor inducing insulin resistance in primary human skeletal muscle cells. Diabetes 58, 2731–2740. Sell, H., et al., 2010. Chemerin correlates with markers for fatty liver in morbidly obese patients and strongly decreases after weight loss induced by bariatric surgery. Journal of Clinical Endocrinology and Metabolism 95, 2892–2896. Sellam, J., Berenbaum, F., 2010. The role of synovitis in pathophysiology and clinical symptoms of osteoarthritis. Nature Reviews. Rheumatology 6, 625–635.
Senolt, L., et al., 2010. Vaspin and omentin: new adipokines differentially regulated at the site of inflammation in rheumatoid arthritis. Annals of the Rheumatic Diseases 69, 1410–1411. van den Berg, W.B., et al., 2007. Amplifying elements of arthritis and joint destruction. Annals of the Rheumatic Diseases 66 (Suppl. 3), iii45–iii48. Weigert, J., et al., 2010a. Systemic chemerin is related to inflammation rather than obesity in type 2 diabetes. Clinical Endocrinology 72, 342–348. Weigert, J., et al., 2010b. Circulating levels of chemerin and adiponectin are higher in ulcerative colitis and chemerin is elevated in Crohn's disease. Inflammatory Bowel Diseases 16, 630–637. Wittamer, V., et al., 2003. Specific recruitment of antigen-presenting cells by chemerin, a novel processed ligand from human inflammatory fluids. The Journal of Experimental Medicine 198, 977–985. Wittamer, V., et al., 2005. Neutrophil-mediated maturation of chemerin: a link between innate and adaptive immunity. Journal of Immunology 175, 487–493. Yoshimura, T., Oppenheim, J.J., 2008. Chemerin reveals its chimeric nature. The Journal of Experimental Medicine 205, 2187–2190.