© 2013 Verlag der Zeitschrift für Naturforschung, Tübingen · http://znaturforsch.com
Introduction
The pholyphenolic phytoalexin resveratrol (3,4´,5-trihydroxystilbene) is abundant in grape skins (with higher concentration in red as com- pared to white wine), peanuts, the roots of the weed Polygonum cuspidatum as well as in other fruits and vegetables (Cardile et al., 2007; Csaki et al., 2008). It is produced by plants in response to stress and has a wide range of pharmacologi- cal activities. Resveratrol possesses several phar- macological activities, such as anti-infl ammatory (Csaki et al., 2008; Bereswill et al., 2010), antipro- liferative (Cardile et al., 2007; Gao et al., 2001,
Glehr et al. 2013), pro-apoptotic (Byun et al., 2008; Glehr et al., 2013), antioxidant (Bhat et al., 2001), and cardioprotective (Lakota et al., 2009), with no obvious toxicity (Jang et al., 1997; Bhat et al., 2001; Baur and Sinclair, 2006).
Rheumatoid arthritis (RA) is a chronic infl am- matory autoimmune disease characterized by synovial proliferation leading to cartilage and bone destruction (Lee et al., 2006). Contributing to the local production of cytokines, fi broblast- like synoviocytes (FLS) play a pivotal role in the pathogenesis of RA (Sipe et al., 1994; Burger et al., 2006). Cytokines such as interleukin-1β (IL-
Metalloproteinases and Receptor Activator of NF-κB Ligand in Rheumatoid Arthritis Fibroblast-Like Synoviocytes
Mathias Glehra,*, Margherita Breisacha, Sonja Walzerb, Birgit Lohbergera, Florentine Fürstc, Joerg Friesenbichlera, Beate Rinnerd, Alexander Aviane, Reinhard Windhagerb, and Andreas Leithnera
a Department of Orthopaedic Surgery, Medical University of Graz,
Auenbruggerplatz 5, A-8036 Graz, Austria. E-mail: mathias.glehr@medunigraz.at
b Department of Orthopaedic Surgery, Medical University of Vienna, Währinger Gürtel 18 – 20, A-1090 Wien, Austria
c Department of Rheumatology, Medical University of Graz, Auenbruggerplatz 15, A-8036 Graz, Austria
d Center of Medical Research, Medical University of Graz, Stiftingtalstraße 24, A-8036 Graz, Austria
e Institute for Medical Informatics, Statistics and Documentation, Medical University of Graz, Auenbruggerplatz 2, A-8036 Graz, Austria
* Author for correspondence and reprint requests
Z. Naturforsch. 68 c, 336 – 342 (2013); received July 30, 2012/May 21, 2013
Medication of rheumatoid arthritis (RA) remains challenging and often controversial con- cerning side effects or long-term complications. We investigated the effect of resveratrol, a phytoalexin discussed for its chondro-protective and anti-infl ammatory qualities, on the synovial expression of matrix-degrading enzymes like matrix metalloproteinases (MMPs) and bone-remodelling proteins in RA fi broblast-like synoviocytes (FLS). Interleukin-1β- stimulated RA-FLS were treated with 100 μM resveratrol for 24 h. To evaluate the effect of resveratrol on the amount of bound/combined MMPs, a Luminex® xMAP multiplexing technology was used. The alteration in expression of receptor activator of nuclear factor- κB ligand (RANKL) and osteoprotegrin (OPG) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Resveratrol reduced the expression of MMP-1 (p = 0.022), MMP-3 (p = 0.021), and MMP-9 (p = 0.047). qRT-PCR showed a signifi cant reduction in the relative abundance of the transcripts of OPG (p = 0.012) and RANKL (p = 0.018).
Our in vitro fi ndings indicate that resveratrol could be a new target for further pharmaco- logical studies in the fi eld of RA. In the future it could play a role as a possible substitute or supplement to currently used drugs against RA to prevent cartilage matrix degradation and pathological bone resorption due to inhibition of MMPs and RANKL.
Key words: Resveratrol, Rheumatoid Arthritis, Matrix Metalloproteinases
1β) up-regulate matrix-degrading enzymes like matrix metalloproteinases (MMPs) leading to cartilage matrix destruction and joint infl amma- tion (Burrage et al., 2006). RA-FLS also release large amounts of receptor activator of nuclear factor-κB ligand (RANKL), which mediates the differentiation of bone-resorbing osteoclasts from their macrophage precursors (Susa et al., 2004).
Osteoclasts are highly specialized cells particular- ly involved in cartilage degradation and bone re- modelling throughout life (Teitelbaum, 2000). Os- teoprotegrin (OPG) functions as a decoy receptor by blocking the interaction between RANKL and its cognate receptor RANK. The RANK/RANKL system is necessary for osteoclast development and function (Yasuda et al., 1998).
It has been demonstrated that intra-articular injection of resveratrol reduces the severity of cartilage lesions and synovial infl ammation in an experimental infl ammatory arthritis rabbit model (Elmali et al., 2005). Wang et al. (2012) further- more observed the effect of resveratrol on carti- lage and chondrocytes apoptosis in experimental osteoarthritis (OA) in rabbits. Histological evalu- ation of cartilage tissue revealed signifi cantly re- duced cartilage destruction. Resveratrol reduced the apoptosis rate of chondrocytes and the pro- duction of NO. The protective effects of resvera- trol were enhanced as a function of its concentra- tion up to 50 μmol/kg (Wang et al., 2012).
The present study was designed to investigate the effect of resveratrol on the expression of MMPs, OPG, and RANKL in RA-FLS.
Material and Methods
Harvest of the synovial tissue and expansion of RA-FLS
RA-FLS were isolated from synovium of ten RA patients (average age, 64.5 years; range, 52 – 77 years; 3 males and 7 females) undergoing synovectomy or joint replacement. Individual participants in this study provided written in- formed consent. The study protocol was approved by the local ethics committee (number of ethics proposal 19-111 ex 07/08; Ethical Committee of the Medical University of Graz, Graz, Austria).
Synovial membrane tissue was cut into 1-mm slices, rinsed several times with phosphate-buff- ered saline (PBS), and then digested with 0.2%
collagenase B (Roche Diagnostics, Mannheim, Germany) in high glucose Dulbecco’s-modifi ed
Eagle’s medium (DMEM-HG; GIBCO® Invit- rogen, Darmstadt, Germany) containing 10%
foetal bovine serum (FBS; GIBCO® Invitrogen), 1% L-glutamine (GIBCO® Invitrogen), 100 units/
ml penicillin (GIBCO® Invitrogen), 100 μg/ml streptomycin (GIBCO® Invitrogen), and 0.25 μg amphotericin B (PAA Laboratory, Pasching, Aus- tria). After overnight incubation at 37 °C, the cell suspension was fi ltered with a nylon membrane (Cell Strainer 40 μm; BD Biosciences, Franklin Lakes, NJ, USA) and plated in T75 culture fl asks (TPP, Trasadingen, Switzerland). Cells were cul- tured in growth medium at 37 °C in a humidifi ed atmosphere with 5% CO2 for expansion. Each bi- ological sample was cultured separately, and the biological replicates were obtained from differ- ent cultures and different patients. The medium was replaced every third day, and cells were pas- saged after reaching 70 – 90% confl uence. Cells were cultured from three to a maximum of fi ve passages in order to establish homogeneity.
Characterization of FLS using fl uorescence- activated cell sorting (FACS) analysis
1 · 106 cells were counterstained with 1 μg/ml of propidium iodide (PI; Molecular Probes, Invitro- gen, Vienna, Austria), and nonviable cells were ex- cluded from living cells. Commercial monoclonal antibodies, CD44PE, CD14FITC, CD90APC, and CD68PE (BD Biosciences, San Jose, CA, USA), were applied for characterization of FLS, and each experiment contained isotype-matched con- trol antibodies. The multicolour fl ow cytometric analysis was performed on a BD LSR II system (BD Biosciences). Data were acquired using BD FACS DivaTMsoftware (BD Biosciences). The daily consistency of measurements was checked with a cytometer set-up and tacking beads (BD Biosciences). To exclude debris, a forward scatter (FSC) versus side scatter (SSC) gate was used and analysed on a linear scale. FLS were defi ned by their phenotype (CD90+, CD44+, CD68–, CD14–) and analysed on a logarithmic scale (according to Rosengren et al., 2007).
Human MMP Fluorokine® MAP
The cell culture supernatant was fi ve-fold di- luted with PBS. All subsequent steps were carried out in the same way. One hundred μl of calibra- tor diluent RD%-37 were added and mixed thor- oughly. RA-FLS from ten RA patients (n = 10)
were pre-treated in 6-well plates with the MMP- inducing agent IL-1β at 10 ng/ml for 2 h in 2 ml of culture medium and then co-stimulated with 100 μM resveratrol (R5010; Sigma-Aldrich, Vien- na, Austria) for 24 h [in accordance to the dos- ages used by Byunet al. (2008)]. The samples for the test with the human MMP base kit were pre- pared according to the manufacturer’s instruction (R & D Sytems, Vienna, Austria). The micropar- ticles were detected using the Luminex® analyzer (BioPlex TM dual laser; BioRad, Munich, Germa- ny) within 90 min. One laser classifi ed micropar- ticles and determined the detected MMPs which were detected, and the second laser determined the magnitude of the signal derived from the streptavidin conjugated to phycoerythrin (PE), which was in direct proportion to the amount of bound MMPs.
qRT-PCR
Quantitative real-time polymerase chain re- action (qRT-PCR) was performed to determine the relative expression of TNFSF11 [tumour necrosis factor (ligand) superfamily, member 11; RANKL] and TNFRSF11B (tumour necro- sis factor receptor superfamily, member 11b;
OPG). FLS from six RA-patients (n = 6) were pre-treated in 6-well plates with 10 ng/ml IL-1β for 2 h and co-stimulated with 100 μM resveratrol for 24 h. Total RNA was obtained from resvera- trol-treated RA-FLS and control cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. To- tal RNA was reverse transcribed using the high- capacity RNA-to-cDNA Master Mix (Applied
Biosystems, Carlsbad, CA, USA). TaqMan® gene expression assays with pre-designed, validated, gene-specifi c primers and probes help to perform quantitative gene expression in the 7900HT re- al-time PCR system (Applied Biosystems). The data were normalized to the expression of the housekeeping gene GAPDH (glyceraldehyde- 3-phosphate dehydrogenase).
Statistical analysis
All values are presented as median and fi rst and third quartiles (Q1, Q3). The signifi cance of changes was determined using the Wilcoxon signed-rank test. We considered p < 0.05 as sig- nifi cant. Data were analysed using PASW 18 soft- ware (SPSS Inc., Chicago, IL, USA). The average outcome of the obtained data was recorded.
Results
Phenotypical characteristics
Fluorescence-activated cell sorting (FACS) analysis was performed for surface antigen ex- pression of FLS by their phenotype. For each experiment, 10,000 events were analysed, and (93 ± 6.2)% viable cells were gated on forward scatter (FSC) versus side scatter (SSC). Fig. 1 shows representative examples of three inde- pendent FACS experiments. In the analysis, the negative staining for CD68 [(0.45 ± 0.49)%;
Fig. 1A] and CD14 [(0.1 ± 0.14)%; Fig. 1C], which are both macrophage markers (Edwards, 2000), verifi ed the absence of synovial macrophages, while positive staining for CD44 [(99.7 ± 0.14)%;
Fig. 1B] and CD90 [(90.2 ± 6.73)%; Fig. 1A] veri- fi ed the presence of FLS in our in vitro culture.
Fig. 1. Characterization of FLS by multicolour fl ow cytometric analysis: (A) positive CD90 and negative CD68 staining; (B) positive CD44 staining; (C) cells were identifi ed by negative CD14 staining. Each experiment con- tained isotype-matched antibodies (Glehr et al., 2013).
CD90 is specifi cally expressed on FLS in the synovial membrane (Palmer et al., 1985; Zimmer- mann et al., 2001), and CD44 is highly expressed on both cultured FLS and synovial macrophages (Johnson et al., 1993). Since macrophages were excluded, it can be assumed that FLS were cul- tured.
Resveratrol decreases different MMP expression levels in RA-FLS
Resveratrol reduced the expression of IL- 1β-induced MMP-1, MMP-3, and MMP-9 pro- duction (Fig. 2) in the cell culture supernatant.
MMP-2, -8, -12, and -13 expression was low and was not reduced further. Median fl uorescence intensity (MFI) of MMP-1 decreased by 30.9%
(Fig. 2A) after the treatment with 100 μM res- veratrol for 24 h (p = 0.013). Fluorescence in- tensities of MMP-3 and MMP-9 decreased by 9.9% (Fig. 2B; p = 0.021) and 28.6% (Fig. 2C;
p = 0.028), respectively. Table I offers detailed information.
qRT-PCR of RANKL and OPG expression
To investigate the effect of resveratrol on TNFSF11 (RANKL) and TNFRSF11B (OPG) expression, we conducted a qRT-PCR. Stimula- tion with IL-1β resulted in a signifi cant increase of RANKL and OPG expression in RA-FLS (data not shown).
Expression of the RANKL gene (Fig. 3A) was reduced by 91% after co-treatment with resvera- trol (p = 0.018), while under these conditions ex- pression of the OPG gene (Fig. 3B) decreased by 82% with a statistical signifi cance of p = 0.012.
Discussion
Chronic exposure to infl ammatory cytokines and growth factors are likely key factors mediat- ing the transformation of mesenchymal cells or their progenitors into stably activated RA-FLS (Keffer et al., 1991).
MMPs are primarily produced in RA-FLS and are highly elevated in the serum of patients with early RA (Fiedorczyk et al., 2006). They partici-
Table I. Additional data of the effect of resveratrol on expression levels of different MMPs.
Group NK IL IL+RES p Value
n Q1 Median Q3 Q1 Median Q3
MMP-1 10 3846.9 9521.6 27822.9 761.5 3719.0 17036.9 0.013*
MMP-3 9 8955.5 8955.5 26368.6 2895.3 13636.0 23617.8 0.021*
MMP-9 10 118.3 268.5 987.3 56.4 184.2 428.1 0.028*
NK, control group, unstimulated cells. IL, cells stimulated with interleukin-1β to increase the MMP expression level. IL+RES, on an interleukin-1 β stimulation followed a co-treatment with resveratrol. Median, the average of the middle two in a set with an even number of values. Q1, Q3, the two ends of the rectangles represent the fi rst quartile (Q1) and the third quartile (Q3).
Fig. 2. Boxplots of the effect of resveratrol on expression levels of different MMP levels (n = 10), i. e. the median of the fl uorescence intensity after the treatment with resveratrol. (A) MMP-1 (p = 0.013); (B) MMP-3 (p = 0.021);
(C) MMP-9 (p = 0.028). In part C an MMP-9 outliner is not shown, because of its high magnitude (IL, 11300;
IL+Res, 11374).
pate in irreversible proteolytic degradation and remodelling of the extracellular matrix. Accord- ing to their substrate specifi city, primary structure, and cellular localization, MMPs can be classifi ed into fi ve main groups: collagenases, gelatinases, stromelysins, matrilysins, and membrane-bound MT-MMPs (Murphy et al., 2002).MMP-1 (colla- genase) and MMP-3 (stromelysin 1) are the ma- jor enzymes involved in tissue destruction (Cur- ran and Murray, 1999). High levels are detected in synovial fl uids of RA patients (Yoshihara et al., 2000).Moreover, MMP-1 preferentially degrades fi brillar collagens, whereas MMP-3 degrades a broad array of extracellular matrix substrates (Curran and Murray, 1999).MMP-9 (gelatinase B) is markedly elevated in the sera and joints of RA patients, and correlates positively with disease progression and severity (Gruber et al., 1996).MMP-9 cleaves denaturated collagen (gel- atins) and type IV collagen, which is the major component of basement membranes (Ram et al., 2006).
MMP inhibition through the action of various components has been described in the literature, e.g. ajulemic acid – a synthetic nonpsychoactive cannabinoid acid – inhibited the release of MMP- 1, MMP-3, and MMP-9 from TNF-α-stimulated human RA-FLS in vitro (Johnson et al., 2007).
Also the fl avonoid nobiletin down-regulated the production of pro-MMP-1 and -3 in human FLS, while on the other hand it up-regulated the ex- pression of tissue inhibitor of matrix metallopro- teinase-1 (TIMP-1) (Lin et al., 2003).
The results of our MMP multiplex study showed that resveratrol has a tendency to suppress the IL-1β-induced production of MMP-1, -3, and -9 by RA-FLS. This signifi es a cartilage-protective
effect of resveratrol. Also Elmali et al. reported in 2005 that resveratrol reduced the severity of cartilage lesions in experimentally induced osteo- arthritis in rabbits.
This study also investigated the effect of res- veratrol on RANKL and its decoy receptor OPG.
RANKL is a key molecule in the pathogenesis of the osteolytic process in RA (Gravallese et al., 2000).OPG is the natural negative regulator of RANKL, which competes with RANKL for binding to RANK as a decoy receptor. The rela- tive levels of RANKL and OPG are possibly key factors in the determination of the rate of osteo- clast formation in vitro (Bucay et al., 1998). Our qRT-PCR analysis revealed that resveratrol sup- presses the IL-1β-induced expression of RANKL and OPG expression.
It has previously been demonstrated that res- veratrol analogues inhibit the differentiation and bone-resorbing activity of osteoclasts and pro- mote the formation of osteoblasts from mesen- chymal precursors in cultures (Mizutani et al., 1998; Boissy et al., 2005; Kupisiewicz et al., 2010).
Resveratrol also enhanced the proliferation and osteoblastic differentiation in human mesenchy- mal stem cells via ER-dependent ERK1/2 activa- tion (Dai et al., 2007).
Our in vitro study showed resveratrol affects human RA-FLS by reducing MMPs, RANKL, and infl ammatory cytokines. This could provide a further explanation for the in vivo fi ndings of Elmali et al. (2005) and Wang et al. (2012) which established the cartilage protective potency of resveratrol in OA. This study could be the basis for further examinations of the infl uence of res- veratrol on OA- and RA-FLS.
Fig. 3. Effect of resveratrol on gene expression of IL-1β-stimulated RA-FLS (n = 6). (A) Signifi cant decrease of RANKL gene expression after a 24-h treatment with resveratrol (p = 0.018); (B) OPG gene expression also de- clined after a 24-h treatment with resveratrol (p = 0.012).
Conclusion
Our results indicate that resveratrol may pro- vide a new therapeutic target for further stud- ies and an alternative approach to conventional drugs against RA by virtue of its inhibition of the overproduction of MMPs and RANKL which cause chondrocyte degeneration and pathological bone resorption in RA.
Acknowledgements
This work was supported by the Association for Orthopaedic Research (AFOR) scientifi c
fund and by the Jubilee Fund of the Austrian National Bank (OENB 12953). The funding agencies had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors would like to thank Heike Kaltenegger and Bettina Turner for excellent technical assistance with cell cultures, Alexandra Novak for perfect technical assistance with the fl ow cytometric analysis, and Wolfgang Karl for perfect techni- cal assistance in qRT-PCR. The authors would also like to thank Jenny Chapman for language editing.
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