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Proc Natl Acad Sci U S A. Sep 23, 2008; 105(38): 14692–14697.
Published online Sep 16, 2008. doi:  10.1073/pnas.0802675105
PMCID: PMC2567200
Pharmacology

Epigallocatechin-3-gallate inhibits IL-6 synthesis and suppresses transsignaling by enhancing soluble gp130 production

Abstract

Regulation of IL-6 transsignaling by the administration of soluble gp130 (sgp130) receptor to capture the IL-6/soluble IL-6R complex has shown promise for the treatment of rheumatoid arthritis (RA). However, enhancing endogenous sgp130 via alternative splicing of the gp130 gene has not yet been tested. We found that epigallocatechin-3-gallate (EGCG), an anti-inflammatory compound found in green tea, inhibits IL-1β–induced IL-6 production and transsignaling in RA synovial fibroblasts by inducing alternative splicing of gp130 mRNA, resulting in enhanced sgp130 production. Results from in vivo studies using a rat adjuvant-induced arthritis model showed specific inhibition of IL-6 levels in the serum and joints of EGCG-treated rats by 28% and 40%, respectively, with concomitant amelioration of rat adjuvant-induced arthritis. We also observed a marked decrease in membrane-bound gp130 protein expression in the joint homogenates of the EGCG-treated group. In contrast, quantitative RT-PCR showed that the gp130/IL-6Rα mRNA ratio increased by ~2-fold, suggesting a possible mechanism of sgp130 activation by EGCG. Gelatin zymography results showed EGCG inhibits IL-6/soluble IL-6R–induced matrix metalloproteinase-2 activity in RA synovial fibroblasts and in joint homogenates, possibly via up-regulation of sgp130 synthesis. The results of these studies provide previously undescribed evidence of IL-6 synthesis and transsignaling inhibition by EGCG with a unique mechanism of sgp130 up-regulation, and thus hold promise as a potential therapeutic agent for RA.

Keywords: rheumatoid arthritis, alternative splicing, cytokine therapy, pharmacology, therapeutics

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by persistent synovial inflammation and progressive destruction of cartilage and underlying bone (1). Recent progress in cytokine biology has provided a considerable amount of evidence for the pathological role of IL-6 in RA (2). IL-6 is a pleiotropic cytokine with a wide range of biological activities, including immunoregulation, mediation of acute-phase responses, and effects on bone metabolism (3). Animal studies have shown IL-6 deficiency resulted in DBA1/J mice becoming resistant to collagen-induced arthritis, and this deficiency suppressed the development of arthritis in SKG mice (46). Despite its important physiological roles, dysregulated overproduction of IL-6 is responsible for many systemic inflammatory manifestations observed in patients with RA (7). The elevated levels of IL-6 in serum and synovial fluid of RA patients have been shown to correlate with clinical and laboratory indices of disease activity (8). In humans, Tocilizumab, a humanized monoclonal antibody against the IL-6 receptor α (IL-6Rα) was effective in RA clinical trials (9).

Conventionally, IL-6 transmits signals by binding to its IL-6Rα on the cell surface and then associates with membrane-bound gp130 (mgp130), a signal-transducing membrane protein, resulting in the dimerization of gp130 and consequent signal transduction (10). However, the expression of the transmembrane form of IL-6R is confined mainly to hepatocytes, monocytes/macrophages, and some lymphocytes (11). In contrast, IL-6 transsignaling utilizes a soluble form of IL-6R (sIL-6R) to provide an alternative mechanism of gp130 activation. With mgp130 ubiquitously expressed and sIL-6R found in abundance in most body fluids, including RA serum and synovial fluids, the IL-6/sIL-6R complex can bind directly to cellular mgp130 to activate cells that would not intrinsically respond to IL-6 itself (11). Interestingly, the soluble form of gp130 (sgp130) acts as a natural inhibitor of IL-6 signaling (12). The complex of (IL-6/sIL-6R)/sgp130 also inhibits IL-6 activity and limits systemic responses to IL-6, suggesting its importance in regulating IL-6 signaling and as a potential therapeutic agent in RA (10, 13).

Despite sufficient evidence for the therapeutic efficacy of sgp130 in animal models of RA and its antagonistic role in blocking IL-6 transsignaling (14), the mechanisms and consequences of its alternative splicing that generates this soluble form have not yet been tested for potential therapeutic implications. With this aim, we tested the efficacy of epigallocatechin-3-gallate (EGCG), a potent anti-inflammatory molecule found in green tea, on IL-6 activity and transsignaling. EGCG has recently attracted attention for its emerging antiarthritic and immune-modulating properties (1517). Our previous studies on EGCG defined the mechanisms of its inhibitory potential against the mediators of inflammation and cartilage degradation in human chondrocytes (16, 18, 19). Expanding our understanding of this molecule in RA, we recently found that EGCG pretreatment blocks chemokine production and matrix metalloproteinase-2 (MMP-2) activity in RA synovial fibroblasts (15). In the present study, we tested the efficacy of EGCG in inhibiting IL-6 synthesis and transsignaling in human RA synovial fibroblasts and rat adjuvant-induced arthritis (AIA).

Results

EGCG Inhibits IL-1β–Induced RA Synovial Fibroblast IL-6 Production.

IL-1β (10 ng/ml) stimulation resulted in a 160-fold induction in IL-6 production (Fig. 1A; P < 0.05). Pretreatment with EGCG (10 and 20 μM) resulted in inhibition of IL-1β–induced IL-6 production by 16% and 49%, respectively, when compared with IL-1β–treated samples (P < 0.05 at 20 μM). IL-1β stimulation of RA synovial fibroblasts resulted in a more than 873-fold induction in IL-6 mRNA levels (Fig. 1B; P < 0.05). EGCG pretreatment at 10 and 20 μM inhibited IL-1β–induced IL-6 mRNA expression by 60% and 67%, respectively, and when given alone, it had no modulating effect on IL-6 mRNA expression in RA synovial fibroblasts.

Fig. 1.
Inhibition of RA synovial fibroblast IL-6 production by EGCG. RA synovial fibroblasts were pretreated with EGCG (10–20 μM) for 12 h, followed by stimulation with IL-1β (10 ng/ml) for 24 h. (A) Production of IL-6 was measured in ...

The results from the inhibitor study showed that the PKCδ, NF-κB, and p38 inhibitors significantly inhibited IL-1β–induced IL-6 production [supporting information (SI) Fig. S1A; P < 0.05]. We have shown earlier that EGCG inhibits IL-1β–induced NF-κBp65 nuclear translocation and PKCδ activation (15). In the present study, pretreatment with EGCG inhibited IL-1β–induced phosphorylation of JNK and p38 MAPK, resulting in the reduced accumulation of p-c-Jun and p-ATF-2 (Fig. S1B).

EGCG Up-Regulates sgp130 Synthesis in RA Synovial Fibroblasts.

In the present study, IL-1β significantly increased the expression of mgp130 by ~2.2-fold compared with untreated samples (Fig. 2A; P < 0.05). Pretreatment of RA synovial fibroblasts with EGCG (10 and 20 μM) caused a marked inhibition of mgp130 expression by ~47% and ~74%, respectively (Fig. 2A; P < 0.05). EGCG alone at these concentrations downregulated the expression of constitutive mgp130 in RA synovial fibroblasts. Western blot analysis on the concentrated conditioned medium from the samples showed a marked up-regulation of sgp130 production by EGCG treatment (Fig. 2B; P < 0.05). To test the specificity of sgp130 up-regulation by EGCG, we examined the effect of EGCG on sIL-6R production using human immortalized osteoblast-like cells (MG-63), because RA synovial fibroblasts lack the IL-6R. MG-63 cells express IL-6R and shed sIL-6R (i.e., production of sIL-6R) when stimulated. The results showed that EGCG is incapable of inducing sIL-6R production in MG-63 cells at the similar concentrations (Fig. S2). Amplification of gp130 mRNA using full-length mgp130, alternatively spliced sgp130, and gp130-RAPS mRNA transcripts was performed by qualitative RT-PCR (qRT-PCR) (20, 21). The results showed that EGCG inhibited gp130 mRNA encoding full-length gp130 but enhanced sgp130 and gp130-RAPS mRNA when used alone or in the presence of IL-1β (Fig. 2C), suggesting its ability to activate the desired pretranscriptional modifications causing enhanced sgp130 synthesis.

Fig. 2.
Differential effect of EGCG on IL-1β–induced mgp130 and sgp130 expression. (A) RA synovial fibroblasts were incubated with EGCG (10 or 20 μM) for 12 h, followed by stimulation with IL-1β (10 ng/ml) for 24 h. The amount ...

EGCG Preferentially Inhibits IL-6 in Vivo.

We used a rat AIA model in which IL-6 production peaks in serum around day 16 and coincides with maximum inflammation and joint destruction (22). To study in vivo modulation of IL-6 by EGCG in rats, a daily i.p. injection of EGCG (100 mg/kg) based on an earlier study, which corresponds to 960 mg of human equivalent dose, was administered starting from day 7 to day 16 (23, 24). Interestingly, the evaluation of serum IL-6, IL-1β, and TNF-α levels at day 17 showed that EGCG treatment significantly inhibited IL-6 levels by almost 28% in serum with no change in the levels of IL-1β and TNF-α (Fig. 3A; P < 0.05). When compared with the PBS group, EGCG administration resulted in a marked decrease in IL-6 (39% decrease) and IL-1β (42% decrease) levels but not in TNF-α levels in the joint homogenates (Fig. 3B).

Fig. 3.
EGCG downregulates IL-6 in vivo and ameliorates AIA in rats. (A and B) The levels of IL-6, TNF-α, and IL-1β in serum and joint homogenates of PBS- or EGCG (100 mg/kg, i.p. daily)-treated rats from day 17 were determined using a commercially ...

With the inhibition of IL-6 attributable to EGCG administration, we also observed a significant reduction in the clinical measurements of joint inflammation and arthritis (Fig. 3 C and D; P < 0.05; also see Figs. S3 A–E and S4). The articular index and the Δ circumference of the ankles of EGCG-treated animals were significantly lower than those of the PBS group (Fig. 3 C and D; P < 0.05). Overall, these results suggest that EGCG ameliorates rat AIA by preferentially downregulating both the circulating and joint concentrations of IL-6 and, in part, by reducing IL-1β levels in the joints. Histological analysis of the joints showed markedly reduced infiltrates, angiogenesis, and bone destruction in EGCG-treated rats as compared with the PBS group (Fig. S3 A–E).

IL-6 has been shown to induce VEGF production (25, 26). We also observed that IL-6/sIL-6R induced VEGF production by ~28% of the basal levels in RA synovial fibroblasts (Fig. S5). Preincubation with EGCG (2–20 μM) inhibited IL-6/sIL-6R–induced VEGF production in a dose-dependent manner (Fig. S5A). Evaluation of the signaling pathways showed that blockade of the JNK pathway reduced IL-6/sIL-6R–induced VEGF production by ~32% (Fig. S5B). However, in the rat AIA study, no reduction in serum and joint homogenate VEGF in EGCG-treated rats was observed when compared with the PBS group (Fig. S5C).

EGCG Inhibits mgp130 Expression but Enhances gp130 mRNA Expression in Vivo.

Western blot analysis showed that mgp130 expression, recognized as a dimer by the rabbit anti-gp130 polyclonal antibody, was markedly reduced in the joint homogenates from EGCG-treated rats when compared with the PBS group (Fig. 4A). To understand the effects of EGCG further, we determined the levels of gp130 and IL-6Rα mRNA in the joints of EGCG and PBS groups. The result of qRT-PCR showed that the gp130 mean mRNA level in the EGCG group increased by ~2.5-fold as compared with the PBS group (Fig. 4B). An inverse association between decreased mgp130 protein expression and increased gp130 mRNA expression suggests a plausible mechanism of sgp130 synthesis by EGCG in vivo. Interestingly, only a moderate increase in IL-6Rα mRNA expression was observed in the joints of EGCG-treated rats as compared with the PBS group (Fig. 4B). However, further comparative analysis showed that EGCG treatment caused almost a 1.8-fold increase in the gp130/IL-6Rα mRNA ratio (Fig. 4B, insert), suggesting a possible mechanism of capturing the IL-6/sIL-6R complex by triggering the synthesis of sgp130.

Fig. 4.
EGCG administration suppresses mgp130 expression but upregulates gp130 mRNA expression in rat AIA joints. (A) In joint homogenates, mgp130 expression was determined by Western blotting. The blots were probed with β-actin to ensure equal protein ...

sgp130 Prevents IL-6/sIL-6R–Induced MMP-2 Activity: A Possible Mechanism of Regulation by EGCG.

Synovial fibroblasts invade tissue and cause joint destruction in RA via MMP-2 activation, thus making MMP-2 a reasonable therapeutic target (15, 27). RA synovial fibroblasts constitutively possessed MMP-2 activity, which was further induced ~2-fold when stimulated by either IL-6 (100 ng/ml) or sIL-6R (100 ng/ml) at 24 h (Fig. 5A; P < 0.05). Interestingly, treatment of synovial fibroblasts with the IL-6/sIL-6R complex caused a significant additive increase in MMP-2 activity when compared with IL-6 stimulation (Fig. 5A; P < 0.05). EGCG at 20 μM inhibited IL-6– or IL-6/sIL-6R–induced MMP-2 activity by almost 70% and 78%, respectively (Fig. 5A; P < 0.001). EGCG at lower concentrations was not effective in inhibiting IL-6/sIL-6R–induced MMP-2 activity (data not shown). Interestingly, the same conditioned medium showed detectable sgp130 levels with EGCG treatment, suggesting a possible mechanism of MMP-2 inhibition by sgp130 up-regulation (Fig. 5A). Results from the study using the signaling inhibitors suggested that the blockade of NF-κB and PKCδ pathways suppressed IL-6/sIL-6R–induced MMP-2 activity in RA synovial fibroblasts (Fig. S6; P < 0.05).

Fig. 5.
Effect of EGCG, inhibitors, and recombinant human sgp130 on MMP-2 activity. (A) RA synovial fibroblasts (2 × 105 per well) were incubated with EGCG (20 μM) for 12 h, followed by stimulation with IL-6 (100 ng/ml), sIL-6R (100 ng/ml), or ...

When pretreated with human recombinant sgp130 (1 μg/ml) or anti-IL-6 antibody (2 μg/ml) for 30 min, followed by IL-6/sIL-6R stimulation for 24 h, RA synovial fibroblasts showed a moderate reduction in IL-6/sIL-6R–induced MMP-2 activity (Fig. 5B), which provides evidence of the mechanism of MMP-2 regulation by EGCG in RA synovial fibroblasts. Furthermore, MMP-2 activity in the joint homogenates from the EGCG-treated group showed ~50% decrease when compared with the activity in the PBS group (Fig. 5C; P < 0.05).

Discussion

This study provides evidence for the ability of EGCG to regulate IL-6 signaling in human RA synovial fibroblasts and rat AIA with the previously undescribed concept of endogenous sgp130 production. Importantly, our results also provide an argument for further testing of EGCG or structurally related molecules as inhibitors of IL-6 synthesis and transsignaling in RA and possibly other IL-6–regulated diseases.

In the present study, EGCG significantly blocked IL-6 synthesis in IL-1β–stimulated RA synovial fibroblasts. Extending our previous study, we provide evidence that EGCG also inhibits the JNK and p38 pathways found to be involved in mediating IL-1β–induced IL-6 production (15). Clinically, the serum and synovial fluid levels of IL-6 in RA patients correlate with the level of acute-phase proteins and disease severity (8, 28). Synovial fibroblast IL-6 production has been shown to inhibit bone formation and concomitantly stimulate bone resorption and pannus formation (8, 29, 30). In a recent study, EGCG inhibited IL-6–induced apoptosis in primary cultured mouse osteoblast-like cells, suggesting its bone-preserving activity in rodents (31). However, the present observation of EGCG-mediated IL-6 inhibition is an evaluation of EGCG's efficacy in cells isolated from RA synovial tissue and emphasizes its ability to reduce the circulating and joint concentrations of IL-6 available for sIL-6R binding.

This study shows that EGCG ameliorated rat AIA along with the specific inhibition of IL-6 levels in serum and joint homogenates. IL-1β levels decreased only in the joints, whereas TNF-α levels remained unchanged with EGCG administration. These results are supported by earlier studies in which IL-6 deficiency or IL-6R blockade ameliorated joint destruction and suggest that controlling IL-6 production may produce a beneficial outcome in RA (32, 33). A previous study by Haqqi et al. (34) showed that administration of green tea polyphenols prevented collagen-induced arthritis in mice. However, this study precisely provides insight into one of the potential mechanisms of EGCG's antirheumatic activity.

In classic signaling, IL-6 exerts its activity on target cells via binding to the cell surface IL-6R and initiates homodimerization of mgp130 to finally transduce its signaling (10). Of particular interest is the transsignaling mechanism, in which IL-6 activates cells that do not express IL-6R by forming a complex with sIL-6R and directly binding to mgp130 (10). Because of lack of the IL-6R, synovial fibroblasts use IL-6 transsignaling in orchestrating tissue invasion, inflammation, and apoptosis (2, 14). It is now well established that sgp130 functions as a natural inhibitor of IL-6 signaling by effectively capturing the IL-6/sIL-6R complex to inhibit IL-6 activity and systemic responses (10, 12). Thus, EGCG-mediated up-regulation of sgp130 production prompted us to study the mechanism of its transcriptional regulation.

Alternative splicing is widely recognized as a mechanism of controlling the expression, diversity, and functions of proteins (35). This is achieved by the production of multiple distinct mRNA transcripts from a given gene that have the potential to encode a unique protein, often with distinct or opposing function (36). Regardless of the pattern of alternative splicing, the production of a soluble form of a cytokine receptor is thought to be tightly regulated and can significantly modulate cytokine-signaling depending on its competition or synergy with the membrane-bound receptor for binding to the cognate cytokine (37). For instance, a recent study showed that an alternatively spliced variant of hyaluronidase, an enzyme involved in the propagation of bladder cancer, negatively regulated bladder tumor growth, invasion, and angiogenesis (38). To understand the mechanism of sgp130 synthesis by EGCG, we amplified sequences that encode mgp130 (full-length) and sgp130 molecules. The results showed that EGCG inhibited mgp130 mRNA expression and concomitantly upregulated sgp130 mRNA expression, a pattern clearly reflected in the synthesis of functionally distinct mgp130 and sgp130 proteins. These results suggest that EGCG might influence pre-mRNA splicing of gp130 toward formation of mRNA transcripts that synthesize sgp130 and warrants further testing. This notion is further supported by recent studies in which EGCG was found to inhibit the loss of survival motor neuron-1 protein and also to produce a full-length functional protein by correcting an aberrant alternative mRNA splicing in the cells from familial dysautonomia patients (39, 40). However, our study demonstrates that EGCG can induce alternative mRNA splicing to produce a truncated form (i.e., sgp130) with potent antagonistic activity.

RA synovial fibroblasts invade through the basal lamina into the vasculature and extravasate into surrounding tissues causing joint destruction, executed, in part, by MMP-2 (41, 42). However, the role of IL-6 and/or sIL-6R in inducing MMP-2 activity in RA synovial fibroblasts is not well studied. We found that the IL-6/sIL-6R complex markedly induces MMP-2 activity, which was inhibited by EGCG, at least in part, by enhancing sgp130 synthesis, resulting in the blockade of MMP-2 activity in RA synovial fibroblasts. The level of IL-6/sIL-6R–induced MMP-2 activity was comparable to that of IL-1β–induced MMP-2 activity observed in our previous study (15). This suggests that reduction in MMP-2 activity in the joints by EGCG might be an overall outcome of the suppression of circulating and joint IL-6 concentrations and possibly enhanced sgp130 synthesis.

In conclusion, we demonstrate that EGCG inhibits IL-6 synthesis and suppresses transsignaling through enhanced synthesis of sgp130. There are certain limitations to this study that must be recognized, including a valid method of measuring rat serum sgp130 levels and the availability of rat mRNA transcripts that encode sgp130. Nevertheless, the results of our study demonstrated EGCG's efficacy in inhibiting IL-6 synthesis and transsignaling in human RA synovial fibroblasts and in rat AIA. Although genetic manipulation to induce selective disruption or constitutive overexpression of gp130 has been shown to cause myocardial and hematological disorders in mice (43, 44), systemic up-regulation of sgp130 as achieved in the present study by EGCG administration might prove beneficial and warrants further testing for its efficacy in RA.

Materials and Methods

Antibodies and Reagents.

EGCG and rabbit anti-β-actin were purchased from Sigma-Aldrich. Recombinant human IL-1β, IL-6, sgp130, sIL-6R, IL-6 neutralizing antibody and human IL-6, and VEGF Duoset ELISA systems were purchased from R&D Systems. Rabbit polyclonal antibodies against human gp130 (Millipore) and rat gp130 (Santa Cruz Biotechnology) were used. MG-63 cells were a gift from R. Taichman (University of Michigan, Ann Arbor, MI).

Culture of Human RA Synovial Fibroblasts.

Fibroblasts were isolated from synovium obtained from RA patients who had undergone total joint replacement or synovectomy according to an institutional review board-approved protocol and processed as described previously (15). RA synovial fibroblasts were grown in RPMI medium 1640 with 10% FBS supplementation and used at passage 5 or older (15). All the treatments were performed in serum-free media.

Preparation of the EGCG Solution.

A stock solution of EGCG was prepared as described previously (15).

Treatment of RA Synovial Fibroblasts.

To study the effect of EGCG on IL-6 production, RA synovial fibroblasts (2 × 105 per well) in six-well plates were incubated with or without EGCG (10 and 20 μM) in serum-free media for 12 h, followed by stimulation with IL-1β (10 ng/ml) for 24 h. To study the mechanism of IL-6 production, cells were incubated with the inhibitors as described in SI Materials and Methods. To study the effect of EGCG on signaling events, cells were incubated with or without EGCG (10–20 μM) for 12 h in serum-free RPMI 1640, followed by stimulation with IL-1β for 30 min. Cells were lysed in cell lysis buffer as described and processed for Western blot analysis using rabbit polyclonal antibodies specific for phospho (*p)-p38, *p-JNK, *p-ATF-2, and *p-c-Jun (Cell Signaling Technology) (15). The blots were reprobed with rabbit polyclonal anti-β-actin to ensure equal protein loading.

To study the mechanism of IL-6/sIL-6R–induced MMP-2 activity and VEGF production, RA synovial fibroblasts were treated as described in SI Materials and Methods.

Western Immunoblotting and Analysis.

Western blot analysis was performed as described in SI Materials and Methods.

Induction of Arthritis by Adjuvant.

Female Lewis rats weighing ~100 g were injected s.c. at the base of the tail with 300 μl (5 mg/ml) of lyophilized Mycobacterium butyricum (Difco Laboratories) in sterile mineral oil. The day of adjuvant injection was considered day 0 for all time points. Clinical parameters measured included articular index and ankle circumference. Articular index scores were recorded for each hind joint by a consistent observer blinded to the treatment regimen and were then averaged for each animal. Scoring was performed on a 0–4 scale, where 0 = no swelling or erythema, 1 = slight swelling and/or erythema, 2 = low to moderate edema, 3 = pronounced edema with limited joint usage, and 4 = excess edema with joint rigidity. Ankle circumferences were measured by the same blinded observer as described previously (45). The increase in ankle circumference (day 17 minus day 0 measurements) was presented as delta (Δ) ankle circumference. The Δ ankle circumferences of both hind ankles from each animal were averaged and “n” is represented as the number of animals used in each of the experimental groups.

Treatment of Animals with EGCG.

EGCG was brought into suspension in PBS. EGCG (100 mg/kg) or PBS was administered i.p., with treatment initiated on day 7 after arthritis induction when the first signs of joint inflammation and pain are usually noted and continued until day 16 (46, 47). On day 17, animals were anesthetized for blood collection by cardiac puncture and then killed for biochemical and cytokine analysis. For all comparisons, the PBS group served as a control. EGCG (100 mg/kg) in rats corresponds to 960 mg of human equivalent dose (24). A daily dose of 800 mg of EGCG for 4 weeks in humans has been found to be safe and, interestingly, provided higher circulating concentrations of EGCG than lower doses (48).

Tissue Processing.

Ankles from the PBS- and EGCG-treated groups were processed for ELISA and Western blotting as described in SI Materials and Methods.

Histological Analysis of the Joints.

For histological analysis of the joints, fresh-frozen sections (6 μm) of the joints from the PBS and EGCG groups were stained with H&E and evaluated for the presence of infiltrates, angiogenesis, and bone destruction.

Assay for Rat IL-6, TNF-α, and IL-1β.

The serum and joint homogenates were analyzed for rat IL-6 levels using colorimetric ELISA kits (R&D Systems) and for TNF-α and IL-1β using fluorescence-based Luminex assay kits (Millipore) according to the manufacturer's protocol. The values obtained from the joint homogenates were normalized to protein content.

RNA Extraction and qRT-PCR.

qRT-PCR was performed using Platinum SYBR Green for detection, and the primer sequences for the human and rat are described in SI Materials and Methods.

Gelatin Zymography.

MMP-2 activity in conditioned medium was measured as previously described (15). To determine MMP-2 activity in joint homogenates, 10 μg of protein from each sample was reconstituted up to 15 μl of PBS and resolved as described for the conditioned medium.

Statistical Analysis.

Student t tests were performed to calculate statistical differences between the results of the different variables. P-values of less than 0.05 with two-tailed analysis were considered statistically significant.

Supplementary Material

Supporting Information:

Acknowledgments.

This study was supported by National Institutes of Health grants AT-003633 (S.A.) and AI-40987 and AR-48267 (A.E.K.); the Frederick G.L. Huetwell and William D. Robinson, M.D. Professorship in Rheumatology (A.E.K.); the Office of Research and Development, Medical Research Service, Department of Veterans Affairs (A.E.K.); and the French Society of Rheumatology (H.M.), Lavoisier Foundation (H.M.), and Philippe Foundation (H.M.).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. D.N. is a guest editor invited by the Editorial Board.

This article contains supporting information online at www.pnas.org/cgi/content/full/0802675105/DCSupplemental.

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