TLR-3 enhances osteoclastogenesis through upregulation of RANKL expression from fibroblast-like synoviocytes in patients with rheumatoid arthritis

Immunology Letters 124 (2009) 9–17

Contents lists available at ScienceDirect

Immunology Letters journal homepage: www.elsevier.com/locate/

TLR-3 enhances osteoclastogenesis through upregulation of RANKL expression from fibroblast-like synoviocytes in patients with rheumatoid arthritis Kyoung-Woon Kim a,1 , Mi-La Cho b,1 , Hye-Joa Oh b , Hae-Rim Kim a , Chang-Min Kang b , Yang-Mi Heo b , Sang-Heon Lee a,∗∗,2 , Ho-Youn Kim b,∗,2 a b

Division of Rheumatology, Department of Internal Medicine, The Konkuk University of Korea, Seoul, South Korea The Center for Rheumatic diseases in Kang-Nam St. Mary’s Hospital, and Rheumatism Research Center, College of Medicine, The Catholic University of Korea, Seoul, South Korea

a r t i c l e

i n f o

Article history: Received 9 November 2008 Received in revised form 2 February 2009 Accepted 8 February 2009 Available online 20 February 2009 Keywords: Toll-like receptor Osteoclastogenesis Fibroblast-like synoviocyte (FLS) RANKL Rheumatoid arthritis

a b s t r a c t This study was undertaken to determine the effect of toll-like receptor-3 (TLR3) on the regulation of osteoclastogenic activity in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). The expression of receptor activator of nuclear factor kappa B ligand (RANKL) mRNA and protein in RA-FLS after TLR3 activation was determined using RT-PCR, real-time PCR, western blot analysis, and immunohistochemistry. Human monocytes were cocultured with RA-FLS that had been prestimulated by the TLR3 ligand polyriboinosinic–polyribocytidylic acid and then stained for tartrate-resistant acid phosphatase (TRAP) activity. Other markers of osteoclasts were measured using RT-PCR and real-time PCR. The expression of TLR3 and RANKL was much higher in the RA synovium than in the osteoarthritis (OA) synovium. TLR3 activation induced RANKL expression in RA-FLS, but not in OA-FLS or in normal skin fibroblasts. TLR3 activation also induced the production of IL-1␤ but had no effect on IL-17 or TNF-␣ production in RA-FLS. Inhibition of IL-1␤ reversed the TLR3-induced upregulation of RANKL expression. Coculture of human monocytes with TLR3-activated RA-FLS or TLR3 ligand-stimulated human monocytes increased the expression of TRAP, RANK, cathepsin K, calcitonin receptor, and MMP-9, reflecting the differentiation of monocytes into osteoclasts. Our results suggest that TLR3 promotes osteoclastogenesis in the RA synovium both directly and indirectly. TLR3 stimulates human monocytes directly to promote osteoclast differentiation. TLR3 induces RANKL expression indirectly in RA-FLS, and the expression of RANKL promotes the differentiation of osteoclasts in the RA synovium. Targeting the TLR3 pathway may be a promising approach to preventing inflammatory bone destruction in RA. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by abnormal synovial hyperplasia with local infiltration of various inflammatory cells. Joint destruction causes joint deformities, one of the most serious problems in RA patients. Joint

∗ Corresponding author at: Department of Internal Medicine, Kangnam St. Mary’s hospital, The Catholic University of Korea, Banpo-Dong 505, Seocho-Gu, Seoul 137701, South Korea. Tel.: +82 2 2030 7541; fax: +82 2 2030 7748. ∗∗ Corresponding author at: Department of Internal Medicine, Konkuk University Hospital, 4-12 Hwayang-dong, Gwangjin-gu, Seoul 143-729, South Korea. E-mail addresses: [email protected] (K.-W. Kim), [email protected] (M.-L. Cho), [email protected] (H.-J. Oh), [email protected] (H.-R. Kim), [email protected] (C.-M. Kang), [email protected] (Y.-M. Heo), [email protected] (S.-H. Lee), [email protected] (H.-Y. Kim). 1 Contributed equally to this work. 2 Contributed equally to this work. 0165-2478/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2009.02.006

inflammatory parenchymal lesions are characterized by synovial hyperplasia, with increased numbers of synovial fibroblasts and cellular infiltrates that are composed predominantly of macrophages and lymphocytes [1]. Although histologic analyses often demonstrate increased osteoclastic bone resorption at the bone–pannus interface in RA joints [2], the precise mechanism of joint destruction remains unknown. During RA, the synovial tissue undergoes many changes, including neoangiogenesis, cellular hyperplasia, and influx of inflammatory cells, which are potentially orchestrated by a complex interplay of proinflammatory mediators such as cytokines and chemokines [3–6]. TLRs are phylogenetically conserved receptors that recognize pathogen-associated molecular patterns (PAMPs) and are involved in the uptake and processing of various exogenous and endogenous antigens [7]. To date, 11 human TLRs have been described, of which TLR2 and TLR-4 are the most thoroughly investigated. In addition to

10

K.-W. Kim et al. / Immunology Letters 124 (2009) 9–17

being involved in the recognition of lipopolysaccharide (LPS), TLR-4 also interacts with endogenous ligands such as heat-shock proteins (HSPs) [8,22], fragments of hyaluronic acid [9], and fibronectin [10]. These endogenous ligands are probably released by cells undergoing stress, damage, or necrotic death and are present in the stressed synovium. Increased cell turnover in conditions of minor trauma or inflammation, leading to increased levels of endogenous TLR ligands, is therefore likely to initiate a reaction leading to TLRmediated triggering of inflammatory cells in RA. Recent evidence underscores such a critical role of TLRs in the onset of autoimmunity [11–13]. We also found that activation of synovial fibroblasts in culture with TLR-2 and TLR-4 ligands promote osteoclastogenic activity by activating RANKL [14]. Receptor activator of NF-␬B (RANK) and its ligand, RANKL, are members of the TNF receptor and TNF superfamilies, respectively [15,16]. The RANKL/RANK system is essential for osteoclast formation. Targeted mutation in either gene prevents the development of osteoclasts, resulting in the development of osteopetrosis [17]. RANKL was characterized initially as a T lymphocyte-specific protein [18] and was detected subsequently in bone marrow stromal cells and osteoblasts [15,16]. In the immune system, RANKL is expressed by activated T cells, B cells, and DCs. In patients with RA, both activated T cells and fibroblast-like synoviocytes (FLS) express RANKL, and it has been proposed that expression of RANKL promotes osteoclast development [19,20]. Page et al. reported that treatment of synoviocytes with TNF-␣ or IL-1␤ in combination with IL-17 is particularly potent for inducing RANKL expression [21]. A recent study reported that RNA released by necrotic synovial fluid cells derived from patients with RA can act as an endogenous ligand for TLR3 in cultured RA-FLS [23]. Injection of dsRNA into mice causes a self-limited arthritis, suggesting that TLR3 signaling contributes to the pathogenesis of arthritis [24]. A number of lines of evidence demonstrate the importance of TLR signaling in RA pathogenesis, but the direct involvement of the TLR pathway in osteoclastogenesis-induced bone destruction remains obscure. Because of the importance of TLR signaling in the development of arthritis and the expression of RANKL in RA-FLS, we hypothesized that activation of the innate immune system through TLRs may affect the osteoclastogenic activity of RA-FLS by modulating RANKL expression and by regulating osteoclast differentiation. In the present study, we investigated the possible role of TLR-3 in RA by analyzing TLR-3 expression in synovial tissues. Our results indicate higher TLR-3 protein expression in RA synovial tissues than in OA synovial tissues. To explore the functional consequences of TLR signaling in the RA joint, we assessed the effect of TLR3 ligation on the expression of RANKL in RA-FLS and on osteoclast differentiation from human monocytes. We also evaluated the influence of proinflammatory cytokines on the RANKL expression induced by TLR3 ligation. 2. Materials and methods 2.1. Patients Seven patients having RA fulfilling the 1987 revised criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) were included. Seven age- and sex-matched patients with osteoarthritis (OA) were included as a control group. Informed consent was obtained from all patients, and the experimental protocol was approved by the Catholic University of Korea Human Research Ethics Committee. Samples of rheumatoid synovium were isolated from the RA patients (mean age, 59.9 ± 12.3 years; range, 42–75 years) and the OA patients (mean age, 62.5 ± 8.2 years; range 46–77 years) who were undergoing total knee replacement surgery.

2.2. Reagents Poly IC was purchased from Sigma (St. Louis, MO, USA). Anti-human IL-17, TNF-␣, IL-1␤, recombinant macrophage colonystimulating factor (M-CSF), and RANKL were purchased from R&D Systems (Minneapolis, MN, USA). 2.3. Isolation of FLS FLS were isolated by enzymatic digestion of synovial tissues obtained from RA and OA patients undergoing total knee replacement surgery, as described previously [25]. 2.4. Immunohistochemistry of RA synovium and FLS Immunohistochemical staining for RANKL and TLR3 was performed on sections of synovium. Briefly, the synovium samples were obtained from the patients, fixed in 4% paraformaldehyde solution overnight at 4 ◦ C, dehydrated with alcohol, washed, embedded in paraffin, and sectioned into slices 7 ␮m thick. The sections were depleted of endogenous peroxidase activity by adding methanolic H2 O2 and were blocked with normal serum for 30 min. After overnight incubation at 4 ◦ C with polyclonal anti-human RANKL and TLR3 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the samples were incubated with the secondary antibodies, biotinylated anti-rabbit IgG or biotinylated anti-goat IgG, for 20 min and then incubated with streptavidin–peroxidase complex (Vector, Peterborough, UK) for 1 h followed by incubation with 3,3 -diaminobenzidine (Dako, Glostrup, Denmark) for 5 min. The sections were counterstained with hematoxylin. Samples were photographed with an Olympus photomicroscope (Tokyo, Japan). FLS were grown in 150 mm dishes in DMEM complete medium, plated at a density of 1 × 104 cells/cm2 onto glass coverslips (12 mm diameter), and stimulated with poly IC 10 ␮g/ml (Sigma). Cells were fixed in 4% paraformaldehyde for immunocytochemical analysis using anti-RANKL antibody 72 h after the addition the of poly IC [21]. 2.5. Expression of RANKL mRNA measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) FLS were incubated with various concentrations of poly IC for 72 h, and mRNA was extracted using RNAzol B (Biotex Laboratories, Houston, TX, USA) according to the manufacturer’s instructions. Reverse transcription of 2 ␮g of total mRNA was carried out at 42 ◦ C using the SuperscriptTM reverse transcription system (Takara, Shiga, Japan). PCR amplification of cDNA aliquots was performed by adding 2.5 mM of dNTPs, 2.5 U of Taq DNA polymerase (Takara), and 0.25 ␮M of sense and antisense primers. The reaction was done in PCR buffer (1.5 mM MgCl2 , 50 mM KCl, 10 mM Tris–HCl, pH 8.3) in a total volume of 25 ␮l. Reactions were processed in a DNA thermal cycler (PerkinElmer Cetus, Wellesley, MA, USA). PCR products were run on a 2% agarose gel and stained with ethidium bromide. The electrophoresis bands were taken photo using Gel Doc XR System and analyzed by Quantity One Gel Analysis software (Bio-Rad Laboratories, CA, USA). The results are expressed as the ratio of target PCR product relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or ␤-actin products (Table 1). 2.6. Real-time PCR and amplification protocol PCR amplification and analysis were achieved using a LightCycler 2.0 instrument (Roche Applied Science) with software version 4.0. All reactions were performed with the LightCycler FastStart DNA master SYBR green I (Roche Applied Science) in a 20 ␮l volume

K.-W. Kim et al. / Immunology Letters 124 (2009) 9–17

11

Table 1 Primers used in the RT-PCR and real-time PCR to detect different targets in FLS. Primers (5 –3 )

RANKL TLR-3 IL-17 IL-1␤ TNF-␣ TRAP Cathepsin K CTR RANK GAPDH Beta-actin

Sense

Antisense

ACC AUC ATC AAA ATC CCA AG GAT CTG TCT CAT AAT GGC TTG atg act cct ggg aag acc tca ttg ATG GCA GAA GTA CCT GAG CTC AGC ACT GAA AGC ATG ATC CGG GAC CAC CTT GGC AATGTC TCTG TGA GGC TTC TCT TGG TGT CCA TAC TGG TGC CAA CCA CTA TCC ATG C GCT GTA ACA AAT GTG AAC CAG GA CGA TGC TGG GCG TGA GTA C GGA CTT CGA GCA AGA GAT GG

CCC CAA AGT ATG TTG CAT CC GAC AGA TTC CGA ATG CTT GTG TTA GGC CAC ATG GTG GAC AAT CGG TTA GGA AGA CAC AAA TTG CAT GGT GAA CAT GGG CTA CAG GCT TGTCAC TGG CTG AGG AAG TCA TCT GAG TTG AAA GGG TGTCATTAC TGC GGG CAC AAG TGC CGC CAT GAC AG GCC TTG CCT GTA TCA CAA ACT CGT TCA GCT CAG GGA TGA CC TGT GTT GGC GTA CAG GTC TTT G

in each reaction capillary. To quantify the cytokines, 2 ␮l of DNA standard dilution or 2 ␮l of cDNA was added before the capillaries were capped, centrifuged, and placed in the LightCycler sample carousel. Amplification conditions comprised an initial preincubation at 95 ◦ C for 10 min (FastStart Taq DNA polymerase activation), followed by amplification of the target DNA for 45 cycles (95 ◦ C for 10 s, target annealing temperatures for 10 s, and 72 ◦ C for 10 s). Melting curve analysis was performed immediately after amplification at a linear temperature transition rate of 0.1 ◦ C/s from 65 ◦ C to 95 ◦ C with continuous fluorescence acquisition. In our protocol, the results are normalized systematically to the expression levels of reference genes to correct for variations in nucleic acid quality and quantity [21]. The concentration of an unknown sample was calculated by comparing its crossing point (Cp) with the corresponding standard curve. The cycle number at the Cp (y-axis) was plotted versus the log of the initial template amount (x-axis) of the standards. After amplification, three analyses were performed sequentially with the data: (i) absolute quantification, which gave an absolute value in copies/␮l; (ii) relative quantification, expressed as a ratio between the cytokine gene and the reference gene; and (iii) results normalized to a calibrator sample. Finally, a calibrator-normalized ratio was obtained, which provided indirect information on the changes in the cytokine mRNA level by adjusting for experimental variations in the different samples [26]. 2.7. Western blot analysis of RANKL expression FLS were incubated with poly IC for 72 h, a whole cell lysate was prepared from about 2 × 105 cells by homogenization in the lysis buffer, and the lysate was centrifuged at 14,000 rpm for 15 min. The protein concentration in the supernatant was determined using the Bradford method (Bio-Rad, Hercules, CA, USA). Protein samples were separated on 10% SDS-PAGE and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Uppsala, Sweden). For western hybridization, the membrane was preincubated with 0.5% skim milk in Tris-buffered saline (TBS) with 0.1% Tween 20 (TTBS) at room temperature for 2 h. The primary antibody to RANKL (R&D Systems), diluted 1:1000 in 5% BSA–0.1% Tween 20/TBS, was added and incubated overnight at 4 ◦ C. The membrane was washed four times with TTBS, horseradish peroxidase-conjugated secondary antibody was added, and the membrane was incubated for 1 h at room temperature. After TTBS washing, the hybridized bands were detected using an ECL detection kit and Hyperfilm-ECL reagents (Amersham Pharmacia). 2.8. Magnetofection of oligonucleotides Short interfering ribonucleic acids (siRNAs) were transfected using magnetofection, as described previously [27,28], and the

Template (bp)

Annealing (◦ C)

196 305 468 807 486 176 134 119 154 412 198

59 56 56 59 59 55 55 55 55 56 60

assays were performed 24 h later. MyD88 was purchased from Dharmacon Inc. (Chicago, IL, USA). 2.9. Monocyte isolation Mononuclear cells were separated from buffy coats obtained from healthy volunteers by Ficoll-Hypaque (Sigma Chemicals, Poole, Dorset, UK) density gradient centrifugation. The cells were washed three times with sterile PBS and resuspended in RPMI 1640 (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, and 1% penicillin–streptomycin, henceforth called complete medium. Freshly isolated peripheral blood mononuclear cells were incubated at 37 ◦ C in complete medium and allowed to adhere for 45 min. The nonadherent cells were removed, and the adherent cells were washed with sterile PBS, harvested with a rubber policeman, and stained with monocyte-specific anti-CD14 monoclonal antibody to assess the purity of the preparation. Ninety percent of the isolated cells expressed CD14 [29]. 2.10. Osteoclast formation RA-FLS were seeded into 12-well multiwell dishes (5 × 103 cells/well) and stimulated by poly IC for 3 days. As described above, isolated human monocytes (5 × 104 cells/well) were added to the stimulated FLS in 12-well multiwell dishes with fresh media. The cells were cocultured for 3 weeks in ␣-MEM and 10% heatinactivated FBS in the presence of 2 ng/ml of M-CSF. The medium was changed on day 3 and then every other day. The addition of recombinant RANKL protein, prepared as described previously [23], was used as a positive control. On day 21, tartrate-resistant acid phosphatase (TRAP)-positive cells were identified using a leukocyte acid phosphatase kit according to the manufacturer’s recommended protocol (Sigma–Aldrich) [30]. 2.11. Statistical analysis Data are expressed as the mean ± SEM. The data were analyzed using the Mann–Whitney U test for independent samples and the Wilcox signed test for paired samples. P < 0.05 was considered significant. 3. Results 3.1. Effect of TLR3 ligation on RANKL mRNA expression in RA-FLS We measured RANKL expression in RA-FLS after 72 h of in vitro pretreatment with the TLR3 ligand poly IC. In our preliminary experiments, we observed RANKL expression at 24, 48, and 72 h after treatment with poly IC and a maximal response at 72 h (data not

12

K.-W. Kim et al. / Immunology Letters 124 (2009) 9–17

Fig. 1. The expression of RANKL mRNA is upregulated by poly IC stimulation in RA FLS. (A) RA-FLS, OA-FLS, and normal skin fibroblasts were cultured with poly IC (10 (g/ml) for 72 h. The expression of RANKL mRNA was analyzed using semi-quantitative RT-PCR. PCR products were run on a 2% agarose gel and stained with ethidium bromide. Results are presented as mean ± SEM of three patients. (B) RA-FLS were cultured with poly IC 10 (g/ml for 72 h. RANKL mRNA expression was measured using quantitative real-time PCR. Results are presented as mean ± SEM of three patients. * p < 0.05, ** p < 0.01.

Fig. 2. (A) TLR3 and RANKL expression increases in the RA synovium and RANKL production increases significantly after activation of TLR3 by poly IC in RA-FLS. The expression of TLR3 and RANKL in the RA and OA synovium was detected using immunohistochemical staining. All tissues were counterstained with hematoxylin (original magnification 400×). (B) RA-FLS were cultured in the presence or absence of poly IC (10 ␮g/ml) for 72 h. RANKL protein expression was measured in cell lysates by western blot analysis using goat polyclonal anti-RANKL antibody. The results are expressed as the ratio of the densitometric intensity of the RANKL product to that of the ␤-actin product. The data represent one of three independent experiments. The graphs present the means ± SEM of three patients. ** p < 0.01. (C) RA-FLS were cultured with poly IC (10 ␮g/ml) for 72 h and then stained with an anti-RANKL antibodies (red) (original magnification 400×).

K.-W. Kim et al. / Immunology Letters 124 (2009) 9–17

13

Fig. 3. IL-1␤ may mediate the induction of RANKL through TLR3. (A) Effects of neutralization of proinflammatory cytokines on TLR3-induced RANKL expression. RA-FLS were treated with Poly IC (10 ␮g/ml) for 72 h in the presence or absence of anti-IL-17, anti-IL-1(, or anti-TNF-( neutralizing antibodies. The expression of RANKL mRNA was detected using RT-PCR. Results are presented as the mean ± SEM of three separate experiments. (B) RA-FLS were treated with Poly IC (10 ␮g/ml) for 72 h, and then, the expression of RANKL, IL-17, IL-1␤, and TNF-␣ mRNA was detected using RT-PCR. The data represent the mean ± SEM of three separate experiments. Results are expressed as the ratio of the densitometric intensity of the RANKL, IL-17, IL-1␤, and TNF-␣ product to that of the GAPDH product. ** p < 0.01. (C) Effect of MyD88 knockdown on Poly IC-induced expression of RANKL. RA-FLS were transfected with MyD88 siRNA. After 24 h, cells were treated with 10 ␮g/ml Poly IC for 72 h, and RNA extracts were subjected to real-time PCR for RANKL and MyD88. (D) Expression of TRIF on Poly IC-induced expression of RANKL. FLS were treated with 10 ␮g/ml Poly IC for 72 h, and RNA extracts were subjected to real-time PCR for TRIF.

shown). Fig. 1A shows that the 72-h stimulation of TLR3 by poly IC strongly induced the expression of RANKL mRNA using semiquantitive RT-PCR in RA-FLS, whereas RANKL expression was not induced in OA-FLS or skin fibroblasts from healthy volunteers. Moreover, RA-FLS constitutively expressed RANKL mRNA, whereas RANKL mRNA was not expressed in the control cells. The expression of RANKL mRNA increased in a dose-dependent manner (data not shown). The inductions of RANKL expression at the most efficient dose of Poly IC (10 ␮g/ml) were confirmed in real time PCR analysis (Fig. 1B). 3.2. Expression of TLR3 and RANKL in RA synovium and FLS Before examining the role of TLR3 in RA osteoclastogenesis, we observed the constitutional expression of TLR3 in RA synovial tissues using immunohistochemical staining. In the synovial tissues, more intense staining of TLR3 and RANKL was observed in the RA synovium compared with the OA synovium (Fig. 2A). Next, we used western blot analysis and immunostaining to examine RANKL production after stimulation of RA-FLS with poly IC. As shown in Fig. 2B, RANKL expression increased significantly after stimulation of TLR3 by 10 ␮g/ml of poly IC (P < 0.01). In vitro cellular immunostaining 72 h after TLR3 stimulation showed that RANKL expression also increased in cultured RA-FLS (Fig. 2C).

3.3. Effects of proinflammatory cytokines on the TLR3-induced RANKL expression by RA-FLS TLR stimulation induces the production of proinflammatory cytokines, such as TNF-␣ and IL-1␤, by RA-FLS. Stimulation of RA-FLS with a combination of IL-1␤, TNF-␣, and IL-17 induces RANKL expression more effectively than does stimulation by a single cytokine [21]. To evaluate whether the TLR3-induced RANKL expression by RA-FLS is a direct effect or an indirect effect mediated by proinflammatory cytokines, we used neutralizing antibodies to IL-1␤, TNF-␣, or IL-17. The expression of RANKL by TLR3 stimulation was inhibited by neutralization of IL-1␤ but not by neutralization of TNF-␣ or IL-17 (Fig. 3A). We also examined the expression of IL-1␤, TNF-␣, and IL-17 after TLR3 stimulation of RA-FLS. Poly IC stimulation significantly increased IL-1␤ and RANKL mRNA expression (P < 0.01) but did not change the expression of TNF-␣ or IL-17 mRNA (Fig. 3B). These data suggest that the TLR3-stimulated induction of RANKL expression is mediated by IL-1␤. 3.4. Intracellular signaling pathways of TLR3-induced RANKL expression To identify the signal pathways of TLR3-induced RANKL expression, we knocked down MyD88 expression using RNA-mediated interference, and we confirmed the expression of MyD88 and

14

K.-W. Kim et al. / Immunology Letters 124 (2009) 9–17

RANKL through TLR3 ligation in RA-FLS using real-time PCR. siMyD88 completely reduced RANKL and MyD88 expression induced by poly IC (Fig. 3C). TLR3 ligation induced TRIF(Toll/ IL-1 receptor domain-containing adaptor inducing interferon-␤) expression (Fig. 3D). 3.5. RA-FLS promote osteoclastogenic activity by activating RANKL via TLR3 ligation Osteoclast precursors of the monocyte lineage exist within the bone marrow and peripheral blood, and their differentiation depends on macrophage colony-stimulating factor (M-CSF) and the engagement of receptor activator of nuclear factor-kB (RANK) and its ligand (RANKL) [31]. It is believed that differentiation of precursors into mature osteoclasts requires accessory cells expressing RANKL, such as osteoblasts, bone marrow stromal cells and activated T cells [32]. At first, we confirmed the direct effects of the Poly IC on osteoclast differentiation by adding Poly IC in monocytes (Fig. 4A). Interestingly, Poly IC increased the number of TRAP-positive osteoclasts. Because we demonstrated that the stimulation of TLR-3 upregulates the expression of RANKL, we examined whether activated RA-FLS through TLR-3 stimulation could play a role in promoting osteoclast differentiation from human monocytes (Fig. 4B). RA-FLS were pretreated with poly IC and then cocultured with human monocytes for 21 days. TRAP staining showed abundant TRAP-

positive multinucleated cells, indicating their differentiation into osteoclasts. This osteoclastogenic effect of TLR3-activated RA-FLS was reversed by addition of osteoprotegerin (OPG) (Fig. 4B). Using real-time PCR analysis, we observed the expression of the surface markers of osteoclasts, such as TRAP, RANK, cathepsin K, calcitonin receptor, and MMP-9 in the cultured monocytes. The cultured monocytes that were treated with M-CSF in the presence of poly IC-stimulated RA-FLS showed increased expression of TRAP, RANK, cathepsin K, calcitonin receptor, and MMP-9. The expression of TRAP increased the most (Fig. 4C). These findings suggest that Poly IC is direct activator of osteoclast differentiation and that it mediates osteoclastogenesis in cocultures of monocytes and Poly IC-stimulated RA-FLS. 4. Discussion We investigated the effect of TLR3 signaling on osteoclastogenesis in RA-FLS. Our data suggest that TLR3 stimulation augments the expression of RANKL in synovial fibroblasts and that this increased expression facilitates osteoclastogenesis from osteoclast precursor cells. This effect may be mediated partly by increased IL-1␤ production through TLR3 signaling, but not by TNF-␣. TLR3 ligation appears to upregulate the expression of RANKL in RA synovial fibroblasts via MyD88-mediated and TRIF-mediated pathways. We used real-time PCR analysis to confirm the previously observed increased expression of TLR3 in FLS. We also found higher levels of RANKL expression

Fig. 4. Human monocytes cocultured with TLR3 activate RA-FLS and differentiate into TRAP-positive multinucleated cells. (A) Schematic representation of the RANKL + M-CSF culture system for osteoclast differentiation. Isolated human monocytes were cultured with M-CSF and RANKL or Poly IC, and the TRAP-positive multinucleated cells were detected and quantified. (B) Schematic representation of the coculture system with monocytes and Poly IC-stimulated RA-FLS to study osteoclast differentiation. Isolated human monocytes were cultured with M-CSF and Poly IC-stimulated RA-FLS in the presence or absence of OPG, and the TRAP-positive multinucleated cells were detected and quantified. The graphs present the mean ± SEM of three separate experiments. * p < 0.05, ** p < 0.001. (C) Schematic representation of the coculture system to study osteoclast differentiation. In the coculture system, RA-FLS were pretreated with Poly IC (10 ␮g/ml) for 3 days, monocytes were added to each well, and the cocultures were maintained for 3 weeks in (-MEM containing 10% horse serum with 100 ng/ml M-CSF in the presence or absence of OPG. The expression of TRAP, RANK, cathepsin K, calcitonin receptor, and MMP-9, and the expression of ␤-actin mRNA from monocytes were quantified using real-time PCR. The results are presented as the mean ± SEM of three separate experiments. * p < 0.05, ** p < 0.001.

K.-W. Kim et al. / Immunology Letters 124 (2009) 9–17

15

Fig. 4. (Continued) .

at both the mRNA and protein levels in RA-FLS than in OA-FLS and normal skin fibroblast stimulated with a known TLR3 ligand. To explore the consequences of TLR activation in the clinical setting in more detail, the endogenous ligands should be used in future studies. RA-FLS exhibit various abnormalities, including increased proliferation, increased production of matrix metalloproteinases [33],

and resistance to apoptosis. Fibroblasts can exhibit abnormal upregulation of numerous genes, including cytokines, chemokines, inflammation mediators, and members of the TNF receptor superfamily, including RANKL [34]. Because macrophages and fibroblasts are present throughout the synovium, additional factors may be responsible for the development of macrophages into osteoclasts at the site of bone erosion. Several cytokines have been implicated

16

K.-W. Kim et al. / Immunology Letters 124 (2009) 9–17

in the regulation of RANKL. TNF-␣ and IL-1␤ are major components of inflammatory synovial fluid and are involved in inflammatory processes through RANKL-independent mechanisms [35]. The recent identification of endogenous ligands for several different TLRs has generated great interest because of their potential role in the pathogenesis of autoimmunity. A recent study showed that RNA released by necrotic synovial fluid cells derived from RA patients can act as an endogenous ligand for TLR3 in cultured RAFLS [23]. We used a synthetic TLR3 ligand, poly IC. Many studies have identified bacterial components such as bacterial DNA and peptidoglycan in synovial membrane specimens from patients with active or inactive inflammatory joint disease, including RA [36,37]. We have demonstrated previously the expression of functional TLR2 in RA-FLS and that TLR2 induces osteoclastogenic activity in RA-FLS [14]. Our finding that TLR2 expression was higher in RA synovial tissue than in OA synovial tissue suggests that TLR signaling is active and is involved in osteoclastogenesis in RA. Interestingly, a recent study showed that RA-FLS express TLRs, particularly TLR3 [23]. Activation of cultured RA-FLS with the TLR3 ligand poly IC results in the production of high levels of IFN-␤, CXCL10, CCL5, and IL-6 protein [23]. However, the direct involvement of innate immunity in the activation of osteoclasts in the rheumatoid synovium has not been confirmed, although proinflammatory cytokines released from RAFLS upon activation of TLRs may affect osteoclastogenesis indirectly. The TIR-containing adaptor protein MyD88 was proposed originally as the crucial mediator of all TLR activities, including those of TLR3 and TLR4. However, analysis of MyD88-deficient mice shows that the TLR3- and TLR4-induced activation of NF-␬B and JNK is not abolished but only delayed, whereas the IFN-␤ responses are unaffected, indicating the existence of parallel MyD88-independent pathways in these mice [38]. This second signaling pathway is dependent on the TIR domain-containing adaptor protein TRIF (also called TICAM-1) [39]. TRIF associates with TLR3 and indirectly with TLR4 through another adaptor protein, TRAM [40]. Multinucleated osteoclast-like cells are characterized by their definitive osteoclast markers such as TRAP, cathepsin K, and calcitonin receptors [41–43]. Synovial macrophages are also capable of differentiating into osteoclasts when cocultured with stromal cells, indicating that the synovial macrophage population may contain osteoclast precursor cells [44]. To determine the functional consequence for osteoclastogenesis of TLR3 activation in RA-FLS, we cocultured peripheral blood monocytes and RA-FLS. In these cocultures, activation of TLRs significantly increased the formation of TRAP-positive multinucleated cells, suggesting the promotion of osteoclast differentiation from monocytes. Our results demonstrate that activation of innate immunity through TLRs can promote osteoclastogenesis by upregulating RANKL in RA-FLS and may thereby contribute directly to bone destruction. To our knowledge, our data provide the first evidence of the involvement of the TLR3 pathway in bone destruction in RA. In summary, our results suggest that the signaling pathway through TLR3 generates osteoclasts directly from monocytes and induces RANKL expression in RA-FLS. This suggests that innate immunity is involved in the initiation and amplification of the inflammatory response and in the bone destruction associated with RA pathogenesis. Effective blockade of specific TLR signaling leading to RANKL expression along with inhibition of proinflammatory cytokines might be an interesting therapeutic option to protect joints from the destructive nature of the rheumatoid synovium. Conflict of interest The authors state that they have no conflicts of interest.

Acknowledgments This work was supported by a grant (R11-2002-098-05001-0) from the Korea Science & Engineering Foundation through the RhRC (Rheumatism Research Center) at Catholic University of Korea. References [1] Harris Jr ED. Rheumatoid arthritis: pathophysiology and implications for therapy. N Engl J Med 1990;322:1277–89. [2] Shimizu S, Shiozawa S, Shiozawa K, Imura S, Fujita T. Quantitative histologic studies on the pathogenesis of periarticular osteoporosis in rheumatoid arthritis. Arthritis Rheum 1985;28:25–31. [3] Feldmann M, Brennan FM, Maini RN. Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 1996;14:397–440. [4] Joosten LA, Radstake TR, Lubberts E, van den Bersselaar LA, van Riel PL, van Lent PL, et al. Association of interleukin-18 expression with enhanced levels of both interleukin-1 and tumor necrosis factor in knee synovial tissue of patients with rheumatoid arthritis. Arthritis Rheum 2003;48:339–47. [5] Miossec P. Pro- and antiinflammatory cytokine balance in rheumatoid arthritis. Clin Exp Rheumatol 1995;13(Suppl. 12):S13–6. [6] Vervoordeldonk MJ, Tak PP. Cytokines in rheumatoid arthritis. Curr Rheumatol Rep 2002;4:208–17. [7] Akira S, Hemmi H. Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett 2003;85:85–95. [8] Ohashi K, Burkart V, Flohe S, Kolb H. Cutting edge: heat shock protein 60 is a putative endogenous ligand of the Toll-like receptor-4 complex. J Immunol 2000;164:558–61. [9] Termeer C, Benedix F, Sleeman J, Fieber C, Voith U, Ahrens T, et al. Oligosaccharides of hyaluronan activate dendritic cells via Toll-like receptor 4. J Exp Med 2002;195:99–111. [10] Okamura Y, Watari M, Jerud ES, Young DW, Ishizaka ST, Rose J, et al. The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem 2001;276:10229–33. [11] Leadbetter EA, Rifkin IR, Hohlbaum AM, Beaudette BC, Shlomchik MJ, MarshakRothstein A. Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature 2002;416:603–7. [12] Ichikawa HT, Williams LP, Segal BM. Activation of APCs through CD40 or Tolllike receptor 9 overcomes tolerance and precipitates autoimmune disease. J Immunol 2002;169:2781–7. [13] Krieg AM. A role for Toll in autoimmunity. Nat Immunol 2002;3:423–4. [14] Kim KW, Cho ML, Lee SH, Oh HJ, Kang CM, Ju JH, et al. Human rheumatoid synovial fibroblasts promote osteoclastogenic activity by activating RANKL via TLR2 and TLR4 activation. Immunol Lett 2007;110:54–64. [15] Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A 1998;95:3597–602. [16] Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998;93:165–76. [17] Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli C, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 1999;397:315–23. [18] Wong Brian R, Rho Jaerang, Arron Joseph, Robinson Elizabeth, Orlinick Jason, Chao Moses, et al. TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J Biol Chem 1997;272:25190–4. [19] Kotake S, Udagawa N, Hakoda M, Mogi M, Yano K, Tsuda E, et al. Activated human T cells directly induce osteoclastogenesis from human monocytes: Possible role of T cells in bone destruction in rheumatoid arthritis patients. Arthritis Rheum 2001;44:1003–12. [20] Takayanagi H, Iizuka H, Juji T, Nakagawa T, Yamamoto A, Miyazaki T, et al. Involvement of receptor activator of nuclear factor ␬B ligand/osteoclast differentiation factor in osteoclastogenesis from synoviocytes in rheumatoid arthritis. Arthritis Rheum 2000;43:259–69. [21] Page G, Miossec P. RANK and RANKL expression as markers of dendritic cell-T cell interactions in paired samples of rheumatoid synovium and lymph nodes. Arthritis Rheum 2005;52:2307–12. [22] Martin Carla A, Carsons Steven E, Kowalewski Robert, Bernstein David, Valentino Michael, Santiago-Schwarz Frances. Aberrant extracellular and dendritic cell (DC) surface expression of heat shock protein (hsp) 70 in the rheumatoid joint: Possible mechanisms of hsp/DC-mediated cross-priming. J Immunol 2003;171:5736–42. [23] Brentano F, Schorr O, Gay RE, Gay S, Kyburz D. RNA released from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via Tolllike receptor 3. Arthritis Rheum 2005;52:2656–65. [24] Zare F, Bokarewa M, Nenonen N, Bergström T, Alexopoulou L, Flavell RA, et al. Arthritogenic properties of double-stranded (Viral) RNA. J Immunol 2004;172:5656–63. [25] Kim HR, Cho ML, Kim KW, Juhn JY, Hwang SY, Yoon CH, et al. Up-regulation of IL-23 p19 expression in rheumatoid arthritis synovial fibroblasts by IL-17 through PI3-kinase-, NF-␬B- and p38 MAPK-dependent signalling pathways. Rheumatology 2007;46:57–64.

K.-W. Kim et al. / Immunology Letters 124 (2009) 9–17 [26] Overbergh L, Giulietti A, Valckx D, Decallonne R, Bouillon R, Mathieu C. The use of real-time reverse transcriptase PCR for the quantification of cytokine gene expression. J Biomol Tech 2003;14:33–43. [27] Krötz F, de Wit C, Sohn HY, Zahler S, Gloe T, Pohl U, et al. Magnetofection—A highly efficient tool for antisense oligonucleotide delivery in vitro and in vivo. Mol Ther 2003;7:700–10. [28] Krötz F, Sohn HY, Gloe T, Plank C, Pohl U. Magnetofection potentiates gene delivery to cultured endothelial cells. J Vasc Res 2003;40:425–34. [29] Sperber K, Bauer J, Pizzimenti A, Najfeld V, Mayer L. Identification of subpopulations of human macrophages through the generation of human macrophage hybridomas. J Immunol Methods 1990;129:31–40. [30] Wu Y, Liu J, Feng X, Yang P, Xu X, Hsu HC, et al. Synovial fibroblasts promote osteoclast formation by RANKL in a novel model of spontaneous erosive arthritis. Arthritis Rheum 2005;52:3257–68. [31] Massey HM, Flanagan AM. Human osteoclasts derive from CD14-positive monocytes. Br J Haematol 1999 Jul;106(1):167–70. [32] Horwood NJ, Kartsogiannis V, Quinn JM, Romas E, Martin TJ, Gillespie MT. Activated T lymphocytes support osteoclast formation in vitro. Biochem Biophys Res Commun 1999;265(November (1)):144–50. [33] Zucker S, Hymowitz M, Conner C, Zarrabi HM, Hurewitz AN, Matrisian L, et al. Measurement of matrix metalloproteinases and tissue inhibitors of metalloproteinases in blood and tissues: clinical and experimental applications. Ann N Y Acad Sci 1999;878:212–27. [34] Zhang HG, Hyde K, Page GP, Brand JP, Zhou J, Yu S, et al. Novel tumor necrosis factor ␣-regulated genes in rheumatoid arthritis. Arthritis Rheum 2004;50:420–31. [35] Seibl R, Birchler T, Loeliger S, Hossle JP, Gay RE, Saurenmann T, et al. Expression and regulation of Toll-like receptor 2 in rheumatoid arthritis synovium. Am J Pathol 2003;162:1221–7. [36] Van der Heijden IM, Wilbrink B, Tchetverikov I, Schrijver IA, Schouls LM, Hazenberg MP, et al. Presence of bacterial DNA and bacterial peptidoglycans in joints

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

17

of patients with rheumatoid arthritis and other arthritides. Arthritis Rheum 2000;43:593–8. Schrijver IA, Melief MJ, Tak PP, Hazenberg MP, Laman JD. Antigen-presenting cells containing bacterial peptidoglycan in synovial tissues of rheumatoid arthritis patients coexpress costimulatory molecules and cytokines. Arthritis Rheum 2000;43:2160–8. Kawai T, Takeuchi O, Fujita T, Inoue J, Mühlradt PF, Sato S, et al. Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharideinducible genes. J Immunol 2001;167:5887–94. Oshiumi H, Matsumoto M, Funami K, Akazawa T, Seya T. TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-␤ induction. Nature Immunol 2003;4:161–7. Yamamoto M, Sato S, Hemmi H, Uematsu S, Hoshino K, Kaisho T, et al. TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway. Nat Immunol 2003;4:1144–50. Gravallese EM, Harada Y, Wang JT, Gorn AH, Thornhill TS, Goldring SR. Identification of cell types responsible for bone resorption in rheumatoid arthritis and juvenile rheumatoid arthritis. Am J Pathol 1998;152:943–51. Gravallese EM, Manning C, Tsay A, Naito A, Pan C, Amento E, et al. Synovial tissue in rheumatoid arthritis is a source of osteoclast differentiation factor. Arthritis Rheum 2000;43:250–8. Hummel KM, Petrow PK, Franz JK, Muller-Ladner U, Aicher WK, Gay RE, et al. Cysteine proteinase cathepsin K mRNA is expressed in synovium of patients with rheumatoid arthritis and is detected at sites of synovial bone destruction. J Rheumatol 1998;25:1887–94. Fujikawa Y, Sabokbar A, Neale S, Athanasou NA. Human osteoclast formation and bone resorption by monocytes and synovial macrophages in rheumatoid arthritis. Ann Rheum Dis 1996;55:816–22.