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Rheumatology Advance Access originally published online on June 12, 2006
Rheumatology 2007 46(1):57-64; doi:10.1093/rheumatology/kel159
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Up-regulation of IL-23p19 expression in rheumatoid arthritis synovial fibroblasts by IL-17 through PI3-kinase-, NF-{kappa}B- and p38 MAPK-dependent signalling pathways

H.-R. Kim1, M.-L. Cho2, K.-W. Kim2, J.-Y. Juhn2, S.-Y. Hwang3, C.-H. Yoon2, S.-H. Park2, S.-H. Lee1 and H.-Y. Kim2

1Department of Internal Medicine, School of Medicine, Konkuk University, Seoul and 2Rheumatism Research Center, Catholic Institutes of Medical Science, The Catholic University of Korea, Seoul and 3Graduate School of Biology and Information Technology, Institute of Genetic Engineering, Hankyung National University, Ansung, Kyonggi-Do, Korea.

Correspondence to: Ho-Youn Kim, MD, PhD, Department of Internal Medicine, Kangnam St., Mary's Hospital, The Catholic University of Korea, Banpo-Dong 505, Seocho-Gu, Seoul, Korea. E-mail: ho{at}catholic.ac.kr


   Abstract
Top
 Abstract
Introduction
 Methods and Materials
 Results
 Discussion
 Acknowledgements
 References
 
Objective. To investigate the expression of interleukin (IL)-23p19 in human rheumatoid arthritis (RA) synovial fibroblasts and its up-regulation by IL-17 stimulation, and to define the signal pathways involved in the regulation of IL-23p19 expression in RA synovial fibroblasts.

Methods. Synovial fluid (SF) and serum levels of IL-23p19 in RA were determined by enzyme-linked immunosorbent assays. The levels of IL-23p19 mRNA and protein were measured after the RA synovial fibroblasts were treated with recombinant human IL-17 and various inhibitors of intracellular signal pathway molecules using reverse transcription (RT) polymerase chain reaction (PCR), real-time PCR and western blotting.

Results. Levels of IL-23p19 in the sera and SF were much higher in RA patients than in osteoarthritis patients or healthy controls. The expression of IL-23p19 mRNA and protein was enhanced in RA synovial fibroblasts by IL-17 stimulation. Such effects of IL-17 were completely blocked by inhibitors of phosphatidylinositol (PI)-kinase/Akt, nuclear factor (NF)-{kappa}B and p38 mitogen-activated protein kinase (MAPK). In accordance with the expression of IL-23p19, the phosphorylation of I{kappa}B, Akt and p38 MAPK in synovial fibroblasts also increased after IL-17 stimulation.

Conclusion. IL-23p19 is over-expressed in RA synovial fibroblasts and IL-17 appears to up-regulate the expression of IL-23p19 in RA synovial fibroblasts via PI3-kinase/Akt, NF-{kappa}B- and p38-MAPK-mediated pathways. These results suggest that a disruption of interaction between IL-17 and IL-23p19 may provide a new therapeutic approach in the treatment of RA.

KEY WORDS: Rheumatoid arthritis, Synovial fibroblast, IL-23, IL-17


   Introduction
Top
 Abstract
 Introduction
Methods and Materials
 Results
 Discussion
 Acknowledgements
 References
 
Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic joint inflammation and subsequent joint destruction. The RA synovium is marked by the hyperplasia of the intimal lining synovial fibroblasts and massive infiltration of the sublining area by mononuclear cells [1]. The predominant T-cell subset in the sublining area of RA synovium is CD4+ T-cells. The majority of these CD4+ T-cells are mature memory CD45RO+ T-cells [1, 2]. Various cytokines from the immune cells participate in the regulation and perpetuation of inflammatory process in RA synovium, and so the coordinate network of the cytokines is essential in RA pathogenesis [3].

Among proinflammatory cytokines that partake in RA inflammation, IL-12 is known as the major inducer of interferon (IFN)-{gamma} production and the Th1 response. IL-12 exists as a heterodimer, and consists of a p35 and a p40 subunit. The serum and synovial fluid (SF) levels of IL-12 are elevated in RA patients and correlated well with RA disease activity [4]. Recently, novel proteins containing the p40 subunit or its relatives have been found and characterized, and together with the prototype IL-12p70, these molecules now establish the IL-12 family. The fact that the new members of the IL-12 family also appear to contribute distinct functions to each stage of Th1 development raises an intriguing concern because the detection and/or blocking of IL-12 heterodimer has relied on antibodies against the p40 subunit [5]. In other words, it is now necessary to re-evaluate the cellular functions that were credited to IL-12 with other members containing p40.

Among the IL-12 family, IL-23 is the first described novel member, and it consists of a common p40 and a unique p19 subunit [6]. The role of IL-23 in the process of Th1 differentiation is particularly interesting in the light of RA pathogenesis because it is known to promote IL-17 production from the memory type CD4 T-cells [7]. IL-17 is a major T-cell-derived cytokine in RA synovium that stimulates synovial fibroblasts to produce inflammatory cytokines and chemokines [8]. Previously, we observed a reciprocal activation of antigen-stimulated T-cells and synovial fibroblasts from RA patients, where the IL-17 production from T-cells increased upon co-culture with the fibroblast-like synoviocytes [9]. A variety of cytokines, including IL-15, IL-6, MCP-1 and IL-23, are known to regulate the release of IL-17 from T-cells. To be more specific, they stimulate memory CD4+ T-lymphocytes to produce IL-17 [7, 10, 11]. It is quite probable that IL-23 is involved in the up-regulation of IL-17; however, the production mechanism of IL-23 has not been assessed in RA synovium.

So far, little is known about the interaction of IL-23 and IL-17 in RA pathogenesis. Although IL-23, like IL-12, is thought to have an important role in autoimmune diseases, the control mechanism of IL-23 production has not yet been fully understood. IL-1ß, tumour necrosis factor (TNF)-{alpha} and prostaglandin E2 (PGE2) are only known to induce the production of IL-23 in human colonic subepithelial myofibroblasts and bone marrow dendritic cells (DC) [12, 13]. Based on the fact that IL-23 regulates the production of IL-17, we formulate a hypothesis that IL-17 may also affect the expression of IL-23p19 as a part of the reciprocal action between two cytokines that accentuate the vicious circle of RA inflammation. To this end, we investigated whether the expression of IL-23p19 is elevated in RA and whether it is up-regulated by IL-17 stimulation. We also determined which signal pathways are involved in the regulation of IL-23p19 expression by IL-17 in RA synovial fibroblasts.


   Methods and Materials
Top
 Abstract
 Introduction
 Methods and Materials
Results
 Discussion
 Acknowledgements
 References
 
Patients
SF and sera were obtained from 38 RA patients fulfilling the 1987 revised criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) [14]. Comparison was made with 44 patients with osteoarthritis (OA) and with 46 healthy volunteers as age- and sex-matched controls. Informed consent was obtained from each patient, and the experimental protocol was approved by the Catholic University of Korea Human Research Ethics Committee. Synovial tissues were isolated from seven RA patients (mean age 56.6 ± 4.7, range 32–70 yrs) undergoing total knee replacement surgery.

Isolation of synovial fibroblasts
Synoviocytes were isolated by the enzymatic digestion of synovial tissues obtained from RA patients undergoing total joint replacement surgery. The tissues were minced into 2–3 mm pieces and treated for 4 h with 4 mg/ml of type I collagenase (Worthington Biochemical, Freehold, NJ, USA) in Dulbecco's modified Eagle's medium (DMEM) at 37°C in 5% CO2. Dissociated cells were then centrifuged at 500 g, re-suspended in DMEM supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin, and plated in 75 cm2 flasks. After overnight culture, non-adherent cells were removed, and adherent cells were cultivated in DMEM supplemented with 20% FCS. The cultures were kept at 37°C in 5% CO2, and the medium was replaced every 3 days. When the cells approached confluence, they were passed after 1:3 diluting with fresh medium. Synoviocytes from passages 4–8 were used in each experiment. The cells were morphologically homogeneous and exhibited the appearance of synovial fibroblasts, with typical bipolar configuration under inverse microscopy. The purity of the cells (1 x 104) was tested by flow-cytometric analysis using phycoerythrin-conjugated anti-CD14 (PharMingen, San Diego, CA, USA) and fluorescein isothiocyanate-conjugated anti-CD3 or anti-Thy-1 (CD90) monoclonal antibodies (PharMingen). At passage 4, most cells (>95%) expressed the surface markers for fibroblasts (Thy-1), whereas 3.5% of the cells were CD14+ and <1% of cells were CD3+.

Reagents
Recombinant IL-17, IL-15 and IFN-{gamma} were purchased from R&D Systems (Minneapolis, MN, USA), and recombinant TNF-{alpha} and IL-1ß from Endogen Inc. (Cambridge, MA, USA). Pyrrolidine dithiocarbamate (PDTC), curcumin and parthenolide were from Sigma Chemical Co. (St Louis, MA, USA), and LY294002, SB203580 and SP600125 from Calbiochem (Schwalbach, Germany).

Concentrations of IL-23p19 determined by sandwich enzyme-linked immunosorbent assays (ELISA)
Concentrations of IL-23p19 in sera and SF were measured by sandwich ELISA, as previously described [15]. Two micrograms per millilitre of antibodies to human IL-23p19 (R&D Systems) was added to a 96-well plate (NUNC, Denmark) and incubated overnight at 4°C. After treating with blocking solution [phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 0.05% Tween-20] for 2 h at room temperature, test samples and the standard recombinant IL-23p19 (R&D Systems) were added to the 96-well plate and incubated at room temperature for 2 h. After washing four times with PBS containing Tween-20, 300 ng/ml of biotinylated IL-12p40 monoclonal antibodies to human cytokines (R&D Systems) were added and the reactions were allowed to proceed for 2 h at room temperature. After washing, 2000-fold diluted streptavidin-alkaline-phosphate (Sigma Bioscience, St Louis, MO, USA) was added and the reaction was again allowed to proceed for 2 h. After washing four times, 50 µl of diluted avidin peroxidase (1:2000 in diluent) was added. After incubating for 2 h at room temperature, tetramethyl benzidine substrate solution (Kirkegaard & Perry Laboratories, Guildford, UK) was added to each well (50 µl) and incubated for 20–30 min. Initially the reaction produced a blue colour that was monitored by absorbance at 595 nm with a microplate reader (MRX Revelation, Dynex Technologies, Chantilly, VA, USA). When the desired intensity was reached [<0.8 optical density (OD)], sulfphuric acid (2.0 mol/l) was added to each well (50 µl) to stop the colour-generating reaction. An automated microplate reader (Vmax, Molecular Devices, Palo Alto, CA, USA) set at 450 nm was used to measure the OD. The sensitivity limit was 15.6 pg/ml for IL-23p19. Recombinant human cytokines diluted in culture medium were used as a calibration standard, ranging from 10 to 2000 pg/ml. A standard curve was drawn by plotting OD vs the log of the concentration of recombinant cytokine.

Expression of IL-23p19 mRNA determined by reverse transcription polymerase chain reaction (RT-PCR)
Synovial fibroblasts were incubated with various concentrations of IL-17 in the presence or absence of various signal inhibitors (LY294002, PDTC, SB203580, parthenolide, curcumin and SP600125). After 12 h of incubation, mRNA was extracted using RNAzol B (Biotex Laboratories, Houston, TX, USA) according to the manufacturer's instructions. RT of 2 µg total mRNA was carried out at 42°C using the SuperscriptTM RT system (Takara, Shiga, Japan). PCR amplification of cDNA aliquots was performed by adding 2.5 mM dNTPs, 2.5 U Taq DNA polymerase (Takara) and 0.25 µM of sense and antisense primers. The reaction was done in 25 µl of PCR buffer (1.5 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl, pH 8.3). The following sense and antisense primers for each molecules were used (5'->3'): IL-23p19 sense GCA GAT TCC AAG CCT CAG TC, IL-23p19 antisense TTC AAC ATA TGC AGG TCC CA, IL-12p35 sense GCC CTG TGC CTT AGT AGT AT, IL-12p35 antisense GCT CGT CAC TCT GTC AAT AG and GAPDH sense CGA TGC TGG GCG TGA GTA C, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antisense CGT TCA GCT CAG GGA TGA CC. Reactions were processed in a DNA thermal cycler (Perkin-Elmer Cetus, Wellesley, MA, USA) through 25 cycles of 30 s of denaturation at 94°C, 1 min of annealing at 55°C for GAPDH, IL-12p35 and at 60°C for IL-23p19, followed by 30 s of elongation at 72°C. PCR products were run on a 1.5% agarose gel and stained with ethidium bromide. Results are expressed as the ratio of IL-23p19 and IL-12p35 products to GAPDH product.

Expression of IL-23p19 mRNA determined by real-time PCR with SYBR Green I
Real-time PCRs were performed in 20 µl final volumes in capillary tubes in a LightCycler instrument (Roche Diagnostic, Mannheim, Germany). Reaction mixtures contained 2 µl of LightCycler FastStart DNA mastermix for SYBR Green I (Roche Diagnostic), 0.5 µM each primer, 4 mM MgCl2 and 2 µl of template DNA. All capillaries were sealed, centrifuged at 500g for 5 s, and then amplified in a LightCycler instrument, with activation of polymerase (95°C for 10 min), followed by 45 cycles of 10 s at 95°C, 10 s at 60°C and 10 s at 72°C. The temperature transition rate was 20°C/s for all steps. Double-stranded PCR product was measured during the 72°C extension step by detection of fluorescence associated with the binding of SYBR Green I to the product. Fluorescence curves were analysed with LightCycler software v. 3.0. For quantification analysis of IL-23p19 mRNA, LightCyclerTM (Roche Diagnostics) was used. Relative levels of expression levels of samples were calculated by IL-23p19 levels normalized to the endogenously expressed housekeeping gene (HPRT). Melting-curve analysis was performed immediately after the amplification protocol under the following conditions: 0 s (hold time) at 95°C, 15 s at 65°C and 0 s (hold time) at 95°C. Temperature change rates were 20°C/s, except in the final step, which was 0.1°C/s. The melt peak generated represented the specific amplified product. The crossing point (Cp) was defined as the maximum of the second derivative from the fluorescence curve. Negative controls were also included and contained all the elements of the reaction mixture except template DNA. All samples were processed in duplicate.

Western blot analysis
Synovial fibroblasts were pretreated for 1 h with signal pathway inhibitors (LY294002, PDTC, SB203580, parthenolide, curcumin and SP600125). In the presence of IL-17 (1 ng/ml), they were cultured for 1 h [for Akt IkB-a and p38 mitogen-activated protein kinase (MAPK)] and 24 h (for IL-23p19). Then, whole-cell lysates were prepared from about 2 x 105 cells by homogenization in the lysis buffer and centrifuged at 14 000 r.p.m. for 15 min. Appropriate concentrations of each inhibitor were based on the methods previously described [16–19]. Protein concentrations in the supernatants were determined using the Bradford method (BioRad, Hercules, CA, USA). Protein samples were separated on 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Uppsala, Sweden). For western hybridization, the membrane was pre-incubated with 0.1% skimmed milk in TTBS (0.1% Tween-20 in Tris-buffered saline) at room temperature for 2 h. Antibodies to Akt, phosphorylated Akt, I{kappa}B-{alpha}, phosphorylated I{kappa}B-{alpha} (Cell Signaling Technology Inc., Beverly, MA, USA), P38 MAPK, phosphorylated P38 MAPK (Santa Cruz, Biotechnology Inc., CA, USA) and IL-23p19 (R&D Systems) diluted 1:1000 in 5% BSA, 0.1% Tween-20/TBS, were added to the membrane and incubated overnight at 4°C. After washing four times with TTBS, 100 ng/ml horseradish-peroxidase-conjugated secondary antibodies were added and incubated for 1 h at room temperature. After TTBS washing, hybridized bands were detected using the enhanced chemiluminescence (ECL) detection kit and Hyperfilm-ECL reagents (Amersham Pharmacia).

Cell viability (Trypan blue dye exclusion assay)
In cell viability assays, the trypan blue dye exclusion method was used to evaluate the potential of direct cytotoxic effect of inhibitors on cells. Following a 24 h incubation, the cells were harvested and the percentage cell viability was expressed by the formula (number of viable cells/number of both viable and dead cells) x 100.

Statistical analysis
Data are expressed as the mean ± SEM. Statistical analysis was performed using Mann–Whitney U-test for independent samples and Wilcoxon signed rank test for related samples. Correlation coefficients were determined by Spearman's rank correlation test. P-values <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Methods and Materials
 Results
Discussion
 Acknowledgements
 References
 
IL-23p19 expression in sera, SF and synovial fibroblasts of RA patients
Serum and SF levels of IL-23p19 from 38 RA patients and 44 OA patients, and serum levels of 46 healthy controls were measured by the sandwich ELISA method. The 38 patients with RA included 6 men and 32 women, with a mean age 50.4 ± 1.5 yrs (range 23–77 yrs) and mean disease duration 71.5 ± 8.2 months (range 3–240 months). Erythrocyte sedimentation rate (ESR) was 40.2 ± 3.8 mm/h, C-reactive protein (CRP) was 2.6 ± 0.4 mg/dl, and 24 patients (63.2%) had positive rheumatoid factor with the mean titre of 110.8 ± 27.7 IU/ml (range 6.7–1060 IU/ml). SF levels of IL-23p19 were much higher in RA patients than in OA patients (9123.8 ± 1632.9 pg/ml vs 507.6 ± 112.1 pg/ml; P < 0.001). The serum IL-23p19 levels were also higher in RA patients than in OA patients (5512.0 ± 548.5 pg/ml vs 1772.9 ± 735.0 pg/ml; P < 0.001) and healthy controls (1553.5 ± 317.7 pg/ml; P < 0.001) (Fig. 1a and b). In the un-stimulated synovial fibroblasts, the basal expression of IL-23p19 mRNA was much higher in RA than in OA as determined by RT-PCR and real-time PCR (Fig. 1c).


Figure 1
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  		FIG. 1. Concentrations of IL-23p19 subunit in patients with RA. (a) Concentrations of SF IL-23p19 in RA and OA patients; (b) serum concentrations of IL-23p19 in RA and OA patients and healthy volunteers were determined by sandwich ELISA. Values are the mean and S.E.M. of duplicates. Both SF and serum concentrations of IL-23p19 were significantly higher in RA patients than in controls. (c) RT-PCR analysis revealed that the expression of IL-23p19 mRNA is higher in unstimulated RA synovial fibroblasts than in OA synovial fibroblasts. The data represent mean ± S.E.M. of three separate experiments. Results are expressed as the ratio of the densitometric intensity of the cytokine product to the densitometric intensity of the GAPDH product. *P < 0.05 and **P < 0.001.

 
Expression of IL-23p19 mRNA inRA synovial fibroblasts
To define what is causing the over-expression of IL-23p19 in RA, the synovial fibroblasts were stimulated with various inflammatory cytokines. RA synovial fibroblasts were cultured in the presence of IL-17 (20 ng/ml), IL-1ß (10 ng/ml), TNF-{alpha} (1 ng/ml), IFN-{gamma} (10 ng/ml) and IL-15 (10 ng/ml), and the expression of IL-23p19 mRNA was determined by RT-PCR and real-time PCR analyses. A low level of constitutive IL-23p19 mRNA expression was detectable in non-stimulated synovial fibroblasts of RA. With the stimulation of exogenous IL-17, as well as TNF-{alpha} and IL-1ß, the expression of IL-23p19 mRNA was increased. But IFN-{gamma} and IL-15 did not increase the expression of IL-23p19 mRNA (Fig. 2a and b).


Figure 2
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  		FIG. 2. Effects of various cytokines on IL-23p19 mRNA expression by RA synovial fibroblasts. RA synovial fibroblasts were incubated for 12 h in the presence of IL-15 (10 ng/ml), IL-1ß (10 ng/ml), TNF-{alpha} (1 ng/ml), IFN-{gamma} (10 ng/ml) and IL-17 (20 ng/ml). Analysis of IL-23p19 mRNA expression was performed using (a) RT-PCR and (b) quantitative real-time PCR. The data represent mean ± S.E.M. of three separate experiments. Results are expressed as the ratio of the densitometric intensity of the cytokine product to the densitometric intensity of the HPRT product. Lane 1: unstimulated, lane 2: stimulated with IL-15 (10 ng/ml), lane 3: stimulated with IL-1ß (10 ng/ml), lane 4: stimulated with IFN-{gamma} (10 ng/ml) and lane 5: stimulated with IL-17 (20 ng/ml). *P < 0.05

 
To better characterize the effects of IL-17 on IL-23p19 expression by RA synovial fibroblasts, we performed the dose–response and time-course studies of IL-17-induced IL-23p19 expression using RT-PCR and real-time PCR. As shown in Fig. 3a, the expression of IL-23p19 mRNA was strongest when IL-17 was given at 1 ng/ml and gradually declined at higher doses. In a time-course analysis, the level of IL-23p19 mRNA was increased 3 h after the addition of recombinant IL-17, and reached the highest level at 12 h (Fig. 3b). Results using real-time PCR were somewhat similar to the results from RT-PCR. The expression of IL-23p19 mRNA was also strongest when IL-17 was given at 1 ng/ml, and the level of IL-23p19 mRNA was increased 3 h after the addition and reached the highest level at 9 h and declined (Fig. 3c and d). On the other hand, the expression of IL-12p35 mRNA remained largely unchanged in RA synovial fibroblasts upon IL-17 stimulation (Fig. 3a and b). Cultured OA synovial fibroblasts did not show any significant increase after IL-17 stimulation (data not shown).


Figure 3
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  		FIG. 3. Dose and time dependent effects of IL-17 stimulated IL-23p19 mRNA expression in synovial fibroblasts from patients with RA. (a, c) Synovial fibroblasts were incubated with rhIL-17 (0.1–50 ng/ml) for 12 h. RNA was then extracted and determined using RT-PCR and real-time PCR. (b, d) Synovial fibroblasts were incubated with 1 ng/ml of rhIL-17 for 0–24 h. mRNA was then extracted and determined using RT-PCR and real-time PCR. The data represent mean ± S.E.M. of three separate experiments. Results are expressed as the ratio of the densitometric intensity of the cytokine product to the densitometric intensity of the GAPDH and HPRT product. *P < 0.05.

 
IL-17-mediated IL-23p19 induction in RA synovial fibroblasts involves the activation of PI3K/Akt, p38 MAPK and NF-{kappa}B signalling pathways
To determine the signal transduction pathways mediating the up-regulation of IL-23p19 by IL-17, we used 1 µM of parthenolide, which antagonizes the activation of nuclear factor (NF)-{kappa}B, 2 µM of LY294002, which antagonizes the activation of phosphatidylinositol (PI) 3-kinase, and 1 µM of SB203580, which antagonizes the activation of p38 MAPK. One micromolar of curcumin was tested as an antagonist of activator protein (AP)-1 and 1µM of SP600125 as an inhibitor of c-Jun N-terminal kinase (JNK). RA synovial fibroblasts were pre-incubated for 1 h with these inhibitors and stimulated with 1 ng/ml of IL-17 for 12 h (for PCR) and 24 h (for western blot). The expression of IL-23p19 mRNA and protein was determined by RT-PCR and western blotting, respectively. The expression of IL-23p19 mRNA and protein was completely blocked after inhibiting the activities of PI3K/Akt, p38 MAPK and NF-{kappa}B (Fig. 4a and b). In contrast, disruption of AP-1 and JNK activities showed no effect on the IL-17-induced IL-23p19 expression. Neither the expression of IL-12p35 mRNA in RA synovial fibroblasts nor the expression of IL-23p19 mRNA in OA synovial fibroblasts was affected by any of the inhibitors tested (data not shown). These results suggest that IL-17 regulates the expression of IL-23p19 in RA synovial fibroblasts via PI3K/Akt-, p38 MAPK- and NF-{kappa}B-mediated pathways. Cytotoxic effects on synovial fibroblasts by the chemical inhibitors at experimental concentrations were not observed (data not shown).


Figure 4
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  		FIG. 4. Effect of inhibitors of signal pathway molecules on IL-17-induced IL-23p19 mRNA and protein expression by RA synovial fibroblasts. RA synovial fibroblasts were pre-treated for 1 h with parthenolide (10 µM), LY294002 (20 µM), SB203580 (10 µM), curcumin (10 µM) and SP604123 (1 µM) then cultured with rhIL-17 (1 ng/ml) for 12 h. (a) mRNA was then extracted and semi-quantitated using RT-PCR. The data represent mean ± SEM of three separate experiments. Results are expressed as the ratio of the densitometric intensity of the cytokine product to the densitometric intensity of the GAPDH product in 3 patients with RA. Lane 1: unstimulated, lane 2: stimulated with IL-17, lane 3: cultured with IL-17 and parthenolide, lane 4: cultured with IL-17 and LY294002, lane 5: cultured with IL-17 and SB203580, lane 6: cultured with IL-17 and curcumin and lane 7: cultured with IL-17 and SP600125. (b) IL-23p19 protein was extracted and assayed using western blot. Results are expressed as the ratio of the densitometric intensity of the cytokine product to the densitometric intensity of the ß-actin product. The data represent one of three independent experiments. *P < 0.05.

 
To confirm the activation of these intracellular signal molecules by the addition of exogenous IL-17, the phosphorylation of Akt and p38 MAPK was determined by western blotting. Activation of NF-{kappa}B was analysed by monitoring the phosphorylation of I{kappa}B. IL-17 activated the phosphorylation of Akt, p38 MAPK and I{kappa}B-{alpha} in synovial fibroblasts and concomitantly increased the production of IL-23p19 by 3-, 3.8- and 5-fold, respectively (Fig. 5), while the amount of total protein levels of Akt, p38 MAPK and I{kappa}B-{alpha} remained unchanged. As expected, co-treatment with chemical inhibitors of PI3-kinase, p38 MAPK and NF-{kappa}B, LY294002, SB203580 and PDTC, respectively, abolished the IL-17-instigated phosphorylation of Akt, p38 MAPK and I{kappa}B-{alpha} (data not shown).


Figure 5
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  		FIG. 5. IL-17 stimulation activates phosphorylation of (a) Akt, (b) I{kappa}B-{alpha} and (c) P38 MAPK in RA synovial fibroblasts. The activated forms of Akt, I{kappa}B and P38 MAPK were detected by western blot analysis in RA synovial fibroblasts stimulated with IL-17, while the amounts of total Akt, I{kappa}B and P38 MAPK were unchanged. Lane 1: unstimulated and lane 2: stimulated with rhIL-17 (1 ng/ml). The data represent one of the three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
Acknowledgements
 References
 
Until now, IL-12 is well-known to have an important role in Th1-mediated autoimmune diseases, such as RA, as it differentiates naïve T-cells into IFN-{gamma}-producing Th1 cells [20]. Recently, IL-23, which shares the common p40 subunit with IL-12, was discovered to be responsible for much of the proinflammatory effects that had been credited to IL-12 [6]. Especially in the experimental RA models, IL-23, not IL-12, has been found to have a central role in the pathogenesis of RA [21]. IL-23 is also gaining much attention for its role in autoimmune inflammation of the brain [22]. IL-23 shares p40 subunit with IL-12 and exhibits IL-12-like immunobiological functions such as the promotion of IFN-{gamma} production and T-cell proliferation. But while IL-12 acts on naïve CD4+ T-cells, IL-23 stimulates mainly memory CD4+ T-cells [6, 23], and induces DC to produce IFN-{gamma} and IL-12 in vitro [24]. IL-23p19 is expressed in monocytes, activated DC, endothelial cells and Th1 cells [6], and up-regulated in inflammatory diseases such as autoimmune encephalitis and psoriasis vulgaris [22, 25]. The fact that IL-23 rather than IL-12 is essential for the development of autoimmune inflammation suggests another aspect of IL-12, which has been characterized as a major cytokine of Th1-skewing autoimmune diseases. The characteristic of IL-23 is completely different from that of IL-12 in the autoimmune disease, and the loop of IL-23 and IL-17, rather than the loop of IL-12 and IFN-{gamma}, is newly known to have a central role in the autoimmune inflammation. However, detailed mechanism of the regulation of IL-23 expression is still to be investigated.

IL-17 is mainly produced by the CD4+CD45RO+ memory T-cells, and over-expression of IL-17 is detected in the SF and synovial tissues of RA patients [29, 26–28]. IL-17 plays a critical role in the inflammatory process of RA, and recently it becomes a subject of special interest in RA pathogenesis. It stimulates the production and expression of proinflammatory cytokines such as IL-1ß and TNF-{alpha} from monocytes or macrophages [30], IL-6 and IL-8 from RA synovial fibroblasts [8, 31] and chemokines, such as CCL20 [32]. Furthermore, IL-17 contributes to bone erosion and tissue destruction. It induces chondrocytes and synovial fibroblasts to produce PGE2 [33] and up-regulates nitric oxide production [34]. IL-17 induces IL-6 and RANKL production of T-cells and osteoblasts [35]. Taken together, IL-17 appears to regulate the inflammatory and destructive process in RA, and great attention has been given to the role of IL-17 in the pathogenesis [30, 32].

The interplay between IL-17 and IL-23 may be critical in RA. IL-23 induces IL-17 production in a particular subset of memory CD4+ T-cells, named ThIL-17 cells [7]. Regarding the elevated expression of IL-17 receptor (IL-17R) mRNA in SF cells in RA patients [36], it is possible to propose that synovial fibroblasts have the potential to respond to IL-17 stimulation, and the mutual activation of IL-17 and IL-23 may cojoin the adaptive and innate parts of the immune system [30]. However, the effect of IL-17 on IL-23-producing cells has not been elucidated. This study addresses the question of whether the expression of IL-23p19 is elevated in human RA and whether IL-17 can act as an inducer of IL-23p19 production. We hypothesized that IL-17, a T-cell cytokine, is one of the inducers of IL-23p19 expression from synovial fibroblasts. Since IL-23 is a potent inducer of IL-17 production in memory T-cells, such reciprocal action of two cytokines is likely to generate a vicious cycle in the joint inflammation. However, the effect of IL-23 on IL-17 expression in RA synovial cells needs to be substantiated by additional research.

We developed the technique of ELISA-based detection of IL-23p19 levels because there is no commercially available method for IL-23p19 detection. Levels of IL-23p19 were measured by homemade ELISA using anti-IL-23p19 antibodies for coating and anti-IL-23p40 antibodies for detection. This system renders a new measurement method specific for human IL-23p19 subunits without the interference by IL-12p35 or p40 subunits. Levels of IL-23p19 were found to be much higher in SF and sera of RA patients than in OA patients or healthy controls, but the clinical importance of elevated IL-23p19 needs to be studied further to understand its role in the development of RA pathogenesis. In our preliminary data, we found that IL-23p19-positive cells were readily detectable in RA synovium, mainly in synovial lining area, when the immunostaining of synovial sections from RA patients was performed using anti-IL-23p19 monoclonal antibodies (data not shown). This result is consistent with the elevated concentrations of the cytokine in synovial fluids of RA patients.

IL-23 is known to be induced by proinflammatory cytokines such as TNF-{alpha} and IL-1ß. In this study, we found that IL-23p19 is also induced by IL-17, a T-cell-derived cytokine with proinflammatory properties, as well as IL-1ß and TNF-{alpha} in RA synovial fibroblasts. Our data suggest that IL-17 from activated T cells (ThIL-17 cells) stimulates synovial fibroblasts to produce IL-23p19, which develops the autoimmune inflammation, resulting in tissue inflammation and joint destruction. Such interplay of two Th1 cytokines seems to have a critical role in the development of RA pathogenesis and joint inflammation. Of special interest is the concentration of IL-17 stimulating the expression IL-23p19 in RA synovial fibroblasts. In one previous experiment, the concentration of IL-17 was elevated in RA SF and the mean concentration of IL-17 was approximately 1 ng/ml [11]. The concentration was virtually identical with the concentration of IL-17 we chose in this study. The maximal effect of IL-17 on IL-23p19 expression is observed with 1 ng/ml and then decreases for higher concentrations. Proceeding from this fact, we could assume that the concentration of IL-17 in RA SF is regulated as the optimal concentration stimulating other cytokines, as well as IL-23p19, maximally.

Next, we investigated the signal pathways involved in the interaction between IL-17 and IL-23p19. The signal pathways instigated by IL-17 has already been well-documented [36, 37], but there is no data on how IL-17 regulates the expression of IL-23 in the inflammatory diseases of humans. Using various signal transduction inhibitors, we tested whether PI3K/Akt, p38 MAPK or NF-{kappa}B are involved in IL-17-induced IL-23p19 mRNA and protein expression. We also analysed whether these signalling mediators undergo phosphorylation after IL-17 stimulation. Our data clearly demonstrated that PI3K/Akt could be the upstream arbitrator of IL-17-mediated up-regulation of IL-23p19 in RA. We postulate that subsequent activation of NF-{kappa}B and p38 MAPK may also have contributed to the increased binding of inflammatory transcription factors to the promoter of IL-23p19 in IL-17-stimulated synovial fibroblasts.

In conclusion, this study has shown that the activity of IL-23p19 is up-regulated in RA and that IL-17 could be responsible for the increased activity of IL-23p19 in RA synovial fibroblasts. We postulate that the interplay of IL-17 of activated T-cells and IL-23p19 of synovial fibroblasts can have an important role in the RA pathogenesis. Interruption of this interaction between IL-17 and IL-23p19 may be considered as a new therapeutic approach to control joint inflammation in patients with RA.


   Acknowledgements
Top
 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
 Acknowledgements
References
 
This work was supported by grant through the RhRC (Rheumatism Research Center) at Catholic University of Korea.

Funding to pay the Open Access publication charges for this article was provided by R11-2002-098-05001-0 from Korea Science & Engineering Foundation.

The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
 Acknowledgements
 References
 

  1. Harris ED Jr. (1990) Rheumatoid arthritis. Pathophysiology and implications for therapy. N Engl J Med 322:1277–89.[Web of Science][Medline]
  2. Hoy MD, O’Donnell JL, Hart DN. (1993) Dual CD45RA, CD45RO positive T-lymphocytes within rheumatoid arthritic joints. Pathology 25:167–73.[Web of Science][Medline]
  3. Miossec P. (2004) An update on the cytokine network in rheumatoid arthritis. Curr Opin Rheumatol 16:218–22.[CrossRef][Web of Science][Medline]
  4. Kim W, Min S, Cho M, et al. (2000) The role of IL-12 in inflammatory activity of patients with rheumatoid arthritis (RA). Clin Exp Immunol 119:175–81.[CrossRef][Web of Science][Medline]
  5. Brombacher F, Kastelein RA, Alber G. (2003) Novel IL-12 family members shed light on the orchestration of Th1 responses. Trends Immunol 24:207–12.[CrossRef][Web of Science][Medline]
  6. Oppmann B, Lesley R, Blom B, et al. (2000) Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13:715–25.[CrossRef][Web of Science][Medline]
  7. Aggarwal S, Ghilardi N, Xie MH, de Sauvage FJ, Gurney AL. (2003) Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17. J Biol Chem 278:1910–4.[Abstract/Free Full Text]
  8. Hwang SY, Kim JY, Kim KW, et al. (2004) IL-17 induces production of IL-6 and IL-8 in rheumatoid arthritis synovial fibroblasts via NF-{kappa}B- and PI3-kinase/Akt-dependent pathways. Arthritis Res Ther 6:R120–8.[CrossRef][Web of Science][Medline]
  9. Cho ML, Yoon CH, Hwang SY, et al. (2004) Effector function of type II collagen-stimulated T cells from rheumatoid arthritis patients: cross-talk between T cells and synovial fibroblasts. Arthritis Rheum 50:776–84.[CrossRef][Web of Science][Medline]
  10. Kim KW, Cho ML, Park MK, et al. (2005) Increased interleukin-17 production via a phosphoinositide 3-kinase/Akt and nuclear factor kappaB-dependent pathway in patients with rheumatoid arthritis. Arthritis Res Ther 7:R139–48.[CrossRef][Web of Science][Medline]
  11. Ziolkowska M, Koc A, Luszczykiewicz G, et al. (2000) High levels of IL-17 in rheumatoid arthritis patients: IL-15 triggers in vitro IL-17 production via cyclosporin A-sensitive mechanism. J Immunol 164:2832–8.[Abstract/Free Full Text]
  12. Zhang Z, Andoh A, Yasui H, et al. (2005) Interleukin-1beta and tumor necrosis factor-alpha upregulate interleukin-23 subunit p19 gene expression in human colonic subepithelial myofibroblasts. Int J Mol Med 15:79–83.[Web of Science][Medline]
  13. Sheibanie AF, Tadmori I, Jing H, Vassiliou E, Ganea D. (2004) Prostaglandin E2 induces IL-23 production in bone marrow-derived dendritic cells. FASEB J 18:1318–20.[Abstract/Free Full Text]
  14. Arnett FC, Edworthy SM, Bloch DA, et al. (1988) The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31:315–24.[Web of Science][Medline]
  15. Verreck FA, de Boer T, Langenberg DM, et al. (2004) Human IL-23-producing type 1 macrophages promote but IL-10-producing type 2 macrophages subvert immunity to (myco)bacteria. Proc Natl Acad Sci USA 101:4560–5.[Abstract/Free Full Text]
  16. Tanaka K, Hasegawa J, Asamitsu K, Okamoto T. (2005) Prevention of the ultraviolet B-mediated skin photoaging by a nuclear factor {kappa}B inhibitor, parthenolide. J Pharmacol Exp Ther 315:624–30.[Abstract/Free Full Text]
  17. Morel J, Audo R, Hahne M, Combe B. (2005) Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces rheumatoid arthritis synovial fibroblast proliferation through mitogen-activated protein kinases and phosphatidylinositol 3-kinase/Akt. J Biol Chem 280:15709–18.
  18. Min SY, Hwang SY, Jung YO, et al. (2004) Increase of cyclooxygenase-2 expression by interleukin 15 in rheumatoid synoviocytes. J Rheumatol 31:875–83.[Abstract/Free Full Text]
  19. Ito H, Iwamoto I, Inaguma Y, et al. (2005) Endoplasmic reticulum stress induces the phosphorylation of small heat shock protein, Hsp27. J Cell Biochem 95:932–41.[CrossRef][Web of Science][Medline]
  20. Morita Y, Yamamura M, Nishida K, et al. (1998) Expression of interleukin-12 in synovial tissue from patients with rheumatoid arthritis. Arthritis Rheum 41:306–14.[CrossRef][Web of Science][Medline]
  21. Murphy CA, Langrish CL, Chen Y, et al. (2003) Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. J Exp Med 198:1951–7.[Abstract/Free Full Text]
  22. Cua DJ, Sherlock J, Chen Y, et al. (2003) Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421:744–8.[CrossRef][Medline]
  23. Frucht DM. (2002) IL-23: a cytokine that acts on memory T cells. Sci STKE 2002:PE1.
  24. Belladonna ML, Renauld JC, Bianchi R, et al. (2002) IL-23 and IL-12 have overlapping, but distinct, effects on murine dendritic cells. J Immunol 168:5448–54.[Abstract/Free Full Text]
  25. Lee E, Trepicchio WL, Oestreicher JL, et al. (2004) Increased expression of interleukin 23 p19 and p40 in lesional skin of patients with psoriasis vulgaris. J Exp Med 199:125–30.[Abstract/Free Full Text]
  26. Chabaud M, Durand JM, Buchs N, et al. (1999) Human interleukin-17: a T cell-derived proinflammatory cytokine produced by the rheumatoid synovium. Arthritis Rheum 42:963–70.[CrossRef][Web of Science][Medline]
  27. Honorati MC, Meliconi R, Pulsatelli L, Cane S, Frizziero L, Facchini A. (2001) High in vivo expression of interleukin-17 receptor in synovial endothelial cells and chondrocytes from arthritis patients. Rheumatology 40:522–7.[Abstract/Free Full Text]
  28. Kotake S, Udagawa N, Takahashi N, et al. (1999) IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis. J Clin Invest 103:1345–52.[Web of Science][Medline]
  29. Jovanovic DV, Di Battista JA, Martel-Pelletier J, et al. (1998) IL-17 stimulates the production and expression of proinflammatory cytokines, IL-beta and TNF-alpha, by human macrophages. J Immunol 160:3513–21.[Abstract/Free Full Text]
  30. Lubberts E, Koenders MI, van den Berg WB. (2005) The role of T cell interleukin-17 in conducting destructive arthritis: lessons from animal models. Arthritis Res Ther 7:29–37.[CrossRef][Web of Science][Medline]
  31. Yamamura Y, Gupta R, Morita Y, et al. (2001) Effector function of resting T cells: activation of synovial fibroblasts. J Immunol 166:2270–5.[Abstract/Free Full Text]
  32. Chabaud M, Page G, Miossec P. (2001) Enhancing effect of IL-1, IL-17, and TNF-alpha on macrophage inflammatory protein-3alpha production in rheumatoid arthritis: regulation by soluble receptors and Th2 cytokines. J Immunol 167:6015–20.[Abstract/Free Full Text]
  33. Stamp LK, Cleland LG, James MJ. (2004) Upregulation of synoviocyte COX-2 through interactions with T lymphocytes: role of interleukin 17 and tumor necrosis factor-alpha. J Rheumatol 31:1246–54.[Abstract/Free Full Text]
  34. Miljkovic D and Trajkovic V. (2004) Inducible nitric oxide synthase activation by interleukin-17. Cytokine Growth Factor Rev 15:21–32.[CrossRef][Web of Science][Medline]
  35. Udagawa N, Kotake S, Kamatani N, Takahashi N, Suda T. (2002) The molecular mechanism of osteoclastogenesis in rheumatoid arthritis. Arthritis Res 4:281–9.[CrossRef][Web of Science][Medline]
  36. Kehlen A, Thiele K, Riemann D, Langner J. (2002) Expression, modulation and signalling of IL-17 receptor in fibroblast-like synoviocytes of patients with rheumatoid arthritis. Clin Exp Immunol 127:539–46.[CrossRef][Web of Science][Medline]
  37. Attur MG, Patel RN, Abramson SB, Amin AR. (1997) Interleukin-17 up-regulation of nitric oxide production in human osteoarthritis cartilage. Arthritis Rheum 40:1050–3.[Web of Science][Medline]
Submitted 15 December 2005; revised version accepted 4 April 2006.
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