Gene expression induced by interleukin-17 in fibroblast-like synoviocytes of patients with rheumatoid arthritis: upregulation of hyaluronan-binding protein TSG-6
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Interleukin-17 (IL-17) has been characterized as a proinflammatory cytokine produced by CD4+ CD45RO+ memory T cells. Overproduction of IL-17 was detected in the synovium of patients with rheumatoid arthritis (RA) compared with patients with osteoarthritis. This study examines differentially expressed genes after the stimulation of fibroblast-like synoviocytes of RA patients by IL-17. Among these genes we identified the following: tumor necrosis factor-stimulated gene-6 (TSG-6), IL-6, IL-8, GRO-β, and bone morphogenetic protein-6 with an expression 3.6–10.6-fold that in the unstimulated control. IL-17 augmented the expression of TSG-6, a hyaluronan-binding protein, in a time- and dose-dependent manner. IL-17 showed additive effects with IL-1β and tumour necrosis factor-α on the expression of TSG-6, IL-6 and IL-8. The mitogen-activated protein kinase p38 seems to be necessary for the regulation of TSG-6 expression by IL-17, as shown by inhibition with SB203580. Our results support the hypothesis that IL-17 is important in the pathogenesis of RA, contributing to an unbalanced production of cytokines as well as participating in connective tissue remodeling.
Keywordsfibroblast-like synoviocytes interleukin-17 rheumatoid arthritis TSG-6
bone morphogenetic protein-6
mitogen-activated protein kinase
reverse transcriptase–polymerase chain reaction
tris-buffered saline containing Tween 20
tumour necrosis factor-α
product of TNF-stimulated gene-6.
Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by a relapsing and remitting course of joint inflammation. The chronic inflammation process leads to an excessive hyperplasia of the synovium with proliferation of the synovial lining cells, the generation of new blood vessels, and diffusely scattered or nodular mononuclear cell infiltrates. The proliferation and invasive growth of fibroblast-like cells of the synovium (fibroblast-like synoviocytes; SFCs) results ultimately in the destruction of the joint [1, 2]. Cytokines such as interleukin (IL)-1β or tumour necrosis factor-α (TNF-α) are known to be involved in the perpetuation of the chronic inflammation in RA [3, 4, 5, 6]. Overproduction of the proinflammatory cytokine IL-17 was detected in the RA synovium compared with patients with osteoarthritis [7, 8]. IL-17 is a 20–30 kDa glycosylated, homodimeric polypeptide secreted by CD4+ activated memory (CD45RO+) T cells [9, 10]. In the context of arthritis, the effects of IL-17 were associated with joint inflammation and destruction because of the IL-17-stimulated production of MMP-1 and MMP-9 and degradation of proteoglycan, and the IL-17-increased expression of IL-6 and leukemia inhibitory factor in SFCs [11, 12, 13, 14]. Recently we showed the increased expression of CXC chemokines such as IL-8, GRO-α and GRO-β after stimulation of SFCs with IL-17 . A blockade of IL-17 in vivo by treatment with a fusion protein of IL-17 receptor with human IgG1 Fc in adjuvant-induced arthritis decreased joint inflammation and bone erosion .
To gain knowledge about the effects of IL-17 in SFCs of patients with RA, we studied the expression and modulation of selected genes differentially expressed after stimulation with IL-17. Our results show that IL-17 is an important member of the cytokine network involved in RA.
Materials and methods
SFCs were obtained from nine patients with classical or definite RA (range of ages 27–71 years, with a mean age (± SEM) of 51.9 ± 16.4 years) undergoing surgical synovectomy, by dissociating the minced tissue enzymatically with HBSS (Hank's buffered saline solution) containing 0.5 mg/ml collagenase type II (Sigma, Deisenhofen, Germany), 0.15 mg/ml DNase I (Boehringer Mannheim, Germany) and 5 mM Ca2+. The cells were cultured in RPMI 1640 medium containing 10% fetal calf serum, antibiotics and glutamine, as described previously . Cells were used at confluence at the third to fifth passage. The inhibitors calphostin C (20 μg/ml; Sigma, Deisenhofen, Germany), PD98059 (10 μM; Alexis, Grünberg, Germany), SB203580 (1 μM; Calbiochem, Schwalbach, Germany) and genistein (10 μg/ml; Gibco BRL, Karlsruhe, Germany) were added to the cultures 30 min before incubation with IL-17. Rabbit neutralizing polyclonal anti-hIL-17 antibody (2 μg/ml; Cell Concepts) was co-incubated for 30 min with recombinant IL-17 and added to the cultures.
cDNA synthesis and cDNA array
Isolation of total cellular RNA was described previously . RNA was treated with DNase I (Qiagen) and resuspended in water. The first strand of DNA was synthesized (after a 10 min incubation at 20°C) at 42°C for 50 min by using 500 ng of total RNA in 5.5 μl of diethyl pyrocarbonate water (0.1% diethyl pyrocarbonate-treated water), 2 μl of 5 × first-strand buffer (250 mM Tris/HCl pH 8.3, 375 mM KCl, 15 mM MgCl2), 0.5 μl of dNTP mix (10 mM of each of dATP, dCTP, dGTP, and dTTP), 1 μl of 0.1 M dithiothreitol, 0.5 μl (50 pmol) of random primer (Roche, Mannheim, Germany), and 0.5 μl of Superscript™ II-RT (200 U/μl; Invitrogen, Karlsruhe, Germany). For quantitative RT–PCR, standard RNA and total RNA were converted into cDNA in separate tubes in triplicate.
Total RNA (4-μg, pooled from cultured SFCs of a female RA patient 41 years old) served as starting material for the preparation of a [α-32P]dCTP-labeled cDNA with the cDNA Synthesis Primer Mix (Clontech). For investigating differential gene expression, cDNA was hybridized to the Atlas™ Human 1.2 Array (Clontech) in accordance with the user manual. This array includes 1176 human cDNAs, housekeeping genes and negative controls immobilized on a nylon membrane. After hybridization and washing, the array membrane was exposed to a phosphorimaging screen. Data analysis was performed with AtlasImage Software 1.0. Expression values of transcripts were normalized to the total signal intensity on the membrane. In agreement with the indications of the manufacturers, transcripts with a ratio of normalized expression levels of more than 2 or less than 0.5 were regarded as modulated.
Construction of RNA standards2
Sequences of primers used for standard construction and PCR amplification
GAT TTA GGT GAC ACT ATA GAA TAC CCA GGC TTC CCA AAT GAG TA
TTG ATT TGG AAA CCT CCA GC
CCA GGC TTC CCA AAT GAG TA
GGC CAT TTT CTT GGA TTC CT
CCG AAG TCA TAG CCA CAC TC
GAA GAG CCC TCA GGC TGG ACT G
AGT AAC TCC TTC TCC ACA AGC GC
TCT CAG CCC TCT TCA AAA ACT TCT C
ATG ACT TCC AAG CTG GCC GTG GCT
CAC CAT GAA GGG CTG CTT AT
GGA CAC CCG TGT AGT ATG GG
GCT GCT ATG GGC AAA GTT TC
AGT AAT TCC AGC GAG AGG CA
Quantitative PCR analysis
For quantification, 1 μl of the reverse transcriptase reaction mixture was added to 25 μl of reaction mixture consisting of 1 × reaction buffer, 1.5 U of Taq polymerase (Qiagen), 1.8 mM MgCl2, 0.1 × SYBR Green (Biozym, Hess. Oldendorf, Germany), dNTPs (each at 200 μM), and primers 3 and 2 (each at 0.5 μM) (Table 1). A negative control without template was included. Samples of six dilutions of the standard cDNA and of the target cDNA were run in triplicate in a Rotor-Gene 2000 (LTF, Wasserburg, Germany). Initial denaturation at 95°C for 300 s was followed by 40 cycles denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and elongation at 72°C for 20 s. The fluorescence intensity of the double-strand-specific SYBR Green, reflecting the amount of PCR product formed, was read after each elongation step at 82°C. RNA amounts were determined with the software Rotor-Gene version 4.04 in quantitation mode.
Supernatants of IL-17-treated SFCs (20 ng/ml) were collected and, after being washed twice with ice-cold PBS, cells were harvested by scraping into ice-cold RIPA buffer (1 × PBS, 1% Nonidet P40, 0.5% sodium deoxycholate). Inhibitors were added in the following concentrations: 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mM Na3VO4, and 1 mM NaF (Sigma). The cell lysate was transferred to microcentrifuge tubes and incubated on ice for 60 min; centrifugation was for 20 min at 14,000 r.p.m. and 4°C. Protein concentration in the supernatant was quantified with the BCA (bicinchoninic acid) Protein Assay Reagent Kit (KMF, Leipzig, Germany), and 40 μg of cell lysate protein or cell culture supernatant was used for Western blot analysis. Proteins were electroblotted from NuPAGE gels (NOVEX, Frankfurt-Hoechst, Germany) onto Hybond ECL (enhanced chemiluminescence) membrane (Amersham, Freiburg, Germany). The membrane was blocked for 1 hour with 5% milk in Tris-buffered saline containing Tween 20 (TBST; pH 7.5, 0.1% Tween 20) at 23 ± 2°C. Blots were incubated with the primary antibody (against TNF-stimulated gene-6 [anti-TSG-6], 1:1000 dilution; kindly provided by Dr MT Bayliss, Oxford, UK) in TBST with 5% milk at 23 ± 2°C for 2 hours. Blots were washed three times and then incubated for 1 hour with the secondary antibody (1:1000 dilution; Dianova, Hamburg, Germany) coupled with horseradish peroxidase. Immunodetection was accomplished with ECL Western blotting detection reagents (Amersham) for chemiluminescent detection. Immunoreactivity was quantified by scanning densitometry with the software Scan Pack version 3.0 (Biometra).
Measurement of IL-6 and IL-8
SFCs were cultured for 72 hours in the presence of 20 ng/ml IL-17. Supernatants were collected and assayed for IL-6 and IL-8 with the Chemiluminescence-Enzyme Immunoassay System, Immulite (DPC Biermann, Bad Nauheim, Germany).
Data are expressed as means ± SEM. The Wilcoxon rank sum test was used to determine whether two experimental values were significantly different.
Transcriptional activation by IL-17
IL-17 shows additive effects with IL-1β and TNF-α on the expression of IL-6 and IL-8
Upregulation of hyaluronan-binding protein TSG-6 by IL-17
IL-17 was found at high levels in the RA synovium, and the concentration of this cytokine in synovial fluid of RA patients is elevated [7, 8]. For the first time we identified an increase in TSG-6 after stimulation of SFCs with IL-17. TSG-6 is a hyaluronan-binding protein found in the synovial fluids of arthritis patients. TSG-6 has a significant homology to the hyaluronan-binding regions present in cartilage link protein, aggrecan, and the adhesion receptor CD44 . The 35 kDa glycoprotein has a role in extracellular matrix remodeling, leucocyte migration, and cell proliferation [20, 21, 22]. TSG-6 forms a covalent complex with the serine protease inhibitor inter-α-inhibitor, and thereby increases its anti-plasmin activity. This suggests a role for TSG-6 in the regulation of the plasmin/plasminogen activator system and therefore the control of growth factor and matrix metalloproteinase activation [23, 24]. In TSG-6 transgenic mice with an antigen-induced arthritis, TSG-6 shows a cartilage-specific constitutive expression and provides chondroprotective, but not anti-inflammatory, effects . Similar results were obtained in TSG-6 transgenic mice with collagen type II-induced arthritis. TSG-6 was locally expressed at sites of inflammation and joint destruction, and resulted in potent inhibition of joint destruction . These findings support the hypothesis that endogenously produced TSG-6 can be part of a negative feedback loop in the inflammatory response . IL-17 most probably has a dual role at sites of inflammation, supporting the local inflammatory response but simultaneously delivering the anti-inflammatory TSG-6 protein.
Among the set of upregulated genes after treatment with IL-17, as expected we found IL-6 and the CXC chemokines IL-8 and GRO-β. IL-6, a pleiotropic cytokine in rheumatoid joints produced predominantly by SFCs and synovial macrophages, has a major role in cellular activation [27, 28]. CXC chemokines are potent neutrophil chemoattractants and have been detected in synovial fluids, synovial tissues, and sera of RA patients (reviewed in ). With the use of cDNA microarray technology in RA tissue, high chemokine expression has been verified, including that of the CXC chemokines IL-8 and GRO-α . IL-8 induces synovial inflammation , might be involved in the regulation of collagen turnover in SFCs , and can stimulate angiogenesis .
Furthermore, we identified the BMP-6 gene as a gene upregulated by IL-17. BMP-6 is a member of the transforming growth factor-β superfamily with a role in chondrogenesis and the osteoblastic diffentiation process and also serves as a mediator of estrogen's osteogenic action [33, 34, 35]. The expression of the proto-oncogene c-myc is not disease-specific in synovial cells because equal levels in samples of patients with RA or osteoarthritis have been reported . Interestingly, antisense oligonucleotides of c-myc can induce apoptosis and downregulation of Fas expression in synoviocytes .
The present study demonstrates the potency of IL-17, IL-1β, and TNF-α alone and also in combination to induce the synthesis of IL-6, IL-8, and TSG-6 by SFCs. The three cytokines activate the common transcription factor NF-κB in SFCs and in a variety of other cell types . Indeed, combination of IL-17 with IL-1β often leads to synergistic or additive effects [9, 11, 38, 39]. In contrast, Lubberts et al. reported an IL-1-independent role of IL-17 in synovial inflammation and joint destruction in the autoimmune collagen-induced arthritis model. Local overexpression of IL-17 in the knee joint of mice immunized with collagen type II resulted in elevated levels of IL-1β in the synovium. Blocking IL-1 with neutralizing antibodies had no effect on the IL-17-induced inflammation and joint damage, implying a pathway independent of IL-1 . The interaction of the cytokines IL-1β, IL-17, and TNF-α sustained inflammatory processes within the joint and amplified the involvement of T cells in the pathogenesis of RA.
We and others have found that IL-17 is capable of stimulating the mitogen-activated protein kinase (MAPK) signaling pathways ERK1/2 and p38 as well as the NF-κB pathway [41, 42, 43]. However, with the inhibitors genistein, calphostin C, or PD98059 we observed no significant decrease in the amounts of TSG-6 mRNA. MAPK p38 seems necessary for the expression of TSG-6, shown by inhibition with SB203580, and is involved in the IL-17-enhanced production of inducible nitric oxide synthase and secretion of chemokines [15, 42], and has a role in the MMP-9 expression induced by IL-17 .
Our results support the hypothesis that IL-17 might have a significant role in the pathogenesis of RA and might contribute to an unbalanced production of cytokines as well as participating in connective tissue remodeling. However, a deeper understanding of the effects of the IL-17 seems necessary for the understanding of its functions as well as for a development of therapeutic approaches including IL-17 and IL-17 receptor as a target.
We thank Sandra Fuhrmann for technical assistance, Kersten Fischer (Department of Urology, Halle) for quantification of IL-6 and IL-8 protein, Mike Schlicker (Department of Human Genetics, Halle) for support in the array procedure, and Markus Benicke (Department of Pathology, Leipzig) for help with the AtlasImage software.
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