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. 2002 Mar;127(3):547–552. doi: 10.1046/j.1365-2249.2002.01785.x

Increased expression of pro-inflammatory cytokines and metalloproteinase-1 by TGF-β1 in synovial fibroblasts from rheumatoid arthritis and normal individuals

H CHEON *,†, S-J YU *,†, D H YOO ‡, I J CHAE §, G G SONG , J SOHN *,*†
PMCID: PMC1906321  PMID: 11966774

Abstract

Transforming growth factor (TGF)-β1 is expressed abundantly in the rheumatoid synovium. In this study, the inflammatory effect of TGF-β1 in rheumatoid arthritis (RA) was investigated using cultured fibroblast-like synoviocytes (FLS) from RA and osteoarthritis (OA) patients, as well as non-arthritic individuals. mRNA expressions of IL-1β, tumour necrosis factor (TNF)-α, IL-8, macrophage inflammatory protein (MIP)-1α and metalloproteinase (MMP)-1 were increased in RA and OA FLS by TGF-β1 treatment, but not in non-arthritic FLS. Enhanced protein expression of IL-1β, IL-8 and MMP-1 was also observed in RA FLS. Moreover, TGF-β1 showed a synergistic effect in increasing protein expression of IL-1β and matrix metalloproteinase (MMP)-1 with TNFα and IL-1β, respectively. Biological activity of IL-1 determined by mouse thymocyte proliferation assay was also enhanced by 50% in response to TGF-β1 in the culture supernatant of RA FLS. DNA binding activities of nuclear factor (NF)-κB and activator protein (AP)-1 were shown to increase by TGF-β1 as well. These results suggest that TGF-β1 contributes for the progression of inflammation and joint destruction in RA, and this effect is specific for the arthritic synovial fibroblasts.

Keywords: IL-1β, IL-8, MIP-1α, rheumatoid arthritis, TGF-β1, TNFα, metalloproteinase-1

INTRODUCTION

Rheumatoid arthritis (RA) is a chronic and systemic inflammatory disease characterized by progressive destruction of joints. Hypertrophic RA synovial tissue is comprised of large numbers of infiltrating inflammatory cells and excessively proliferating synovial lining cells which produce several inflammatory as well as anti-inflammatory cytokines. Analyses of the cytokine mRNA and proteins in the RA synovial tissue revealed that TNFα, IL-1, IL-6, GM-CSF and TGF-β were expressed at high levels in RA patients [1–3]. Among these cytokines, TNFα and IL-1β are known to be the pivotal pro-inflammatory cytokines in the pathogenesis of RA. However, the role of other cytokines in RA has not yet been fully understood.

The presence of TGF-β1 at a high level in the RA synovium suggests that TGF-β1 per se or in combination with other cytokines plays an important role in the progression of RA. Although TGF-β1 is well known for its immune-suppressive and anti-inflammatory properties, it is also capable of promoting inflammation [4]. In a RA animal model, injections of TGF-β into the synovium induced an inflammatory response with accumulation of neutrophils, and exacerbated arthritic responses [5,6]. Moreover, anti-TGF-β antibody blocked accumulation of inflammatory cells and tissue pathology in an experimental model of chronic erosive polyarthritis [4].

Although it is not clearly understood, there are several ways by which TGF-β can regulate RA pathogenesis. First, TGF-β can modulate expression of inflammatory cytokines such as TNFα and IL-1β[7]. Secondly, production and activity of metalloproteinases are regulated by TGF-β[8–10]. Thirdly, TGF-β1 is strongly chemotactic and may attract inflammatory cells to synovial tissue [4,6]. Fourthly, synovial hypertrophy can be accelerated by TGF-β since it induces proliferation of fibroblasts [7] and may also modulate apoptosis of synovial fibroblasts. Finally, VEGF is strongly induced by TGF-β, and therefore, TGF-β can contribute indirectly to angiogenesis in arthritic synovium [11].

In the present study, we have investigated the effect of TGF-β1 on the expression of inflammatory cytokines and MMP-1 in RA FLS. The results indicated that TGF-β1 induced or increased the expressions of IL-1β, TNFα, IL-8, MIP-1α and MMP-1, and synergized with other proinflammatory cytokines in RA FLS. These effects of TGF-β1 were similar in RA and osteoarthritis (OA) FLS, but not in non-arthritic FLS.

MATERIALS AND METHODS

Primary culture of human FLS and cytokine treatment

Synovial tissues were obtained from RA and OA patients during the total joint replacement surgery. RA and OA were diagnosed according to the criteria of the American College of Rheumatology [12,13]. Non-arthritic synovial tissues were obtained from the knee joint of two trauma patients undergoing arthroscopic examination and the unaffected knee joint of a sarcoma patient undergoing amputation. The synovial tissues were minced and digested with 500 units/ml of type II collagenase (Sigma, St Louis, MO, USA) and 3 mg/ml of dispase (grade II) (Boehringer Mannheim, Indianapolis, IN, USA) in MEM by shaking vigorously at 37°C for 30 min. Supernatant containing the released cells was removed and the digestion procedure was repeated four times. Isolated cells were cultured in RPMI-1640 (Gibco BRL, Grand Island, NY, USA) containing 15% FBS and antibiotics (100 μg/ml streptomycin, 100 units/ml penicillin G, and 0·25 μg amphotericin B). When the cells had grown to confluence, they were split at a 1:2 ratio. FLS were used for experiments at passages 4–10. TGF-β1 was purchased from R&D systems (Minneapolis, MN, USA) and TNFα and IL-1β from Biosource (Camarillo, CA, USA). Cytokines (TGF-β1, TNFα or IL-1β) were added to the cultures to a final concentration of 10 ng/ml.

Reverse transcription-polymerase chain reaction (RT-PCR)

Total cellular RNA was extracted from synovial cells as described previously [14]. Reverse transcription was performed using oligo(dT)17 primer (Bioneer, Taejun, Korea) and Molony murine leukaemia virus (M-MuLV) reverse transcriptase (Gibco BRL, Grand Island, NY, USA) at 37°C for 1h cDNA synthesized from total RNA (0·5 μg unless otherwise indicated) was amplified with a specific primer pair in a 25-μl reaction mixture. PCR primers for IL-1β and TNFα were purchased from Clontech (Palo Alto, CA, USA) and PCR was carried out according to the manufacturer’s instruction. Sequences of other PCR primers were as follows: IL-8 forward, 5′atg-act-tcc-aag-ctg-gcc-gtg 3′; IL-8 reverse, 5′ tta-tga-att-ctc-agc-cct-ctt-caa-aaa-ctt-ctc 3′; MIP-1α forward, 5′ cgc-ctg-ctg-ctt-cag-cta-cac 3′; MIP-1α reverse, 5′ tgt-gga-ggt-cac-acg-cat-gtt 3′; MMP-1 forward, 5′ gca-cag-ctt-tcc-tcc-act 3′; and MMP-1 reverse, 5′cat-ccc-ctc-caa-tac-ctg 3′. The thermocycling programmes consisted of 30 cycles at 94°C for 1 min, 60°C 1min, and 72°C 1 min for IL-8 and MIP-1α, and at 94°C 1 min, 52°C 1 min, and 72°C 1 min for MMP-1. As a negative control, RT-PCR was performed in parallel without a template. mRNA from RA synovial tissue or RA FLS stimulated with TNFα or IL-1β was used as a positive control.

Immunoblotting

Cells treated with TGF-β1 were lysed in 1× lysis buffer (31·25 mm Tris-HCl, pH 6·8, 10% glycerol, 1% SDS, 2·5% β-mercaptoethanol) and heated for 10 min at 100°C. The cell lysate (80 μg of protein) was subjected to electrophoresis on a 10% polyacrylamide gel and the proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH, USA). The membrane was incubated with a primary antibody for 1h followed by a horseradish peroxide-conjugated secondary antibody for 1 h at room temperature. Bands of interest were detected by enhanced chemiluminescence (ECL) (Amersham, Buckinghamshire, UK) according to the manufacturer’s instruction. Antibody for IL-1β was purchased from R&D systems (Minneapolis, MN, USA) and antibodies for IL-8 and MIP-1α from Santa Cruz Biotechnology (Santa Cruz, CA, USA), while antibody for MMP-1 was from Oncogene (Cambridge, MA, USA). Densitometry of the data was carried out using LabWorks software produced by UVP Inc. (Cambridge, UK).

Biological activity assay of IL-1

Biological activity of IL-1 in the culture supernatant was determined by mouse thymocyte proliferation assay in triplicate. FLS were incubated in serum-depleted RPMI-1640 for 12 h, and further incubated in RPMI-1640 containing 10 ng/ml of TGF-β1 and 0·2% FBS at 37°C for 72 h, and the culture supernatant was harvested. Thymocytes were obtained from 6–8-week-old female DBA2 mice (Korean Chemical Institute, Taejun, Korea). Culture supernatant (50 μl) was added to thymocytes (2×106 cells in 150 μl) in RPMI-1640:F12 (1:1) medium containing 5% FBS, 1 mm glutamine, antibiotics, 50 μmβ-mercaptoethanol, 0·7 μg/ml concanavalin A (Con A) and 10 μg/ml polymixin B (Sigma, St Louis, MO, USA). Thymocytes were cultured for 30 h before adding [3H]thymidine (1 μCi/well). After 18 h, thymocytes were harvested onto a glass fibre filter and [3H]thymidine incorporation was measured by liquid scintillation counting.

Electrophoretic mobility shift assay (EMSA)

Nuclear extracts were prepared from cells treated with TGF-β1. The oligonucleotide with the NF-κB or AP-1 binding consensus sequence (Promega, Madison, WI, USA) was end-labelled with γ-32P]dATP (Amersham, Buckinghamshire, UK) using T4 DNA polynucleotide kinase. Labelled oligonucleotide probes were incubated with nuclear extracts (3 μg of proteins) in the binding buffer 50 mm HEPES, pH 7·5, 0·5 mm EDTA, 0·5 mm DTT, 1% NP-40, 5% glycerol, 50 mm NaCl, 1 μg of poly(dI:dC)] at room temperature for 40 min. For the cold competition experiment, unlabelled oligonucleotide in 100-fold molar excess was added to the binding reaction. For supershift, antibodies specific for p65 or Fos (Santa Cruz, Santa Cruz, CA, USA) were incubated with the nuclear extract for 30 min at 4°C before the labelled oligonucleotide was added. The samples were electrophoresed on a 6% native polyacrylamide gel in 0·5 × TBE buffer, and the gels were dried and visualized by autoradiography.

RESULTS

mRNA expression of IL-1β and TNFα in response to TGF-β1

The cultured synovial cells were fibroblast-like: CD68+, CD64–, CD14–, HLA-DQ+ and HLA-DR– (data not shown). TGF-β1 mRNA expression was readily detected not only in RA and OA, but also in non-arthritic FLS (data not shown). The effect of TGF-β1 on expression of the major proinflammatory cytokines, IL-1β and TNFα, was assessed in these cells at 2–4h post-stimulation. Competitive PCR showed that the level of IL-1β mRNA increased by 25–50-fold by TGF-β1 treatment in RA FLS (Fig. 1a). In non-arthritic FLS and MRC5, the basal levels of IL-1β mRNA were >10 and >100 times higher than that in RA FLS, respectively. However, IL-1β expression did not change by TGF-β1 treatment in non-arthritic FLS (NS1), and only mildly increased (by 2·5-fold) in MRC-5. We analysed additional FLS cultures by RT-PCR and confirmed enhanced expression of IL-1β mRNA by TGF-β1 stimulation in 4 RA FLS (Fig. 1b). Three OA FLS showed similar results as RA. Two additional nonarthritic FLS did not increase IL-1β expression upon TGF-β1 stimulation, again confirming the competitive PCR results.

Fig. 1.

Fig. 1

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Effect of TGF-β1 on the mRNA expression of IL-1β. (a) IL-1β mRNA expression was analysed quantitatively by competitive PCR. Three RA and one non-arthritic FLS cultures as well as MRC-5 were treated with TGF-β1 for 2h. cDNA synthesized from the total RNA and the PCR competitor in a 10-fold serial dilution were co-amplified with IL-1β primers. The amount of the IL-1β competitor ranged from 5 × 10–6 pmole (lane 1) to 5 × 10–9 pmole (lane 4). The upper bands (*) are products amplified from IL-1β cDNA and the lower bands (**) are products from the competitor. Arrowheads indicate where the intensities of upper and lower bands are equal. (b) IL-1β expression was analysed in additional RA, OA and non-arthritic FLS stimulated with TGF-β1 for 2h. (c) Effect of TGF-β1 on the mRNA expression of TNFα. TGF-β1 treatment and RT-PCR were carried out as described in (b), except that the amplification was performed for 35 cycles.

Expression of TNFα mRNA was also shown to increase significantly in RA and OA FLS (Fig. 1c). In non-arthritic FLS, the TNFα expression was only mildly increased. TNFα-treated FLS was used as positive controls for RT-PCR and no bands were observed in negative controls where no templates were added (data not shown).

Expression and secretion of IL-1β in response to TGF-β1

To assess changes in IL-1β protein expression, immunoblot analysis was carried out in RA5 FLS. In the absence of brefeldin A (BFA), induction of IL-1β protein by TGF-β1 was not detectable, whereas that by TNFα could be observed (Fig. 2a). In the presence of BFA, however, the 35 kDa precursor form of IL-1β was observed in RA FLS treated with TGF-β1 (Fig. 2a, lane 5), suggesting that the IL-1β protein induced by TGF-β1 was mostly secreted. A synergistic effect between TGF-β1 and TNFα on IL-1β expression was also found (Fig. 2b). IL-1 bioactivity was measured in triplicate by thymocyte proliferation assay. In the culture supernatant of RA FLS treated with TGF-β1 for 72 h, the bioactivity of IL-1 was enhanced by 50% compared to that in the control supernatant (P≤ 0·05, Student’s t-test) (Fig. 2c). This experiment was repeated four times and similar results were obtained.

Fig. 2.

Fig. 2

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Effect of TGF-β1 on the expression of IL-1β protein in RA FLS. (a) RA5 FLS were stimulated with TGF-β1 (10 ng/ml) or TNFα (10 ng/ml) for 12h in the absence (lanes 1–3) or presence (lanes 4–6) of BFA (0·5 μg/ml). IL-1β protein was analysed by immunoblotting using IL-1β specific antibody. (b) RA FLS were stimulated with TGF-β1 and/or TNFα for 24h and the IL-1β protein expression was analysed by immunoblotting. (c) Bioactivity of IL-1 in the culture supernatant of RA2 FLS was measured by thymocyte proliferation assay in triplicate. Error bars represent standard deviation. *P≤ 0·05 (Student’s t-test).

IL-8 and MIP-1α expression in response to TGF-β1

TGF-β1 is a strong chemoattractant for monocytes and neutrophils [7]. However, as yet it has not been shown clearly whether TGF-β induces expression of other chemokines. Therefore, the TGF-β1 effect on expression of IL-8, a prototype CXC chemokine and MIP-1α, a CC chemokine, was examined. Similar to its effect on IL-1β expression, TGF-β1 enhanced the level of IL-8 mRNA in RA and OA, but not in the non-arthritic FLS (Fig. 3a). TNFα-treated FLS was used as a positive control for RT-PCR. The IL-8 protein expression in RA FLS was also markedly induced (Fig. 3). The level of IL-8 protein induced by TGF-β1 was comparable to that induced by TNF-α. As for the IL-1β, increased expression of IL-8 protein by TGF-β1 was revealed only after BFA treatment. The MIP-1α mRNA expression was also elevated in both RA and OA FLS (Fig. 3c). Again, in non-arthritic FLS, TGF-β1 did not augment MIP-1α mRNA expression. Despite the increase in the steady state mRNA level, the protein expression of MIP-1α was not induced by TGF-β1 in RA FLS (data not shown).

Fig. 3.

Fig. 3

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Effect of TGF-β1 on IL-8 and MIP-1α expression. (a) IL-8 mRNA expression was analysed in RA, OA and non-arthritic FLS treated with TGF-β1 for 2 or 4 h. RT-PCR was performed as described in Materials and methods. (b) IL-8 protein expression was analysed by immunoblotting using anti-IL-8 antibody. RA5 FLS were stimulated with TGF-β and/or TNFα for 12h in the absence (lanes 1–4) or presence (lanes 5–8) of BFA (0·5 μg/ml). (c) MIP-1α mRNA expression was analysed by RT-PCR as in (a).

MMP-1 expression in response to TGF-β1

MMP-1 (interstitial collagenase, collagenase-1) plays an important role in destruction of joints in arthritis. The effect of TGF-β on MMP-1 expression has been contradictory. We showed here that TGF-β1 increased MMP-1 expression in RA and OA FLS (Fig. 4). Expression of MMP-1 mRNA reached to a peak at 2–4 h post-stimulation and started to decline after 12 h. In OA FLS, MMP-1 expression was also increased by TGF-β1, but with a slower rate (Fig. 4b). However, in non-arthritic FLS, the mRNA level of MMP-1 did not change. Similar results were obtained in additional RA, OA and non-arthritic FLS cultures (Fig. 4b). For a positive control of MMP-1 RT-PCR, IL-1β treated RA FLS was used (data not shown). Protein expression of MMP-1 was also enhanced in RA FLS by TGF-β1 stimulation (Fig. 4c). There was a remarkable synergism between TGF-β1 and IL-1β for the protein expression of MMP-1, suggesting the importance of TGF-β1 in inducing MMP-1 expression in vivo. In OA FLS, TGF-β1 significantly enhanced MMP-1 expression induced by IL-1β. However, TGF-β1 did not increase the MMP-1 protein expression either alone or together with IL-1β in non-arthritic FLS (Fig. 4c).

Fig. 4.

Fig. 4

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Effect of TGF-β1 on MMP-1 expression. (a) Time-course analysis of MMP-1 mRNA expression was carried out by RT-PCR. (b) MMP-1 mRNA expression was analysed by RT-PCR in additional RA, OA and non-arthritic FLS cultures treated with TGF-β1 for 2h (c) Protein expression of MMP-1 was analysed by immunoblotting. RA5 FLS were cultured in a low serum condition (0·2% FBS) for 24 h before stimulating with TGF-β1 and/or IL-1β. After 48 h, cell lysates were prepared and immunoblot analysis was performed using anti-MMP-1 antibody. The numbers below are the relative intensities of the bands measured by densitometry.

Activity of NF-κB and AP-1 in response to TGF-β1

Since NF-κB and AP-1 were transcription factors required for expression of many genes mediating inflammation, their activities were analysed after TGF-β1 treatment. TGF-β1 activated NF-κB and AP-1 in both RA and OA synoviocytes within 15 min and the activity of NF-κB and AP-1 lasted up to 60 min (Fig. 5a). Anti-p65 and anti-Fos antibodies caused supershift of the NF-κB and AP-1 bands, respectively (Fig. 5b). The specificity of DNA binding activity was also shown by the competition with unlabelled specific or irrelevant oligonucleotides.

Fig. 5.

Fig. 5

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Activation of NF-κB and AP-1 in response to TGF-β1. (a) After stimulation of RA and OA FLS with TGF-β1, nuclear extracts were prepared. EMSA was carried out as described in Materials and methods. (b) For supershift assay, nuclear extracts of RA5 were incubated with anti-p65 or anti-Fos antibodies for 30 min at 4°C before 32P-labelled oligonucleotides were added. For cold competition, unlabelled NF-κB or AP-1 oligonucleotide was used in 100-fold molar excess.

DISCUSSION

TGF-β1 and its receptors are known to be expressed in RA synovial tissue. TGF-β exerts diverse and even opposite effects depending on the cell types and conditions. In the present study, we provided evidence that TGF-β1 could contribute to the inflammation and progression of the disease in RA and OA.

First, TGF-β1 increased mRNA expression of several inflammatory cytokines such as IL-1β, TNFα, IL-8 and MIP-1α in RA and OA FLS. This effect, interestingly, was specific to the arthritic FLS, since there was no or minimal increase in the mRNA expression of these cytokines in non-arthritic FLS. Protein expression of IL-1β and IL-8 was also enhanced by TGF-β1 in RA FLS. Moreover, TGF-β1 synergized with TNFα to increase expression of the IL-1β protein. This synergistic effect is potentially significant in in vivo situations, since both TNFα and TGF-β1 are present in the RA synovial tissue. mRNA expression of other members of CC chemokines such as MCP-1 and RANTES was not changed by TGF-β1 stimulation (data not shown).

Secondly, the level of MMP-1 expression was up-regulated by TGF-β1 in RA FLS. Synergistic effect of TGF-β1 and IL-1β in inducing MMP-1 protein expression was also observed. We also observed that MMP-3 mRNA expression was increased by TGF-β1 in RA FLS (data not shown). Thus, in RA, TGF-β1 appears to promote degradation of cartilages and the extracellular matrix proteins, rather than exerting an anabolic effect. Although TGF-β has been shown to reduce MMP-1 expression in many cases, up-regulation of MMP-1 by TGF-β1 is also reported [9,10,15,16]. In a ras-transformed HaCaT cell line, MMP-1 expression was stimulated by TGF-β and this effect was blocked by inhibitors of Erk1, 2 and p38 MAP kinases. Smad 3 and Smad 4 were shown to act together with c-Jun and c-Fos to activate the MMP-1 promoter.

Thirdly, TGF-β1 activated NF-κB and AP-1 in both RA and OA FLS. These transcription factors are required for the expression of many genes involved in the inflammatory process including IL-1β, TNFα and MMP-1 [17,18]. Therefore, TGF-β1 seems to increase the expression of inflammatory cytokines and MMP-1 through activation of these transcription factors. The significance of NF-κB and AP-1 in RA pathogenesis has been well documented [17–20]. Activation of these transcription factors was observed in RA synovial lining cells, which preceded arthritis development in collagen-induced arthritis (CIA).

Altogether, our results indicate that TGF-β1 can contribute for the inflammation and destruction of joints in RA and OA. The pro-inflammatory effects of TGF-β1 were specific to arthritic FLS. In non-arthritic FLS, TGF-β1 did not significantly induce expression of pro-inflammatory cytokines or MMP-1. It is well established that the effect of TGF-β is determined in the cellular context. The many different effects of TGF-β can be explained, in part, by interaction of the Smad complex in the nucleus with a set of partner proteins that are specific to a particular cell type under a particular condition [21]. These partners determine which genes the Smad complex will activate and how long this will last. Therefore, it is possible that a condition such as inflammation activates certain transcription factors that interact with Smad complex and influence the outcome of TGF-β stimulation.

Accumulating evidence suggests that RA FLS possesses unique transformed characteristics such as an anchorage-independent growth, lack of contact inhibition, elevated expression of proto-oncogenes and mutations in p53 [22,23]. However, these permanent changes do not seem to explain the differential response of RA FLS to TGF-β1. The inflammatory effect of TGF-β1 was also observed in OA FLS and while FLS cultures between the 4th and 10th passages gave similar responses to TGF-β1, the RA and OA FLS start to lose the proinflammatory responses to TGF-β1 after the 10th to 15th passages. Therefore, it is tempting to speculate that the inflammatory environment where the RA and OA FLS reside for a prolonged period might bring the proinflammatory effect of TGF-β1. An alternative explanation that cellular senescence affects the outcome of TGF-β stimulation cannot be ruled out. RA and OA FLS might be younger than the non-arthritic cells, since arthritic FLS can be maintained in culture much longer than the non-arthritic FLS.

Acknowledgments

We thank Dr Sangduk Kim for critical reading of the manuscript. This work was supported by grants from the Ministry of Health and Welfare, Republic of Korea (Good Health R&D Project, HMP-96-M-2–1035), the Brain Korea 21 Project in 2001 and the Medical Science Research Center at Korea University (1999–204).

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