Increased expression of pro-inflammatory cytokines and metalloproteinase-1 by TGF-beta1 in synovial fibroblasts from rheumatoid arthritis and normal individuals

Abstract

Transforming growth factor (TGF)-beta1 is expressed abundantly in the rheumatoid synovium. In this study, the inflammatory effect of TGF-beta1 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-1beta, tumour necrosis factor (TNF)-alpha, IL-8, macrophage inflammatory protein (MIP)-1alpha and metalloproteinase (MMP)-1 were increased in RA and OA FLS by TGF-beta1 treatment, but not in non-arthritic FLS. Enhanced protein expression of IL-1beta, IL-8 and MMP-1 was also observed in RA FLS. Moreover, TGF-beta1 showed a synergistic effect in increasing protein expression of IL-1beta and matrix metalloproteinase (MMP)-1 with TNFalpha and IL-1beta, respectively. Biological activity of IL-1 determined by mouse thymocyte proliferation assay was also enhanced by 50% in response to TGF-beta1 in the culture supernatant of RA FLS. DNA binding activities of nuclear factor (NF)-kappaB and activator protein (AP)-1 were shown to increase by TGF-beta1 as well. These results suggest that TGF-beta1 contributes for the progression of inflammation and joint destruction in RA, and this effect is specific for the arthritic synovial fibroblasts.

Fig. 1

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.

Fig. 2

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).

Fig. 3

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).

Fig. 4

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.

Fig. 5

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.