Suppression of IL-6 biological activities by activin A and implications for inflammatory arthropathies

Abstract

Activin A is a cytokine whose multiple functions have yet to be fully determined. In this study, the role of proinflammatory cytokines in regulatory control of activin A production was shown in synoviocytes and chondrocytes. Additional facets of functional inflammation-related activities of activin A were also determined. Results showed that activin A concentrations in the synovial fluid of patients with rheumatoid arthritis and gout were elevated relative to those in patients with osteoarthritis. Further studies showed that production of activin A by synoviocytes and chondrocytes in culture was stimulated by cytokines such as IL-1, transforming growth factor-beta (TGF-beta), interferon-gamma (IFN-gamma), and IL-8, consistent with previous studies in regard to the control of activin A production in marrow stromal cells and monocytes by cytokines, glucocorticoids and retinoic acid. In addition, the relationship of activin A to IL-6-induced biological activities was investigated. Three major IL-6 activities involved in inflammatory responses were found to be suppressed by activin A. In a dose-dependent manner, activin A efficiently suppressed IL-6-induced proliferation of 7TD1 B lymphoid cells, phagocytic activity of monocytic M1 cells, and fibrinogen production in HepG2. Therefore, it is likely that activin A serves as a suppressor for IL-6, dampening inflammatory responses, and has the potential to perform some previously unrecognized roles in inflammation.

Fig. 1

Expression of activin A mRNA in synoviocytes and chondrocytes. Synoviocytes and chondrocytes were cultured in the presence of the following cytokines: IL-1 (5 ng/ml), transforming growth factor-beta (TGF-β; 10 ng/ml), IFN-γ (500 U/ml), IL-8 and IL-10. Polymerase chain reaction (PCR) amplification of activin A from equal amounts of quantified cDNA prepared from synoviocytes and chondrocytes was performed as described in Materials and Methods. The 1003 bp PCR product of the activin β_A subunit is shown after amplification in synoviocytes (a) and chondrocytes (b) which were stimulated with various cytokines and regulators. Pst I digestion of PCR product yielded fragments of 626 bp and 378 bp characteristic of the activin A cDNA (not shown).

Fig. 2

Level of activin A in synovial fluid. Synovial fluid was collected from patients with rheumatoid arthritis (RA), gout, and osteoarthritis (OA). Activin A content was determined by ELISA [3,22]. Arrows indicate the means of the values for activin A content in synovial fluid. One-way analysis of variance (ANOVA) indicates that there is a significant increase of activin A level in synovial fluid from patients with RA and gout, compared with that from patients with OA (P < 0.02 and 0.01, Tukey–Kramer multiple comparison test). No differences were observed between patients with RA and gout (P > 0.36).

Fig. 3

Effect of activin A on IL-6-induced proliferation of B lymphoid cells. 7TD1 B lymphoid cells were incubated with (a) medium alone, (b) 0.01 ng/ml IL-6, (c) 0.1 ng/ml IL-6, (d) 0.5 ng/ml activin A alone, (e) 0.01 ng/ml IL-6 + 0.1 ng/ml activin A, and (f) 0.1 ng/ml IL-6 + 0.5 ng/ml activin A. After 2 days of incubation, cell density was assessed under the phase contrast inverted microscope.

Fig. 4

MTT assay for the effect of activin A on 7TD1 cell proliferation. 7TD1 cells (1 × 104) were plated in triplicate, and IL-6 ranging in concentration from 0.0001 to 10 ng/ml was added in the absence or presence of activin A (0, 0.5, 5.0 and 25 ng/ml). After 2 days of incubation, a MTT assay was performed and optical density (OD) was measured as described in Materials and Methods. These results are representative of at least five independent experiments.

Fig. 5

Effect of activin A on IL-6-induced phagocytic activity in M1 monocyte cells. M1 cells were incubated with medium, 10 ng/ml IL-6 and/or 25 ng/ml activin A for 2 days. Differentiation was measured based on the number of polystyrene beads ingested after incubation with the beads for 4 h. Phagocytic activity determinations were made under the phase contrast microscope. These results are representative of three independent experiments.

Fig. 6

Effect of activin A on IL-6-induced production of fibrinogen. HepG2 cells were incubated for 24 h with medium alone, IL-6 (0.01–10 ng/ml), and/or activin A (25 ng/ml). The cells were then measured for production of fibrinogen using the ELISA as described. These results are representative of four independent experiments.