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Lee, Cho, Park, Oh, Park, and Kim: IL-10 Inihibits VEGF Production in the Synovial Fibroblasts of RA Patients via Down-regulation of the ERK and AP-1 Pathways
Original Article

J Korean Rheum Assoc 2009;16(3):213-222.

Published online: 20 September 2009

DOI: https://doi.org/10.4078/jkra.2009.16.3.213

IL-10 Inihibits VEGF Production in the Synovial Fibroblasts of RA Patients via Down-regulation of the ERK and AP-1 Pathways

Seon-Yeong Lee, Mi-La Cho, Mi-Kyung Park, Hye-Jwa Oh, Sung-Hwan Park, Ho-Youn Kim

Rheumatism Research Center, Catholic Institutes of Medical Science, The Catholic University of Korea, College of Medicine, Seoul, Korea

Received 8 August 2009       Revised 1 September 2009       Accepted 2 September 2009

Copyright © 2009 The Korean Rheumatism Association

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Objective

Interleukin (IL)-10 has been demonstrated to have anti-inflammatory and anti-tumour activity. Because aberrant angiogenesis is a significant pathogenic component of tumor growth and chronic inflammation, we investigated the effect of IL-10 on the production of vascular endothelial growth factor (VEGF) by the synovial fibroblasts derived from the patients with rheumatoid arthritis (RA).

Methods

Fibroblast-like synoviocytes (FLS) were cultured with transforming growth factor (TGF-β) alone or with IL-10. The level of VEGF was measured by RT-PCR and enzyme-linked immunosorbent assay (using the 24, 48 and 72 h culture supernatants). The FLSs were cultured with TGF-b for 48 hr in the presence of PD98059 (an ERK inhibitor), curcumin and SP600125 (a JNK and Ap-1 inhibitor, respectively). The level of VEGF in the supernatants was measured by ELISA. Cell viability was assessed using MTT assay. The expressions of VEGF, ERK, AP-1 and IL-10 in the synovial tissue were quantified by immunohistochemistry.

Results

IL-10 exhibited the inhibitory effect on VEGF production when the FLSs were stimulated with TGF-β. ERK and AP-1 inhibitors inhibited the TGF-β induced VEGF production. Moreover, TGF-β increased the phosphorylation of ERK and C-Jun, which was significantly inhibited by the IL-10.

Conclusion

IL-10 may exert an antiangiogenic effect by inhibiting the ERK- and AP-1 mediated VEGF expression in rheumatoid synovial fibroblasts.

Keywords: IL-10, VEGF, ERK, AP-1, Rheuamtoid arthritis synovial fibroblast

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Fig. 1.
IL-10 inhibits the VEGF mRNA in the TGF-β stimulated RA synovial fibroblasts. The FLSs were cultured with TGF-β (10 ng/mL) alone or with the indicated concentrations of IL-10 (1∼50 ng/mL) for 12 h. The mRNA level of VEGF was measured by RT-PCR. The data is expressed as the mean±s.d. of three independent experiments (∗p＜0·05, ∗∗p＜0.01 versus the that were well treated with TGF-β alone).
jkra-16-213f1.tif
Fig. 2.
IL-10 inhibits the TGF-β induced VEGF production in synovial fibroblasts. The FLSs were cultured for 24, 48 and 72 h with TGF-β (10 ng/mL) in the presence of increasing doses of IL-10 (1∼50 ng/mL). The level of VEGF in the supernatants was measured by enzyme-linked immunosorbent assay (ELISA). Cell viability was assessed using MTT assay. The data is expressed as the mean±s.d. of three independent experiments (∗p＜0.05, ∗∗p＜0.01 versus the that were well treated with TGF-β alone).
jkra-16-213f2.tif
Fig. 3.
IL-10 inhibits the TGF-β-induced phospho ERK and phospho C-Jun expressions in synovial fibroblasts. The FLSs were cultured for 48 h with medium alone or with 10 ng/mL of TGF-β in the presence of PD98059 (an ERK inhibitor), curcumin and SP600125 (a JNK and Ap-1 inhibitor, respectively). The level of VEGF in the supernatants was measured by ELISA. The levels of phosphor ERK and phosphor C-Jun in the cell lysates were measured by western blotting. This data shows the dose-dependent inhibition of the phospho ERK and phospho C-Jun expressions by IL-10 (10 ng/mL, 50 ng/mL). The data is expressed as the mean±s.d. of three independent experiments (∗∗p＜0.01 versus the that were well treated with TGF-β alone).
jkra-16-213f3.tif
Fig. 4.
VEGF was highly expresses by RA patients, as compared to the OA patients, in the synovial tissue and synovial fluid mononuclear cells. Immunostaining was performed using the specific antibodies against VEGF, ERK, AP-1 and IL-10 in the synovial tissue samples from the patients with RA and OA. VEGF, ERK, AP-1 and IL-10 stained a brown color. All the tissues were counterstained with hematoxylin (original magnification ×400). The levels of mRNA VEGF and IL-10 in the synovial fluid were measured by RT-PCR. The data is expressed as the mean±s.d. of three independent experiments (∗∗p＜0.01 versus OA SFMC).
jkra-16-213f4.tif
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