Upregulation of Macrophage Migration Inhibitory Factor (MIF) Production by Engagement of Toll-like Receptor 3 (TLR3) on Fibroblast-like Synoviocyte (FLS) from Patients with Rheumatoid Arthritis

Yang Mi Her; Sung-Hwan Park; Mi Kyung Park; Hye-Jwa Oh; Kwi Young Kang; Mi-La Cho

doi:10.4078/jkra.2009.16.2.123

Journal List > J Korean Rheum Assoc > v.16(2) > 1003659

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Her, Park, Park, Oh, Kang, and Cho: Upregulation of Macrophage Migration Inhibitory Factor (MIF) Production by Engagement of Toll-like Receptor 3 (TLR3) on Fibroblast-like Synoviocyte (FLS) from Patients with Rheumatoid Arthritis

Original Article

J Korean Rheum Assoc 2009;16(2):123-132.

Published online: 20 June 2009

DOI: https://doi.org/10.4078/jkra.2009.16.2.123

Upregulation of Macrophage Migration Inhibitory Factor (MIF) Production by Engagement of Toll-like Receptor 3 (TLR3) on Fibroblast-like Synoviocyte (FLS) from Patients with Rheumatoid Arthritis

Yang Mi Her¹, Sung-Hwan Park², Mi Kyung Park¹, Hye-Jwa Oh¹, Kwi Young Kang², Mi-La Cho¹

¹The Rheumatism Resarch Center, Catholic Research Institute of Medical Science, Division of Rheumatology, Seoul, Korea

²Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea

Received 25 May 2009 Revised 1 June 2009 Accepted 2 June 2009

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

Rheumatoid arthritis (RA) is a chronic autoimmune disease. Macrophage migration inhibitory factor (MIF) has been shown to be an important pro-inflammatory cytokine in RA. The aim of this study was to determine if the engagement of toll-like receptor 3 (TLR3) induces the production of MIF in the fibroblast-like synoviocytes (FLS) of patients with RA.

Methods

The expression of inflammatory cytokines (e.g. MIF, IL-6, IL-1β and TNFα) and toll-like receptors (e.g. TLR2, TLR3 and TLR4) in the synovial tissue were quantified by immunohistochemistry. FLS were isolated from the synovial tissues of patients with RA and stimulated with TLR-3 ligand polyI:C, in the presence of a neutralizing antibody against IL-6. The concentrations of MIF and IL-6 in the culture supernatants from the FLS were measured using sandwich ELISA.

Results

The engagement of TLR3 with PolyI:C increased the production of MIF in FLS. The stimulatory effect of these TLR ligands showed a dose-dependent trend. The combination of TLR3 and TLR4 synergistically increased the level of MIF and IL-6 production. The addition of neutralizing antibodies against IL-6 abrogated the stimulatory effect of the ligands of TLR3 and TLR4 on the production of MIF.

Conclusion

These results show that TLR3 engagement stimulates the production of MIF and IL-6. Therefore, the TLRs help perpetuate of RA pathogenesis through production of MIF from the FLS in patients with RA, and might provide a new therapeutic approach for the treatment of rheumatoid arthritis.

Keywords: Rheumatoid arthritis, Fibroblast-like synoviocytes, Macrophage migration inhibitory factor, Toll-like receptor engagement

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Fig. 1.

Increased expression of MIF, IL-6, TNFα and IL-1β in RA synovium. Immunostaining was performed using the specific antibodies against MIF, IL-6, TNFα and IL-1β in synovial biopsy samples from patients with rheumatoid arthritis (RA) and osteoarhtirits (OA). MIF, IL-6, TNFα and IL-1β stained a brown color. All tissues were counterstained with hematoxylin (original magnification ×400).

Fig. 2.

Increased expression of TLR 2, 3 and 4 in RA synovium. Immunostaining was performed using the specific antibody against TLR 2, 3 and 4 in synovial biopsy samples from patients with RA and OA. TLR 2, 3 and 4 stained a brown color. All tissues were counterstained with hematoxylin (original magnification ×400).

Fig. 3.

Enhancement of MIF production by the stimulation of TLR3 or TLR4 engagement in FLS from patients with RA. The FLS were cultured with the TLR3 (1 μg/mL, 10 μg/mL, 50 μg/mL) and TLR4 ligands (10 ng/mL) only or combination for 72 hr. The culture supernatants were assayed for MIF by ELISA. ∗p＜0.01.

Fig. 4.

Enhancement of IL-6 production by the stimulation of TLR3 or TLR4 engagement in the FLS from patients with RA. FLS were cultured with TLR3 (1 μg/mL, 10 μg/mL, 50 μg/mL) and TLR4 ligands (10 ng/mL) only or a combination for 72 hr. The culture supernatants were assayed for IL-6 by ELISA. ∗p＜0.05, ∗∗p＜0.01.

Fig. 5.

Blockade of the IL-6-induced decrease in MIF by the engagement of TLR3 and TLR4 in RA FLS. The RA FLS were stimulated with TLR3 (1 ug/mL) and TLR4 ligands (10 ng/mL) alone and in combination for 72 hr. The culture supernatants were assayed for MIF by ELISA. ∗p＜0.05, ∗∗p＜0.01.

Fig. 6.

Increased level of MIF and IL-6 in the synovial fluid and sera derived from patients with RA, OA and HC. The concentrations of MIF and IL-6 in RA and OA synovial fluids and RA, OA and HC sera determined by ELISA. ∗p＜0.05, ∗∗p＜0.01.

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