Methotrexate Suppresses Inflammatory Agonist Induced Interleukin 6 Synthesis in Osteoblasts

MINORU YOSHIDA, YOSUKE KANNO, AKIRA ISHISAKI, HARUHIKO TOKUDA, KOUSEKI HIRADE, KEIICHI NAKAJIMA, YOSHIHIRO KATAGIRI, KATSUJI SHIMIZU, and OSAMU KOZAWA

ABSTRACT.

Objective. Interleukin 6 (IL-6) is a pleiotropic cytokine that plays a crucial role in the pathogenesis of rheumatoid arthritis (RA). In bone metabolism, it is known that IL-6 is produced and secreted by osteoblasts, and that IL-6 induces osteoclast formation and stimulates bone resorption. Various bone inflammatory agonists such as tumor necrosis factor-a (TNF-a), IL-1a, prostaglandin D₂ (PGD₂), PGE₂, and PGF_2a, which play important roles in the pathogenesis of RA, induce IL-6 synthesis in osteoblast-like MC3T3-E1 cells. Low dose methotrexate (MTX) is currently used for treatment of patients with RA. We investigated the effect of MTX on IL-6 synthesis induced by these agents in MC3T3-E1 cells.

Methods. Cultured cells were pretreated with various doses of MTX, and then stimulated by these inflammatory agonists. The IL-6 in the conditioned medium was measured by IL-6 enzyme immunoassay.

Results. MTX significantly suppressed IL-6 synthesis stimulated by these agonists in a dose-dependent manner, although MTX alone had no effect on the levels of IL-6. In addition, MTX significantly inhibited the enhancement by IL-17 of TNF-a-stimulated IL-6 synthesis. MTX reduced the levels of IL-6 induced by 12-O-tetradecanoylphorbol 13-acetate, a direct activator of protein kinase C (PKC), suggesting that MTX inhibits PKC signals for IL-6 synthesis.

Conclusion. MTX suppresses IL-6 synthesis stimulated by various inflammatory agonists in osteoblasts. (J Rheumatol 2005;32:787-95)

Key Indexing Terms:

METHOTREXATE
INTERLEUKIN 6
OSTEOBLAST
RHEUMATOID ARTHRITIS
INFLAMMATION

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From the Departments of Pharmacology and Orthopaedic Surgery, Gifu University Graduate School of Medicine; and Department of Pharmacy, Gifu University Hospital, Gifu, Japan.

M. Yoshida, MD, Fellow, Departments of Pharmacology and Orthopaedic Surgery; Y. Kanno, PhD Student, Department of Pharmacology; A. Ishisaki, PhD, Assistant Professor, Department of Pharmacology; H. Tokuda, MD, PhD, Department of Clinical Laboratory, National Center for Geriatrics and Gerontology; K. Hirade, PhD, Fellow, Department of Pharmacology; Y. Katagiri, PhD, Professor, Department of Pharmacy; K. Nakajima, PhD, Assistant Professor, Department of Pharmacology; K. Shimizu, MD, PhD, Professor, Department of Orthopaedic Surgery; O. Kozawa, MD, PhD, Professor, Department of Pharmacology.

Address reprint requests to Dr. O. Kozawa, Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan. E-mail: okozawa@cc.gifu-u.ac.jp

Accepted for publication December 13, 2004.

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