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. 2002 Oct;61(10):870–876. doi: 10.1136/ard.61.10.870

Interleukin 17 synergises with tumour necrosis factor α to induce cartilage destruction in vitro

R L van Bezooijen 1, L van der Wee-Pals 1, S Papapoulos 1, C Lowik 1
PMCID: PMC1753923  PMID: 12228154

Abstract

Background: Interleukin 17 (IL17) is produced by activated T cells and has been implicated in the development of bone lesions and cartilage degradation in rheumatoid arthritis (RA).

Objective: To determine whether IL17, alone or together with tumour necrosis factor α (TNFα), induces cartilage destruction in vitro.

Methods: Fetal mouse metatarsals stripped of endogenous osteoclast precursors were used to study the effect of IL17 on cartilage degradation independently of osteoclastic resorption. Cartilage destruction was analysed histologically by Alcian blue staining.

Results: IL17 alone, up to 100 ng/ml, had no effect on the cartilage of fetal mouse metatarsals. IL17 (≥0.1 ng/ml), however, induced severe cartilage degradation when given together with TNFα (≥1 ng/ml). The cytokine combination decreased Alcian blue staining, a marker of proteoglycans, throughout the metatarsals and induced loss of the proliferating and early hypertrophic chondrocyte zones. TNFα alone also decreased Alcian blue staining, but not as dramatically as the cytokine combination. In addition, it did not induce loss of chondrocyte zones. Treatment with inhibitors of matrix metalloproteinase (MMP) activity and nitric oxide synthesis showed that MMP activity played a part in cartilage degradation, whereas nitric oxide production did not.

Conclusions: IL17, together with TNFα, induced cartilage degradation in fetal mouse metatarsals in vitro. IL17 may, therefore, participate in the development of cartilage destruction associated with RA by enhancing the effects of TNFα and may provide a potential therapeutic target.

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Figure 1 .

Figure 1

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Histological staining of 19 day old fetal mouse metatarsals cultured in the absence or presence of IL17, TNFα, or a combination of the two cytokines. 19 Day old fetal mouse metatarsals were cultured for seven days in the absence (A, B) or presence of IL17 (100 ng/ml) (C, D), TNFα (100 ng/ml) (E, F), or a combination of the two cytokines (G, H). The presence of mononuclear and multinuclear osteoclasts was visualised using TRAP staining. Magnification: A, C, E, G x40; B, D, F, H x200. Cultures were performed in quintuplicate (n=5).

Figure 2 .

Figure 2

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Zones of chondrocyte differentiation in fetal mouse metatarsals. 17 Day old fetal mouse metatarsals were fixed directly after isolation and stained for Alcian blue (A) or von Kossa (B). Four different zones of chondrocyte differentiation are shown: lhc, mineralised late hypertrophic chondrocytes (black von Kossa staining); ehc, unmineralised early hypertrophic chondrocytes; pc, proliferating chondrocytes; rc, resting chondrocytes. Magnification x40.

Figure 3 .

Figure 3

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Effect of IL17, TNFα, and a combination of the two cytokines on Alcian blue staining. Osteoclast-free 17 day old fetal mouse metatarsals were cultured for 11 days in the absence (A, B) or presence of IL17 (50 ng/ml) (C, D), TNFα (50 ng/ml) (E, F), or a combination of the two cytokines (G, H) and stained with Alcian blue. lhc, late hypertrophic chondrocytes; ehc, early hypertrophic chondrocytes; pc, proliferating chondrocytes; rc, resting chondrocytes. Magnification: A, C, E, G x40; B, D, F, H x200. Cultures were performed in quadruplicate (n=4).

Figure 4 .

Figure 4

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Effect of IL17, TNFα, and a combination of the two cytokines on von Kossa staining. Osteoclast-free 17 day old fetal mouse metatarsals were cultured for 11 days in the absence (A) or presence of IL17 (50 ng/ml) (B), TNFα (50 ng/ml) (C), or a combination of the two cytokines (D) and stained with von Kossa. Magnification x100. Cultures were performed in quadruplicate (n=4).

Figure 5 .

Figure 5

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Effect of an NO synthesis inhibitor and an MMP inhibitor on cartilage destruction induced by IL17 + TNFα. Osteoclast-free 17 day old fetal mouse metatarsals were treated with IL17 (100 ng/ml) + TNFα (100 ng/ml) for 11 days in the presence of the nitric oxide synthesis inhibitor L-NMMA (1 mM) (A, B) or the MMP inhibitor marimastat (10-6 M) (C, D) and stained with Alcian blue (A, C) or von Kossa (B, D). Marimastat was dissolved in DMSO and the final concentration of DMSO in culture medium was 0.05%. This DMSO concentration had no effect on the histological picture of control and cytokine treated cultures (data not shown). DMSO, dimethylsulphoxide. Magnification x40. Cultures were performed in quadruplicate (n=4).

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