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. 2002 Aug;61(8):704–713. doi: 10.1136/ard.61.8.704

Interleukin 17 induces cartilage collagen breakdown: novel synergistic effects in combination with proinflammatory cytokines

P Koshy 1, N Henderson 1, C Logan 1, P Life 1, T Cawston 1, A Rowan 1
PMCID: PMC1754191  PMID: 12117676

Abstract

Objective: To investigate whether interleukin 17 (IL17), derived specifically from T cells, can promote type II collagen release from cartilage. The ability of IL17 to synergise with other proinflammatory mediators to induce collagen release from cartilage, and what effect anti-inflammatory agents had on this process, was also assessed.

Methods: IL17 alone, or in combination with IL1, IL6, oncostatin M (OSM), or tumour necrosis factor α (TNFα), was added to bovine nasal cartilage explant cultures. Proteoglycan and collagen release were determined. Collagenolytic activity was determined by bioassay. Chondroprotective effects of IL4, IL13, transforming growth factor ß1 (TGFß1) and insulin-like growth factor-1 (IGF1) were assessed by inclusion in the explant cultures.

Results: IL17 alone stimulated a dose dependent release of proteoglycan and type II collagen from bovine nasal cartilage explants. Suboptimal doses of IL17 synergised potently with TNFα, IL1, OSM, and IL6 to promote collagen degradation. This collagen release was completely inhibited by tissue inhibitor of metalloproteinase-1 and BB-94 (a synthetic metalloproteinase inhibitor), and was significantly reduced by IL4, IL13, TGFß1, and IGF1. In IL17 treated chondrocytes, mRNA expression for matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 was detected. Moreover, a synergistic induction of these MMPs was seen when IL17 was combined with other proinflammatory cytokines.

Conclusions: IL17 can, alone and synergistically in combination with other proinflammatory cytokines, promote chondrocyte mediated MMP dependent type II collagen release from cartilage. Because levels of all these proinflammatory cytokines are raised in rheumatoid synovial fluids, this study suggests that IL17 may act as a potent upstream mediator of cartilage collagen breakdown in inflammatory joint diseases.

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Figure 1 .

Figure 1

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Effect of IL17 on the release of proteoglycan and collagen, and the production of collagenolytic activity, from bovine nasal cartilage in explant culture. Three discs of cartilage per well in quadruplicate were cultured for 14 days in control medium ± IL17 (0.4–50 ng/ml). Supernates were harvested at day 7, replenished with identical treatments and harvested on day 14. The levels of (A) GAGs (as a measure of proteoglycan), and (B) OHPro (as a measure of collagen) released into the medium by day 7 and day 14, respectively, were determined as described in the "Materials and methods". Results were expressed as a percentage of the total (mean (SD)). (C) Active and total collagenolytic activities (open and closed bars, respectively) present in day 14 media were determined by bioassay. (D) TIMP-1 (70 U/ml) was included in some bioassays of day 14 media to assess its effect on the collagenolytic activity present. Bonferroni's multiple comparison test was used to compare IL17 with control (A–D), and the same test was used to compare IL17 with control or IL17 + TIMP-1 in the bioassay (D), where ***p≤0.001; **p≤0.01

Figure 2 .

Figure 2

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Effect of metalloproteinase inhibitors on IL17-induced collagen release from cartilage. Bovine nasal cartilage explants were cultured as described in fig 1 except that IL17 was at 100 ng/ml and either TIMP-1 (100 U/ml) or BB-94 (10 µmol/l) were also included. Day 14 culture supernates were assayed for OHPro as a measure of collagen, and the results expressed as a percentage release of the total (mean (SD)). Bonferroni's multiple comparison test was used to compare IL17 alone with control or IL17 + inhibitor, where ***p≤0.001.

Figure 3 .

Figure 3

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Effect of IL17 in combination with TNFα, IL1, or OSM on proteoglycan release from cartilage. Bovine nasal cartilage explants were cultured as described in fig 1 with IL17 (0.01–50 ng/ml) ± (A) TNFα (2 and 10 ng/ml), (B) IL1α (0.1 ng/ml) or OSM (10 ng/ml). Culture supernates (day 7) were assayed for the release of proteoglycan as described in "Materials and methods", and the results expressed as a percentage release of the total (mean (SD)). Bonferroni's multiple comparison test was used to compare IL17 alone with control, and IL17 + TNFα or IL1 or OSM with TNFα or IL1 or OSM alone, where ***p≤0.001; **p≤0.01; NS, not significant. For the selected comparisons shown, the statistical significance relates to all the treatments within the lines.

Figure 4 .

Figure 4

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Effect of IL17 in combination with TNFα, IL1, or OSM on the release of collagen from cartilage. Bovine nasal cartilage explants were cultured as described in fig 3 with IL17 (0.01–50 ng/ml) ± (A) TNFα (2 and 10 ng/ml), (B) IL1α (0.1 ng/ml) or OSM (10 ng/ml). Collagen release was determined as a measure of OHPro as described in "Materials and methods", and the release by day 14 expressed as a percentage of the total (mean (SD)). Bonferroni's multiple comparison test was used as described in fig 3, where ***p≤0.001; *p≤0.05. For the selected comparisons shown, the statistical significance relates to all the treatments within the lines.

Figure 5 .

Figure 5

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Effect of IL17 in combination with gp130 binding cytokines on collagen release from cartilage. Bovine nasal cartilage explants were cultured as described in fig 1 with (A) IL17 (50 ng/ml) ± gp130 binding cytokines (OSM, 10 ng/ml; IL6, IL11, LIF, CNTF, CT-1, all at 50 ng/ml), or (B) IL17 (50 ng/ml) ± OSM (10 ng/ml) or IL6 (50 ng/ml) ± sIL6R (200 ng/ml). Collagen release was determined as a measure of OHPro as described in "Materials and methods", and the release by day 14 expressed as a percentage of the total (mean (SD)). Bonferroni's multiple comparison test was used to compare IL17 alone with control, and IL17 + test reagent(s) with IL17 alone or IL17 + test reagent, where ***p≤0.001.

Figure 6 .

Figure 6

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Effect of metalloproteinase inhibitors on the synergistic release of collagen from cartilage stimulated with IL17 in combination with TNFα, IL1, or OSM. Bovine nasal cartilage explants were cultured as described in fig 3 except that TIMP-1 (100 U/ml) or BB-94 (10 µmol/l) were included. (A) IL17 (50 ng/ml) was combined with TNFα (2 ng/ml), and (B) IL17 (20 ng/ml) was combined with IL1α (0.2 ng/ml) or OSM (10 ng/ml). Collagen release was determined as a measure of OHPro as described in "Materials and methods", and the release by day 14 expressed as a percentage of the total (mean (SD)). Bonferroni's multiple comparison test was used to compare each cytokine combination with the same combination + inhibitor or control, where ***p≤0.001.

Figure 7 .

Figure 7

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Effects of IL17 in combination with TNFα or IL1α on MMP and TIMP mRNA expression in chondrocytes. Total cellular RNA (20 µg) from primary bovine nasal cartilage chondrocytes was harvested after stimulation for 24 hours with IL17 (5–100 ng/ml) ± TNFα (10 ng/ml) or IL1α (0.2 ng/ml) in serum free medium. GAPDH was used to assess RNA loading.

Figure 8 .

Figure 8

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Effects of IL17 in combination with OSM or IL6/sIL6R on MMP and TIMP mRNA expression in chondrocytes. Total cellular RNA (20 µg) from primary bovine nasal cartilage chondrocytes was harvested after stimulation for 24 hours with (A) IL17 (2–50 ng/ml) ± OSM (10 ng/ml), or (B) IL17 (50 ng/ml) ± IL6 (50 ng/ml) ± sIL6R (200 ng/ml) in serum-free medium. GAPDH was used to assess RNA loading.

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