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. 2001 Feb;60(2):150–157. doi: 10.1136/ard.60.2.150

Interleukin 13 blocks the release of collagen from bovine nasal cartilage treated with proinflammatory cytokines

C Cleaver 1, A Rowan 1, T Cawston 1
PMCID: PMC1753472  PMID: 11156549

Abstract

OBJECTIVE—To investigate whether interleukin 13 (IL13) could act in a chondroprotective manner and protect cartilage stimulated to resorb with a combination of IL1α and oncostatin M (OSM), in a similar way to the anti-inflammatory cytokine, IL4.
METHODS—IL13 was added to explant cultures of bovine nasal cartilage stimulated to resorb with IL1α and OSM, and the release of collagen and proteoglycan determined. Collagenolytic and tissue inhibitors of metalloproteinase (TIMP) activities were determined by bioassay. Northern blot analyses were performed to determine the effects of IL13 on the induction of matrix metalloproteinase-1 (MMP-1), MMP-3, MMP-13, and TIMP-1 gene expression.
RESULTS—IL13 can prevent the release of collagen from bovine nasal cartilage in a dose dependent manner. This was accompanied by a concomitant decrease in measurable collagenolytic activity in the culture supernates and an increase in TIMP activity. Northern blot analysis showed that IL13 down regulated MMP-3 and MMP-13 levels but up regulated MMP-1 and TIMP-1 gene expression in bovine nasal chondrocytes at 24 hours.
CONCLUSION—This study showed for the first time that IL13 can block collagen release from resorbing cartilage in a similar manner to IL4. This is accompanied by a reduction in detectable collagenolytic activity, a decrease in MMP-3 and MMP-13 mRNA levels, and an up regulation of TIMP-1 expression.



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Figure 1  .

Figure 1  

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The effect of interleukin 1α (IL1α) in combination with oncostatin M (OSM), with or without IL13, on the release of proteoglycan and collagen from bovine nasal cartilage in explant culture. Three discs of cartilage per well in quadruplicate were cultured in 600 µl control medium alone, IL1α (1 ng/ml), OSM (10 ng/ml), IL1α + OSM, with and without IL13 (2-50 ng/ml), for 0-7 days and the media removed. Each well was replenished under identical conditions and left for a further seven days. At day 14 media were removed and the remaining cartilage digested with papain. The levels of glycosaminoglycan (GAG) and hydroxyproline (OHPro) released into the medium on days 7 and 14 were determined and the results expressed as a percentage of the total. Results are expressed as mean (SD). The data shown are representative of three independent experiments. Student's unpaired two tailed t test was used to compare IL1α and IL1α + IL13, where †††p<0.001 and †p<0.05. The same test was used to compare IL1α + OSM with IL1α + OSM + IL13, where ***p<0.001 and **p<0.01.

Figure 2  .

Figure 2  

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Levels of active and total collagenase activity in media samples removed from cartilage cultures at day 14 after stimulation with interleukin 1α (IL1α), oncostatin M (OSM), IL1α + OSM with and without IL13 (50 ng/ml). Cartilage was incubated and treated as described in fig 1. The levels of active and total collagenase released into the medium on day 14 were measured and the results expressed as U/ml (mean (SD)). Active and total collagenase were measured as described in "Materials and methods". Student's unpaired two tailed t test was used to compare IL1α and IL1α + IL13, where †††p<0.001. The same test was used to compare IL1α + OSM with IL1α + OSM + IL13, where ***p<0.001, **p<0.01, and *p<0.05.

Figure 3  .

Figure 3  

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Levels of tissue inhibitors of metalloproteinase (TIMP) inhibitory activity in media samples removed from cartilage cultures at day 14 after stimulation with interleukin 1α ( IL1α), oncostatin M (OSM), IL1α + OSM, with and without IL13. Cartilage was incubated and treated as described in fig 1. The levels of TIMP released into the medium on day 14 were measured and the results expressed as U/ml (mean (SD)). Student's unpaired two tailed t test was used to compare IL1α and IL1α + IL13, where †††p<0.001. The same test was used to compare IL1α + OSM with IL1α + OSM + IL13, where ***p<0.001.

Figure 4  .

Figure 4  

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Cartilage collagen release inhibited by interleukin 13 (IL13) and IL4 can be recovered by addition of an exogenous activator of procollagenases. Three discs of cartilage per well in quadruplicate were cultured in 600 µl control medium alone, IL1α (1 ng/ml) + oncostatin M (OSM) (10 ng/ml), with and without IL4/13 (2-50 ng/ml), for 0-7 days, and the media removed. Media containing identical cytokine combinations to day 1 were replenished. One set received no further additions (A), whereas others had exogenous active matrix metalloproteinase-3 (MMP-3) added at 0.83 µg/ml (B), or 3.33 µg/ml (C). All cultures were left for a further seven days. At day 14 media were removed and the remaining cartilage digested with papain. The levels of hydroxyproline (OHPro) released into the medium on days 7 and 14 were determined and the results expressed as a percentage of the total released. Results are expressed as mean (SD). The data shown are representative of three independent experiments. Student's unpaired two tailed t test was used to analyse data, firstly, where the addition of MMP-3 at 0.83 µg/ml (B) was compared with its identical treatment in the absence of exogenous MMP-3 (A), where ***p<0.001, and, secondly, where the addition of MMP-3 at 3.33 µg/ml (C) was compared with its identical treatment in the presence of MMP-3 at 0.83 µg/ml (B), where †††p<0.001 and ††p<0.01.

Figure 5  .

Figure 5  

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Interleukin 13 (IL13) inhibits IL1α and IL1α + oncostatin M (OSM) induction of metalloproteinase-3 (MMP-3) and MMP-13 mRNA but not MMP-1 or tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) mRNA in bovine nasal chondrocytes at 24 hours. Total RNA was isolated at 24 hours after the addition of IL1α (1 ng/ml), OSM (10 ng/ml), IL1α + OSM, alone or in combination with IL13 in cultures of bovine nasal chondrocytes. Northern blots were hybridised to cDNA probes corresponding to MMP-1 (A), MMP-3 (B), MMP-13 (C), and TIMP-1 (D). Ethidium bromide stained 28 S rRNA was used for normalisation of RNA loading. The northern blots are shown on the left hand side of the figure and were measured by scanning densitometry. The results are expressed as fold induction over control on the right hand side. The data shown are representative of three independent experiments.

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