Human articular chondrocytes produce IL-7 and respond to IL-7 with increased production of matrix metalloproteinase-13 - PubMed (original) (raw)

Human articular chondrocytes produce IL-7 and respond to IL-7 with increased production of matrix metalloproteinase-13

David Long et al. Arthritis Res Ther. 2008.

Abstract

Introduction: Fibronectin fragments have been found in the articular cartilage and synovial fluid of patients with osteoarthritis and rheumatoid arthritis. These matrix fragments can stimulate production of multiple mediators of matrix destruction, including various cytokines and metalloproteinases. The purpose of this study was to discover novel mediators of cartilage destruction using fibronectin fragments as a stimulus.

Methods: Human articular cartilage was obtained from tissue donors and from osteoarthritic cartilage removed at the time of knee replacement surgery. Enzymatically isolated chondrocytes in serum-free cultures were stimulated overnight with the 110 kDa alpha5beta1 integrin-binding fibronectin fragment or with IL-1, IL-6, or IL-7. Cytokines and matrix metalloproteinases released into the media were detected using antibody arrays and quantified by ELISA. IL-7 receptor expression was evaluated by flow cytometry, immunocytochemical staining, and PCR.

Results: IL-7 was found to be produced by chondrocytes treated with fibronectin fragments. Compared with cells isolated from normal young adult human articular cartilage, increased IL-7 production was noted in cells isolated from older adult tissue donors and from osteoarthritic cartilage. Chondrocyte IL-7 production was also stimulated by combined treatment with the catabolic cytokines IL-1 and IL-6. Chondrocytes were found to express IL-7 receptors and to respond to IL-7 stimulation with increased production of matrix metalloproteinase-13 and with proteoglycan release from cartilage explants.

Conclusion: These novel findings indicate that IL-7 may contribute to cartilage destruction in joint diseases, including osteoarthritis.

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Figures

Figure 1

Figure 1

Chondrocytes produce IL-7 in response to stimulation with fibronectin fragments. Human articular chondrocytes obtained from normal articular cartilage and cultured in serum-free media were treated overnight with 500 nmol/l of the 110 kDa fibronectin fragment (FN-f). Media was collected and analyzed for cytokine production using (a) an inflammation antibody array or (b) an IL-7 ELISA. Results are representative of three experiments for each result with different donor cells used in each experiment. The IL-7 spots on the array are shown in the red circles. (Other spots that were shown to change after fibronectin fragment stimulation included IL-6, soluble IL-6 receptor [sIL-6R], interferon-inducible protein [IP]-10, and monocyte chemotactic protein [MCP]-1.)

Figure 2

Figure 2

Effects of age and OA on chondrocyte production of IL-7. Media was collected 48 hours after changing to serum-free conditions in chondrocyte cultures established from (a) nonarthritic cartilage from 10 donors of different ages or from (b) cartilage from age-matched nonarthritic (n = 7) and osteoarthritic cartilage (n = 5). IL-7 was measured in the media using ELISA. The relationship of age to IL-7 levels was analyzed by Spearman correlation. The numbers in parentheses above the data points in panel a are the Collin's scores for the donor samples. OA, osteoarthritis.

Figure 3

Figure 3

Chondrocyte expression of IL-7 receptors. (a) Chondrocytes isolated from normal cartilage (n = 1) were incubated with a fluorescently labeled recombinant IL-7 to demonstrate binding of IL-7 to the cell surface. Labeled cells were examined by flow cytometry. The peak that is shaded purple with the black line shows cells stained with IL-7, the peak with the pink line shows blocking antibody negative control, and the peak with the green line shows cells stained with the biotin negative control. (b) Chondrocytes isolated from normal cartilage were incubated with a fluorescently labeled recombinant IL-7 as above. Labeled cells were examined by confocal microscopy. IL-7 staining is shown in green. Top left is the green channel, top right is differential intermittent contrast, and bottom left is the merged image. Chondrocytes from eight different donors showed similar results. (c) Pooled RNA isolated from 10 different sets of cultured chondrocytes was subjected to reverse transcription and real-time PCR with an IL-7 receptor primer set. An amplification plot is shown to demonstrate positive signal. Amplified chondrocyte cDNA in triplicate is shown with the blue lines. Negative control with no reverse transcription of RNA before real-time PCR is shown with a red line. Negative control with no cDNA is shown with the black line.

Figure 4

Figure 4

Chondrocytes respond to IL-7 stimulation with increased PYK2 phosphorylation and production of MMP-13. (a) Chondrocytes isolated from normal adult cartilage were stimulated with 10 ng/mL recombinant IL-7 and lysates were made at indicated time points for immunoblotting with an antibody to phosphorylated proline-rich tyrosine kinase (PYK)2 (Tyr402). The blot was then stripped and probed with total PYK2 antibody to confirm equal loading. (b) Densitometric scanning of the blot shown in panel a. (c) Medium was collected from serum-free chondrocyte cultures after overnight stimulation with 10 ng/ml recombinant IL-7 and examined for the presence of multiple matrix metalloproteinase (MMP) family members using an MMP antibody array. MMP-13 spots are shown in circles. (d,e) Media was collected from serum-free chondrocyte cultures after overnight stimulation with 10 ng/ml recombinant IL-7 or IL-1β, or the two together, and examined for the presence of MMP-13 using a commercially available ELISA. Results are the mean of seven experiments.

Figure 5

Figure 5

IL-7 causes proteoglycan release, but not nitric oxide production, in cartilage explants. Cartilage explants were stimulated for 72 hours with 10 ng/ml recombinant human IL-7 before media collection. (a) Medium was analyed for sulfated glycosaminoglycan (sGAG) using the dimethylmethylene blue assay and normalized for the wet weight of the tissue. (b) Total nitrite was measured in the media as a marker for nitric oxide production using commercially available colorimetric nitrate/nitrite assay kit. Results represent four experiments.

Figure 6

Figure 6

Role for IL-1 and IL-6 in stimulation of IL-7 production by chondrocytes. (a) Chondrocytes were pretreated with either an IL-6 neutralizing antibody or the IL-1 receptor antagonist, or the combination of the two inhibitors, and then subsequently stimulated with fibronectin fragment. After overnight stimulation media samples were collected and used for an inflammation antibody array. IL-7 spots are shown in red circles. (b) Chondrocytes were stimulated with either IL-1β (10 ng/ml) or IL-6/soluble IL-6 receptor (10 ng/ml and 20 ng/ml) or the combination of cytokines. Medium was collected and subsequently analyzed with an IL-7 ELISA.

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References

    1. Goldring MB. The role of the chondrocyte in osteoarthritis. Arthritis Rheum. 2000;43:1916–1926. doi: 10.1002/1529-0131(200009)43:9<1916::AID-ANR2>3.0.CO;2-I. - DOI - PubMed
    1. Pelletier JP, Martel-Pelletier J, Abramson SB. Osteoarthritis, an inflammatory disease: potential implication for the selection of new therapeutic targets. Arthritis Rheum. 2001;44:1237–1247. doi: 10.1002/1529-0131(200106)44:6<1237::AID-ART214>3.0.CO;2-F. - DOI - PubMed
    1. Nietfeld JJ, Wilbrink B, Helle M, van Roy JL, den Otter W, Swaak AJ, Huber-Bruning O. Interleukin-1-induced interleukin-6 is required for the inhibition of proteoglycan synthesis by interleukin-1 in human articular cartilage. Arthritis Rheum. 1990;33:1695–1701. doi: 10.1002/art.1780331113. - DOI - PubMed
    1. Henrotin YE, De Groote DD, Labasse AH, Gaspar SE, Zheng SX, Geenen VG, Reginster JY. Effects of exogenous IL-1 beta, TNF alpha, IL-6, IL-8 and LIF on cytokine production by human articular chondrocytes. Osteoarthritis Cartilage. 1996;4:163–173. doi: 10.1016/S1063-4584(96)80012-4. - DOI - PubMed
    1. Pulai JI, Chen H, Im HJ, Kumar S, Hanning C, Hegde PS, Loeser RF. NF-κB mediates the stimulation of cytokine and chemokine expression by human articular chondrocytes in response to fibronectin fragments. J Immunol. 2005;174:5781–5788. - PMC - PubMed

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