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Regulation of osteoarthritis-associated key mediators by TNFα and IL-10: effects of IL-10 overexpression in human synovial fibroblasts and a synovial cell line

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Abstract

Synovial fibroblasts (SF) contribute to the pathogenesis of osteoarthritis (OA), but the effects of intra-articular cytokines on SF are not completely understood. The aim of this study was to characterize the interplay between tumor necrosis factor (TNF)α and the anti-inflammatory interleukin (IL)-10. Non-immortalized human SF and SF of the human cell line K4IM were stimulated with recombinant TNFα, IL-10, or TNFα + IL-10 (10 ng/ml each) for 24 h or transduced with an adenoviral vector overexpressing human IL-10 (hIL-10) and subsequently treated with 10 ng/ml TNFα for 24 h. Effects on the gene expression and protein synthesis of IL-6, IL-10, matrix metalloproteinases (MMP)-1, −3, type I collagen, β1-integrin, and CD44 were investigated via real-time detection polymerase chain reaction, immunofluorescence labeling, flow cytometry, and Western blotting. IL-10 release by transduced SF was confirmed with enzyme-linked immunosorbent assay. Both cell populations were activated by TNFα and by TNFα + IL-10, increasing their gene expression and protein synthesis of IL-6, IL-10, MMP-1, and MMP−3 and altering the synthesis of type I collagen, β1-integrin, and CD44. hIL-10 overexpression greatly elevated the gene expression and protein synthesis of IL-10. However, transduction did not significantly affect the gene expression of IL-6, MMP-1, and MMP−3 in SF. The increased expression of pro-inflammatory and catabolic mediators in TNFα-activated SF indicates their role in OA pathogenesis, suggesting they are a potential therapeutic target. Although the vigorousness of the responses of non-immortalized SF and K4IM clearly differ, the K4IM cell line seems to be a suitable model for non-immortalized human SF.

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Acknowledgements

We are grateful to Dr. Michaela Endres for providing us with the K4IM cell line and Dr. Beth Hutchins and Dr. Drake LaFace for supplying the adenoviral vectors. We also thank Kristin Moderzynski and Prof. Dr. Marcus Frohme for their support, and Owen Godkin and Hannah Gough for proof reading the manuscript.

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Authors and Affiliations

  1. Department for Orthopaedic, Trauma and Reconstructive Surgery, Charité-University of Medicine, Campus Benjamin Franklin, FEM Garystrasse 5, 14195, Berlin, Germany

    I. Mrosewski, N. Jork, K. Gorte, C. Conrad, E. Wiegand, B. Kohl, W. Ertel & G. Schulze-Tanzil

  2. Department for Orthopaedics and Trauma Surgery, DRK Kliniken Berlin Westend, Berlin, Germany

    T. John

  3. Zentrum für Gelenk- und Sportchirurgie, Klinik Pyramide am See, Zürich, Switzerland

    A. Oberholzer

  4. Department of Rheumatology, Charité-University of Medicine, Campus Charité Mitte, Berlin, Germany

    C. Kaps

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  1. I. Mrosewski
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  3. K. Gorte
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  4. C. Conrad
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  9. A. Oberholzer
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  11. G. Schulze-Tanzil
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Correspondence to G. Schulze-Tanzil.

Additional information

This study was supported by a grant from the ENDO-Stiftung, and the equipment used was funded by the Sonnenfeld foundation; we express our gratitude for this support.

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Supplementary Figure 1

Protein synthesis of non-immortalized human SF during expansion in passage 2 and 7. Comparison of the synthesis of CD44 (a, b), CD55 (c, d), UDPGD (e, f), tenascin C (g, h), fibronectin (i, j), type I collagen (coll. type I, k, l), and proteoglycans (m, n) between passage 2 (a, c, e, g, i, k, m) and 7 (b, d, f, h, j, l, n) is depicted. The cell nuclei have been counter-stained with DAPI. Bar 50 μm. (TIFF 4065 kb)

Supplementary Figure 2

Protein synthesis profile of human non-immortalized SF and K4IM SF with regard to additional markers. Non-immortalized (NI-SF) human synovial fibroblasts (a, d, g, j), K4IM cells (b, e, h, k), and paraffin sections of human synovium (c, f, i, l) were immunolabeled as indicated left for CD34 (a-c), CD106 (d-f), lubricin (g-i), and αSMA (j-l). The cell nuclei have been counter-stained with DAPI. Bar 200 μm. (TIFF 2477 kb)

Supplementary Figure 3

Protein synthesis profile of human non-immortalized SF and K4IM SF with regard to additional markers. Non-immortalized (NI-SF) human synovial fibroblasts (a, c, e, g) and K4IM cells (b, d, f, h) were immunolabelled for CD54, CD59, CD90, and vimentin. Bar 50 μm. (TIFF 5113 kb)

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Mrosewski, I., Jork, N., Gorte, K. et al. Regulation of osteoarthritis-associated key mediators by TNFα and IL-10: effects of IL-10 overexpression in human synovial fibroblasts and a synovial cell line. Cell Tissue Res 357, 207–223 (2014). https://doi.org/10.1007/s00441-014-1868-y

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