Temporal effects of cytokine treatment on lubricant synthesis and matrix metalloproteinase activity of fibroblast-like synoviocytes
Ahmad E. Abu-Hakmeh
Laboratory for Tissue Engineering and Morphogenesis, Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York
Search for more papers by this authorAllison K. M. Fleck
Laboratory for Tissue Engineering and Morphogenesis, Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York
Search for more papers by this authorCorresponding Author
Leo Q. Wan
Laboratory for Tissue Engineering and Morphogenesis, Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York
Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York
Center for Modeling, Simulation and Imaging in Medicine, Rensselaer Polytechnic Institute, Troy, New York
Correspondence
Leo Q. Wan, Laboratory for Tissue Engineering and Morphogenesis, Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Biotech 2147, 110 8th Street, Troy, NY 12180.
Email: [email protected]
Search for more papers by this authorAhmad E. Abu-Hakmeh
Laboratory for Tissue Engineering and Morphogenesis, Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York
Search for more papers by this authorAllison K. M. Fleck
Laboratory for Tissue Engineering and Morphogenesis, Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York
Search for more papers by this authorCorresponding Author
Leo Q. Wan
Laboratory for Tissue Engineering and Morphogenesis, Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York
Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York
Center for Modeling, Simulation and Imaging in Medicine, Rensselaer Polytechnic Institute, Troy, New York
Correspondence
Leo Q. Wan, Laboratory for Tissue Engineering and Morphogenesis, Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Biotech 2147, 110 8th Street, Troy, NY 12180.
Email: [email protected]
Search for more papers by this authorAbstract
Fibroblast-like synoviocytes (FLS) are major contributors to the composition and function of synovial fluid (SF). In disease, changes to important SF molecules such as hyaluronic acid (HA), lubricin, and numerous inflammatory markers contribute to a loss of SF functional properties. Previous studies characterized the ability of FLS to produce SF molecules in short-term cultures using continuous cytokine supplementation. This study assessed the HA, lubricin, and matrix metalloproteinase-2 (MMP-2) secretion profile of FLS over 12 days of culture. FLS were subjected to continuous, intermittent, and sequential cytokine treatments of interleukin-1 beta (IL-1β), tumour necrosis factor-alpha (TNF-α), and transforming growth factor-beta 1 (TGF-β1). HA was assessed by an enzyme-linked immunosorbent assay (ELISA) for content and agarose gel electrophoresis for molecular weight distribution. Relative lubricin content was determined by western blot. Pro MMP-2 and active MMP-2 were quantified by gelatin zymography. All intermittent and sequential treatments significantly increased secretion of high-molecular-weight (>3 MDa) HA for the duration of the culture. Sequentially treated groups elevated lubricin synthesis, whereas only groups receiving IL-1β and TNF-α for 2 days followed by TGF-β1 for 1 day reduced active MMP-2 to unstimulated control levels. These data provide important information on the long-term functional potential of cytokine-stimulated FLS and suggest that temporal regulation of cytokine exposure can be a powerful tool to guide healthy synovial secretions.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Supporting Information
| Filename | Description |
|---|---|
| term2771-sup-0001-sf1.tifTIFF image, 8.3 MB |
Figure S1. 10x phase contrast images showing morphology at day 12 of cytokine stimulated fibroblast-like synoviocytes (FLS). (A) Control; (B) Continuous; (C) Intermittent (M); (D) Intermittent (H); (E) Sequential (1+2); (F) Sequential (2+1). All stimulations show enhanced proliferation of FLS compared to controls. Scale bar: 50μm. |
| term2771-sup-0002-sf2.tifTIFF image, 4 MB |
Figure S2. Representative agarose gel for HA molecular weight distribution for continuous, intermittent, and sequential cytokine stimulation of fibroblast-like synoviocytes (FLS). Plus (+) signs indicate negative controls digested with Streptomyces hyaluronidase. |
| term2771-sup-0003-sf3.tifTIFF image, 2.8 MB |
Figure S3. Representative gelatin zymography of pro and active MMP-2 secretion for continuous, intermittent, and sequential cytokine stimulation of fibroblast-like synoviocytes (FLS). |
| term2771-sup-0004-sf4.tifTIFF image, 1.3 MB |
Figure S4. Representative lubricin western blot for continuous, intermittent, and sequential cytokine stimulation of fibroblast-like synoviocytes (FLS). |
| term2771-sup-0005-sf5.tifTIFF image, 683.4 KB |
Figure S5. mRNA levels of lubricin determined by qPCR from continuous, intermittent (Int (M)), and sequential cytokine stimulation (i.e., Seq (1+2) and Seq (2+1) groups) of fibroblast-like synoviocytes (FLS). Figure inset more appreciably shows differences between treatments from day 6–12. Data reported as mean ± SD. Differences within a time point are indicated by different letters, and differences within a group from day 3 basal values indicated by asterisk (*). Data are normalized to day 3 continuous lubricin levels. |
| term2771-sup-0006-sf6.tifTIFF image, 589.2 KB |
Figure S6. mRNA levels of MMP-2 determined by qPCR for continuous, intermittent (Int (M)), and sequential cytokine stimulation (i.e., Seq (1+2) and Seq (2+1) groups) of fibroblast-like synoviocytes (FLS). Data reported as mean ± SD. Differences within a time point are indicated by different letters, and differences within a group from day 3 basal values indicated by asterisk (*). Data are normalized to day 3 continuous MMP-2 levels. |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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