Temporal effects of cytokine treatment on lubricant synthesis and matrix metalloproteinase activity of fibroblast-like synoviocytes

Abstract

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 12days 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 (>3MDa) HA for the duration of the culture. Sequentially treated groups elevated lubricin synthesis, whereas only groups receiving IL-1β and TNF-α for 2days followed by TGF-β1 for 1day 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.