37. INTERACTIVE CYTOKINE REGULATION OF SYNOVIOCYTE HYALURONAN AND PRG4 SECRETION RATES
Name: Megan Elizabeth Blewis
Grad Year: 2008
Introduction: Synovial joints constitute complex, low-friction, low-wear load-bearing systems normally in homeostasis. However, cartilage undergoes erosion in arthritis and post-injury degeneration, in association with decreases in boundary lubricating properties of synovial fluid (SF). In abnormal SFs, the concentration of lubricants hyaluronan (HA) and proteoglycan 4 (PRG4) are altered, although mechanisms are unknown. One source of HA and PRG4 is the fibroblast-like synoviocyte (FLS) in synovium, which is regulated by many SF cytokines in counterbalancing and synergistic manners. How cytokines regulate FLS lubricant secretion remains to be established, in health, injury, and arthritis. In the latter, IL-1β, IL-17, IL-32, TGF-β1, and TNF-alpha are often elevated. The hypothesis was that these cytokines regulate FLS lubricant secretion individually and in combination, additively or synergistically. Methods: Synoviocytes from human patients were obtained (IRB approval). After passaging to obtain FLS, cells were incubated under basal conditions, then with addition of individual or combinations of cytokines for 3d. Individual cytokines were applied over concentration ranges, and combinations applied at high concentrations. Secretion rates (rHASYN, rPRG4SYN) were assessed by analyzing medium for HA and PRG4, and normalizing to cell number and duration. Individual cytokine effects were analyzed by 1-way ANOVA and Dunnett?s post-hoc. For combinations of cytokines, 5-way ANOVA was used to assess individual and interactive effects Results: rHASYN was affected markedly by IL-1β, and to a lesser extent by TNF-alpha and TGF-β1 (p<0.05), with ctrl of 0.67±0.15 µg/(10^6 cell?day) increased 22x, 4.2x, and 2.8x. rPRG4SYN was affected by TGF-β1 (p<0.05), with ctrl of 0.08±0.09 µg/(10^6 cell?day) increased ~80x. Non-additive upregulation of rHASYN occurred when TNF-alpha was present with IL-17, TGF-β1, or IL-1β, increasing rHASYN 6.7x, 2.4x, and 1.3x over additive effects; IL-1β also acted synergistically with TGF-β1, increasing rHASYN 2.0x over additive effects (p<0.05). TGF-β1 effects on rPRG4SYN were counterbalanced by IL-1β and TNF-alpha (p<0.05). Discussion: This is the first report of synergistic upregulation of FLS HA secretion by TNF-alpha with IL-17. This role of IL-17 suggests involvement of T cells in HA regulation. Differential regulation of HA and PRG4 by IL-1β and TGF-β1, and synergistic regulation of HA by other cytokines may allow modulation of lubricant concentration to desired levels in bioreactors. Marked upregulation in HA secretion from synergistic effects of inflammatory cytokines may explain marked SF volume increases in injured/diseased joints, with only moderate decreases in SF HA concentration. Synergistic effects of TNF-alpha with multiple cytokines suggest therapies targeting TNF-alpha may have potent effects.