Background/Purpose:   Gout is an inflammatory arthritis caused by precipitation of monosodium urate (MSU) crystals in synovial joints. MSU crystals interact with resident macrophages that release pro-inflammatory cytokines, interleukin-1 beta (IL-1β) and chemokines, interleukin-8 (IL-8).  MSU crystals are phagocytosed in a process mediated by toll-like receptor (TLR2). Proteoglycan-4 (PRG4) is a lubricating mucinous glycoprotein released by synovial fibroblasts and exhibits a multifaceted homeostatic role in the synovial joint.  We have recently shown that recombinant human PRG4 (rhPRG4) can inhibit agonist-induced TLR2 activation. The objective is to evaluate the efficacy of rhPRG4 in modulating MSU activation of macrophages.  We hypothesized that rhPRG4 inhibits MSU phagocytosis by human macrophages dose-dependently and inhibits MSU-induced IL-1β and IL-8 gene expression.

Methods: THP1 human monocytes were differentiated into macrophages using 5ng/ml phorbol 12-myristate13-acetate for 48 hours at 37^oC. A total of 500,000 macrophages were plated per well in sterile tissue culture plates and were treated with MSU crystals (100μg/mL) for 6 hours at 37^oC, and afterwards in the absence or presence of rhPRG4 (25, 50, 100, 200μg/mL) for 24 hours at 37^oC.  Following incubation, the percent of macrophages that phagocytosed MSU was quantitatively determined using flow cytometry, based on an increase in cellular side-scatter.  RNA was extracted and cDNA was synthesized followed by qRT-PCR using TaqMan Fast Advanced Master Mix and primers for human IL-1β and IL-8.  IL-1β and IL-8 media supernatant concentrations were determined by commercially available ELISA.

Results: Flow cytometry scatter plots of MSU phagocytosis by macrophages in the absence or presence of rhPRG4 is shown in figure 1A.  rhPRG4 treatment significantly inhibited MSU phagocytosis (p<0.05) (fig. 1B).  MSU treatment resulted in a significant induction of IL-1β gene expression which was inhibited by rhPRG4 treatment (100 and 200μg/ml) (p<0.05; fig. 2A), and a significant reduction in IL-1β protein concentration at the 200μg/ml rhPRG4 concentration (p<0.05; fig. 2C).  Likewise, MSU treatment resulted in a significant induction of IL-8 gene expression which was inhibited by rhPRG4 treatment (50, 100 and 200μg/ml) (p<0.05; fig. 2B), and a significant reduction in IL-1β protein concentration at the 100 and 200μg/ml rhPRG4 concentrations (p<0.05 and p<0.001; fig. 2D).   

Conclusion: rhPRG4 inhibits MSU phagocytosis and the resultant inflammatory response by human macrophages and may be useful as a biological treatment for acute gout exacerbations.