Recombinant Human Proteoglycan 4 Regulates Phagocytic Activation of Monocytes and Reduces IL-1ß Secretion by Urate Crystal Stimulated Gout PBMCs.

ABSTRACT

Objectives: To compare phagocytic activities of monocytes in peripheral blood mononuclear cells (PBMCs) from acute gout patients and normal subjects, examine monosodium urate monohydrate (MSU) crystal-induced IL-1ß secretion ± recombinant human proteoglycan 4 (rhPRG4) or interleukin-1 receptor antagonist (IL-1RA), and study the anti-inflammatory mechanism of rhPRG4 in MSU stimulated monocytes. Methods: Acute gout PBMCs were collected from patients in the Emergency Department and normal PBMCs were obtained from a commercial source. Monocytes in PBMCs were identified by flow cytometry. PBMCs were primed with Pam3CSK4 (1µg/mL) for 24h and phagocytic activation of monocytes was determined using fluorescently labeled latex beads. MSU (200µg/mL) stimulated IL-1ß secretion was determined by ELISA. Reactive oxygen species (ROS) generation in monocytes was determined fluorometrically. PBMCs were incubated with IL-1RA (250ng/mL) or rhPRG4 (200µg/mL) and bead phagocytosis by monocytes was determined. THP-1 monocytes were treated with MSU crystals ± rhPRG4 and cellular levels of NLRP3 protein, pro-IL-1ß, secreted IL-1ß, and activities of caspase-1 and protein phosphatase-2A (PP2A) were quantified. The peritoneal influx of inflammatory and anti-inflammatory monocytes and neutrophils in Prg4 deficient mice was studied and the impact of rhPRG4 on immune cell trafficking was assessed. Results: Enhanced phagocytic activation of gout monocytes under basal conditions (p<0.001) was associated with ROS generation and MSU stimulated IL-1ß secretion (p<0.05). rhPRG4 reduced bead phagocytosis by normal and gout monocytes compared to IL-1RA and both treatments were efficacious in reducing IL-1ß secretion (p<0.05). rhPRG4 reduced pro-IL-1ß content, caspase-1 activity, conversion of pro-IL-1ß to mature IL-1ß and restored PP2A activity in monocytes (p<0.05). PP2A inhibition reversed rhPRG4's effects on pro-IL-1ß and mature IL-1ß in MSU stimulated monocytes. Neutrophils accumulated in peritoneal cavities of Prg4 deficient mice (p<0.01) and rhPRG4 treatment reduced neutrophil accumulation and enhanced anti-inflammatory monocyte influx (p<0.05). Conclusions: MSU phagocytosis was higher in gout monocytes resulting in higher ROS and IL-1ß secretion. rhPRG4 reduced monocyte phagocytic activation to a greater extent than IL-1RA and reduced IL-1ß secretion. The anti-inflammatory activity of rhPRG4 in monocytes is partially mediated by PP2A, and in vivo, PRG4 plays a role in regulating the trafficking of immune cells into the site of a gout flare.