Background/Purpose: Basic calcium phosphate (BCP) crystals are uniquely associated with osteoarthritis (OA). They are found in the majority of affected joints and closely correlate with the extent of joint destruction, suggesting a pathogenic role in driving disease.  They have been shown to induce pro-inflammatory cytokine production and activate synovial fibroblasts (SF) and articular chondrocytes, resulting in increased cell proliferation and matrix metalloprotease expression. These combined processes eventually lead to an imbalance in anabolic versus catabolic mediators of cartilage turnover and ultimately to extracellular matrix degradation. At the molecular level, BCP crystals were shown to activate protein kinase C, ERK1/2, MAP kinase and NF-κB in SF. Recent studies have demonstrated that both mechanical stress and activation of purinergic receptors drives the release of nucleotides such as ATP and UTP from osteocytes which then activate signalling pathways that can negatively impact bone remodelling. It has also been proposed that particulate matter such as alum and monosodium urate (MSU) crystals drive interleukin-1 production via ATP release and purinergic signalling. Furthermore, it has been reported that the P2Y6 receptor and its downstream signalling molecule phospholipase C (PLC) mediate the inflammatory responses induced by MSU crystals. In this study, we sought 1) to determine whether BCP crystals induce the release of ATP from murine macrophages and 2) to investigate the role of purinergic signalling in BCP-induced inflammation in order to identify novel targets for the treatment of BCP-related arthropathies, such as OA. 

Methods: Murine macrophages were stimulated with BCP crystals over the course of 5 hours, and ATP release was measured using the ATPlite luminescence assay system. BCP crystals are known to drive IL-1β production in vitro. Therefore in order to investigate the role of purinergic receptors in BCP-induced cell activation, murine macrophages were primed with lipopolysaccharide, a toll-like receptor agonist prior to treatment with the broad-spectrum P2 receptor inhibitor oATP, the P2Y6-specific inhibitor MRS2578, or the PLC inhibitor U73122. Alternatively, siRNA was used to knock down the expression of P2Y6. The cells were then stimulated with BCP crystals and IL-1β production was quantified by enzyme linked immunosorbent assay (ELISA).    

Results: We have found that physiological concentrations of BCP crystals (50μg/ml) induced the release of approximately 100 nM ATP by murine macrophages, a concentration sufficient to activate purinergic receptors. Inhibition of the P2Y6 receptor or PLC dose-dependently reduced IL-1β production following BCP stimulation, with full abrogation observed with the top dose of each inhibitor. Furthermore a 40% knockdown of the P2Y6 receptor led to an equal reduction in BCP-induced IL-1β production. 

Conclusion: Based on these studies we propose that nucleotides released from BCP-activated cells act in an autocrine or paracrine manner via purinergic receptors to enhance BCP-induced inflammation. Released nucleotides may also act on neighbouring osteoblasts/osteoclasts to impact on bone remodelling.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-role-for-purinergic-receptor-signalling-in-basic-calcium-phosphate-crystal-induced-inflammation/