Background/Purpose: Intraarticular basic calcium phosphate (BCP) crystals are present in the majority of osteoarthritic (OA) joints and are associated with severe degeneration. In vitro, BCP promote pro-inflammatory cytokine production and matrix metalloprotease (MMP) expression, suggesting a pathogenic role in OA. Recently it has been demonstrated that BCP crystals drive IL-1β and IL-18 production following activation of the NOD-like receptor, NLRP3. Here, we sought to determine whether uptake of BCP crystals leads to the activation of the membrane proximal kinases, Syk and PI3 kinase and to further characterise events downstream of Syk activation in order to identify novel molecular targets for the treatment of BCP related arthropathies.

Methods: Murine  macrophages were stimulated with BCP crystals, with or without priming with a Toll-like receptor (TLR) agonist and IL-1β and IL-18 production was quantified by enzyme linked immunosorbent assay (ELISA). A role for Syk and PI3 kinase was determined with the use of the inhibitors, piceatannol and LY294002, respectively. Activation of the kinases was confirmed by western blotting using phospho-specific antibodies to Syk and PI3 kinase following treatment of cells with the crystals over a 30 minute time course. Finally, activation of the downstream kinase, ERK, and production of the damage associated molecule, S100A8, was assessed in the presence of piceatannol in order to determine if these events are associated with BCP dependent Syk activation.  

Results:

Physiological concentrations of BCP crystals (50mg/ml) induced robust IL-1β and IL-18 production in a Syk and PI3 kinase dependent manner. Treatment with the inhibitors piceatannol and Ly294002 led to a significant reduction in cytokine levels (>80%) and activation of Syk and PI3 kinase was apparent after approximately 5 minutes treatment with the crystals. Phosphorylation of the downstream kinase, ERK, was prevented following treatment with the Syk inhibitor, piceatannol, thus identifying Syk kinase activation as a proximal event in BCP induced pro-inflammatory cytokine production. Finally, treatment of cells with BCP crystals led directly to the production of the danger-associated molecule, S100A8 and this was also dependent on activation of Syk.

Conclusion: Since S100A8 is considered a TLR 4 ligand, we propose a model whereby BCP crystals drive the production of S100A8 which in turn leads to the expression of pro-IL-1β and pro-IL-18 (Signal 1). In macrophages, BCP crystals can also induce the activation of the NLRP3 inflammasome (Signal 2) leading to the production of the mature forms of these cytokines. These events are dependent on tyrosine phosphorylation and we identify Syk kinase as a potential target for the treatment of BCP related pathologies

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/osteoarthritis-associated-basic-calcium-phosphate-crystals-induce-il-1%ce%b2-il-18-and-s100a8-production-in-a-tyrosine-kinase-dependent-manner/