Background Calcium pyrophosphate (CPP) crystals deposition is frequently associated with acute arthritis and its self-resolution. Interleukin-1b (IL-1β) plays a pivotal role in CPP crystal-induced inflammation process in vivo. However, the mechanisms of CPP crystal-mediated IL-1β production and inflammation remain unknown. One possible pathway could imply the reactive oxygen species (ROS) reported as an intracellular mediator of IL-1β production through the inflammasome activation. Although 4 different types of CPP crystals can be synthesized in vitro [amorphous CPP (a-CPP), monoclinic CPP dihydrate (m-CPPD) or tetrahydrate (m-CPPT) and triclinic CPP dihydrate (t-CPPD) crystals], only m-CPPD and t-CPP crystals have been identified in human samples and the effect of these different types of CPP crystals on IL-1β production has never been explored.
Objectives To evaluate the inflammatory properties of 4 types of CPP crystals and to elucidate the cellular pathways involved in IL-1β production.
Methods a-CPP, m-CPPD, m-CPPT and t-CPP crystals were synthesized as previously described  and sterilized by gamma irradiation. Monosodium urate crystals (MSU) were used as positive control. In vitro, PMA-activated THP-1 cells, a human monocyte cell line, were stimulated with CPP or MSU crystals (200 μg/ml) in the presence or not of N-acetyl-L-cystein (NAC, 25 mM) – an antioxidant molecule counteracting ROS production. At different times of culture, IL-1β production was quantified by ELISA in cell supernatants and expression of pro- and anti-inflammatory genes (IL-1β, IL-6, TNF-α, IL-8, COX-2, IL-10) was determined by quantitative PCR (qPCR). In vivo CPP crystal-mediated inflammation was assessed using the air pouch model in WT mice (n=6/groups). IL-1β production and cell infiltrate were evaluated in air pouch lavages by ELISA and flow cytometry respectively. IL-1β, IL-6 and TNF-α gene expression were quantified in air pouch membranes by qPCR.
Results In vitro m-CPPD crystals induced a higher release of IL-1β than t-CPPD or MSU while a-CPP and m-CPPT crystals did not induce it. IL-1β production in response to m-CPPD, t-CPPD and MSU crystals significantly increased from 2 to 48 hrs of stimulation (figure). The pre-incubation with NAC significantly inhibited IL-1βproduction at 6 hrs after m-CPPD crystal stimulation. Similarly, m-CPPD and t-CPPD crystals increased the expression of pro-inflammatory cytokine and COX-2 genes with a maximum effect after 24 hrs of stimulation. Interestingly, maximum gene induction of anti-inflammatory cytokine IL-10 occurred at 6 hrs stimulation. In vivo studies confirmed initiation of inflammatory parameters in response to m-CPPD and t-CPPD crystals. Indeed, after CPP crystal injection compared to PBS, IL-1β concentrations increased in air pouch lavages as well as neutrophil and monocyte infiltrates. Similarly, IL-1β-air pouch membrane gene expression increased in treated mice.
Conclusions Diverse forms of CPP crystals displayed differential cellular responses; m-CPPD representing the most inflammatory CPP crystals in vitro. Preliminary results highlighted a role of ROS production in CPP crystal-induced IL-1β production. Investigations of their sources and mechanisms of generation are ongoing.
Gras P et al. Acta Crystallogr C Struct Chem. 2014 Sep 15; 70:862-6.
Disclosure of Interest None declared