Synovial Fluid from Patients with Osteoarthritis Shows Different Inflammatory Features Depending on the Presence of Calcium Pyrophosphate Crystals

Abstract

The role of calcium pyrophosphate (CPP) crystals in osteoarthritis (OA) is still a matter of debate. With this study we aimed to investigate the inflammatory features of synovial fluid (SF) collected from patients with OA with CPP crystals compared with those without crystals. We also explored the effect of OA SF on monocytes response. SFs were collected from adult patients with OA and subdivided according to the presence of crystals. Local cellular and humoral inflammatory mediators were analysed in the SF samples. The expression levels of IL-1β, IL-18, CASP-1, NLRP3, and GAPDH were measured by RT-PCR in the cells obtained by pelleting the SF samples. For the in vitro study, a monocytic cell line was treated with selected SF samples. SF with CPP crystals showed a significant increase in inflammatory cellular indices and higher levels of IL-1β, IL-8, and caspase-1 transcript with respect to SF without crystals. Higher concentrations of VEGF were also observed in the early stages of the whole OA patients. THP-1 cells stimulated with OA SF released a significant amount of IL-1 β in culture supernatants. This study demonstrated that SF collected from patients with OA shows different inflammatory features depending on the presence of CPP crystals.

Keywords: calcium pyrophosphate crystals; inflammation; interleukin-1; osteoarthritis; synovial fluid.

Figure 1

Cytokines and chemokines in SFs from OA CPP+ and OA CPP−. Measurement of IL-1β (A), IL-1RA (B), IL-1β/IL-1RA (C), IL-18 (D), IL-8 (E), CCL-2 (F), IL-6 (G), IL-10 (H), and OSMR (I) levels was performed in 29 SFs from OA CPP+ patients and 39 SFs from OA CPP− patients as described in Materials and Methods. Data are shown as the median (IQR). p values calculated according to the Kruskal–Wallis test and Dunn’s post hoc test: *** p <0.001, **** p <0.0001. Abbreviations are as follows: OA, osteoarthritis; CPP−, SF negative to CPP crystals; CPP+, SF positive to CPP crystals; ns, not significant.

Figure 2

VEGF, BMP-2, MMP-1, and TIMP-1 levels in SFs from OA CPP+ and OA CPP−. Measurement of VEGF (A), BMP-2 (B), MMP-1 (C), and TIMP-1 (D) levels was performed in 29 SFs from OA CPP+ patients and 39 SFs from OA CPP− patients as described in Materials and Methods. Data are shown as the median (IQR); BMP-2 and TIMP-1 are shown as logarithmic scale due to their very variable values. p values calculated according to the Kruskal–Wallis test and Dunn’s post hoc test: ** p < 0.01. Abbreviations are as follows: OA, osteoarthritis; CPP−, SF negative to CPP crystals; CPP+, SF positive to CPP crystals; ns, not significant.

Figure 3

Association between KL values and VEGF and MMP-1 in SFs from OA patients. (A) VEGF and (B) MMP-1 levels evaluated in 35 SFs from OA patients as described in Materials and Methods. Data are shown as the median (IQR). p values calculated according to the Kruskal–Wallis test and Dunn’s post hoc test: ** p < 0.01. Abbreviations are as follows: KL, Kellgren and Lawrence classification.

Figure 4

IL-1β released from monocytes treated with SFs from OA patients. THP-1 cells were treated with 20% of SFs (n = 10) for 24h as described in Materials and Methods. (A) Levels of IL-1β released from monocytes treated with SFs from OA patients (n = 10). IL-1 β levels were also evaluated in SFs at 20% to stimulate monocytes. Data are shown as the median (IQR). p values calculated according to the Kruskal–Wallis test and Dunn’s post hoc test: * p < 0.05. (B) Intracellular and extracellular IL-1β levels released by monocytes treated with CPP+ OA SFs and with CPP− OA SFs (n = 5). Data are shown as the median (IQR). p values calculated according to the Kruskal–Wallis test and Dunn’s post hoc test. Abbreviations are as follows: OA, osteoarthritis; SF, synovial fluid; CPP−, SF negative to CPP crystals; CPP+, SF positive to CPP crystals; ns: not significant.

Figure 5

Intracellular CPP crystal observed in OA patient using a pre-stained slide. An extracellular rhomboid crystal is also visible. Ordinary light microscopy. 1000× magnification.