OCP crystal-induced inflammation and cartilage degradation is NLRP3 inflammasome- and IL-1 independent.

<p>WT (n = 6), ASC-/- (n = 4), NLRP3-/- (n = 6), IL-1α-/- (n = 5) and IL-1β-/- (n = 6) mice were injected i.a. with OCP crystals (200 µg in 20 µl) or PBS. In a second set of experiment, anakinra, the recombinant form of IL-1Ra, or PBS were injected for 4 days (7 mice per group), the first injection being 30 min prior to OCP injection into the knee of WT (F). Ratio of isotope uptake into OCP injected knee versus PBS-injected ones was calculated at different time points (A). Synovial inflammation (B, E, F), cartilage PG loss (C, E, F) and VDIPEN immunohistochemistries (D, F) were assessed. Results are expressed as % of scores against WT (B,C,D) or in arbitrary units (E, F), and represent mean ± S.E.M. of at least n = 4 mice per group. For p values, *  =  p<0.05, **  =  p<0.01, ***  =  p<0.001.</p

OCP crystals induce macrophage expression of genes involved in inflammation and cartilage degradation.

<p>Bone marrow derived macrophages were stimulated <i>in vitro</i> with 500 µg/ml of OCP crystals for 4 hours. RNA was extracted, reverse transcribed and qRT-PCR performed using gene specific primers with Tbp, and Gapdh as reference genes. Results are expressed as the fold induction of OCP treated over unstimulated macrophages, using the mean ± S.E.M of triplicate samples.</p

OCP crystals induce cartilage degradation.

<p>C57BL/6 mice were injected with OCP crystals (OCP+) or PBS (OCP-). Knees harvested at different times (day 4, 17 and 30 n = 8 mice per group) were assessed for cartilage PGs with Safranin-O (A), aggrecan degradation via VDIPEN immunohistochemistry (B) and apoptosis (C). Since at all time points, data from PBS-injected control knees were similar, only data from PBS-injected knees at day 4 were shown in D, E, and F. Scoring of PG loss and VDIPEN staining was performed on sections, using a scale of 0 to 6 and 0 to 3, respectively (D and E). Apoptotic chondrocytes were counted per field of view (F). Results are expressed as mean ± S.E.M with significance being at * p<0.05, ** p<0.01, *** p<0.001</p

Intra-articular BCP crystals induce synovial inflammation and cartilage proteoglycan loss in mice.

<p>OCP crystals (200 µg/20 µl) were injected into right knees of C57BL/6 mice whereas 20 µl PBS was injected into the left knees (A–E). Knees were harvested at different times (day 4, 17 and 30 n = 8 mice per group). Sections were stained with fast green/iron hematoxylin (A) and the degree of inflammation was assessed at the different time points (B). Since the inflammation was very low and similar at all time points in the PBS-injected control knees, only data from PBS-injected knees at day 4 was shown in B. OCP crystal deposition in the synovial membrane was evidenced at day 30 after OCP crystals injection by Von Kossa staining (see arrows) (C). Macrophage, endothelial and PMN cells were detected using antibodies for MAC-2, ICAM, and MPO, respectively, at day 4 after OCP injection (D). Isotype controls allowed the identification of giant cells that had engulfed tissue crystal deposits (*) (D). Ratio of Tc uptake between OCP-injected (n = 8) versus PBS controls was calculated (E). Fast green/iron hematoxylin staining of knees injected with 20 µg/20 µl of HA or OCP crystal at day 4 (F). Results are expressed as mean ± S.E.M with significance being at * p<0.05, ** p<0.01, *** p<0.001.</p