The inflammasome links the sensing of pathogen and danger signals

The inflammasome links the sensing of pathogen and danger signals to pro-IL-1β processing. The NALP3 inflammasome is the best-known inflammasome, detecting bacterial wall components or the bacteria themselves. In addition, NALP3 can be activated by signals that induce potassium efflux, such as MAPK Inhibitor Library ATP, via its P2X7 receptor.3 The importance of the inflammasomes in human disease is illustrated by the discovery that cryopyrin-associated

periodic syndromes are the result of mutations in the NALP3 gene4 and that monosodium urate (MSU) crystals induce inflammation through the NALP3 inflammasome.5 There are scant data on inflammasome expression in RA. Rosengren et al. showed that NALP3 RNA levels were increased in RA synovium and that macrophages differentiated in vitro increased NALP3 expression when stimulated by tumour necrosis factor (TNF).6 We therefore analysed the expression NALP3 and ASC in the synovium as well as examining the capacity of RA synovial fibroblasts to produce active IL-1β. Synovial tissues from patients with RA and patients with osteoarthritis (OA) were also compared for the expression of NLR proteins and their production of IL-1β and caspase-1. Synovial tissues were obtained see more from nine patients

with RA (nine women, mean age 58·6 ± 11·6 years) and 11 patients with OA (five women, six men, mean age 74·6 ± 11·7 years) undergoing joint replacement surgery of the knee or the hip Mirabegron (Department of Orthopaedics, CHUV). Osteoarthritis was diagnosed by clinical and radiological

criteria and RA patients fulfilled the American Rheumatism Association revised criteria for RA. All tissues were cut into small pieces and immediately frozen in pre-cooled hexane and stored at −70° until use, or fixed in formol and embedded in paraffin. Ethical committee approval was obtained for these experiments. Fibroblast-like synoviocyte (FLS) lines were established as described previously.7 Cells were used between the third and seventh passages. Synoviocyte cell cultures or, as positive control, THP-1 cells (2 × 105 cells/well) were incubated in Dulbecco’s modified Eagle’s minimal essential medium or RPMI-1640 medium containing 0·5% fetal calf serum, with or without the following stimuli: lipopolysaccharide (LPS; 10 μg/ml), ATP (5 mm), H2O2 (30 μm), TNF-α (10 ng/ml) and MSU (200 μg/ml). After 24 hr incubation, culture supernatants were harvested, and cells were suspended for 20 min in 200 μl ice-cold lysis buffer [50 mm Tris–HCl pH 7·4, 110 mm NaCl, 10 mm ethylenediaminetetraacetic acid (EDTA), 0·1% nonidet P-40 (NP-40)] containing a protease inhibitor cocktail (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland). The detergent-soluble proteins were separated by centrifugation (14 000 g for 15 min at 4°).

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