Kivonat:
OBJECTIVE AND DESIGN: We studied the involvement of calcium and calcium-activated NADPH oxidases in NLRP3 inflammasome activation and IL-1beta release to better understand inflammasome signaling in macrophages. MATERIAL OR SUBJECTS: Human volunteer blood donors were recruited to isolate monocytes to differentiate them into macrophages. Wild-type or DUOX1-deficient C57/B6 mice were used to prepare bone marrow-derived macrophages. TREATMENT: Murine or human macrophages were treated in vitro with NLRP3 inflammasome agonists (ATP, silica crystals) or calcium agonists (thapsigargin, ionomycin) in calcium-containing or calcium-free medium. METHODS: Intracellular calcium changes were followed by measuring FURA2-based fluorescence. Gene expression changes were measured by quantitative real-time PCR. Protein expression was assessed by western blotting. Enzymatic activity was measured by fluorescence caspase-1 activity assay. IL-1beta release was determined by ELISA. ELISA data were analyzed by ANOVA and Tukey's post hoc test. RESULTS: Our data show that calcium is essential for IL-1beta release in human macrophages. Increases in cytosolic calcium alone lead to IL-1beta secretion. Calcium removal blocks caspase-1 activation. Human macrophages express Duox1, a calcium-regulated NADPH oxidase that produces reactive oxygen species. However, Duox1-deficient murine macrophages show normal IL-1beta release. CONCLUSIONS: Human macrophage inflammasome activation and IL-1beta secretion requires calcium but does not involve NADPH oxidases.
