Balázs Rada1, Jonathan J Park, Payel Sil, Miklós Geiszt, Thomas L Leto. 1. Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD, 20852, USA, radab@uga.edu.
Abstract
OBJECTIVE AND DESIGN: We studied the involvement of calcium and calcium-activated NADPH oxidases in NLRP3 inflammasome activation and IL-1β 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-1β 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-1β release in human macrophages. Increases in cytosolic calcium alone lead to IL-1β 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-1β release. CONCLUSIONS: Human macrophage inflammasome activation and IL-1β secretion requires calcium but does not involve NADPH oxidases.
OBJECTIVE AND DESIGN: We studied the involvement of calcium and calcium-activated NADPH oxidases in NLRP3 inflammasome activation and IL-1β 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-1β 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-1β release in human macrophages. Increases in cytosolic calcium alone lead to IL-1β 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-1β release. CONCLUSIONS:Human macrophage inflammasome activation and IL-1β secretion requires calcium but does not involve NADPH oxidases.
Authors: Rinke Stienstra; Leo A B Joosten; Tim Koenen; Berry van Tits; Janna A van Diepen; Sjoerd A A van den Berg; Patrick C N Rensen; Peter J Voshol; Giamilla Fantuzzi; Anneke Hijmans; Sander Kersten; Michael Müller; Wim B van den Berg; Nico van Rooijen; Martin Wabitsch; Bart-Jan Kullberg; Jos W M van der Meer; Thirumala Kanneganti; Cees J Tack; Mihai G Netea Journal: Cell Metab Date: 2010-12-01 Impact factor: 27.287
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