Figure 2

Zn²⁺ depletion drives processing and secretion of IL-1β from macrophages. (a) Mouse primary peritoneal macrophages were primed with LPS (1 μg/ml, 2 h), followed by 4 h treatment with 10 μM TPEN, the copper chelator TTM, or the iron chelator SIH with effects on cell death measured by LDH release (i), IL-1β release measured by ELISA (ii), and IL-1β processing measured by western blot analysis (iii). The band at 31 kDa is pro-IL-1β and the band at 17 kDa is IL-1β. (b) Mouse primary peritoneal macrophages were primed with LPS (1 μg/ml, 2 h), followed by treatment with TPEN (10 μM, 4 h)±50 μM ZnCl₂ with release of IL-1β measured by ELISA (i) and cell death by LDH release (ii). (c) Mouse primary peritoneal macrophages were primed with LPS (1 μg/ml, 2 h), followed by 4 h treatment with the non-cell permeable Zn²⁺ chelator DTPA (1 mM) with release of IL-1β measured by ELISA. (d) J774 macrophages were loaded with the Zn²⁺ indicator FluoZin-3 and Lysotracker Red and then treated with 1 μM of the Zn²⁺ ionophore, 1-hydroxypyridine-2-thione (zinc salt) (ZnPyr), to induce increases in cellular Zn²⁺. Addition of TPEN to a Zn²⁺-loaded cell caused a rapid drop in FluoZin-3 fluorescence. Shown are snap shot images taken with a BD Pathway illustrating the fluorescence changes over the course of the experiment. Scale bar=50 μm. (e) Representative fluorescent traces (F/Fo) of FluoZin-3 (i) and lysotracker red (ii) of the experiment shown in d. All data are presented as the mean±S.D. from at least four separate experiments. Blots shown are representative. ***P<0.001, **P<0.01, *P<0.05