Figure 4.

Lysosomal damage triggers the release of cathepsin B, which is involved in the IL-1β pathway. (a) Confocal microscopy of microglial cells left unstimulated or stimulated for 1 or 4 h with FITC-labeled Aβ (green), and then stained with anti–cathepsin B (red) or anti-LAMP-1 (cyan; far right); nuclei were stained with Hoechst 33258 (blue). Arrows indicate colocalization of cathepsin B and Aβ. Scale bars, 5 µm. (b) Effect of various concentrations (0, 5 or 10 µM) of inhibitors of cathepsin D (CathD inh), cathepsin L (CathL inh) or cathepsin B (CathB inh) on the Aβ-induced release of IL-1β from LPS-primed immortalized microglia (left) and the effect of cathepsin inhibitors at the highest concentration (10 µM) on IL-1β release induced by ATP (right). (c) ELISA of the release of IL-1β by wild-type and cathepsin B–deficient primary macrophages left unstimulated or after stimulation with Aβ, ATP or poly(dA:dT). (d) Caspase-1 activation in microglial cells after stimulation with Aβ (above) or ATP (below) and treatment with the cathepsin B inhibitor (20 µM), assessed by flow cytometry with a fluorescent cell-permeable probe that binds only to activated caspase-1. MFI, mean fluorescence intensity. Data are representative of two (a,c) or three (b,d) separate experiments (error bars (b–d), s.e.m.).