Silica-Induced Apoptosis in Mouse Alveolar Macrophages Is Initiated by Lysosomal Enzyme Activity

Abstract

Past studies in our laboratory have shown that silica (-quartz) particle exposure of a mouse alveolar macrophage cell line (MH-S) elicits mitochondrial depolarization and caspase 3 and 9 activation, contributing to apoptosis. However, cellular pathways leading to these outcomes have not been extensively investigated. Initial studies revealed that silica exposure elicits lysosomal permeability after 1 h, as evidenced by leakage of FITC-conjugated dextran and acridine orange. We next evaluated a role for the lysosomal acidic compartment in apoptosis. Cells pretreated with the lysosomotropic weak base ammonium chloride, to increase lysosomal pH, showed decreased caspase activation and apoptotic DNA fragmentation. MH-S cells pretreated with pepstatin A, an inhibitor of lysosomal cathepsin D, showed decreased caspase 9 and 3 activation as well as a decreased percentage of cells that became apoptotic. DNA fragmentation and caspase 9 and 3 activation were also decreased in cells pretreated with despiramine, an inhibitor of lysosomal acidic sphingomyelinase. Silica pretreated with aluminum lactate (to blunt surface active sites) reduced caspase activation and apoptosis. Although aluminum lactate-treated silica still induced lysosomal permeability (by FITC-dextran leakage), one measure of lysosome integrity and function suggested a reduction in the extent and/or nature of lysosomal injury (by acridine orange retention). A role for reactive oxygen species (ROS) was investigated to explore another pathway for silica-induced apoptosis in addition to lysosomal enzymes; however, no role for ROS was apparent. Thus, following silica exposure, lysosomal injury precedes apoptosis, and the apoptotic signaling pathway includes cathepsin D and acidic sphingomyelinase.

Silica binding and toxicity in alveolar macrophages

“…al., using the murine macrophage cell line MH-S, showed lysosomal permeability after only 1 hour of silica exposure using fluorescent markers. A variety of inhibitors of the lysosomal processing resulted in reduced caspase activation and apoptosis [106]. This study concluded that the apoptotic signaling pathway involved lysosomal leaking, cathepsin D and acidic sphingomyelinase, which preceded the mitochondrial depolarization and caspase 3 and 9 activation (reported earlier by the same group) caused by silica exposure [105].…”

“…This study concluded that the apoptotic signaling pathway involved lysosomal leaking, cathepsin D and acidic sphingomyelinase, which preceded the mitochondrial depolarization and caspase 3 and 9 activation (reported earlier by the same group) caused by silica exposure [105]. It was further determined that ROS was not the cause of the lysosomal permeability [106].…”

“…The silica particle cannot be broken down in the lysosomal compartment, and the resulting failure can lead to a loss of membrane integrity in the process. Some researchers believe that the failed attempt at silica digestion in the AM leads to caspase activation and apoptosis [105,106]. A model illustrating this hypothesis can be found in Figure 4.…”

Lysosomal membrane permeabilization in cell death

“…Another pathogenic situation in which macrophages have been implicated is lung damage induced by silica exposure. Silica particles enter cells by phagocytosis and their reactive surface may interact with phagolysosomal membranes, thus triggering the translocation of lysosomal enzymes to the cytosol and consequent cell death (Thibodeau et al, 2004).…”

Protein kinase C-δ isoform mediates lysosome labilization in DNA damage-induced apoptosis

“…The lysosomal compartment is associated with apoptotic signaling in a wide diversity of cells and with stimuli, such as oxidative stress (2-4), lysomotropic (5)(6)(7)(8) and photosensitizing agents (9)(10)(11), photodamage (12), serum withdrawal (13), Fas and tumor necrosis factor-· (TNF-·) ligation (13)(14)(15), phospholipid and sphingolipid analogues (16)(17)(18), DNA-damaging (19,20) and microtubule-stabilizing agents (21,22), the quinolone class of antibiotics (23), artificial retinoids (24), zinc chloride (25) and silica (26). Although participation of the lysosomal compartment in apoptosis is cell type-and stimuli-specific, a shared feature among all models is the apparition of moderate lysosomal membrane ruptures, referred to here as labilization of lysosomal membranes (LLM), and the subsequent release from the lysosomal lumen to the cytosol of lysosomal peptidases, the cathepsins, which contribute to cell death through various targets (27)(28)(29)(30).…”