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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.

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Although silica has been documented to cause apoptotic cell death, the cellular pathways leading to caspase activation have not been extensively investigated. Here we demonstrate in a mouse macrophage cell line (MH-S cells) that alpha-quartz silica exposure (12.5 mug/cm2 to 50 mug/cm2) elicited activation of both caspase 3 and caspase 9, whereas anatase titanium dioxide (TiO2), a non-fibrogenic particle, did not. Silica exposure in vitro also induced apoptosis after 6 h, as measured by the appearance of subdiploid cell fragments in a flow cytometric analysis. Exposure to TiO 2 did not elicit significant apoptosis. Silica-induced apoptosis and caspase 3 activation were, in part, caspase 9 dependent, as determined by their sensitivity to either a general caspase inhibitor (Z-VAD-FMK) or a specific caspase 9 inhibitor (Z-LEHD-FMK). Silica exposure in vitro also elicited significant mitochondrial depolarization after 2 and 6 h of exposure. Cyclosporin A, an inhibitor of the mitochondrial permeability pore, partially decreased mitochondrial depolarization, caspase 3 activation, and caspase 9 activation, suggesting a role for mitochondrial dysfunction in these events. Pepstatin A, an inhibitor of cathepsin D, also decreased mitochondrial depolarization, caspase 3 activation, and caspase 9 activation, whereas leupeptin, an inhibitor of cathepsin B, had no effect. These data suggest that short-term silica exposure in vitro induces both caspase 3 and caspase 9 activity, which appears to participate in apoptosis. Activation of these caspases seems to be dependent, in part, on aspartic proteolysis and loss of mitochondrial integrity.

Occupational exposure to crystalline silica is associated with the development of pulmonary inflammation and silicosis, yet how silica initiates pulmonary fibrosis and which cell types are involved are unclear. In studies here, we hypothesized that silica particles interact initially with pulmonary epithelial cells and alveolar macrophages (AMs) to cause transcriptional activation of nuclear factor (NF)-kappaB-regulated genes encoding inflammatory cytokines. Exposure of NF-kappaB luciferase reporter mice intratracheally to silica or lipopolysaccharide (LPS), but not the nonfibrogenic particle titanium dioxide (TiO(2)), increased immunoreactivity of luciferase protein in bronchiolar epithelial cells and AMs. Ribonuclease protection assays revealed significant (P < or = 0.05) increases in mRNA levels of inducible nitric oxide synthase, tumor necrosis factor-alpha, macrophage inflammatory protein-2, macrophage chemotactic protein-1 (MCP-1), interferon-gamma, interleukin (IL)-6, and IL-12 in lung homogenates of reporter mice after exposures to silica or LPS. Immunoreactivity of MCP-1 in these animals was localized to AMs and epithelial cells. These data are the first to show activation of NF-kappaB in situ by fibrogenic particles in pulmonary epithelial cells and AMs. Increased expression of NF-kappaB-related inflammatory cytokines by these cell types, which first encounter silica after inhalation, may be critical to the initiation of silica-associated lung diseases, thus providing a rationale for focusing on NF-kappaB in preventive and therapeutic strategies.

. Immediate sensory nerve-mediated respiratory responses to irritants in healthy and allergic airway-diseased mice.