Silica phagocytosis causes apoptosis and necrosis by different temporal and molecular pathways in alveolar macrophages
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Chronic inhalation of crystalline silica is an occupational hazard that results in silicosis due to the toxicity of silica particles to lung cells. Alveolar macrophages play an important role in clearance of these particles, and exposure of macrophages to silica particles causes cell death and induction of markers of apoptosis. Using time-lapse imaging of MH-S alveolar macrophages, a temporal sequence was established for key molecular events mediating cell death. The results demonstrate that 80 % of macrophages die by apoptosis and 20 % by necrosis by clearly distinguishable pathways. The earliest detectable cellular event is phago-lysosomal leakage, which occurs between 30 and 120 min after particle uptake in both modes of death. Between 3 and 6 h later, cells undergoing apoptosis showed a dramatic increase in mitochondrial transmembrane potential, closely correlated with activation of both caspase-3 and 9 and cell blebbing. Externalization of phosphatidyl serine and nuclear condensation occurred 30 min–2 h after the initiation of cell blebbing. Cells undergoing necrosis demonstrated mitochondrial membrane depolarization but not hyperpolarization and no caspase activation. Cell swelling followed the decrease in mitochondrial membrane potential, distinguishing necrosis from apoptosis. All cells undergoing apoptosis followed the same temporal sequence, but the time lag between phago-lysosomal leakage and the other events was highly variable from cell to cell. These results demonstrate that crystalline silica exposure can result in either apoptosis or necrosis and each occurs in a well-defined but temporally variable order. The long time gap between phago-lysosomal leakage and hyperpolarization is not consistent with a simple scenario of phago-lysosomal leakage leading directly to cell death. The results highlight the importance of using a cell by cell time-lapse analysis to investigate a complex pathway such as silica induced cell death.
KeywordsSilicosis Apoptosis Lysosome Mitochondria Caspase Necrosis
We are thankful to Dr. Toru Kawanishi at NIHS, Japan and Dr. Gavin Welsh at University of Bristol, UK for their gift of Initiator and Effector FRET caspase probes. We are also thankful to Dr. Carol Norris at UConn microscopy facility for her assistance.
Supplementary material 9 (MP4 519 kb) Cells undergoing apoptosis show nuclear condensation Cells labeled with nuclear dye Hoechst 33342 shows nuclear condensation at 420 minutes whereas cell blebbing was initiated at 300 minutes
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