Uptake and Trafficking of Opsonized and Non-Opsonized Silica Particles During Silica-Induced Cell Death
Silicosis is a fibrotic lung disease induced by prolonged exposure to silica dust. Upon inhalation from the surrounding air, silica particles that are smaller than 5 micrometers in diameter reach the alveoli. One type of cell that resides in the alveoli is the alveolar macrophage. The role of the alveolar macrophage is to clear debris and destroy pathogens. Alveolar macrophages express a number of cell-surface receptors, such as FcγRIIA, that aid in the rapid recognition and internalization of foreign particles. Alveolar macrophages clear inhaled silica particles by phagocytosis, but this causes the macrophages to die by apoptosis. One of the first abnormalities associated with silica uptake is the leakage of phago-lysosomal contents into the cytoplasm, which may be the trigger for activation of the apoptotic pathway. Our objective is to understand silica particle uptake into phagosomes, phago-lysosomal fusion, and how the resulting leakage causes subsequent apoptosis.
Silica particles were coated with protein (non-opsonized) or antibody (opsonized) and added to cultured alveolar macrophages or Cos7 kidney fibroblasts. While alveolar macrophages internalize both non-opsonized and opsonized particles within 10 to 15 minutes, Cos7 cells only internalize 10% of opsonized and non-opsonized particles within 2 hours and continue to gradually internalize particles over the next 48 hours. Cos7 cells expressing the Fc-receptor (FcR-Cos7) internalize 70% of opsonized particles within two hours, while uptake of non-opsonized particles is similar to that of Cos7 cells that do not express the Fc-receptor. In order to demonstrate phago-lysosomal fusion, macrophages, Cos7 fibroblasts and FcR-Cos7 fibroblasts were loaded with Tritc-dextran (TD) prior to particle addition. Vesicles containing Tritc-dextran fused with opsonized and non-opsonized particle phagosomes in each of these cell lines. In order to demonstrate phago-lysosomal leakage, macrophages, Cos7s and FcR-Cos7s were loaded with FITC-dextran. After the vesicles containing FITC-dextran fuse with phagosomes containing opsonized or non-opsonized silica particles, macrophages and FcR-Cos7s undergo phago-lysosomal leakage. It was also determined that phago-lysosomal leakage could be detected in each of these cell lines when loaded with Tritc-dextran. This finding indicates that phago-lysosomal leakage is not specific to macrophages, and future experiments will determine if and how phago-lysosomal leakage leads to silica-induced cell death in Cos7s and FcR-Cos7s, as it does in macrophages.