Dependence of NF-κB activation and free radical generation on silica-induced TNF-α production in macrophages

Tumor necrosis factor α (TNFα) plays an important role in the pathogenesis of silicosis and other chronic inflammatory lung diseases. The present study investigates the role nuclear transcription factor κB (NF-κB) and oxygen free radicals in silica-induced TNFα production in primary alveolar macrophages and RAW 264.7 cells. Using electrophoretic mobility shift assay (EMSA) and enzyme-linked immunoadsorbent assay (ELISA), we have demonstrated that silica can induce NF-κB activation and TNFα expression in a dose-dependent manner. Transient transfection assays with a plasmid construct containing NF-κB binding sites linked to a reporter gene further show that silica is able to induce the transcriptional activation of NF-κB-dependent gene. Inhibition of NF-κB activation by SN50, a specific NF-κB blocker, abolishes silica-induced TNFα production. Pretreatment of the cells with catalase (H₂O₂ scavenger) or deferoxamine (·OH scavenger) effectively inhibits NF-κB and TNFα activation, whereas superoxide dismutase (O₂ scavenger) has an opposite effect. These results indicate that silica mediated free radical generation and NF-κB activation play important roles in silica-induced TNFα gene expression.