We have shown previously that epithelial cells may contribute to the inflammatory response in the lung after exposure to crystalline silica through the production of and response to specific chemokines and cytokines. However, the exact cellular and molecular responses of epithelial cells to silica exposure remain unclear. We hypothesize that non-oxidant-mediated silica-cell interactions lead to the upregulation of tumor necrosis factor- (TNF-), whereby TNF--induced generation of reactive oxygen species (ROS) leads to the activation of the monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-2 genes. Using a murine alveolar type II cell line, murine lung epithelial (MLE)-15, we measured the early changes in TNF-, MCP-1, and MIP-2 mRNA species after exposure of the cells to 18 µg/cm² silica (cristobalite) in combination with various antioxidants. Total mRNA was isolated and assayed using an RNase protection assay after 6 h of particle exposure. We found that extracellular GSH could completely attenuate the cristobalite-induced expression of MCP-1 and MIP-2 mRNAs, whereas TNF- mRNA levels were unaltered. We also found using the oxidant-sensitive dye 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate di(acetoxymethyl ester) that treatment of MLE-15 cells with cristobalite and TNF- (1 ng/ml) resulted in ROS production. This ROS production could be inhibited with extracellular GSH treatment, and in the case of cristobalite-induced ROS, inhibition was also achieved with an anti-TNF- antibody. The results support the hypothesis that TNF- mediates cristobalite-induced MCP-1 and MIP-2 expression through the generation of ROS.

airway epithelium; reactive oxygen species; monocyte chemotactic protein-1; macrophage inflammatory protein-2; tumor necrosis factor-

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