Silica-induced chemokine expression in alveolar type II cells is mediated by TNF-alpha-induced oxidant stress

We have shown previously that epithelial cells may contribute to the inflam matory response in the lung after exposure to crystalline silica through th e production of and response to specific chemokines and cytokines. However, the exact cellular and molecular responses of epithelial cells to silica e xposure remain unclear. We hypothesize that non-oxidant-mediated silica-cel l interactions lead to the upregulation of tumor necrosis factor-alpha (TNF -alpha), whereby TNF-alpha-induced generation of reactive oxygen species (R OS) leads to the activation of the monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-8 genes. Using a murine alveolar typ e II cell line, murine lung epithelial (MLE)-15, we measured the early chan ges in TNF-alpha, MCP-1, and MIP-2 mRNA species after exposure of the cells to 18 mu g/cm(2) silica (cristobalite) in combination with various antioxi dants. Total mRNA was isolated and assayed using an RNase protection assay after 6 h of particle exposure. We found that extracellular GSH could compl etely attenuate the cristobalite-induced expression of MCP-1 and MIP-2 mRNA s, whereas TNF-alpha mRNA levels were unaltered. We also found using the ox idant-sensitive dye 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate di (acetoxymethyl ester) that treatment of MLE-15 cells with cristobalite and TNF-alpha (1 ng/ml) resulted in ROS production. This ROS production could b e inhibited with extracellular GSH treatment, and in the case of cristobali te-induced ROS, inhibition was also achieved with an anti-TNF-alpha antibod y. The results support the hypothesis that TNF-alpha mediates cristobalite- induced MCP-1 and MIP-2 expression through the generation of ROS.