Intracellular oxidant production and cytokine responses in lung macrophages: evaluation of fluorescent probes.

The fluorescent probes dichlorofluorescin (DCFH), dihydrorhodamine (DHR), and hydroethidine (HE) allow convenient assay of alveolar macrophage (AM) oxidant responses to enviromental particulates and pathogens. We sought to more precisely define the relationship of these measures of oxidant stress to production of pro-inflammatory cytokines. Normal AMs were challenged in vitro with a panel of soluble or particulate stimuli in the presence of DCFH, HE, or DHR. Flow cytometry measured cell-associated fluorescence and relative particle uptake. Tumor necrosis factor alpha and macrophage inflammatory protein 2 expression were quantitated in the same experiments. We observed variable and complex correlations between intracellular oxidant production as reported by these probes and subsequent cytokine response, including examples of striking discordance (e.g., lipopolysaccharide induced large cytokine responses with minimal probe oxidation, whereas fly ash particles caused marked oxidation of DCFH but trivial TNF release; TiO2 caused oxidation of DHR and HE, but not DCFH, and also did not increase cytokine production). Although fluorescent probes offer many advantages in analysis of intracellular oxidant responses, the data indicate that they cannot be used reliably as quantitative predictors of AM cytokine responses to environmental particulates or other stimuli.