Flow cytometric analysis of oxidative product formation in phytohemagglutinin-stimulated ethanol-treated immune mononuclear cells.

Oxidative products formed by immune mononuclear cells were studied by flow cytometry. JURKAT T cells and peripheral blood mononuclear cells were incubated with 2,7-dichlorofluorescin diacetate. This substance was hydrolysed in the cells, leading to a non-fluorescent product which was oxidized into highly fluorescent 2,7-dichlorofluorescein by oxygen reactive species. These latter products were analysed by flow cytometry in phytohemagglutinin (PHA)-stimulated ethanol (ETH)-treated mononuclear cells. The level of fluorescence intensity (FI) was found higher in stimulated cells than in non-stimulated cells. ETH displayed two different effects on the cells: either a decrease of FI associated with a decrease of the number of fluorescent cells (FC) or an increase in FI. Both effects were dose-dependent. ETH is an effective scavenger of .OH radicals, but it is also oxidized by the microsomal ETH oxidizing system with production of oxygen reactive species, which probably explains the opposite effects of ETH. In the presence of desferal, an iron-chelating agent, and nordihydroguaiaretic acid, an inhibitor of the lipooxygenase pathway, the cells showed a decrease of FI and FC. These results suggest that .OH and other oxygen reactive species are involved in stimulation by PHA of ETH-treated immune mononuclear cells.