Proliferative capacity of human peripheral blood lymphocytes sorted on the basis of glutathione content

Glutathione (GSH) is important in defense against oxygen free radical damage, in detoxification of xenobiotics, and in mitogenesis. The reducing conditions provided by low molecular weight thiols such as 2-mercaptoethanol (ME) have been shown to promote the growth of lymphocytes in culture. We wished to determine the effects of 2-ME on GSH content, and to determine to what extent GSH status affected lymphocyte proliferation. GSH content was quantitated in human peripheral blood lymphocytes (PBL) using a flow cytometric assay with monochlorobimane. This analysis was performed on PBL as well as on the CD4+ T-cell subset, as identified with fluorescent anti-CD4 monoclonal antibodies (mAb). Cells were viably sorted on the basis of their GSH content, and incubated for 3 days with mitogenic concentrations of PHA (for PBL) or anti-CD3 mAb (for CD4+ cells) in the presence of bromodeoxyuridine (BrdU). BrdU/Hoechst cell cycle analysis was then performed on these cells. High GSH sorted cells had a higher percentage of cells capable of entering the cell cycle than low GSH sorted cells. This data indicates that some of the heterogeneity in proliferative capacity within PBL in culture is directly or indirectly related to GSH content. Incubation of cells in 2-ME prevented the loss of GSH that occurs when cells are cultured. 2-ME improved the proliferative capacity of unsorted cells, and of cells sorted for high and low GSH. Acridine orange staining of anti-CD3 mAb stimulated cells sorted for high and low GSH indicated that an early event in cell activation was affected by GSH content.