Abstract:
Intracellular reactive oxygen species (ROS) production by activated murine T lymphocytes was investigated
by analyzing intracellular dichlorofluorescin (DCFH2) oxidation in lymph node cells (LNC). An increase in DCFH2
oxidation in LNC induced by phorbol myristate acetate (PMA) was detected by flow cytometry. It was confirmed that
this increase was present in Thy11 LNC. We examined the contribution to intracellular DCFH2 oxidation of ROS
released by leukocytes other than T cells present in the LNC suspension. Superoxide dismutase, catalase, and
glutathione/glutathione peroxidase inhibited the PMA-induced increase in intracellular DCFH2 oxidation. Furthermore,
PMA failed to elicit DCFH2 oxidation in LNC isolated from mice lacking a functional NADPH oxidase (gp91phox gene
knockout mice), but this response could be restored in these cells by the addition of T cell-depleted LNC from wild-type
litter mates. This study highlights the necessity for caution in using the DCFH2 assay to demonstrate specific
intracellular ROS production in heterogeneous cell populations. It also suggests that cells other than T cells in lymph
node populations may, through production of ROS, influence the intracellular redox state of T lymphocytes.
