Oxidative phenomena in T lymphocyte activation

Murine lymph node cells (LNC) were used as model for the assessment of a role for reactive oxygen species (ROS) in T lymphocyte activation. When 2’7’- dichlorofluorescin (DCFHz) is oxidised it becomes the fluorescent compound dichlorofluorescein. Analysis of DCFHz-loaded LNC by flow cytometry identified an increase in DCFH2 oxidation upon stimulation with a mitogenic dose of the phorbol ester, phorbol myristate acetate. This could also be seen, although to a lesser extent, with a mitogenic dose of the lectin concanavalin A. The phorbol ester-induced increase in DCFH2 oxidation was inhibited by chelerythrine and desferrioxamine (the latter at concentrations lower than that required for maximal inhibition of lymphoproliferation in vitro), indicating a role in DCFHZ oxidation for protein kinase C and iron, respectively. Analysis of LNC prelabelled with an antibody against a pan-T lymphocyte marker, Thy-1, established that phorbol ester treatment of LNC induces an increase in DCFHZ oxidation in murine T lymphocytes. The inhibition of DCFH2 oxidation in LNC by superoxide dismutase, catalase and glutathione/glutathione peroxidase suggested that the source of oxidants may have been B lymphocytes and/or phagocytic cells within the population and that the oxidation in T lymphocytes essentially represented a "bystander effect". This was supported by preliminary studies where there was little or no response to phorbol ester stimulation in DCFHz-loaded LNC from mice lacking a functional NADPH oxidase (gp91Ph0X gene knockout mice). Studies of cell-free oxidation of DCFH2 demonstrated that the fluorogen could be oxidised by peroxyl radicals from either chemical (2,2'-azobis(2-amidinopropane) dihydrochloride) or enzymatic (soybean lipoxygenase) sources. Finally, the role of iron in T lymphocyte activation was investigated using the iron chelators desferrioxamine and a set of novel pyridoxal-based compounds. The novel iron chelators were of comparable or greater potency compared to desferrioxamine with regard to inhibition of lymphoproliferation in vitro. Time course studies confirmed previous reports by showing that a major target of iron chelators in activated T lymphocytes are events late in G1 or at the G1/S transition of the cell cycle. These studies: highlight the technical difficulties of assaying oxidant production using mixed populations of cells; support the notion that DCFH2 is a general target for radical-mediated oxidation; and confirm a critical role for iron in DNA synthesis in activated T lymphocytes