Antioxidants inhibit proliferation and cell surface expression of receptors for interleukin-2 and transferrin in T lymphocytes stimulated with phorbol myristate acetate and ionomycin

We have previously shown that several antioxidant compounds inhibit the proliferation of T lymphocytes stimulated with alloantigen (Chaudhri, G., Clark, I. A., Hunt, N. H., Cowden, W. B., and Ceredig, R., J. Immunol. 136, 2646, 1986). We concluded from these studies that free oxygen radicals are positive mediators in T-lymphocyte activation and proliferation. In order to extend these studies we examined the effects of antioxidants on T cells stimulated with a combination of phorbol myristate acetate (PMA) and ionomycin. The following antioxidants were used: ferricyanide, an inhibitor of superoxide production; iron chelators, which block hydroxyl radical formation; and butylated hydroxyanisole, a free radical scavenger. Responder cells included purified peripheral T cells (Lyt-2⁺ or L3T4⁺ cells) and immature (Lyt-2^-/L3T4^-) thymocytes. All agents, in the micromolar range, caused a dose-dependent inhibition of proliferation of each T-cell subset studied. Flow microfluorometric analysis of T cells stimulated for 48 hr showed that the expression of interleukin-2 (IL-2) receptors and transferrin receptors was inhibited by all the antioxidants tested but not by hydroxyurea (HU), an inhibitor of the enzyme ribonucleotide reductase. In contrast, the expression of a third activation marker, phagocytic glycoprotein-1 (Pgp-1 or Lyt-24), was not affected by any of the agents. Furthermore, while both the antioxidants and HU inhibited T-cell cycling, analysis of a light-scattering parameter related to cell size indicated that the antioxidant-treated cells remained small while the HU-treated and control cells were larger and blast-like. Therefore, the mechanism of action of the three classes of antioxidants is similar, but quite distinct from the inhibition of proliferation caused by HU. Taken together, these results suggest that free radicals are involved in specific early events in T-cell activation.