Effect of in vitro generation of oxygen free radicals on T cell function in young and old rats

Abstract

T cells from young (6 months) and old (24 months) male Fischer 344 rats were isolated and exposed to three different oxidative stress conditions: (a) reactive oxygen species generated by xanthine-xanthine oxidase (X/XO), (b) hydrogen peroxide (H 2O 2), and (c) hyperthermia (43°C for 1 h). After oxidative stress treatment, the induction of proliferation and IL-2 production by concanavalin A (Con A) was measured. Exposure of T cells to X/XO or H 2O 2 resulted in suppression of proliferation and IL-2 expression, and the suppressive effect was more pronounced in T cells from young rats than in T cells from old rats. Similarly, hyperthermia caused inhibition of proliferation and IL-2 expression in T cells from young and old rats. Addition of antioxidant to cultured cells only slightly attenuated the effects of X/XO and H 2O 2 on T cell function; however, antioxidant had no effect on heat shock-mediated inhibition of proliferation in young or old rats. Because IL-2 plays a crucial role in T cell proliferation and because the transcription factor NFAT (nuclear factor of activated T cell) plays a major role in the regulation of IL-2 transcription, the induction of NFAT as well as NF-κB and AP-1 DNA binding activities in nuclear extracts of the X/XO-treated and untreated control cells was measured using a gel shift assay. The ability of nuclear extracts to bind NFAT or NF-κB oligonucleotide decreased in the X/XO-treated cells from young and old rats compared to the untreated controls. Therefore, these data imply that reactive oxygen species generated by the X/XO system alter the distal step of mitogen-mediated signal transduction, i.e., transcription factors that regulate IL-2 transcription.