Hydrogen peroxide and tumour necrosis factor-α induce NF-κB-DNA binding in primary human T lymphocytes in addition to T cell lines

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Abstract
Reactive oxygen intermediates (ROIs), such as hydrogen peroxide (H2O2), have been implicated as second messengers in the activation of NF-κB by a variety of stimuli, including tumour necrosis factor-alpha (TNF-α). The aim of the present study was to examine the effects of ROIs on NF-κB activation in primary human CD3+ T lymphocytes and human peripheral blood mononuclear cells (PBMCs). For comparison purposes, Jurkat T cells (subclones JR and JE6.1) were also investigated. Cells were incubated in the presence of either H2O2 or TNF-α and nuclear proteins were extracted. NF-κB binding was assessed by electrophoretic mobility shift assays (EMSAs). The concentration of H2O2 required to activate NF-κB in human primary CD3+ T lymphocytes was as low as 1 μM. In contrast, much higher concentrations of H2O2 were required to activate NF-κB in PBMCs and in the JR subclone of Jurkat T cells. H2O2-induced NF-κB activation was not observed in the JE6.1 subclone of Jurkat T cells. NF-κB was activated by TNF-α in all four cell types tested. In PBMCs and Jurkat T cells (subclones JR and JE6.1), this activation could be inhibited by pre-treatment with the antioxidants, pyrrolidine dithiocarbamate (PDTC) and N-acetyl-l-cysteine (NAC). Our results support a role for ROIs in NF-κB-DNA binding in human primary T lymphocytes.

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