Hydrogen peroxide and tumour necrosis factor-alpha induce NF-kappaB-DNA binding in primary human T lymphocytes in addition to T cell lines.
Reactive oxygen intermediates (ROIs), such as hydrogen peroxide (H2O2), have been implicated as second messengers in the activation of NF-kappaB by a variety of stimuli, including tumour necrosis factor-alpha (TNF-alpha). The aim of the present study was to examine the effects of ROIs on NF-kappaB 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-alpha and nuclear proteins were extracted. NF-kappaB binding was assessed by electrophoretic mobility shift assays (EMSAs). The concentration of H2O2 required to activate NF-kappaB in human primary CD3+ T lymphocytes was as low as 1 microM. In contrast, much higher concentrations of H2O2 were required to activate NF-kappaB in PBMCs and in the JR subclone of Jurkat T cells. H2O2-induced NF-kappaB activation was not observed in the JE6.1 subclone of Jurkat T cells. NF-kappaB was activated by TNF-alpha 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-kappaB-DNA binding in human primary T lymphocytes.
Gilston
V
V
Bone and Joint Research Unit, St. Bartholomew's and the Royal London School of Medicine and Dentistry, UK.
Williams
M A
MA
Newland
A C
AC
Winyard
P G
PG
D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D019169:Jurkat Cells; D016328:NF-kappa B / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D013601:T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D004247:DNA; D006861:Hydrogen Peroxide