Pyrrolidine dithiocarbamate inhibits NF-kappa B mobilization and TNF production in human monocytes

Abstract

The human TNF promoter contains four potential nuclear factor-kappa B (NF-kappa B)-binding sites, with the strongest binding seen for the -605 motif. Nuclear extracts from unstimulated cells of the human monocytic cell line, Mono Mac 6, contain one specific binding protein (complex II), consistent with a constitutive p50 homodimer. Stimulation of Mono Mac 6 cells with LPS will increase complex II and will strongly induce a second specific complex (complex I), which represents the p50/65 heterodimer. Treatment of Mono Mac 6 cells with pyrrolidine-dithiocarbamate (PDTC) at 300 microM will block the LPS-induced complex I almost completely and will reduce complex II to the constitutive level. Binding activity of other nuclear factors that recognize the SP-1 and c/EBP motifs of the human TNF promoter is not affected by such treatment. Northern blot analysis demonstrates that PDTC treatment will strongly reduce LPS-induced TNF transcripts. Secreted TNF protein as detected in the Wehi 164S/ActD bioassay and in a sandwich immunoassay was similarly reduced by PDTC. Kinetic analyses show that after LPS stimulation, NF-kappa B will peak at 1 h, TNF transcript prevalence at 2 h, and TNF protein at 4 h. PDTC did not shift this response to LPS to a later time, but suppressed NF-kappa B mobilization, TNF transcripts, and TNF protein over the entire 8-h observation period. Analysis of freshly isolated, LPS-stimulated blood monocytes showed a similar blockade of NF-kappa B. Furthermore, in these primary cells, induction of TNF transcripts, as determined by Northern blot analysis and by quantitative polymerase chain reaction, was prevented by PDTC as was TNF protein production. These data show that dithiocarbamates can profoundly affect cytokine expression and suggest that NF-kappa B is involved in LPS-induced TNF gene expression in human monocytes.