Inhibitory kappaBalpha control of nuclear factor-kappaB is dysregulated in endotoxin tolerant macrophages.
The transcription factor nuclear factor (NF)-kappaB is thought to be required for endotoxin-stimulated tumor necrosis factor (TNF) and interleukin (IL)-1 gene transcription. Nuclear translocation of NF-kappaB is regulated by the cytoplasmic inhibitory factor IkappaBalpha. Low-dose lipopolysaccharide (LPS) pretreatment modulates cytokine release by altering subsequent LPS-activated signal transduction pathways. In this study, we examined the effect of LPS pretreatment exposure on IkappaBalpha and NF-kappaB following activation with LPS. Murine macrophages (Mphi were exposed to a range of LPS concentrations +/-24 h PreRx with 10 ng/mL LPS pretreatment. Cytoplasmic IkappaBalpha (Western immunoblot) and NF-kappaB (gel-shift assay) were assayed 30 min after LPS activation. Gene transcription for TNF was measured 6 h after LPS activation using RT-PCR. In the absence of LPS pretreatment, IkappaBalpha disappeared from the cytoplasm coincident with nuclear translocation of NF-kappaB. Tolerant Mphi had markedly enhanced levels of IkappaBalpha and normal to increased levels of NF-kappaB translocation with a different electrophoretic shift. LPS activation enhanced cytokine gene transcription in a dose-dependent manner, and this was unaltered by LPS pretreatment. Endotoxin-tolerant Mphi also had increased cytoplasmic levels of the p65 subunit of NF-kappaB. LPS tolerance is associated with increases of cytoplasmic IkappaBalpha p65, as well as enhanced NF-kappaB. We conclude that control of NF-kappaB translocation by IkappaBalpha is dysregulated in endotoxin-tolerant Mphi.
Wahlstrom
K
K
Department of Surgery, Hennepin County Medical Center, University of Minnesota, Minneapolis 55415, USA.
Bellingham
J
J
Rodriguez
J L
JL
West
M A
MA
D000818:Animals; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004731:Endotoxins / Q000633:toxicity*; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides / Q000633:toxicity; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016328:NF-kappa B / Q000378:metabolism*; D012333:RNA, Messenger; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D004731:Endotoxins; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha