Inhibitory κBα control of nuclear factor-κB is dysregulated in endotoxin tolerant macrophages

The transcription factor nuclear factor (NF)-kappa B is thought to be required for endotoxin-stimulated tumor necrosis factor (TNF) and interleukin (IL)-1 gene transcription. Nuclear translocation of NF-kappa B is regulated by the cytoplasmic inhibitory factor I kappa B alpha. 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 I kappa B alpha and NF-kappa B following activation with LPS. Murine macrophages (Mg) were exposed to a range of LPS concentrations +/- 24 h PreRx with 10 ng/mL LPS pretreatment. Cytoplasmic I kappa B alpha (Western immunoblot) and NF-kappa B (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, I kappa B alpha disappeared from the cytoplasm coincident with nuclear translocation of NF-kappa B. Tolerant M phi had markedly enhanced levels of I kappa B alpha and normal to increased levels of NF-kappa B 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 M phi also had increased cytoplasmic levels of the p65 subunit of NF-kappa B. LPS tolerance is associated with increases of cytoplasmic I kappa B alpha p65, as well as enhanced NF-kappa B. We conclude that control of NF-kappa B translocation by I kappa B alpha is dysregulated in endotoxin-tolerant M phi.