Inhibitory kappa B alpha control of nuclear factor-kappa B is dysregulatedin endotoxin tolerant macrophages

The transcription factor nuclear factor (NF)-kappa B is thought to be requi red for endotoxin-stimulated tumor necrosis factor (TNF) and interleukin (I L)-1 gene transcription. Nuclear translocation of NF-kappa B is regulated b y the cytoplasmic inhibitory factor I kappa B alpha. Low-dose lipopolysacch aride (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 follo wing activation with LPS. Murine macrophages (Mg) were exposed to a range o f LPS concentrations +/- 24 h PreRx with 10 ng/mL LPS pretreatment. Cytopla smic 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 me asured 6 h after LPS activation using RT-PCR. In the absence of LPS pretrea tment, I kappa B alpha disappeared from the cytoplasm coincident with nucle ar 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 translocat ion with a different electrophoretic shift. LPS activation enhanced cytokin e gene transcription in a dose-dependent manner, and this was unaltered by LPS pretreatment, Endotoxin-tolerant M phi also had increased cytoplasmic l evels of the p65 subunit of NF-kappa B. LPS tolerance is associated with in creases 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.