Rapid up-regulation of IκBβ and abrogation of NF-κB activity in peritoneal macrophages stimulated with lipopolysaccharide

Abstract

Lipopolysaccharide (LPS) administration to mice elicited the activation of nuclear factor κB (NF-κB) in several tissues including liver and macrophages. Maximal activation was observed 1 h after treatment but declined at 3 and 6 h. The levels of IκBα and IκBβ were analyzed during this period in an attempt to correlate NF-κB activity with IκB resynthesis. Degradation of IκBα was very rapid and was followed by recovery 1 h after LPS administration. IκBβ degradation, which has been associated with persistent NF-κB activation, was complete at 1 h. However, a rapid recovery of IκBβ in these tissues was observed at 3 h in parallel with the abrogation of NF-κB activity. Immunolocalization of newly synthesis IκBβ by confocal microscopy revealed its preferential accumulation in the cytosol. Analysis of IκBβ by Western blot using high resolution polyacrylamide gel electrophoresis showed the presence of two bands in cytosolic extracts of LPS-treated macrophages at 3 h, but only one band with the same mobility as the control was detected at 6 h. Moreover, treatment of extracts of resynthesized IκBβ with alkaline phosphatase resulted in the accumulation of the protein of slightly higher electrophoretic mobility, indicating the prevalence of a rapid phosphorylation of the newly synthesized IκBβ. At the mRNA level, up-regulation of IκBβ was observed in macrophages stimulated for 1 h with LPS. When the effect of pro-inflammatory cytokines was investigated, tumor necrosis factor α, but not interleukin-1 or interferon- γ, promoted an important degradation of IκBβ followed by an increase in the mRNA at 1 h. These results suggest the existence of LPS- and tumor necrosis factor α- specific pathways involved in a rapid IκBβ degradation and resynthesis and might explain the transient period of activation of NF- κB in these tissues upon stimulation with these factors. This rapid control of NF-κB function may contribute to the attenuation of the inflammation response of these cells.