Rapid up-regulation of I kappa B beta and abrogation of NF-kappa B activity in peritoneal macrophages stimulated with lipopolysaccharide

Lipopolysaccharide (LPS) administration to mice elicited the activation of nuclear factor kappa B (NF-kappa B) 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 I kappa B alpha and I kappa B beta were analyzed during this period in an attempt to correlate NF-kappa B activity with I kappa B resynthesis. Degradation of I kappa B alpha was very rapid and was followed by recovery 1 h after LPS administration. I kappa B beta degradation, which has been associated with persistent NF-kappa B activation, was complete at 1 h. However, a rapid recovery of I kappa B beta in these tissues was observed at 3 h in parallel with the abrogation of NF-kappa B activity. Immunolocalization of newly synthesized I kappa B beta by confocal microscopy revealed its preferential accumulation in the cytosol. Analysis of I kappa B beta 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 I kappa B beta 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 I kappa B beta. At the mRNA level, up-regulation of I kappa B beta was observed in macrophages stimulated for 1 h with LPS. When the effect of pro-inflammatory cytokines was investigated, tumor necrosis factor alpha, but not interleukin-1 or interferon-gamma, promoted an important degradation of I kappa B beta followed by an increase in the mRNA at 1 h. These results suggest the existence of LPS and tumor necrosis factor gamma-specific pathways involved in a rapid I kappa B beta degradation and resynthesis and might explain the transient period of activation of NF-kappa B in these tissues upon stimulation with these factors. This rapid control of NF-kappa B function may contribute to the attenuation of the inflammatory response of these cells.