NF-kappa B1 (p50) is upregulated in lipopolysaccharide tolerance and can block tumor necrosis factor gene expression

Monocytes respond to lipopolysaccharide (LPS) stimulation with a rapid expr ession of the tumor necrosis factor (TNF) gene. Upon repeated LPS stimulati on there is, however, little production of TNF mRNA and protein; i.e., the cells are tolerant to LPS, Analysis of NF-kappa B proteins in gel shift ass ays demonstrated that the DNA binding activity that is induced by LPS stimu lation in tolerant cells consists mainly of p50-p50 homodimers, Since p50 c an bind to DNA but lacks a transactivation domain, this may explain the blo ckade of TNF gene expression. We now show that in the monocytic cell line M ono Mac 6, this inability to respond can be largely ascribed to NF-kappa B, since a reporter construct directed by a trimeric NF-kappa B motif is stro ngly transactivated by LPS stimulation of naive cells whereas LPS-tolerant cells exhibit only low activity. Also, Western blot analyses of proteins ex tracted from purified nuclei showed mobilization of threefold-higher levels of p50 protein in tolerant compared to naive cells, while mobilization of p65 was unaltered. Overexpression of p50 in HEK 293 cells resulted in a str ong reduction of p65-driven TNF promoter activity at the levels of both luc iferase mRNA and protein. These data support the concept that an upregulati on of p50 is instrumental in LPS tolerance in human monocytes.