Role of p52 (NF-kappa B2) in LPS tolerance in a human B cell line

Cells of the weakly CD14 positive human B cell line RPMI 8226, clone 1, wil l mobilize NF-kappa B (p50/p65 and p50/p50) proteins and produce TNF mRNA w hen stimulated with lipopolysaccharide (LPS), When such cells are precultur ed with a low amount of LPS (50 - 250 ng/ml) for 3 - 4 days followed by a s econdary stimulation with a high dose of LPS (1 mu g/ml) then the cytokine expression is strongly reduced, i.e, the cells have become tolerant. Western blot analysis of proteins of the NF-kappa B/rel family demonstrates cytoplasmic p50 and p65 for naive B cells plus a low level of p52. While w ith tolerance induction the pattern of p50 and p65 proteins remains essenti ally unchanged, the LPS tolerant 8226 cells show a dramatic increase of bot h p52 protein and its p100 precursor in the cytosol. This p52 is found stro ngly upregulated in Western blots of extracts from purified nuclei of toler ant cells, Also, gelshift analysis with the -605 kappa B motif Of the human TNF 5'-region shows an additional high mobility complex in LPS tolerant ce lls - a complex that is supershifted with an anti-p52 antibody, Functional analysis with the -1064 TNF 5'-region in front of the luciferase reporter gene demonstrates that transactivation of the TNF promoter is str ongly reduced in tolerant cells, Also, overexpression of p52 will suppress activity of TNF promoter reporter gene constructs. Taken together these data show that tolerance to LPS in the human RPM1 8226 a cell line involves upregulation of the p52 (NF-kappa B2) gene, which app ears to be instrumental in the blockade of TNF gene expression.