TOLERANCE TO LIPOPOLYSACCHARIDE INVOLVES MOBILIZATION OF NUCLEAR FACTOR KAPPA-B WITH PREDOMINANCE OF P50 HOMODIMERS

Stimulation of the human monocytic cell line Mono Mac 6 with lipopolys accharide (LPS) leads to rapid and transient expression of cytokines l ike tumor necrosis factor (TNF). When such cells are precultured for 2 days with a low dose of LPS (20 ng/ml) followed by stimulation with a high dose of LPS (1 mu g/ml), expression of the TNF gene is minimal, i.e. the cells are tolerant. In nuclear run-on analysis, such tolerant cells show only a low degree of transcription, indicating that tolera nce operates at or upstream of the transcription level, The CD14 LPS r eceptor is, however, up-regulated (not down-regulated) in tolerant cel ls, and LPS can, in fact, still lead to activation of tolerant cells a s evidenced by mobilization of the transcription factor nuclear factor kappa B (NF-kappa B). Resolution of the NF-kappa B complex in gel shi ft analysis shows that the binding protein, mobilized in naive Mono Ma c 6 cells, consists mainly of p50-p65 heterodimers, while in tolerant cells, the p50 homodimer is predominant. This increase in p50 homodime rs coincides with an increase in p105 mRNA, suggestive of a transcript ional up-regulation of p50. Reporter gene analysis reveals that the NF -kappa B complex mobilized in tolerant cells is functionally inactive in that NF-kappa B dependent luciferase constructs containing the huma n immunodeficiency virus long terminal repeat or the TNF 5'-region sho w only minimal transactivation after LPS stimulation. Similar to Mono Mac 6 cells, primary blood monocytes, when precultured with a low dose of LPS, also become tolerant and produce little TNF after LPS stimula tion. The tolerant blood monocytes also up regulate CD14, and they mob ilize NF-kappa B with a predominance of p50 homodimers. Taken together , these results demonstrate that tolerance to LPS is determined by pos t-receptor mechanisms that involve an altered composition of the NF-ka ppa B complex.