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 lipopolysaccharide (LPS) leads to rapid and transient expression of cytokines like 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 microgram/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 tolerance operates at or upstream of the transcription level. The CD14 LPS receptor is, however, up-regulated (not down-regulated) in tolerant cells, and LPS can, in fact, still lead to activation of tolerant cells as evidenced by mobilization of the transcription factor nuclear factor kappa B (NF-kappa B). Resolution of the NF-kappa B complex in gel shift analysis shows that the binding protein, mobilized in naive Mono Mac 6 cells, consists mainly of p50-p65 heterodimers, while in tolerant cells, the p50 homodimer is predominant. This increase in p50 homodimers coincides with an increase in p105 mRNA, suggestive of a transcriptional 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 human immunodeficiency virus long terminal repeat or the TNF 5'-region show 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 stimulation. The tolerant blood monocytes also up-regulate CD14, and they mobilize NF-kappa B with a predominance of p50 homodimers. Taken together, these results demonstrate that tolerance to LPS is determined by post-receptor mechanisms that involve an altered composition of the NF-kappa B complex.
Ziegler-Heitbrock
H W
HW
Institute for Immunology, München, Federal Republic of Germany.
Wedel
A
A
Schraut
W
W
Ströbel
M
M
Wendelgass
P
P
Sternsdorf
T
T
Bäuerle
P A
PA
Haas
J G
JG
Riethmüller
G
G
D015703:Antigens, CD / Q000378:metabolism*; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism*; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D004361:Drug Tolerance; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000633:toxicity*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009000:Monocytes / Q000187:drug effects; D016328:NF-kappa B / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D015854:Up-Regulation
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D017931:DNA Primers; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha