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  • Title: Construction of a lipopolysaccharide reporter cell line and its use in identifying mutants defective in endotoxin, but not TNF-alpha, signal transduction.
    Author: Delude RL, Yoshimura A, Ingalls RR, Golenbock DT.
    Journal: J Immunol; 1998 Sep 15; 161(6):3001-9. PubMed ID: 9743364.
    Abstract:
    Gram-negative bacterial LPS is a potent activator of inflammatory responses. The binding of LPS to CD14 initiates signal transduction; however, the molecular processes immediately following this event remain unclear. We engineered an LPS-inducible fibroblast reporter cell line to facilitate the use of molecular genetic techniques to study the LPS signaling pathway. A plasmid containing the human Tac Ag cDNA under transcriptional control of the human E selectin promoter was cotransfected into Chinese hamster ovary (CHO)-K1 cells together with a CD14 expression plasmid. A cell line was obtained, 3E10, which upregulated expression of Tac following stimulation with LPS. Pools of mutagenized cells were exposed to LPS and then labeled with anti-Tac mAb. Cells that failed to up-regulate Tac expression were enriched by flow cytometry. Thirty clonal mutant cell lines were identified that continued to express CD14 and bind LPS, but failed to express Tac or translocate nuclear factor-kappaB (NF-kappaB) following LPS exposure. TNF-alpha-treated mutant cells continued to express Tac and translocate NF-kappaB. An analysis of LPS-induced NF-kappaB activity in heterokaryons derived from polyethylene glycol-fused cell lines indicated that recessive mutations in genes encoding components of the LPS signaling pathway accounted for the signaling defects. To date, two complementation groups have been identified from 11 cell lines analyzed. These data demonstrate that the TNF-alpha signaling pathway diverges from the LPS pathway early in the signal-transduction cascade despite similarities in LPS- and TNF-alpha-induced responses. Identification of the genes affected in these mutant reporter cells should identify heretofore-elusive components of the LPS signaling cascade.
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