| Construction of a lipopolysaccharide reporter cell line and its use in identifying mutants defective in endotoxin, but not TNF-alpha, signal transduction. | |
| | |
MedLine Citation:
|
PMID: 9743364 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
|
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. |
| | |
Authors:
|
R L Delude; A Yoshimura; R R Ingalls; D T Golenbock |
Related Documents
:
|
17434724 - N-3 pufa attenuate lipopolysaccharide-induced down-regulation of toll-like receptor 4 e... 8955874 - Identification of macrophage migration inhibitory factor in human leukemia hl-60 cells ... 15312234 - Endotoxin leads to rapid subcellular re-localization of hepatic rxralpha: a novel mecha... 9545534 - Abnormalities of floor plate, notochord and somite differentiation in the loop-tail (lp... 11290514 - Hyperoxia upregulates the no pathway in alveolar macrophages in vitro: role of ap-1 and... 11955914 - Downregulated expression of adam9 in anterior polar cataracts. |
Publication Detail:
|
Type: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
|
Title: Journal of immunology (Baltimore, Md. : 1950) Volume: 161 ISSN: 0022-1767 ISO Abbreviation: J. Immunol. Publication Date: 1998 Sep |
Date Detail:
|
Created Date: 1998-10-06 Completed Date: 1998-10-06 Revised Date: 2007-11-14 |
Medline Journal Info:
|
Nlm Unique ID: 2985117R Medline TA: J Immunol Country: UNITED STATES |
Other Details:
|
Languages: eng Pagination: 3001-9 Citation Subset: AIM; IM |
Affiliation:
|
Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA. rdelude@bidmc.harvard.edu |
Export Citation:
|
APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
|
Acute-Phase Proteins* Animals Biological Transport / immunology Blotting, Northern CHO Cells Carrier Proteins / physiology Cell Culture Techniques / methods Cell Fusion / genetics, immunology Cell Line Cell Separation Cricetinae Endotoxins / deficiency, genetics* Flow Cytometry Genes, Reporter / immunology* Genetic Complementation Test Humans Lipopolysaccharides / immunology*, metabolism, pharmacology Membrane Glycoproteins* Mutagenesis / immunology* NF-kappa B / metabolism Phenotype Receptors, Interleukin-2 / biosynthesis, drug effects, genetics Signal Transduction / genetics*, immunology Transfection / immunology Tumor Necrosis Factor-alpha / genetics*, pharmacology |
| Grant Support | |
ID/Acronym/Agency:
|
R01-GM54060/GM/NIGMS NIH HHS |
| Chemical | |
Reg. No./Substance:
|
0/Acute-Phase Proteins; 0/Carrier Proteins; 0/Endotoxins; 0/Lipopolysaccharides; 0/Membrane Glycoproteins; 0/NF-kappa B; 0/Receptors, Interleukin-2; 0/Tumor Necrosis Factor-alpha; 0/lipopolysaccharide-binding protein |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
Previous Document: Inducible nitric oxide synthase is not required for long-term vaccine-based immunity against Toxopla...
Next Document: Two distinct phospholipases C of Listeria monocytogenes induce ceramide generation, nuclear factor-k...