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Abrupt expression of TLR4 in TLR4-deficient macrophages imposes a selective disadvantage: genetic evidence for TLR4-dependent responses to endogenous, nonmicrobial stimuli.

TitleAbrupt expression of TLR4 in TLR4-deficient macrophages imposes a selective disadvantage: genetic evidence for TLR4-dependent responses to endogenous, nonmicrobial stimuli.
Publication TypeJournal Article
Year of Publication2006
AuthorsCohn EF, Nathan C, Radzioch D, Yu H, Xiang Z, Ding A
JournalJ Immunol
Volume176
Issue2
Pagination1185-94
Date Published2006 Jan 15
ISSN0022-1767
KeywordsAnimals, Base Sequence, Cell Line, Clone Cells, Culture Media, Conditioned, DNA, Complementary, Gene Expression, Lipopolysaccharides, Macrophage Activation, Macrophages, Mice, Mice, Inbred C57BL, Mice, Knockout, RNA, Messenger, Signal Transduction, Toll-Like Receptor 4, Transfection
Abstract

TLR4 is crucial for macrophage responses to LPS. It is less clear whether TLR4 may also transduce signals from host factors, and if so, with what consequences. Immortalized bone marrow-derived macrophage cell lines, termed T4Cr and T4ko, were established from TLR4null strains, C57BL/10ScNCr and TLR4 knockout mice, respectively. Multiple transfections and selections were conducted to stably introduce TLR4 into these cell lines. Among 196 individual clones isolated, 48 expressed TLR4 on the cell surface but did not respond to LPS due to a deletion in the MyD88 gene. The remaining clones integrated TLR4 DNA into the genome but expressed neither detectable TLR4 mRNA nor TLR4 protein. To test the possibility that TLR4null cells lack modulating factors to protect against a harmful effect of TLR4, 15 stably transfected clones were generated in the presence of conditioned media from wild-type macrophages. Some of these cells expressed a small amount of TLR4 and regained responsiveness to LPS. Because no microbial ligands were available to the cell lines during their generation, signaling via endogenous ligands is likely to have occurred in TLR4-expressing, signal-competent macrophages and imposed a proliferative or other selective disadvantage. These studies support the existence of constitutive signaling via TLR4 during in vitro culture of macrophages without microbial products, and help account for the lack of reports of restoration of TLR4 expression in normally TLR4-expressing types of cells in vitro whose TLR4 genes are deleted or disrupted.

Alternate JournalJ. Immunol.
PubMed ID16394008
Grant ListR01-AI30165 / AI / NIAID NIH HHS / United States
R01-GM61710 / GM / NIGMS NIH HHS / United States

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