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Cigarette smoke activates NFκB‐mediated Tnf‐α release from mouse middle ear cells
Author(s) -
Preciado Diego,
Kuo Elaine,
Ashktorab Samaneh,
Manes Peter,
Rose Mary
Publication year - 2010
Publication title -
the laryngoscope
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.181
H-Index - 148
eISSN - 1531-4995
pISSN - 0023-852X
DOI - 10.1002/lary.21014
Subject(s) - cigarette smoke , nf κb , tumor necrosis factor alpha , smoke , microbiology and biotechnology , chemistry , medicine , biology , immunology , signal transduction , environmental health , organic chemistry
Objectives/Hypothesis: Cigarette smoke exposure is a significant risk factor in the development of otitis media. NF‐κB is a transcription factor known to mediate cigarette smoke effects in multiple cell types. We hypothesized that stimulation of murine middle ear epithelial cells (MEEC) with cigarette smoke condensate (CSC) activates NF‐κB resulting in upregulation of proinflammatory cytokines. Study Design: In vitro model of cultured murine middle ear epithelial cells. Methods: Time course CSC stimulation of MEEC was performed. Antibody microarrays were then utilized to simultaneously measure 40 inflammatory cytokines. Enzyme‐linked immunosorbent assay (ELISA) and quantitative reverse transcriptase‐polymerase chain reaction were performed to validate and further evaluate array results. Luciferase reporter assays were performed to evaluate NF‐κB activation with CSC in MEEC. Chromatin immunoprecipitation (ChIP) assays were performed to determine whether CSC induces NF‐κB interaction with the Tnf ‐α promoter. Results: Multiple cytokines showed significant increases with CSC exposure. ELISA studies demonstrated that Tnf‐α secretion increased the most. CSC stimulation likewise increased Tnf‐α mRNA abundance and induced promoter activity 4.8‐fold in a Tnf ‐α reporter plasmid. Reporter assays demonstrated 4.84‐fold activation of NF‐κB with CSC. ChIP assays demonstrated NF‐κB binding to canonical κB sites in the Tnf ‐α promoter with CSC stimulation. Conclusions: CSC activates NF‐κB in MEEC. Furthermore, this activation results in CSC induced Tnf ‐α promoter activation, gene expression, and levels in cell secretions. Laryngoscope, 120:2508–2515, 2010