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Exposure to Mexicali PM10 Induces IL-8 Expression Through an Alternative NFKB Mechanism In Human Airway Epithelial Cells
Citation:
SILBAJORIS, R. A., L. A. DAILEY, S. SIMMONS, A. R. Osornio-Vargas, AND J. M. SAMET. Exposure to Mexicali PM10 Induces IL-8 Expression Through an Alternative NFKB Mechanism In Human Airway Epithelial Cells. Presented at American Thoracic Society meeting, New Orleans, LA, May 14 - 19, 2010.
Impact/Purpose:
The aim of this study was to examine the signaling events involved in the expression of inflammatory genes in cultured human airway epithelial cells (HAEC) exposed to PM IOderived from vehicle emissions in an urban area of Mexicali, Mexico.
Description:
Studies have shown associations between exposure to ambient air particulate matter (PM) and increased rates of cardio-pulmonary morbidity and mortality. The aim of this study was to examine the signaling events involved in the expression of inflammatory genes in cultured human airway epithelial cells (HAEC) exposed to PM IOderived from vehicle emissions in an urban area of Mexicali, Mexico. PM IO fractions were collected on cellulose membranes using High-Vol samplers between October 2005 and March 2006. Real Time (RT)-PCR showed that HAEC exposed for 4 h to 10-80 ug/crrr' PM resuspended in media resulted in a dose-dependent increase in IL-8 mRNA expression that was 2-8-fold higher than media controls. PM exposure induced IL-8 transcriptional activity in BEAS-2B cells expressing an IL-8 promoter reporter construct. Mutation of the NFKB response element in the IL-8 promoter resulted in a significant impairment in IL-8 transcriptional activity. The activation ofNFKB-dependent transcriptional activity was confirmed using a synthetic luciferase reporter driven by a tandem repeat ofthe canonical NFKB binding site. Chromatin Immunoprecipitation (CHiP) assay was used to immunoprecipitate Nfxfs-bound DNA fragments. RT-PCR, employing primers and a fluorescent probe directed against the NFKB sequence in the IL-8 gene promoter region, was then used to measure % of input, comparing signals of CHiP samples to total input DNA. We found a 3-fold enrichment of IL-8 promoter sequence in HAEC exposed to PM compared to media controls. Expression of IL-8 mRNA in PM treated HAEC was reduced when cells were pretreated with BAY 11-7082, an NFKB inhibitor, but not with the IKK-2 inhibitor BMS-345541, and Western blot analyses showed increased NFKBp65 phosphorylation independent of IKBa degradation or phosphorylation. Taken together, this data suggests that activation ofNFKB occurs through a non-canonical pathway. We conclude that the increase in IL-8 mRNA expression in HAEC exposed to PMIO is mediated through an NFKB-dependent signaling mechanism that involves direct phosphorylation ofthe transcription factor p65. These findings show that PM IO exposure can induce inflammatory responses by activating specific signaling mechanisms in human airway epithelial cells. THIS ABSTRACT OF A PROPOSED PRESENTATION DOES NOT NECESSARILY REFLECT EPA POLICY.