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This Article Full Text Full Text (PDF) All Versions of this Article: jimmunol.0803832v1 183/7/4682    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Google Scholar Articles by John, A. E. Articles by Knox, A. J. PubMed PubMed Citation Articles by John, A. E. Articles by Knox, A. J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information Asthma

Human Airway Smooth Muscle Cells from Asthmatic Individuals Have CXCL8 Hypersecretion Due to Increased NF-B p65, C/EBPβ, and RNA Polymerase II Binding to the CXCL8 Promoter¹

Alison E. John^*, Yong M. Zhu, Christopher E. Brightling, Linhua Pang^* and Alan J. Knox^2,*

^* Centre for Respiratory Research and Nottingham Respiratory Biomedical Research Unit, University of Nottingham, Nottingham, United Kingdom; Institute for Cancer Studies, School of Medicine and Biomedical Science, University of Sheffield, Sheffield, United Kingdom; and Institute for Lung Health, Department of Infection, Inflammation and Immunity, University of Leicester, Leicester, United Kingdom

CXCL8 is a neutrophil and mast cell chemoattractant that is involved in regulating inflammatory cell influx in asthma. Here, we investigated the transcriptional mechanism involved in CXCL8 induction by TNF- in cultured human airway smooth muscle (HASM) cells and compared these in cells from nonasthmatic and asthmatic individuals. Transfection studies with mutated CXCL8 promoter constructs identified NF-B, activating protein-1, and CAAT/enhancer binding protein (C/EBP)β as key transcription factors, and binding of these three transcription factors to the CXCL8 promoter after TNF- stimulation was confirmed by chromatin immunoprecipitation analysis. Cells derived from asthmatic individuals produced significantly higher levels of CXCL8 than nonasthmatic cells both basally and following 24 h of stimulation with TNF- (p < 0.001). Furthermore, chromatin immunoprecipitation studies detected increased binding of NF-B p65 and RNA polymerase II to the CXCL8 promoter of asthmatic HASM cells both in the presence and absence of TNF- stimulation. This was not due to either an increased activation or phosphorylation of NF-B per se or to an increase in its translocation to the nucleus. Increased binding of C/EBPβ to the CXCL8 promoter of unstimulated cells was also detected in the asthmatic HASM cells. Collectively these studies show that HASM cells from asthmatic individuals have increased CXCL8 production due to the presence of a transcription complex on the CXCL8 promoter, which contains NF-B, C/EBPβ, and RNA polymerase II. This is the first description of an abnormality in transcription factor binding altering chemokine expression in airway structural cells in asthma.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Wellcome Trust, Medical Research Council, Asthma UK, and National Institute for Health Research through the Nottingham Respiratory Biomedical Research Unit.

² Address correspondence and reprint requests to Dr. Alan Knox, Centre for Respiratory Research, Clinical Science Building, City Hospital, Hucknall Road, Nottingham, NG5 1PB, U.K. E-mail address: alan.knox{at}nottingham.ac.uk

³ Abbreviations used in this paper: HASM cell, human airway smooth muscle cell; ActD, actinomycin D; AP-1, activating protein-1; BD, bronchodilator; C/EBP, CAAT/enhancer binding protein; ChIP, chromatin immunoprecipitation; ICS, inhaled corticosteroid; IKK2, IB kinase 2; Pol II, polymerase II; TPCA-1, 2-[(aminocarbonyl)amino]-5-[4-fluorophenyl]-3-thiophenecarboxamide; wt, wild-type.