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B Kinase 2, but Not Activation of NF-B, in the Release of CXCR3 Ligands from IFN-
-Stimulated Human Bronchial Epithelial Cells
* Airway Disease, National Heart and Lung Institute, Imperial College, London, United Kingdom;
Heatherwood and Wexham Park National Health Service Trust, Berkshire, United Kingdom;
GlaxoSmithKline, Philadelphia, PA 19406 and
Cell Biology and Anatomy, Respiratory Research Group, University of Calgary, Calgary, Alberta, Canada
The severity of chronic obstructive pulmonary disease correlates with increased numbers of cytotoxic CD8+ T lymphocytes in the lung parenchyma. CD8+ T lymphocytes release IFN-
which stimulates airway epithelial cells to produce CXCR3 chemokines leading to further recruitment of CD8+ T lymphocytes. To evaluate the signaling pathways involved in regulation of CXCR3 ligands, the human bronchial epithelial cell line BEAS-2B was stimulated with IFN- and the release of the CXCR3 ligands was measured by ELISA. The release of CXCL9, CXCL10, and CXCL11 was inhibited by an I
B kinase 2 (IKK2) selective inhibitor 2-[(Aminocarbonyl)amino]-5-[4-fluorophenyl]-3-thiophenecarboxamide (TPCA-1) (EC50 values were 0.50 ± 0.03, 0.17 ± 0.06, and 0.45 ± 0.10 µM, respectively (n = 6)) and an IKK1/2 selective inhibitor 2-amino-6-(2'cyclopropylemethoxy-6'-hydroxy-phenyl)-4-piperidin-3-yl-pyridine-3-carbonitrile (EC50 values 0.74 ± 0.40, 0.27 ± 0.06, and 0.88 ± 0.29 µM, respectively (n = 6)). The glucocorticosteroid dexamethasone had no effect on CXCR3 ligand release. The release of CXCL10 was most sensitive to inhibition by IKK2 and a role for IKK2 in CXCL10 release was confirmed by overexpression of dominant-negative adenoviral constructs to IKK2 (68.2 ± 8.3% n = 5), but not of IKK1. Neither phosphorylation of IB
, translocation of p65 to the nucleus, or activation of a NF-B-dependent reporter (Ad-NF-B-luc) were detected following stimulation of BEAS-2B cells with IFN-. These data suggest that IKK2 is also involved in the IFN--stimulated release of the CXCR3 ligands through a novel mechanism that is independent NF-B.
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 Address correspondence and reprint requests to Dr. Louise E. Donnelly, Airway Disease Section, Guy Scadding Building, National Heart and Lung Institute, Imperial College London, Dovehouse Street, London, SW3 6LY, U.K. E-mail address: l.donnelly{at}imperial.ac.uk
2 Abbreviations used in this paper: COPD, chronic obstructive pulmonary disease; IKK, IB kinase; TPCA, 2-[(aminocarbonyl)amino]-5-[4-fluorophenyl]-3-thiophenecarboxamide; TBK, TANK-binding kinase; K-SFM, keratinocyte serum-free medium; MOI, multiplicity of infection; DAPI, 4',6-diamidino-2-phenyl-indole hydrochloride; MEF, mouse embryo fibroblast; IRF, IFN regulatory factor; APPC, 2-amino-6-(2'cyclopropylmethoxy-6'-hydroxy-phenyl)-4-piperidin-3-yl- pyridine-3-carbonitrile.
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