A role for ikk2 involvement, but not NF-κB, in IFN-γ stimulated release of the cxcr3 ligand IP-10 from human airway epithelial cells

Susan J. Smith, Matthew C. Catley, William L. Rumsey1, Robert Newton2, Peter J. Barnes and Louise E. Donnelly. Airway Disease, NHLI, Imperial College London, London, UK. 1 GlaxoSmithKline, Philapdelphia, USA, 2 University of Calgary, Calgary, Canada.

The numbers of cytotoxic CD8+ T-lymphocytes in the lung parenchyma is correlated with the severity of chronic obstructive pulmonary disease (COPD). CD8+ T-lymphocytes release interferon (IFN)- g that stimulate epithelial cells to produce CXCR3 ligands leading to an enhanced recruitment of CD8+ T-lymphocytes, thus amplifying the inflammatory insult. In order to evaluate the signalling pathways involved in the expression of CXCR3 ligands, the human bronchial epithelial cell line, BEAS-2B, was stimulated with IFN-γ and the release of the CXCR3 ligand interferon-inducible protein (IP)-10 (CXCL10) was measured by ELISA. The glucocorticosteroid, dexamethasone, had no effect on IFN-γ stimulated release of IP-10 however, the release of IP-10 was inhibited with the dominant negative adenoviral constructs to inhibitor of κB kinase (IKK)2 (78.5 ± 21.54% inhibition; n=3), but not with adenoviral constructs to IKK1. Following stimulation of BEAS-2B cells with IFN-γ , neither phosphorylation of IκB- α or translocation of p65 to the nucleus, measured by Western blotting (n=4) and confocal microscopy (n=4), respectively, was detected. In contrast, IL-1β /TNF-α , induced these responses. An adenovirus carrying a NF-κB-dependent reporter (Ad-NF- κB-luc) was infected into Beas2B (n=2) and this, whilst showing responsiveness to IL-1 β/TNF-α , did not respond to stimulation with IFN- g . Nevertheless, a role for IKK2 in the inhibition of IP-10 release was confirmed using a selective IKK2 inhibitor TPCA-1 (Podolin et al., 2005), which completely suppressed IP-10 production at 10 μM; EC50 0.22 ± 0.08 μM (n=8). IFN-γ and TNF- α synergistically enhanced IP-10 production and also stimulated IL-6 release from Beas2B. The IKK2 inhibitor TPCA-1 suppressed the release of both IP-10 and IL-6 in a concentration-dependent manner EC50 0.68 ± 0.09 μM and 0.50 ± 0.05 μM (n=6), respectively and completely inhibited their release at 10 μM, whereas dexamethasone had no significant effect on the release of IP-10 and suppressed IL-6 release by 68.2 ± 10.3 % at 1 μM, EC50 39.74 ± 8.86 nM (n=6). These data suggest that IKK2 not only operates through the classical NF-κB pathway but is also involved in the IFN-γ stimulated release of the CXCR3 ligand IP-10 through a novel mechanism that is independent NF-κB.

Podolin PL et al. , J Pharmacol Exp Ther . 2005; 312(1):373-81.