Investigating the role of NF-kappaB/IKK-2 signalling in cigarette smoke-induced airway inflammation

Joseph MD Rastrick1, Christopher S Stevenson2, Iain Kilty3, Steven M Evans3, Manolis Pasparakis4, Matthew C Catley51, Toby Lawrence6, Ian M Adcock1, Maria G Belvisi1, Mark A Birrell1. 1Imperial College, London, UK, 2Roche, Nutley, New Jersey, USA, 3Pfizer, Sandwich, Kent, UK, 4University of Cologne, Cologne, Germany, 5UCB Celltech, Sough, Berkshire, UK, 6Centre d\'Immunologie Marseille-Luminy, Marseille, France.

Chronic Obstructive Pulmonary Disease (COPD) is an inflammatory lung disease largely associated with smoking. The mechanism by which cigarette smoke (CS) leads to the pathogenesis of COPD is currently unclear, however many inflammatory mediators present in the COPD lung are thought to be regulated by the transcription factor Nuclear Factor-kappaB (NF-κB) and it’s upstream signalling kinase, Inhibitor of κB kinase-2 (IKK-2) (Rajendrasozan S, et al, 2010). The NF-κB/IKK-2 signalling pathway may therefore represent a novel target to attenuate the inflammation associated with this disease. To test this hypothesis, male C57BL/6 mice (16-20g) lacking IKK-2 exclusively in the airway epithelium or in myeloid-derived cells were exposed to aerosolised LPS (1mg ml-1, 30min) or CS (500ml/min CS for 50min, 2xdaily for 3days). CS exposure induced an increase in neutrophil number (Air control: 0.06 ± 0.06x103 vs. Smoke: 39.1 ± 10.0x103) and cytokine release (KC, IL-23/IL-12p40) in the bronchoalveolar lavage fluid (BAL) of wild type mice (n=8); however in contrast to LPS-induced airway inflammation CS exposure did not increase NF-κB (p50/p65/p52/RelB):DNA association in whole lung nuclear extracts. Mice lacking IKK-2 exclusively in the airway epithelium or in myeloid-derived cells were then exposed to LPS or CS as any change in NF-κB:DNA association in these discrete cell types may have been undetectable in whole lung nuclear extracts from CS-exposed mice. The NF-κB-dependent model of LPS-induced airway inflammation confirmed the ablation of IKK-2 in these conditional knockout mice, with both strains showing functional decreases in NF-κB(p65):DNA association (figure 1). In contrast, CS-induced airway inflammation was not modulated by ablation of IKK-2 in either cell type. Furthermore, lung samples taken from human COPD patients (n≥10)also showed no change in NF-κB(p65):DNA association compared to healthy smokers and non-smokers. This data suggests that contrary to previous reports (Di Stefano A, et al, 2002) IKK-2/NF-κB signalling may not play a prominent role in CS-induced lung inflammation.

Figure 1: p65:DNA association in mice lacking IKK-2 exclusively in airway epithelial cells (A) and myeloid-derived cells (B) following treatment with saline or LPS (1mg ml-1, 30mins). Significance tested by Unpaired t-test (#) or one-way ANOVA (*) with appropriate post test (p<0.05).

Di Stefano A, Caramori G, Oates T, et al. Increased expression of nuclear factor-κB in bronchial biopsies from smokers and patients with COPD, Eur Respir J. 2002;20:556-563.
Rajendrasozhan S, Hwang JW, Yao H, et al. Anti-inflammatory effect of a selective IκB kinase-β inhibitor in rat lung in response to LPS and cigarette smoke, Pulm Pharmacol Ther. 2010;23:172-81