Basal, IFN-γ and IL-12 mediated secretion of IP-10 in lung epithelial cell/PBMC co-cultures cultured in transwell chambers with separating filters

<p><b>Copyright information:</b></p><p>Taken from "The role of IFN-γ in regulation of IFN-γ-inducible protein 10 (IP-10) expression in lung epithelial cell and peripheral blood mononuclear cell co-cultures"</p><p>http://respiratory-research.com/content/8/1/80</p><p>Respiratory Research 2007;8(1):80-80.</p><p>Published online 8 Nov 2007</p><p>PMCID:PMC2174934.</p><p></p> Data represent the mean ± SEM of 4 independent experiments. Control (□) values are shown as IP-10 secretion in lung epithelial cell/PBMC co-cultures cultured in transwell chambers without a separating filter

Cigarette Smoke Induced Airway Inflammation Is Independent of NF-κB Signalling

<div><h3>Rationale</h3><p>COPD is an inflammatory lung disease largely associated with exposure to cigarette smoke (CS). The mechanism by which CS leads to the pathogenesis of COPD is currently unclear; it is known however that many of the inflammatory mediators present in the COPD lung can be produced via the actions of the transcription factor Nuclear Factor-kappaB (NF-κB) and its upstream signalling kinase, Inhibitor of κB kinase-2 (IKK-2). Therefore the NF-κB/IKK-2 signalling pathway may represent a therapeutic target to attenuate the inflammation associated with COPD.</p> <h3>Aim</h3><p>To use a range of assays, genetically modified animals and pharmacological tools to determine the role of NF-κB in CS-induced airway inflammation.</p> <h3>Methods</h3><p>NF-κB pathway activation was measured in pre-clinical models of CS-induced airway inflammation and in human lung tissue from COPD patients. This data was complemented by employing mice missing a functional NF-κB pathway in specific cell types (epithelial and myeloid cells) and with systemic inhibitors of IKK-2.</p> <h3>Results</h3><p>We showed in an airway inflammation model known to be NF-κB-dependent that the NF-κB pathway activity assays and modulators were functional in the mouse lung. Then, using the same methods, we demonstrated that the NF-κB pathway appears not to play an important role in the inflammation observed after exposure to CS. Furthermore, assaying human lung tissue revealed that in the clinical samples there was also no increase in NF-κB pathway activation in the COPD lung, suggesting that our pre-clinical data is translational to human disease.</p> <h3>Conclusions</h3><p>In this study we present compelling evidence that the IKK-2/NF-κB signalling pathway does not play a prominent role in the inflammatory response to CS exposure and that this pathway may not be important in COPD pathogenesis.</p> </div

Temporal characterisation of the airway inflammation after LPS or 3 days of CS challenge.

<p>Mice were challenged with LPS (1 mg/ml) or endotoxin free saline for 30 minutes. Samples were collected at increasing time points after challenge. A) LPS-induced BALF neutrophilia. B) LPS-induced NF-κB(p65):DNA association in lung nuclear extract. Mice were challenged for 3 days with CS (500 ml/min, 1 hour, twice daily) or ambient air. Samples were collected at increasing time points after the final challenge. C) CS-induced BALF neutrophilia. D) CS-induced NF-κB (p65):DNA association in lung nuclear extract. Data are presented as mean ± s.e.m. of n = 6–8 observations.</p

Profile of IKK-2^ΔEpi mice in the 3 and 14 day CS-driven models.

<p>IKK-2^ΔEpi mice were challenged with 3 or 14 days CS (500 ml/min, 1 hour, twice daily) or ambient air. BALF samples were collected twenty-four hours after the final challenge. BALF neutrophilia after 3 days of CS challenge (A) BALF neutrophilia, macrophages and lymphocytes after 14 days of CS challenge (B, C and D, respectively). Data are presented as mean ± s.e.m. of n = 8–16 observations. # indicates a statistically significant difference (p<0.05) from air challenged control groups (Mann-Whitney test). Any changes in data collected from the GM control mice i.e. fl/fl, TET alone and CRE alone did not reach statistical significance when compared to the appropriate control group.</p

Temporal characterisation of airway inflammation after 14 days of CS challenge.

<p>Mice were challenged for 14 days with CS (500 ml/min, 1 hour, twice daily) or ambient air. Samples were collected at increasing time points after the final challenge. A) BALF neutrophilia, B) BALF macrophage number, C) BALF lymphocytes and D) NF-κB (p65):DNA association in lung nuclear extract. Data are presented as mean ± s.e.m. of n = 6–8 observations.</p

Characterisation of the IKK-2^ΔEpi mice.

<p>To demonstrate the knockdown of IKK-2 in the airway epithelium, mouse primary epithelial cells were harvested and cultured from control or IKK-2^ΔEpi mice and mRNA levels were determined by RT-PCR. Data is expressed as fold change from wild type control values (A). To demonstrate a function on the knockdown, mice were challenged with 1 mg/ml aerosolised LPS or saline and 2 hours after challenge lung tissue was collected for immunohistochemistry. Levels of p65 in the nuclei were assessed by 2 people blinded to the treatment groups. The score represents the mean ± s.e.m of 10 random fields performed by both scorers (B). To show this knockdown can impact on whole tissue extracts, NF-κB (p65):DNA association in lung nuclear extracts were measured (C). Finally to show the effect of disrupting the NF-κB signalling pathway in epithelial cells, mice were challenged with LPS and BALF was collected 6 hours later. Neutrophilia was measured in the BALF (D). # indicates a statistically significant difference (p<0.05) from saline challenged control groups (Mann-Whitney test). * indicates statistical significance (p<0.05) from LPS treated control groups by Kruskal-Wallis one-way-ANOVA with Dunn's multiple comparison post-hoc test. Any changes in data collected from the GM control mice i.e. fl/fl, TET alone and CRE alone did not reach statistical significance when compared to the appropriate control group.</p

NF-κB:DNA association in human lung tissue.

<p>NF-κB(p65): DNA association in lung tissue from non-smoking donors, smoking donors and emphysema patients. Data are presented as mean ± s.e.m. of n = 10–25 observations. * indicates a statistically significant difference (p<0.05) from non-smoking donors by Kruskal-Wallis one-way-ANOVA with Dunn's multiple comparison post-hoc test.</p

Cytotoxicity and efficacy of p38, PI3K and ROCK inhibition in macrophages.

<p><b>(A-F)</b> Alveolar macrophages (AM) from COPD patients or healthy controls were incubated with either vehicle (-), or incubated with (+) 1μM SCIO469, 1μM VX745, 100nM NVS-PI3K-2/3/5 or 200nM PF4950834 for 20 h, before cultures were assessed for apoptosis (A-C) by nuclear fragmentation, or necrosis (D-F). In all experiments, n = 4, there was no significant differences between groups.</p