Im Adcock

Cellular and molecular mechanisms in chronic obstructive pulmonary disease: an overview

In the last decade, the analysis of bronchial biopsies and lung parenchyma obtained from chronic obstructive pulmonary disease (COPD) patients compared with those from smokers with normal lung function and non‐smokers has provided new insights on the role of the different inflammatory and structural cells, their signalling pathways and mediators, contributing to a better knowledge of the pathogenesis of COPD. This review summarizes and discusses the lung pathology of COPD patients with emphasis on inflammatory cell phenotypes that predominate in different clinical conditions. In bronchial biopsies, a cascade of events takes place during progression from mild‐to‐severe disease. T lymphocytes, particularly CD8+ cells and macrophages are the prevalent inflammatory cells in the lung of healthy smokers and patients with mild COPD, while total and activated neutrophils predominate in severe COPD. The number of CD4+, CD8+ cells and macrophages expressing nuclear factor‐kappa B (NF‐κB), STAT‐4 and IFN‐γ proteins as well as endothelial adhesion molecule‐1 in endothelium is increased in mild/moderate disease. In contrast, activated neutrophils (MPO+ cells) and increased nitrotyrosine immunoreactivity develops in severe COPD. In bronchial biopsies obtained during COPD exacerbations, some studies have shown an increased T cell and granulocyte infiltration. Regular treatment with high doses of inhaled glucocorticoids does not significantly change the number of inflammatory cells in bronchial biopsies from patients with moderate COPD. The profile in lung parenchyma is similar to bronchial biopsies. ‘Healthy’ smokers and mild/moderate diseased patients show increased T lymphocyte infiltration in the peripheral airways. Pulmonary emphysema is associated with a general increase of inflammatory cells in the alveolar septa. The molecular mechanisms driving the lymphocyte and neutrophilic prevalence in mild and severe disease, respectively, needs to be extensively studied. Up‐regulation of pro‐inflammatory transcription factors NF‐κB and STAT‐4 in mild, activated epithelial and endothelial cells in the more severe disease may contribute to this differential prevalence of infiltrating cells.

Association of increased CCL5 and CXCL7 chemokine expression with neutrophil activation in severe stable COPD

Background: Increased numbers of activated neutrophils have been reported in the bronchial mucosa of patients with stable chronic obstructive pulmonary disease (COPD), particularly in severe disease. Objectives: To investigate the expression of neutrophilic chemokines and adhesion molecules in bronchial biopsies from patients with stable COPD of different severity (GOLD stages I–IV) compared with age-matched control subjects, smokers with normal lung function and never smokers. Methods: The expression of CCL5, CXCL1, 5, 6, 7 and 8, CXCR1, CXCR2, CD11b and CD44 was measured in the bronchial mucosa using immunohistochemistry, confocal immunofluorescence, real-time quantitative polymerase chain reaction (RT-QPCR) and Western blotting (WB). Results: The numbers of CCL5+ epithelial cells and CCL5+ and CXCL7+ immunostained cells were increased in the bronchial submucosa of patients with stable severe COPD compared with control never smokers and smokers with normal lung function. This was also confirmed at the level of mRNA expression. The numbers of CCL5+ cells in the submucosa of patients with COPD were 2–15 times higher than any other chemokines. There was no correlation between the number of these cells and the number of neutrophils in the bronchial submucosa. Compared with control smokers, the percentage of neutrophils co-expressing CD11b and CD44 receptors was significantly increased in the submucosa of patients with COPD. Conclusion: The increased expression of CCL5 and CXCL7 in the bronchial mucosa of patients with stable COPD, together with an increased expression of extracellular matrix-binding receptors on neutrophils, may be involved in the pathogenesis of COPD.

Montelukast inhibits tumour necrosis factor-α-mediated interleukin-8 expression through inhibition of nuclear factor-κB p65-associated histone acetyltransferase activity

Background Montelukast is a potent cysteinyl leukotriene‐1 receptor antagonist possessing some anti‐inflammatory effects although the molecular mechanism of these anti‐inflammatory effects is unknown. In this study, we aimed to investigate the effect of montelukast on nuclear factor (NF)‐κB‐associated histone acetylation activity in phorbol myristate acetate (PMA)‐differentiated U937 cells.

Cytokine Regulation of Chronic Inflammation in Asthma

Publisher Summary This chapter discusses the role of cytokines in the induction, coordination, and perpetuation of the immunological and inflammatory responses observed in asthma. There is an increasing recognition that large peptide mediators such as cytokines and growth factors orchestrate and perpetuate the chronic inflammation of asthma. Cytokines are extracellular signalling proteins, usually less than 80 kD in weight, and many are glycosylated. Cytokines usually have an effect on closely adjacent cells and therefore function in a predominantly paracrine manner, although they may also act at a distance and may have effects on the cell of origin. Cytokines may be regarded as a mechanism for cell-cell communication, and they are involved in cell growth and differentiation, inflammation, immunity, and repair, which are all aspects of importance in understanding the cellular events in chronic asthma. Multiple cytokines have been implicated in asthma and they are derived from a variety of cells. Inhibition of cytokines is an important approach to asthma treatment, and it is likely that inhaled corticosteroids are effective, at least in part, by inhibition of cytokine synthesis in airway cells such as epithelial cells and lymphocytes.

S119 Mapping mouse models of severe asthma onto human disease

Introduction Severe asthma represents a significant unmet need in terms of therapeutics. Drug development in asthma has been slow and expensive and one of the reasons for this is that positive findings for drugs tested in preclinical models have not readily translated into successful therapies in man. Aims and Objectives We sought to improve the predictive power of existing models of asthma by using novel bioinformatics techniques to align these models with subsets of human asthma. Methods We applied differential gene expression analysis to transcriptomic data from whole lung samples of 6 murine models of asthma and oxidative stress to produce gene signatures that represented each model. These signatures were then used to calculate enrichment scores (ESs) for transcriptomic data from bronchial biopsies taken from 81 asthmatic and 26 healthy subjects from the U -BIOPRED cohort using gene set variation analysis. These ESs were taken as an index of similarity between each model and each patient and were used to drive further analyses using topological data analysis and goodness of fit modelling. Results We found that no single mouse model was aligned well with all asthmatics. We identified three clusters of patients who were represented to varying degrees by different mouse models and who displayed clinical features that aligned well with phenotypes of asthmatics identified previously by clustering analyses based upon clinical features and biological markers. Patients in cluster χ were defined by neutrophilic sputum, later onset of disease, higher incidence of sinusitis, more frequent exacerbations and more airflow limitation. Patients in cluster Y1 showed significantly lower sputum neutrophils, a trend towards higher sputum eosinophils, a significantly later onset of airways disease and a trend towards higher BMI. Patients in cluster Y2 showed a significantly higher percentage of neutrophils in the blood, a trend towards increased sputum lymphocytes and were more likely to identify aspirin as a trigger (p=0.06). Conclusion Our evidence supports the assertion that it is possible, on a transcriptional level, to align mouse models of asthma to subsets of human asthma and that doing so may have significant implications for the expedience of drug development in asthma.

P159 Peripheral Blood Mononuclear Cells from patients with Idiopathic Pulmonary Arterial Hypertension are Hyporesponsive to Inflammatory stimuli

Background Inflammation is a key feature of pulmonary arterial hypertension (PAH); circulating levels of plasma cytokines and chemokines are raised, some of which (including interleukin (IL)-6 and CXCL8) are associated with increased mortality (1). We wished to determine whether it was possible to detect an altered inflammatory phenotype in circulating inflammatory cells from PAH patients compared to controls. Methods Following ethical approval, patients with idiopathic PAH and age-matched non-smoking control subjects were selected. Peripheral blood mononuclear cells (PBMC) (105 cells) or citrated whole blood (WB) were plated into 96-well plates and treated with E. Coli LPS (0–1µg/ml) (Sigma) or recombinant TNF-a (0–10ng/ml) (R&D systems) for 24h at 37°C. Following centrifugation, plasma was aspirated and samples frozen at -20°c, and IL-6 and CXCL8 ELISAs later performed. Data are mean ± SEM. Results All patients had idiopathic PAH (n = 12) (7 in NYHA class III-IV) with 6MWD 370 ± 18m, mPAP 72 ± 5mmHg). All patients were taking advanced PAH therapies but not conventional anti-inflammatory therapies. There was no age difference between patients and controls (n = 9) (36.5 ± 3.7 vs. 36.2 ± 2.7 years, NS). At baseline, release of IL-6 and CXCL8 was higher from PBMC from PAH patients than controls (918.1 ± 280.5 vs. 195.2 ± 64.3 for IL-6 (p < 0.05) and 4203 ± 1354 vs. 1048 ± 288.4 p < 0.05 for CXCL8), although no difference was seen between groups from the whole blood assays. Following stimulation with LPS, however, release of both IL-6 and CXCL8 was lower from both WB and PBMC assays for PAH patients compared to controls. For example for LPS-induced IL-6 release from PBMC, both the maximal effect (Emax) was lower in PAH vs. controls (5148 ± 1320 vs. 9154 ± 2510 pg/ml, p < 0.05) and the log EC50 (half maximal effective concentration) was higher in PAH vs. controls (-2.70 ± 0.27 vs. -5.90 ± 1.16 µg/ml, p < 0.01) (Figure 1). Similar results were seen following stimulation with TNF-a. Conclusion The main findings of this study are that: (1) baseline CXCL8 and IL6 release was higher from PBMCs of patients with PAH compared to control donors; (2) PBMCs and WB from patients were hyporesponsive to LPS (and TNF-a), suggesting an altered inflammatory phenotype, at least in these models, to these stimuli. References Soon E. et al, Circulation. 2010;122(9):920–7 Abstract P159 Figure 1.

P29 Endothelial cell NF-kB activation is increased in human idiopathic PAH

Background Pulmonary arterial hypertension (PAH) is associated with pulmonary vascular inflammation, and several of the inflammatory genes involved are regulated by nuclear factor-kB (NF-kB). NF-kB is a heterodimer of p65 and p50 subunits which, upon activation, translocate into the nucleus and binds to target gene promoters. NF-kB activation in PAH has not been examined in detail to date. We assessed NF-kB activation by immunohistochemical analysis of nuclear p65. Methods Samples were obtained from South Paris University from patients with severe idiopathic PAH (IPAH) following lung transplantation (n=10) and from control subjects undergoing lobectomy or pneumonectomy (n=10). Tissue blocks were fixed and paraffin-embedded, 4-mm thick sections underwent immunoperoxidase double staining for macrophage (CD68+)/p65, using mouse anti-human CD68 (Dako; 1:100 dilution) and rabbit anti human p65 (Santa Cruz Biotechnology; 1:50 dilution), before detection with chromogen fast red and counterstaining with haematoxylin. Vessels of interest were defined and quantitative scoring of nuclear p65 immunostaining of ten randomly selected pulmonary arteries per slide performed by a researcher blinded to the groups. Data are expressed as median and IQR, and the groups were compared by the Kruskal–Wallis or Mann–Whitney U test. Results (1) PAH specimens showed co-localisation of p65 within CD68+ macrophages in 75.4 (64.8–84.6)% of samples. Airway epithelium, neutrophils and lymphocytes were also positive for p65. (2) Pulmonary arterial medial thickness was increased in PAH compared to controls, at 33.7 (18.8–67.9)% in vessels 100–250u2009mm external diameter (E.D.) and 27.2 (14.8–44.2)% in vessels 250–500u2009mm ED, vs 17.7 (11.2–30.3)% and 14.9 (11.8–17.8)% in controls (p<0.0001 between groups). (3) Nuclear p65 was present in pulmonary artery endothelial cells (EC) but not other vascular cells including pulmonary artery smooth muscle cells in PAH: 53.9 (0–100)% of vessels 100–250u2009mm E.D. and 53.1 (0–100)% of those 250–500u2009mm E.D. scored EC p65 positivity in PAH compared to 7.5 (0–25.0)% in 100–250u2009mm ED and 4.7 (0–21.1)% in 250–500u2009mm ED in controls (p<0.0001 between groups) (Abstract P29 Figure 1).Abstract P29 Figure 1 Percentage of positive endothelial cell (EC) nuclear p65 immunostaining subdivided according to pulmonary artery external diameter (E.D.). Conclusion NF-kB activation is present in macrophages and pulmonary arterial endothelial cells in pulmonary arteries of 100–500u2009mm ED in patients with PAH.

S152 Dexamethasone reverses established monocrotaline-induced pulmonary hypertension in rats and increases pulmonary BMPR2 expression

Background Pulmonary arterial hypertension (PAH) is associated with pulmonary vascular inflammation and dysregulated bone morphogenetic protein receptor type 2 (BMPR2) signalling in both human and experimental PAH. We evaluated the effects of dexamethasone on established monocrotaline-induced PAH in rats for potential reversal of PAH, at time points when pulmonary vascular remodelling has already developed (from day 14 after a single injection of monocrotaline at day 0), and for the effects on pulmonary IL6 and BMPR2 expression. Methods Saline-treated controls, monocrotaline-exposed, monocrotaline-exposed and dexamethasone-treated rats (5u2005mg/kg/day, 1.25u2005mg/kg and 2.5u2005mg/kg/48u2005h given from day 14–28 and day 21–35) were evaluated at day 28 and day 35 following monocrotaline for pulmonary vascular haemodynamic parameters, right ventricular hypertrophy, morphometry, immunohistochemistry, whole lung IL-6 and BMPR2 expression by quantitative real-time PCR (qRT-PCR). Results Dexamethasone significantly improved pulmonary haemodynamics and morphometric indices of pulmonary vascular remodelling, reversing PAH when given at day 14–28, day 21–35 following monocrotaline, as well as improving survival in monocrotaline-exposed rats compared to controls (log rank p<0.0001). Dexamethasone reduced both monocrotaline-induced whole lung IL-6 overexpression (p<0.05), as well as reducing IL-6-expressing adventitial inflammatory cell infiltration as assessed by immunohistochemistry. This was associated with pulmonary BMPR2 down-regulation (p<0.01) following monocrotaline, which was significantly increased following day 14–28 dexamethasone treatment in whole lung (p<0.05) (Abstract S152 Figure 1). Cellular BMPR2 was also increased following in vitro treatment of control pulmonary artery smooth muscle cells (PASMC) with ×10−8 molar dexamethasone (p<0.05), but not in PASMC isolated from pulmonary hypertensive rats. Dexamethasone (×10−8 and 10−7 molar) also reduced proliferation of PASMC isolated from both control and pulmonary hypertensive rats (p<0.05 for both doses).Abstract S152 Figure 1 Effects of monocrotaline and day 14–28 dexamethasone on whole-lung BMPR2 expression by qRT-PCR. Conclusion PAH in this well-characterised experimental model can be reversed by dexamethasone, and survival is improved. In this model, mechanisms may involve reduction of IL-6-expressing inflammatory cells, reduced proliferation of vascular smooth muscle cells, and restoration of pulmonary BMPR2 expression may be important.