Airway Elastin is increased in severe asthma and relates to proximal wall area: histological and computed tomography findings from the U-BIOPRED severe asthma study.

BACKGROUND: Airway remodelling, which may include goblet cell hyperplasia / hypertrophy, changes in epithelial integrity, accumulation of extracellular matrix components, smooth muscle hypertrophy and thickening of the lamina reticularis, is a feature of severe asthma and contributes to the clinical phenotype. OBJECTIVE: Within the U-BIOPRED severe asthma study, we have assessed histological elements of airway remodelling and their relationship to computed tomography (CT) measures of proximal airway dimensions. METHODS: Bronchial biopsies were collected from two severe asthma groups, one non-smoker (SAn, n = 28) and one current/ex-smoker (SAs/ex, n = 13), and a mild-moderate asthma group (MMA, n = 28) classified and treated according to GINA guidelines, plus a healthy control group (HC, n = 33). Movat's pentachrome technique was used to identify mucin, elastin and total collagen in these biopsies. The number of goblet cells (mucin+) was counted as a percentage of the total number of epithelial cells and the percentage mucin epithelial area measured. The percentage area of elastic fibres and total collagen within the submucosa was also measured, and the morphology of the elastic fibres classified. Participants in the asthma groups also had a CT scan to assess large airway morphometry. RESULTS: The submucosal tissue elastin percentage was higher in both severe asthma groups (16.1% SAn, 18.9% SAs/ex) compared with the HC (9.7%) but did not differ between asthma groups. There was a positive relationship between elastin and airway wall area measured by CT (n = 18-20, rho=0.544, p = 0.024), which also related to an increase in elastic fibres with a thickened lamellar morphological appearance. Mucin epithelial area and total collagen were not different between the four groups. Due to small numbers of suitable CT scans, it was not feasible to compare airway morphometry between the asthma groups. CONCLUSION: These findings identify a link between extent of elastin deposition and airway wall thickening in severe asthma.

Effects of selective COX-2 inhibition on allergen-induced bronchoconstriction and airway inflammation in asthma.

BACKGROUND: Prostaglandins that constrict and relax airways are synthesized in reactions catalyzed by either COX-1 or COX-2. It is not known whether selective inhibition of COX-2 makes asthmatic responses better or worse. OBJECTIVE: To determine the effects of the selective COX-2 inhibitor, etoricoxib, on allergen-induced bronchoconstriction in asthmatic subjects. METHODS: Sixteen subjects with mild atopic asthma underwent rising dose inhalation challenges with allergen or methacholine to determine PD20 FEV1 during a control study period or after 10 to 13 days of treatment with etoricoxib (90 mg once daily). The order of study periods was randomized with at least 2-week washout periods. Induced sputum cells and fractional exhaled nitric oxide levels were used to assess airway inflammation and blood assays for COX-1 and COX-2 activity to assess enzyme inhibition. Urinary excretion of lipids was used to assess prostaglandin biosynthesis. RESULTS: Etoricoxib did not change baseline lung function, nor airway responsiveness to allergen or to methacholine. Neither were the allergen-induced increase in sputum eosinophils and fractional exhaled nitric oxide levels affected by treatment. The biochemical effectiveness of the treatment was established both in the blood assays and by the distinct inhibitory effect of etoricoxib on urinary excretion of tetranor-prostaglandin E2 (P < .001). CONCLUSIONS: This first study of COX-2 inhibition in provoked asthma found no negative effects of etoricoxib on allergen-induced airflow obstruction and sputum eosinophils, basal lung function, or methacholine responsiveness. The study suggests that short-term use of COX-2 inhibitors is safe in subjects with asthma.

Compartment differences of inflammatory activity in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is associated with local and systemic inflammation. The knowledge of interaction and co-variation of the inflammatory responses in different compartments is meagre. METHOD: Healthy controls (n = 23), smokers with (n = 28) and without (n = 29) COPD performed spirometry and dental examinations. Saliva, induced sputum, bronchoalveolar lavage (BAL) fluid and serum were collected. Inflammatory markers were assessed in all compartments using ELISA, flow cytometry and RT-PCR. RESULTS: Negative correlations between lung function and saliva IL-8 and matrix metalloproteinase-9 (MMP-9) were found in smokers with COPD. IL-8 and MMP-9 in saliva correlated positively with periodontal disease as assessed by gingival bleeding in non-smokers.Tumor necrosis factor-α (TNF-α) in saliva, serum and TNF-α mRNA expression on macrophages in BAL-fluid were lower in smokers than in non-smokers. There were positive correlations between soluble TNF-α receptor 1 (sTNFR1) and soluble TNF-α receptor 2 (sTNFR2) in sputum, BAL-fluid and serum in all groups. Sputum interleukin-8 (IL-8) or interleukin-6 (IL-6) was positively correlated with sTNFR1 or sTNFR2 in non-smokers and with sTNFR2 in COPD. CONCLUSION: Saliva which is convenient to collect and analyse, may be suitable for biomarker assessment of disease activity in COPD. An attenuated TNF-α expression was demonstrated by both protein and mRNA analyses in different compartments suggesting that TNF-α response is altered in moderate and severe COPD. Shedding of TNFR1 or TNFR2 is similarly regulated irrespective of airflow limitation.

Adhesion molecules in subjects with COPD and healthy non-smokers: a cross sectional parallel group study.

BACKGROUND: The aim of the study was to investigate how the expression of adhesion molecules changes as neutrophils migrate from the circulation to the lung and if these changes differ between non-smoking subjects and smokers with and without COPD. METHODS: Non-smoking healthy subjects (n=22), smokers without (n=21) and with COPD (n=18) were included. Neutrophils from peripheral blood, sputum and bronchial biopsies were analysed for cell surface expression of adhesion molecules (CD11b, CD62L, CD162). Serum, sputum supernatant and BAL-fluid were analysed for soluble adhesion molecules (ICAM-1, -3, E-selectin, P-selectin, VCAM-1, PECAM-1). RESULTS: Expression of CD11b was increased on circulating neutrophils from smokers with COPD. It was also increased on sputum neutrophils in both smokers groups, but not in non-smokers, as compared to circulating neutrophils.Serum ICAM-1 was higher in the COPD group compared to the other two groups (p<0.05) and PECAM-1 was lower in smokers without COPD than in non-smoking controls and the COPD group (p<0.05). In BAL-fluid ICAM-1 was lower in the COPD group than in the other groups (p<0.05). CONCLUSIONS: Thus, our data strongly support the involvement of a systemic component in COPD and demonstrate that in smokers neutrophils are activated to a greater extent at the point of transition from the circulation into the lungs than in non-smokers.

Dendritic cell functional properties in a three-dimensional tissue model of human lung mucosa.

In lung tissue, dendritic cells (DC) are found in close association with the epithelial cell layer, and there is evidence of DC regulation by the epithelium; that epithelial dysfunction leads to overzealous immune cell activation. However, dissecting basic mechanisms of DC interactions with epithelial cells in human tissue is difficult. Here, we describe a method to generate a three-dimensional organotypic model of the human airway mucosa in which we have implanted human DC. The model recapitulates key anatomical and functional features of lung mucosal tissue, including a stratified epithelial cell layer, deposition of extracellular matrix proteins, and the production of tight junction and adherence junction proteins. Labeling of fixed tissue model sections and imaging of live tissue models also revealed that DC distribute in close association with the epithelial layer. As functional properties of DC may be affected by the local tissue microenvironment, this system provides a tool to study human DC function associated with lung mucosal tissue. As an example, we report that the lung tissue model regulates the capacity of DC to produce the chemokines CCL17, CCL18, and CCL22, leading to enhanced CCL18 expression and reduced CCL17 and CCL22 expression. This novel tissue model thus provides a tool well suited for a wide range of studies, including those on the regulation of DC functional properties within the local tissue microenvironment during homeostasis and inflammatory reactions.

Effects of budesonide on toll-like receptor expression in alveolar macrophages from smokers with and without COPD.

INTRODUCTION: Alveolar macrophages (AMs) are equipped with innate immune receptors such as toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4). In primary bronchial epithelial cells, exposure of toll-like receptor (TLR) ligands or tumor necrosis factor-alpha (TNF-α) increased TLR2 mRNA expression and reduced interleukin-8 (IL-8) release when coincubated with glucocorticosteroids. The aim of this study was to compare TLR2 and TLR4 expression levels and the effect of a glucocorticosteroid after stimulation with TLR ligands on AMs from smokers with and without COPD compared with the healthy controls. SUBJECTS AND METHODS: Bronchoalveolar lavage was performed, and AMs were isolated from smokers with (n=10) and without COPD (n=11) and healthy controls (n=10) and stimulated ex vivo with peptidoglycan (PGN), lipopolysaccharide (LPS), or TNF-α ± budesonide (Bud). Blocking antibodies to TLR2 or TLR4 were added before stimulation with LPS or PGN ± Bud, respectively. The release of proinflammatory cytokine (TNF-α), chemoattractant (CXCL8), and TLR expression was analyzed by enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction. RESULTS: LPS, PGN, and TNF-α induced an increased release of IL-8 and TNF-α in the AMs in all the groups independent of smoking or disease. These responses were inhibited by a glucocorticosteroid (Bud) in all the three groups, except PGN-induced IL-8 secretion in smokers without COPD. Bud increased TLR2 expression in the healthy controls and smokers without COPD. Costimulation of TLR ligands and Bud significantly enhanced TLR2 mRNA expression in both groups of smokers compared with TLR ligands alone. In smokers, costimulation with PGN and Bud significantly increased TLR2 expression when compared with Bud alone. On stimulation with the TLR4 agonist, LPS downregulated TLR4 mRNA expression in all the three groups. CONCLUSION: The combination of glucocorticosteroids with TLR ligands can increase TLR2 expression, thereby improving host defense in smokers. Also this combination can decrease the secretion of proinflammatory cytokines and chemokines as an anti-inflammatory response. Our findings indicate that glucocorticosteroid therapy strengthens immune defense pathways, which may have implication during exacerbation caused by microorganisms.

Impact of tobacco smoking on cytokine signaling via interleukin-17A in the peripheral airways.

There is excessive accumulation of neutrophils in the airways in chronic obstructive pulmonary disease (COPD) but the underlying mechanisms remain poorly understood. It is known that extracellular cytokine signaling via interleukin (IL)-17A contributes to neutrophil accumulation in the airways but nothing is known about the impact of tobacco smoking on extracellular signaling via IL-17A. Here, we characterized the impact of tobacco smoking on extracellular cytokine signaling via IL-17A in the peripheral airways in long-term smokers with and without COPD and in occasional smokers before and after short-term exposure to tobacco smoke. We quantified concentrations of IL-17A protein in cell-free bronchoalveolar lavage (BAL) fluid samples (Immuno-quantitative PCR) and cytotoxic T-cells (immunoreactivity for CD8+ and CD3+) in bronchial biopsies. Matrix metalloproteinase-8 and human beta defensin 2 proteins were also quantified (enzyme-linked immunosorbent assay) in the BAL samples. The concentrations of IL-17A in BAL fluid were higher in long-term smokers without COPD compared with nonsmoking healthy controls, whereas those with COPD did not differ significantly from either of the other groups. Short-term exposure to tobacco smoke did not induce sustained alterations in these concentrations in occasional smokers. Long-term smokers displayed higher concentrations of IL-17A than did occasional smokers. Moreover, these concentrations correlated with CD8+ and CD3+ cells in biopsies among long-term smokers with COPD. In healthy nonsmokers, BAL concentrations of matrix metalloproteinase-8 and IL-17A correlated, whereas this was not the case in the pooled group of long-term smokers with and without COPD. In contrast, BAL concentrations of human beta defensin 2 and IL-17A correlated in all study groups. This study implies that long-term but not short-term exposure to tobacco smoke increases extracellular cytokine signaling via IL-17A in the peripheral airways. In the smokers with COPD, this signaling may involve cytotoxic T-cells. Long-term exposure to tobacco smoke leads to a disturbed association of extracellular IL-17A signaling and matrix metalloproteinase-8, of potential importance for the coordination of antibacterial activity.

Toll-like receptor expression in smokers with and without COPD.

INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is characterized by non-reversible airflow limitation and systemic engagement. Bacterial colonization in the lungs is common in COPD-patients and may be associated with frequent acute exacerbations. Pattern-recognition receptors (PRRs), like Toll-like receptor 2 (TLR2), TLR4 and CD14 are expressed on most immunologic active cell types and are most likely of importance in COPD patho-physiology. MATERIAL AND METHODS: Twenty smokers with and 20 without COPD and 20 healthy non-smokers participated in the study. At two visits, induced sputum was collected after spirometry, blood was sampled and bronchoscopy with bronchoalveolar lavage was performed. Expression of TLR2, TLR4 and CD14 on different cell types and soluble receptors were assessed in the different compartments. RESULTS: Expression of TLR2 was lower on sputum neutrophils and soluble TLR2 (sTLR2) was higher in the supernatant in the COPD group, indicating a down regulation of TLR2 at the transit from blood to sputum. Expression of CD14 on sputum neutrophils and gene expression of CD14 on alveolar macrophages was up-regulated in the two smoking groups compared with non-smokers. No differences between the groups were found regarding TLR4 expression. CONCLUSIONS: Pattern-recognition receptors (PRRs), that are expected to make a first line of defense against invading micro-organisms, are differently regulated in smokers with COPD compared with smokers without airflow limitation and non-smokers. This is likely of importance in COPD patho-physiology, in particular for exacerbations, which often are caused by micro-organisms.