Airway smooth muscle NOX4 is upregulated and modulates ROS generation in COPD.

The burden of oxidative stress is increased in chronic obstructive pulmonary disease (COPD). However, whether the intra-cellular mechanisms controlling the oxidant/anti-oxidant balance in structural airway cells such as airway smooth muscle in COPD is altered is unclear. We sought to determine whether the expression of the NADPH oxidase (NOX)-4 is increased in airway smooth muscle in COPD both in vivo and primary cells in vitro and its role in hydrogen peroxide-induced reactive oxygen species generation. We found that in vivo NOX4 expression was up-regulated in the airway smooth muscle bundle in COPD (n = 9) and healthy controls with >20 pack year history (n = 4) compared to control subjects without a significant smoking history (n = 6). In vitro NOX4 expression was increased in airway smooth muscle cells from subjects with COPD (n = 5) compared to asthma (n = 7) and upregulated following TNF-α stimulation. Hydrogen peroxide-induced reactive oxygen species generation by airway smooth muscle cells in COPD (n = 5) was comparable to healthy controls (n = 9) but lower than asthma (n = 5); and was markedly attenuated by NOX4 inhibition. Our findings demonstrate that NOX4 expression is increased in vivo and in vitro in COPD and although we did not observe an intrinsic increase in oxidant-induced reactive oxygen species generation in COPD, it was reduced markedly by NOX4 inhibition supporting a potential therapeutic role for NOX4 in COPD.

NADPH Oxidase-4 Overexpression Is Associated With Epithelial Ciliary Dysfunction in Neutrophilic Asthma.

BACKGROUND: Bronchial epithelial ciliary dysfunction is an important feature of asthma. We sought to determine the role in asthma of neutrophilic inflammation and nicotinamide adenine dinucleotide phosphate (NADPH) oxidases in ciliary dysfunction. METHODS: Bronchial epithelial ciliary function was assessed by using video microscopy in fresh ex vivo epithelial strips from patients with asthma stratified according to their sputum cell differentials and in culture specimens from healthy control subjects and patients with asthma. Bronchial epithelial oxidative damage was determined by 8-oxo-dG expression. Nicotinamide adenine dinucleotide phosphate oxidase (NOX)/dual oxidase (DUOX) expression was assessed in bronchial epithelial cells by using microarrays, with NOX4 and DUOX1/2 expression assessed in bronchial biopsy specimens. Ciliary dysfunction following NADPH oxidase inhibition, using GKT137831, was evaluated in fresh epithelial strips from patients with asthma and a murine model of ovalbumin sensitization and challenge. RESULTS: Ciliary beat frequency was impaired in patients with asthma with sputum neutrophilia (n = 11) vs those without (n = 10) (5.8 [0.6] Hz vs 8.8 [0.5] Hz; P = .003) and was correlated with sputum neutrophil count (r = -0.70; P < .001). Primary bronchial epithelial cells expressed DUOX1/2 and NOX4. Levels of 8-oxo-dG and NOX4 were elevated in patients with neutrophilic vs nonneutrophilic asthma, DUOX1 was elevated in both, and DUOX2 was elevated in nonneutrophilic asthma in vivo. In primary epithelial cultures, ciliary dysfunction did not persist, although NOX4 expression and reactive oxygen species generation was increased from patients with neutrophilic asthma. GKT137831 both improved ciliary function in ex vivo epithelial strips (n = 13), relative to the intensity of neutrophilic inflammation, and abolished ciliary dysfunction in the murine asthma model with no reduction in inflammation. CONCLUSIONS: Ciliary dysfunction is increased in neutrophilic asthma associated with increased NOX4 expression and is attenuated by NADPH oxidase inhibition.

No evidence for altered intracellular calcium-handling in airway smooth muscle cells from human subjects with asthma.

BACKGROUND: Asthma is characterized by airway hyper-responsiveness and variable airflow obstruction, in part as a consequence of hyper-contractile airway smooth muscle, which persists in primary cell culture. One potential mechanism for this hyper-contractility is abnormal intracellular Ca(2+) handling. METHODS: We sought to compare intracellular Ca(2+) handling in airway smooth muscle cells from subjects with asthma compared to non-asthmatic controls by measuring: i) bradykinin-stimulated changes in inositol 1,4,5-trisphosphate (IP3) accumulation and intracellular Ca(2+) concentration, ii) sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) expression, iii) mechanisms of cytoplasmic Ca(2+) clearance assessed following instantaneous flash photolytic release of Ca(2+) into the cytoplasm. RESULTS: We found no differences in airway smooth muscle cell basal intracellular Ca(2+) concentrations, bradykinin-stimulated IP3 accumulation or intracellular Ca(2+) responses. Quantification of SERCA2 mRNA or protein expression levels revealed no differences in ASM cells obtained from subjects with asthma compared to non-asthmatic controls. We did not identify differences in intracellular calcium kinetics assessed by flash photolysis and calcium uncaging independent of agonist-activation with or without SERCA inhibition. However, we did observe some correlations in subjects with asthma between lung function and the different cellular measurements of intracellular Ca(2+) handling, with poorer lung function related to increased rate of recovery following flash photolytic elevation of cytoplasmic Ca(2+) concentration. CONCLUSIONS: Taken together, the experimental results reported in this study do not demonstrate major fundamental differences in Ca(2+) handling between airway smooth muscle cells from non-asthmatic and asthmatic subjects. Therefore, increased contraction of airway smooth muscle cells derived from asthmatic subjects cannot be fully explained by altered Ca(2+) homeostasis.

COPD exacerbation severity and frequency is associated with impaired macrophage efferocytosis of eosinophils.

BACKGROUND: Eosinophilic airway inflammation is observed in 10-30% of COPD subjects. Whether increased eosinophils or impairment in their clearance by macrophages is associated with the severity and frequency of exacerbations is unknown. METHODS: We categorised 103 COPD subjects into 4 groups determined by the upper limit of normal for their cytoplasmic macrophage red hue (<6%), an indirect measure of macrophage efferocytosis of eosinophils, and area under the curve sputum eosinophil count (≥ 3%/year). Eosinophil efferocytosis by monocyte-derived macrophages was studied in 17 COPD subjects and 8 normal controls. RESULTS: There were no differences in baseline lung function, health status or exacerbation frequency between the groups: A-low red hue, high sputum eosinophils (n=10), B-high red hue, high sputum eosinophils (n=16), C-low red hue, low sputum eosinophils (n=19) and D- high red hue, low sputum eosinophils (n=58). Positive bacterial culture was lower in groups A (10%) and B (6%) compared to C (44%) and D (21%) (p=0.01). The fall in FEV1 from stable to exacerbation was greatest in group A (ΔFEV1 [95 % CI] -0.41 L [-0.65 to -0.17]) versus group B (-0.16 L [-0.32 to -0.011]), C (-0.11 L [-0.23 to -0.002]) and D (-0.16 L [-0.22 to -0.10]; p=0.02). Macrophage efferocytosis of eosinophils was impaired in COPD versus controls (86 [75 to 92]% versus 93 [88 to 96]%; p=0.028); was most marked in group A (71 [70 to 84]%; p=0.0295) and was inversely correlated with exacerbation frequency (r=-0.63; p=0.006). CONCLUSIONS: Macrophage efferocytosis of eosinophils is impaired in COPD and is related to the severity and frequency of COPD exacerbations.

[Ca2+]i oscillations in ASM: relationship with persistent airflow obstruction in asthma.

The cause of airway smooth muscle (ASM) hypercontractility in asthma is not fully understood. The relationship of spontaneous intracellular calcium oscillation frequency in ASM to asthma severity was investigated. Oscillations were increased in subjects with impaired lung function abolished by extracellular calcium removal, attenuated by caffeine and unaffected by verapamil or nitrendipine. Whether modulation of increased spontaneous intracellular calcium oscillations in ASM from patients with impaired lung function represents a therapeutic target warrants further investigation.

Airway smooth muscle hypercontractility in asthma.

In recent years, asthma has been defined primarily as an inflammatory disorder with emphasis on inflammation being the principle underlying pathophysiological characteristic driving airway obstruction and remodelling. Morphological abnormalities of asthmatic airway smooth muscle (ASM), the primary structure responsible for airway obstruction seen in asthma, have long been described, but surprisingly, until recently, relatively small number of studies investigated whether asthmatic ASM was also fundamentally different in its functional properties. Evidence from recent studies done on single ASM cells and on ASM-impregnated gel cultures have shown that asthmatic ASM is intrinsically hypercontractile. Several elements of the ASM contraction apparatus in asthmatics and in animal models of asthma have been found to be different from nonasthmatics. These differences include some regulatory contractile proteins and also some components of both the calcium-dependent and calcium-independent contraction signalling pathways. Furthermore, oxidative stress was also found to be heightened in asthmatic ASM and contributes to hypercontractility. Understanding the abnormalities and mechanisms driving asthmatic ASM hypercontractility provides a great potential for the development of new targeted drugs, other than the conventional current anti-inflammatory and bronchodilator therapies, to address the desperate unmet need especially in patients with severe and persistent asthma.

Increased nicotinamide adenine dinucleotide phosphate oxidase 4 expression mediates intrinsic airway smooth muscle hypercontractility in asthma.

RATIONALE: Asthma is characterized by disordered airway physiology as a consequence of increased airway smooth muscle contractility. The underlying cause of this hypercontractility is poorly understood. OBJECTIVES: We sought to investigate whether the burden of oxidative stress in airway smooth muscle in asthma is heightened and mediated by an intrinsic abnormality promoting hypercontractility. METHODS: We examined the oxidative stress burden of airway smooth muscle in bronchial biopsies and primary cells from subjects with asthma and healthy controls. We determined the expression of targets implicated in the control of oxidative stress in airway smooth muscle and their role in contractility. MEASUREMENTS AND MAIN RESULTS: We found that the oxidative stress burden in the airway smooth muscle in individuals with asthma is heightened and related to the degree of airflow obstruction and airway hyperresponsiveness. This was independent of the asthmatic environment as in vitro primary airway smooth muscle from individuals with asthma compared with healthy controls demonstrated increased oxidative stress-induced DNA damage together with an increased production of reactive oxygen species. Genome-wide microarray of primary airway smooth muscle identified increased messenger RNA expression in asthma of NADPH oxidase (NOX) subtype 4. This NOX4 overexpression in asthma was supported by quantitative polymerase chain reaction, confirmed at the protein level. Airway smooth muscle from individuals with asthma exhibited increased agonist-induced contraction. This was abrogated by NOX4 small interfering RNA knockdown and the pharmacological inhibitors diphenyleneiodonium and apocynin. CONCLUSIONS: Our findings support a critical role for NOX4 overexpression in asthma in the promotion of oxidative stress and consequent airway smooth muscle hypercontractility. This implicates NOX4 as a potential novel target for asthma therapy.

Mast cell-airway smooth muscle crosstalk: the role of thymic stromal lymphopoietin.

BACKGROUND: The mast cell localization to airway smooth muscle (ASM) bundle in asthma is important in the development of disordered airway physiology. Thymic stromal lymphopoietin (TSLP) is expressed by airway structural cells. Whether it has a role in the crosstalk between these cells is uncertain. We sought to define TSLP expression in bronchial tissue across the spectrum of asthma severity and to investigate the TSLP and TSLP receptor (TSLPR) expression and function by primary ASM and mast cells alone and in coculture. METHODS: TSLP expression was assessed in bronchial tissue from 18 subjects with mild to moderate asthma, 12 with severe disease, and nine healthy control subjects. TSLP and TSLPR expression in primary mast cells and ASM was assessed by immunofluorescence, flow cytometry, and enzyme-linked immunosorbent assay, and its function was assessed by calcium imaging. The role of TSLP in mast cell and ASM proliferation, survival, differentiation, synthetic function, and contraction was examined. RESULTS: TSLP expression was increased in the ASM bundle in mild-moderate disease. TSLP and TSLPR were expressed by mast cells and ASM and were functional. Mast cell activation by TSLP increased the production of a broad range of chemokines and cytokines, but did not affect mast cell or ASM proliferation, survival, or contraction. CONCLUSIONS: TSLP expression by the bronchial epithelium and ASM was upregulated in asthma. TSLP promoted mast cell synthetic function, but did not contribute to other functional consequences of mast cell-ASM crosstalk.

Immunopathogenesis of severe asthma.

Severe asthma is a complex heterogeneous disease with substantial unmet clinical need. Understanding the immunopathogenesis is likely to provide insights into potential novel therapies. To date researchers have focussed primarily at a single scale for example genome, cell or whole organ physiology. In this review we shall summarise the current knowledge of the immunopathogenesis of severe asthma integrated across multiple scales to provide the insights into the structure function relationships required to begin to unravel the complexity of severe asthma.

Mast cell fibroblastoid differentiation mediated by airway smooth muscle in asthma.

Mast cell microlocalization to the airway smooth muscle (ASM) bundle is a key feature of asthma, but whether these mast cells have an altered phenotype is uncertain. In this paper, we report that in vivo, mast cells within the ASM bundle, in contrast to mast cells in the bronchial submucosa, commonly expressed fibroblast markers and the number of these cells was closely related to the degree of airway hyperresponsiveness. In vitro human lung mast cells and mast cell lines cultured with fibronectin or with primary human ASM cells acquired typical fibroblastic markers and morphology. This differentiation toward a fibroblastoid phenotype was mediated by ASM-derived extracellular matrix proteins, independent of cell adhesion molecule-1, and was attenuated by α5ß1 blockade. Fibroblastoid mast cells demonstrated increased chymase expression and activation with exaggerated spontaneous histamine release. Together these data indicate that in asthma, ASM-derived extracellular matrix proteins mediate human mast cell transition to a fibroblastoid phenotype, suggesting that this may be pivotal in the development of airway dysfunction in asthma.

Eosinophil protein in airway macrophages: a novel biomarker of eosinophilic inflammation in patients with asthma.

BACKGROUND: Noneosinophilic asthma is common across asthma severities. However, in patients with moderate-to-severe disease, the absence of sputum eosinophilia cannot distinguish between asthmatic subjects with eosinophilic inflammation controlled by corticosteroids versus those in whom eosinophilic inflammation is not a component of the disease. OBJECTIVES: We sought to develop a method to quantify eosinophil proteins in airway macrophages as a novel biomarker of eosinophilic airway inflammation. METHODS: Eosinophil proteins in airway macrophages were assessed by means of flow cytometry, immunofluorescence, and cytoplasmic hue change after ingestion of apoptotic eosinophils. Airway macrophage median percentage of red-hued area in stained sputum cytospin preparations was assessed by means of image analysis from (1) subjects with mild-to-severe asthma, subjects with nonasthmatic eosinophilic bronchitis, and healthy control subjects; (2) subjects with eosinophilic severe asthma after treatment with prednisolone; and (3) subject with noneosinophilic asthma before corticosteroid withdrawal. RESULTS: Eosinophil proteins were detected in airway macrophages, and cytoplasmic red hue increased after ingestion of apoptotic eosinophils. Airway macrophage percentage redhued area was increased in subjects with moderate-to-severe asthma compared with that seen in subjects with mild asthma and healthy control subjects, was similar in those with or without a sputum eosinophilia, and was increased after corticosteroid therapy. In asthmatic subjects without sputum eosinophilia, the airway macrophage percentage red-hued area was increased in subjects who did versus those who did not have sputum eosinophilia after corticosteroid withdrawal. CONCLUSIONS: Eosinophil proteins can be reliably measured in airway macrophages. In combination with sputum eosinophilia, the macrophage eosinophil protein content might further define the asthma phenotype and provide an additional tool to direct therapy.

Fibrocyte localization to the airway smooth muscle is a feature of asthma.

BACKGROUND: Airway smooth muscle (ASM) hyperplasia is a hallmark of asthma that is associated with disease severity and persistent airflow obstruction. OBJECTIVES: We sought to investigate whether fibrocytes, a population of peripheral blood mesenchymal progenitors, are recruited to the ASM compartment in asthma. METHODS: We assessed the number of fibrocytes in bronchial biopsy specimens and peripheral blood from subjects with mild-to-severe refractory asthma versus healthy control subjects. In vitro we investigated potential mechanisms controlling fibrocyte migration toward the ASM bundle. RESULTS: Fifty-one subjects with asthma and 33 control subjects were studied. In bronchial biopsy specimens, the number of fibrocytes was increased in the lamina propria of subjects with severe refractory asthma (median [interquartile range] number, 1.9/mm(2) [1.7/mm(2)]) versus healthy control subjects (median [interquartile range] number, 0/mm(2) [0.3/mm(2)], P < .0001) and in the ASM bundle of subjects with asthma of all severities (subjects with severe asthma, median [interquartile range] number, 3.8/mm(2) [9.4/mm(2)]; subjects with mild-to-moderate asthma, median [interquartile range] number, 1.1/mm(2) [2.4/mm(2)]); healthy control subjects, (median [interquartile range] number, 0/mm(2) [0/mm(2)]); P = .0004). In the peripheral blood the fibrocyte number was also increased in subjects with severe refractory asthma (median [interquartile range] number, 1.4 x 10(4)/mL [2.6 x 10(4)/mL]) versus healthy control subjects (median [interquartile range] number, 0.4 x 10(4)/mL [1.0 x 10(4)/mL], P = .002). We identified that in vitro ASM promotes fibrocyte chemotaxis and chemokinesis (distance of migration after 4.5 hours, 31 microm [2.9 microm] vs 17 microm [2.4 microm], P = .0001), which was in part mediated by platelet-derived growth factor (mean inhibition by neutralizing antibody, 16% [95% CI, 2% to 32%], P = .03) but not by activation of chemokine receptors. CONCLUSION: This study provides the first evidence that fibrocytes are present in the ASM compartment in asthma and that ASM can augment fibrocyte migration. The importance of fibrocytes in the development of ASM hyperplasia and airway dysfunction in asthma remains to be determined.

Human airway smooth muscle promotes human lung mast cell survival, proliferation, and constitutive activation: cooperative roles for CADM1, stem cell factor, and IL-6.

The microlocalization of mast cells within specific tissue compartments is thought to be critical for the pathophysiology of many diverse diseases. This is particularly evident in asthma where they localize to the airway smooth muscle (ASM) bundles. Mast cells are recruited to the ASM by numerous chemoattractants and adhere through CADM1, but the functional consequences of this are unknown. In this study, we show that human ASM maintains human lung mast cell (HLMC) survival in vitro and induces rapid HLMC proliferation. This required cell-cell contact and occurred through a cooperative interaction between membrane-bound stem cell factor (SCF) expressed on ASM, soluble IL-6, and CADM1 expressed on HLMC. There was a physical interaction in HLMC between CADM1 and the SCF receptor (CD117), suggesting that CADM1-dependent adhesion facilitates the interaction of membrane-bound SCF with its receptor. HLMC-ASM coculture also enhanced constitutive HLMC degranulation, revealing a novel smooth muscle-driven allergen-independent mechanism of chronic mast cell activation. Targeting these interactions in asthma might offer a new strategy for the treatment of this common disease.