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.

Functional KCa3.1 K+ channels are required for human fibrocyte migration.

BACKGROUND: Fibrocytes are bone marrow-derived CD34(+) collagen I-positive cells present in peripheral blood that develop α-smooth muscle actin expression and contractile activity in tissue culture. They are implicated in the pathogenesis of tissue remodeling and fibrosis in both patients with asthma and those with idiopathic pulmonary fibrosis. Targeting fibrocyte migration might therefore offer a new approach for the treatment of these diseases. Ion channels play key roles in cell function, but the ion-channel repertoire of human fibrocytes is unknown. OBJECTIVE: We sought to examine whether human fibrocytes express the K(Ca)3.1 K(+) channel and to determine its role in cell differentiation, survival, and migration. METHODS: Fibrocytes were cultured from the peripheral blood of healthy subjects and patients with asthma. Whole-cell patch-clamp electrophysiology was used for the measurement of ion currents, whereas mRNA and protein were examined to confirm channel expression. Fibrocyte migration and proliferation assays were performed in the presence of K(Ca)3.1 ion-channel blockers. RESULTS: Human fibrocytes cultured from the peripheral blood of both healthy control subjects and asthmatic patients expressed robust K(Ca)3.1 ion currents together with K(Ca)3.1 mRNA and protein. Two specific and distinct K(Ca)3.1 blockers (TRAM-34 and ICA-17043) markedly inhibited fibrocyte migration in transwell migration assays. Channel blockers had no effect on fibrocyte growth, apoptosis, or differentiation in cell culture. CONCLUSIONS: The K(+) channel K(Ca)3.1 plays a key role in human fibrocyte migration. Currently available K(Ca)3.1-channel blockers might therefore attenuate tissue fibrosis and remodeling in patients with diseases such as idiopathic pulmonary fibrosis and asthma through the inhibition of fibrocyte recruitment.

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.

Expression of the T helper 17-associated cytokines IL-17A and IL-17F in asthma and COPD.

BACKGROUND: Asthma and COPD are characterized by airway dysfunction and inflammation. Neutrophilic airway inflammation is a common feature of COPD and is recognized in asthma, particularly in severe disease. The T helper (Th) 17 cytokines IL-17A and IL-17F have been implicated in the development of neutrophilic airway inflammation, but their expression in asthma and COPD is uncertain. METHODS: We assessed IL-17A and IL-17F expression in the bronchial submucosa from 30 subjects with asthma, 10 ex-smokers with mild to moderate COPD, and 27 nonsmoking and 14 smoking control subjects. Sputum IL-17 concentration was measured in 165 subjects with asthma and 27 with COPD. RESULTS: The median (interquartile range) IL-17A cells/mm² submucosa was increased in mild to moderate asthma (2.1 [2.4]) compared with healthy control subjects (0.4 [2.8]) but not in severe asthma (P = .04). In COPD, IL-17A(+) cells/mm² submucosa were increased (0.5 [3.7]) compared with nonsmoking control subjects (0 [0]) but not compared with smoking control subjects (P = .046). IL-17F(+) cells/mm² submucosa were increased in severe asthma (2.7 [3.6]) and mild to moderate asthma (1.6 [1.0]) compared with healthy controls subjects (0.7 [1.4]) (P = .001) but was not increased in subjects with COPD. IL-17A and IL-17F were not associated with increased neutrophilic inflammation, but IL-17F was correlated with the submucosal eosinophil count (rs = 0.5, P = .005). The sputum IL-17 concentration in COPD was increased compared with asthma (2 [0-7] pg/mL vs 0 [0-2] pg/mL, P < .0001) and was correlated with post-bronchodilator FEV1% predicted (r = -0.5, P = .008) and FEV(1)/FVC (r = -0.4, P = .04). CONCLUSIONS: Our findings support a potential role for the Th17 cytokines IL-17A and IL-17F in asthma and COPD, but do not demonstrate a relationship with neutrophilic inflammation.

Airway wall expression of OX40/OX40L and interleukin-4 in asthma.

BACKGROUND: The costimulatory molecule OX40 and its ligand, OX40L, mediate key aspects of allergic airway inflammation in animal models of asthma, including eosinophilic airway inflammation, airway hyperresponsiveness, and T helper 2 polarization. We sought to examine OX40/OX40L and interleukin (IL)-4 expression in asthma across severities. METHODS: Bronchial biopsies were obtained from 27 subjects with asthma (mild Global Initiative for Asthma [GINA] 1 [n = 10], moderate GINA 2-3 [n = 7], and severe GINA 4-5 [n = 10]) and 13 healthy controls. The number of OX40(+), OX40L(+), IL-4(+), and IL-4 receptor alpha (IL-4Ralpha)(+) cells in the lamina propria and airway smooth muscle (ASM) bundle and the intensity of IL-4Ralpha(+) expression by the ASM were assessed. RESULTS: The number of OX40(+), OX40L(+), and IL-4(+) cells in the lamina propria and OX40(+) and IL-4(+) cells in the ASM bundle was significantly increased in subjects with mild asthma, but not in those with moderate or severe asthma, compared with healthy controls. In the subjects with asthma, OX40/OX40L expression was positively correlated with the number of eosinophils and IL-4(+) cells in the lamina propria. The number of IL-4Ralpha(+) cells in the lamina propria was significantly increased in moderate-to-severe disease, but not in mild asthma, compared with controls. IL-4Ralpha expression by the ASM bundle was not different among groups. CONCLUSIONS: OX40/OX40L expression is increased in the bronchial submucosa in mild asthma, but not in moderate-to-severe disease, and is related to the degree of tissue eosinophilia and IL-4 expression. Whether these costimulatory molecules have a role as targets for asthma requires further investigation.

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.

Airway hyperresponsiveness is dissociated from airway wall structural remodeling.

BACKGROUND: Nonasthmatic eosinophilic bronchitis (EB) has emerged as a useful tool to study the structural and inflammatory mechanisms of airway hyperresponsiveness (AHR) in asthma. We have previously shown that vascular remodeling and reticular basement membrane (RBM) thickening are present in EB. However, it is not known whether other features of structural remodeling including increased airway smooth muscle (ASM) mass, matrix deposition, and glandular hyperplasia are also present in EB. OBJECTIVES: We sought to determine whether structural remodeling occurs in EB and is associated with AHR and airflow limitation. METHODS: Forty-two patients with asthma, 21 patients with EB, and 19 healthy volunteers were recruited. ASM area, RBM thickness, collagen 3 deposition, glandular area, mast cells, and granulocytes were assessed in bronchial biopsy samples. RESULTS: Nonasthmatic eosinophilic bronchitis and asthma were associated with a significant increase in ASM mass and RBM thickness compared with healthy subjects. In contrast, we did not observe any significant differences in collagen 3 deposition in the lamina propria and ASM or the % area of glands in the lamina propria. Univariate analysis demonstrated that mast cell numbers in the ASM were the only feature of remodeling associated with AHR (beta = -0.51; P = .004). Stepwise linear regression revealed that a combination of mast cell numbers in the ASM (beta = -0.43) and disease duration (beta = -0.25; model-adjusted R(2) = 0.26; P = .027) best modeled AHR. CONCLUSION: Mast cell localization to the ASM bundle, but not structural remodeling of the airway wall, is associated with AHR in asthma.

Vascular remodeling is a feature of asthma and nonasthmatic eosinophilic bronchitis.

RATIONALE: Increased vascularity and expression of vascular endothelial growth factor (VEGF) are recognized features of the asthmatic airway. The association of vascular remodeling with airway hyperresponsiveness (AHR) is unclear. OBJECTIVE: To assess vascular remodeling and sputum VEGF concentration in subjects with asthma, subjects with nonasthmatic eosinophilic bronchitis (EB), and healthy controls. METHODS: In cohort 1, 19 patients with asthma (Global Initiative for Asthma [GINA] 1-2, n = 9; GINA 3-5, n = 10), 10 patients with EB, and 11 healthy matched controls were recruited. Expression of the endothelial marker EN4 was assessed in bronchial biopsy samples. Vessels were counted using the validated mean Chalkley count by a blind observer. For cohort 2, a second independent cohort of 31 patients with asthma (GINA 1-2, n = 11; GINA 3-5, n = 20), 14 patients with EB, and 15 matched controls was recruited. Induced sputum supernatant VEGF was measured by ELISA. RESULTS: The mean chalkley count was significantly greater in GINA 3-5 asthma (5.2 [0.4]) and EB (4.8 [0.3]) compared with controls (3.5 [0.5]) and demonstrated a significant inverse correlation with the postbronchodilator FEV(1)% predicted in patients with asthma (R(2) = 0.28; P = .02). Sputum VEGF concentration was also increased in GINA 3-5 asthma (2365 [1361-4110] pg/g) and EB (4699 [2818-7834] pg/g) compared with controls (1094 [676-1774] pg/g) and was inversely related to postbronchodilator FEV(1)% predicted in asthma (R(2) = 0.2; P = .01). CONCLUSION: Vascular remodeling is a feature of asthma, and EB and is inversely associated with the postbronchodilator FEV(1) in asthma, suggesting that vascular remodeling is associated with airflow obstruction but not AHR. CLINICAL IMPLICATIONS: Vascular remodeling is dissociated from AHR in asthma and associated with airflow limitation.

Protonation, photobleaching, and photoactivation of yellow fluorescent protein (YFP 10C): a unifying mechanism.

Yellow fluorescent protein (YFP 10C) is widely used as a probe in biology, but its complex photochemistry gives rise to unusual behavior that requires fuller definition. Here we characterize the kinetics of protonation and reversible bleaching over time scales of picoseconds to hours. Stopped-flow and pressure-jump techniques showed that protonation of the fluorescent YFP(-) anion state is two-step with a slow transition that accounts for blinking of 527 nm emission at the single molecule level on the seconds time scale. Femtosecond spectroscopy revealed that the protonated excited-state (YFPH*) decayed predominantly by a radiationless mechanism, but emission at 460 nm was detected within the first picosecond. Limited excited-state proton transfer leads to 527 nm emission characteristic of the YFP(-*) anion. Prolonged continuous wave illumination at the peak of YFP(-) absorbance (514 nm) yields, irreversibly, a weakly fluorescent product that absorbs at 390 nm. This "photobleaching" process also gives a different species (YFPHrb) that absorbs at 350/430 nm and spontaneously regenerates YFP(-) in the dark on the time scale of hours but can be photoactivated by UV light to regenerate YFP(-) within seconds, via a ground-state protonated intermediate. Using a pulsed laser for photobleaching resulted in decarboxylation of YFP as indicated by the mass spectrum. These observations are accounted for in a unifying kinetic scheme.