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1.  Fibrocyte localization to the airway smooth muscle is a feature of asthma 
Airway smooth muscle (ASM) hyperplasia is a hallmark of asthma that is associated with disease severity and persistent airflow obstruction.
We sought to investigate whether fibrocytes, a population of peripheral blood mesenchymal progenitors, are recruited to the ASM compartment in asthma.
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.
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/mm2 [1.7/mm2]) versus healthy control subjects (median [interquartile range] number, 0/mm2 [0.3/mm2], P < .0001) and in the ASM bundle of subjects with asthma of all severities (subjects with severe asthma, median [interquartile range] number, 3.8/mm2 [9.4/mm2]; subjects with mild-to-moderate asthma, median [interquartile range] number, 1.1/mm2 [2.4/mm2]); healthy control subjects, (median [interquartile range] number, 0/mm2 [0/mm2]); P = .0004). In the peripheral blood the fibrocyte number was also increased in subjects with severe refractory asthma (median [interquartile range] number, 1.4 × 104/mL [2.6 × 104/mL]) versus healthy control subjects (median [interquartile range] number, 0.4 × 104/mL [1.0 × 104/mL], P = .002). We identified that in vitro ASM promotes fibrocyte chemotaxis and chemokinesis (distance of migration after 4.5 hours, 31 μm [2.9 μm] vs 17 μm [2.4 μm], 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.
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.
PMCID: PMC3992369  PMID: 19081612
Asthma; airway smooth muscle; remodeling; mast cells
2.  Eosinophil protein in airway macrophages: A novel biomarker of eosinophilic inflammation in patients with asthma 
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.
We sought to develop a method to quantify eosinophil proteins in airway macrophages as a novel biomarker of eosinophilic airway inflammation.
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.
Eosinophil proteins were detected in airway macrophages, and cytoplasmic red hue increased after ingestion of apoptotic eosinophils. Airway macrophage percentage red-hued 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.
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.
PMCID: PMC3992372  PMID: 20639010
Asthma; macrophage; eosinophil; computer-assisted image analysis; induced sputum
3.  Human Airway Smooth Muscle Promotes Human Lung Mast Cell Survival, Proliferation, and Constitutive Activation: Cooperative Roles for CADM1, Stem Cell Factor, and IL-61 
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.
PMCID: PMC3992374  PMID: 18684968
4.  Mast Cells Promote Airway Smooth Muscle Cell Differentiation via Autocrine Up-Regulation of TGF-β11 
Asthma is a major cause of morbidity and mortality worldwide. It is characterized by airway dysfunction and inflammation. A key determinant of the asthma phenotype is infiltration of airway smooth muscle bundles by activated mast cells. We hypothesized that interactions between these cells promotes airway smooth muscle differentiation into a more contractile phenotype. In vitro coculture of human airway smooth muscle cells with β-tryptase, or mast cells with or without IgE/anti-IgE activation, increased airway smooth muscle-derived TGF-β1 secretion, α-smooth muscle actin expression and agonist-provoked contraction. This promotion to a more contractile phenotype was inhibited by both the serine protease inhibitor leupeptin and TGF-β1 neutralization, suggesting that the observed airway smooth muscle differentiation was driven by the autocrine release of TGF-β1 in response to activation by mast cell β-tryptase. Importantly, in vivo we found that in bronchial mucosal biopsies from asthmatics the intensity of α-smooth muscle actin expression was strongly related to the number of mast cells within or adjacent to an airway smooth muscle bundle. These findings suggest that mast cell localization in the airway smooth muscle bundle promotes airway smooth muscle cell differentiation into a more contractile phenotype, thus contributing to the disordered airway physiology that characterizes asthma.
PMCID: PMC3992381  PMID: 18802103
5.  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.
PMCID: PMC3402550  PMID: 22108207
asthma; airway smooth muscle; airway hyperresponsiveness; NOX4; SOD2

Results 1-5 (5)