Application of functional genomics to study of inflammatory airways disease.
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Asthma↗
Biomedical subjects
S T Holgate
Publications and source records attributed to S T Holgate.
Respiratory symptoms in older people and use of domestic gas appliances.
BACKGROUND: Studies in children and young women have indicated an increased risk of respiratory illness in association with the use of domestic gas appliances, possibly caused by oxides of nitrogen generated when gas is burned. It is not known whether risks are similarly increased in older subjects. METHODS: A questionnaire about respiratory symptoms in the past year and potential risk factors for respiratory disease was mailed to 6000 men and women aged 65 years and older who were selected at random from the lists of general practices in North Bristol, UK. Associations between symptoms and the use of gas appliances were examined by logistic regression with adjustment for age, sex, social class, and smoking habits. RESULTS: Questionnaires were completed by 4792 (80%) of those mailed. The most common symptoms were exercise induced breathlessness, wheeze, or chest tightness (51%); wheeze (27%); morning phlegm (20%); and daytime breathlessness at rest (19%). In an analysis that included all subjects only weak associations were found with use of gas appliances, odds ratios all being 1.2 or less. The risks associated with use of a gas hob tended to be higher in women, with odds ratios of 1.36 (95% CI 1.01 to 1.83) for wheeze and 1.33 (95% CI 0.56 to 3.17) for morning chest tightness, but were lower than had been reported previously in younger women. CONCLUSION: The absence of stronger associations cannot readily be explained by bias or confounding. Gas cookers and fires are unlikely to be an important cause of respiratory illness in the elderly. If they do cause such illness, the largest risks are likely to be in women who use gas hobs.
Aged↗
Lymphocyte and eosinophil influx into alveolar tissue in nocturnal asthma.
We have shown in nocturnal asthma that alveolar tissue eosinophils are increased at night as compared with the proximal airway, and that they correlate with the overnight decrement in lung function. As the CD4+ cell is thought to be the principal orchestrating cell in eosinophil recruitment, we evaluated its presence in the proximal and distal airways in nocturnal asthma. Eleven patients with nocturnal asthma (NA) and 10 patients with non-nocturnal asthma (NNA) underwent two bronchoscopies with proximal airway endobronchial and distal alveolar tissue transbronchial biopsy in a random order at 4:00 P.M. and at 4:00 A.M. separated by 1 wk. Immunohistochemical staining and morphometric analysis were used to determine the number of CD3+, CD4+, and CD8+ cells and EG2+ eosinophils per mm2 in the epithelium, lamina propria, and alveolar tissue. At 4:00 A.M., the NA group had a significantly greater number of CD4+ cells in the alveolar tissue than the NNA group (9.8 cells/ mm2 [5.6-30.8, interquartile (IQ)] versus 1.5 cells/mm2 [0-6. 3, IQ], p = 0.04). Within the NA group, there were significantly greater numbers of CD3+, CD4+, CD8+, and EG2+ cells in the proximal airway lamina propria than in the distal airway at both 4:00 P.M. and 4:00 A.M. There were no differences within the epithelium between the groups at either time point. Only alveolar tissue, not airway tissue, CD4+ cells correlated inversely with the percentage predicted FEV1 at 4:00 A.M. (r = -0.68, p = 0.0018) and positively with the number of alveolar tissue EG2+ cells (r = 0.66, p = 0.01). These findings suggest that the CD4+ lymphocyte is increased in the alveolar tissue at night in nocturnal asthma as compared with non-nocturnal asthma.
Adult↗
Persistent airway inflammation but accommodated antioxidant and lung function responses after repeated daily exposure to nitrogen dioxide.
Nitrogen dioxide (NO2) is a common indoor and outdoor air pollutant that may induce deterioration of respiratory health. In this study the effects of repeated daily exposure to NO2 on airway antioxidant status, inflammatory cell and mediator responses, and lung function were examined. Healthy nonsmoking subjects were exposed under controlled conditions to air (once) and to 2 ppm of NO2 for 4 h on four consecutive days. Lung function measurements were made before and immediately after the end of each exposure. Bronchoscopy with endobronchial biopsies, bronchial wash (BW), and bronchoalveolar lavage (BAL) was carried out 1.5 h after the air exposure and after the last exposure to NO2. Repeated NO2 exposure resulted in a decrease in neutrophil numbers in the bronchial epithelium. The BW revealed a twofold increase in content of neutrophils (p < 0.05) and a 1.5-fold increase in myeloperoxidase (MPO) (p < 0.01) indicative of both migration and activation of neutrophils in the airways. After the fourth NO2 exposure, antioxidant status of the airway fluid was unchanged. Significant decrements in FEV1 and FVC were found after the first exposure to NO2, but these attenuated with repeated exposures. Together, these data indicate that four sequential exposures to NO2 result in a persistent neutrophilic inflammation in the airways, whereas changes in pulmonary function and airway antioxidants are resolved. We conclude that NO2 is a proinflammatory air pollutant under conditions of repeated exposure.
Adult↗
Acute inflammatory responses in the airways and peripheral blood after short-term exposure to diesel exhaust in healthy human volunteers.
Several epidemiologic studies have demonstrated a consistent association between levels of particulate matter (PM) in the ambient air with increases in cardiovascular and respiratory mortality and morbidity. Diesel exhaust (DE), in addition to generating other pollutants, is a major contributor to PM pollution in most places in the world. Although the epidemiologic evidence is strong, there are as yet no established biological mechanisms to explain the toxicity of PM in humans. To determine the impact of DE on human airways, we exposed 15 healthy human volunteers to air and diluted DE under controlled conditions for 1 h with intermittent exercise. Lung functions were measured before and after each exposure. Blood sampling and bronchoscopy were performed 6 h after each exposure to obtain airway lavages and endobronchial biopsies. While standard lung function measures did not change following DE exposure, there was a significant increase in neutrophils and B lymphocytes in airway lavage, along with increases in histamine and fibronectin. The bronchial biopsies obtained 6 h after DE exposure showed a significant increase in neutrophils, mast cells, CD4+ and CD8+ T lymphocytes along with upregulation of the endothelial adhesion molecules ICAM-1 and VCAM-1, with increases in the numbers of LFA-1+ cells in the bronchial tissue. Significant increases in neutrophils and platelets were observed in peripheral blood following DE exposure. This study demonstrates that at high ambient concentrations, acute short-term DE exposure produces a well-defined and marked systemic and pulmonary inflammatory response in healthy human volunteers, which is underestimated by standard lung function measurements.
Adult↗
Suggestive evidence for genetic linkage between IgE phenotypes and chromosome 14q markers.
Chromosome 14q was screened for loci modulating immunoglobulin E (IgE) phenotypes in 15 extended and 45 nuclear asthmatic families using a panel of 14 microsatellite markers. We examined the reported linkage between the TCR A/D locus on 14q11.2 and specific (cognate) allergic responses and observed supportive evidence for linkage between a general skin prick test reactivity trait (but not with total serum IgE) and TCRA microsatellite (in the total sample of informative sib-pairs p = 0.039, in selected sample of one or zero affected parent p = 0.017). We also show suggestive evidence for a novel linkage between markers D14S75 and D14S63 on 14q13-23 and log total serum IgE (p = 0.034 and p = 0.0029). The evidence for linkage with marker D14S63 on 14q23 is strengthened by the finding of association of allele 165 to log IgE (p = 0.0029). We conclude that chromosome 14q may contain a locus close to TCR A/D at 14q11.2 linked to skin prick reactivity and a locus at 14q13- 23 linked to total serum IgE.
Adolescent↗
Expression of c-erbB receptors and ligands in human bronchial mucosa.
The epidermal growth factor receptor (EGFR, c-erbB1) plays a pivotal role in maintenance and repair of epithelial tissues; however, little is known about coexpression of c-erbB receptors and their ligands in human bronchial epithelium. We therefore analyzed the expression of these molecules in cultured bronchial epithelial cells and normal bronchial mucosa, using reverse transcription-polymerase chain reaction (RT- PCR), flow cytometry, and immunohistochemistry. Messenger RNA (mRNA) encoding EGFR, c-erbB2, and c-erbB3, but not c-erbB4, was detected in primary cultures of human bronchial epithelial cells, as well as in the human bronchial epithelial-derived cell lines H292 and 16HBE 14o-. Transcripts encoding epidermal growth factor (EGF), heparin binding epidermal growth factor (HB-EGF), transforming growth factor-alpha (TGF-alpha), and amphiregulin (AR) were also detected, and expression of the three receptors and four ligands was confirmed by immunocytochemical staining of the cultured cells. Immunohistochemical analysis of resin- or paraffin-embedded sections from surgical specimens of bronchial mucosa revealed strong membrane staining for EGFR within the bronchial epithelium; this was particularly evident between basal cells and the basal aspect of columnar cells. The patterns of staining for c-erbB2 and c-erbB3 in the bronchial epithelium were similar to those for EGFR. Immunostaining for EGF, TGF-alpha, AR, HB- EGF, and betacellulin (BTC) was intense in the submucosal glands; with the exception of BTC, EGFR ligand immunoreactivity was also observed in the bronchial epithelium, where it paralleled EGFR staining. Colocalization of c-erbB receptors and ligands demonstrates the potential for productive c-erbB receptor interactions in bronchial epithelium. Further study of these interactions may help to define their role in maintenance and repair of the bronchial epithelium.
Base Sequence↗
Mucosal inflammation in severe glucocorticoid-dependent asthma.
To improve our understanding of the inflammatory mechanisms underlying severe disease, a biopsy study was performed comparing 15 clinically unstable glucocorticoid-dependent asthmatics, 10 mild asthmatics, and 10 control subjects. Compared with mild asthma, severe asthma was characterized by reduced mucosal eosinophilia. Whilst no significant differences were found in the numbers of mast cells, neutrophils, CD3+ and CD4+ T-cells between the three groups, up to a 4-fold increase In the numbers of activated T-lymphocytes bearing the interleukin (IL)-2 receptor (IL-2R) was found in the mucosa in severe asthma compared to mild asthma (p = 0.03) and control subjects (p = 0.003). Compared to control subjects, the mucosa of severe asthmatics contained significantly (p = 0.02) higher numbers of IL-5+ cells, with no differences between mild and severe disease. In contrast, staining for the anti-IL-4 monoclonal antibody 3H4 revealed that biopsies from mild asthmatics contained more IL4+ cells than biopsies from severe asthmatics and control subjects (p = 0.0008). In the severe asthmatics, a close correlation (r(s) = 0.76, p = 0.005) was found between the numbers of IL-2R-bearing cells and the variability in peak expiratory flow. In conclusion, persistent T-cell activation is a prominent feature of severe asthma. These results also indicate that interleukin-5, and not interleukin-4, is upregulated in severe disease.
Administration, Topical↗
Ozone-induced lung function decrements do not correlate with early airway inflammatory or antioxidant responses.
This study sought to clarify the early events occurring within the airways of healthy human subjects performing moderate intermittent exercise following ozone challenge. Thirteen healthy nonsmoking subjects were exposed in a single blinded, crossover control fashion to 0.2 parts per million (ppm) O3 and filtered air for 2 h, using a standard intermittent exercise and rest protocol. Lung function was assessed pre- and immediately post-exposure. Bronchoscopy was performed with endobronchial mucosal biopsies, bronchial wash (BW) and bronchoalveolar lavage (BAL) 1.5 h after the end of the exposure period. Respiratory tract lining fluid (RTLF) redox status was assessed by measuring a range of antioxidants and oxidative damage markers in BW and BAL fluid samples. There was a significant upregulation after O3 exposure in the expression of vascular endothelial P-selectin (p<0.005) and intercellular adhesion molecule-1 (p<0.005). This was associated with a 2-fold increase in submucosal mast cells (p<0.005) in biopsy samples, without evidence of neutrophilic inflammation, and a decrease in BAL fluid macrophage numbers (1.6-fold, p<0.005), with an activation of the remaining macrophage subset (2.5-fold increase in % human leukocyte antigen (HLA)-DR+ cells, p<0.005). In addition, exposure led to a 4.5-fold and 3.1-fold increase of reduced glutathione (GSH) concentrations, in BW and BAL fluid respectively (p<0.05), with alterations in urate and alpha-tocopherol plasma/RTLF partitioning ratios (p<0.05). Spirometry showed reductions in forced vital capacity (p<0.05) and forced expiratory volume in one second (p<0.01), with evidence of small airway narrowing using forced expiratory flow values (p<0.005). Evidence was found of O3-induced early adhesion molecule upregulation, increased submucosal mast cell numbers and alterations to the respiratory tract lining fluid redox status. No clear relationship was demonstrable between changes in these early markers and the lung function decrements observed. The results therefore indicate that the initial lung function decrements are not predictive of, or causally related to the O3-induced inflammatory events in normal human subjects.
Adult↗
Antioxidant consumption and repletion kinetics in nasal lavage fluid following exposure of healthy human volunteers to ozone.
To obtain information on the real-time events occurring within human respiratory tract lining fluids (RTLFs) during ozone exposure, sequential nasal lavage was performed on 13 human volunteers exposed on separate occasions to 0.2 parts per million O3 and filtered air (2-h exposures, with intermittent exercise). Nasal lavage was performed and blood samples obtained at four time points throughout each exposure: pre-exposure (Pre-E), 1 h into exposure (1h-E), immediately post-exposure (0h-PE) and 1 h post-exposure (1h-PE). Endobronchial mucosal biopsies were obtained at 1.5 h-post exposure (1.5h-PE). Nasal RTLF neutrophilia was not apparent during, or 1.5 h after, 03 exposure. Furthermore, activation of the pre-existing neutrophil population did not occur. Airway permeability was not altered by this 03 exposure regimen. Sequential lavage resulted in significant washout of RTLF ascorbic acid, reduced glutathione, extracellular superoxide dismutase and myeloperoxidase at 1h-E, 0h-PE and 1.5h-PE relative to baseline Pre-E values. In contrast, RTLF uric acid (UA), total protein and albumin concentrations did not display washout kinetics. Of the antioxidants examined, only UA was clearly depleted by 03, concentrations, falling by 6.22 micromol x L(-1) at 1h-E, compared with 1.61 micromol x L(-1) (p<0.01) during control air exposure. The establishment of a new pseudo-steady-state concentration of RTLF UA (70% of Pre-E values) during the second hour of O3 exposure was coincident with a small but significant increase in plasma UA concentration (19.27 (O3) versus 1.95 micromol x L(-1) (air), p<0.05). These data demonstrate that inhalation of 0.2 parts per million 03 results in the depletion of nasal respiratory tract lining fluid uric acid and that this regional loss of uric acid leads to a small increase in plasma uric acid concentration. Whilst the reaction of uric acid with inspired 03 may confer protection locally, the role of upper airway uric acid as a sink for inhaled O3 is not supported by these findings.
Adult↗
Growth factors secreted by bronchial epithelial cells control myofibroblast proliferation: an in vitro co-culture model of airway remodeling in asthma.
Remodeling of the bronchial wall is a major determinant of morbidity in asthma. An increased number of myofibroblasts beneath the bronchial epithelial basement membrane has been described in asthma. The production of mediators by epithelial cells in close proximity to myofibroblasts during epithelial repair after repeated damage is one of the possible mechanisms for airway remodeling. In this study, we established a three-dimensional co-culture system in which myofibroblasts derived from human bronchial wall were maintained in collagen gels and a human bronchial epithelial cell line, 16HBE14o-, was grown on the surface of the gels. The epithelial cells were chemically injured by exposure to poly-L-arginine as a surrogate for eosinophil granule cationic protein and the proliferative response of the fibroblasts was examined. Conditioned medium from mechanically damaged epithelial cells was also tested for its effect on fibroblast proliferation. Myofibroblasts in the co-cultures showed significantly enhanced proliferation after poly-L-arginine-induced epithelial damage. Conditioned medium from mechanically damaged epithelial cells also increased fibroblast-proliferation. After epithelial perturbation, basic fibroblast growth factor, platelet-derived growth factor, IGF-1, transforming growth factor-beta2, and endothelin-1 levels increased in culture supernatants. Blockade of these growth factors inhibited fibroblast proliferation by 76% after epithelial injury. This study demonstrates that epithelial cells are an important regulator of airway remodeling by means of paracrine control of bronchial myofibroblasts in response to cell damage and repair.
Asthma↗
The Link Between Asthma and COPD. Foreword, summary and conclusions.
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Animals↗
Antihistamines: back to the future. Preface.
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Animals↗
Antihistamines: back to the future. Summary of the conclusions. BSACI. British Society for Allergy and Clinical Immunology.
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Animals↗
The epidermal growth factor receptor and its ligand family: their potential role in repair and remodelling in asthma.
The bronchial epithelium acts as a physical barrier to the ingress of airborne irritants and plays an active role in the airways immune response, reacting to a range of environmental stimuli to enable interaction with immune and inflammatory cells. It is well established that mucosal damage and epithelial cell shedding are important features of inflammatory airways diseases such as asthma. Evidence now suggests that the repairing epithelium has the potential to contribute to disease chronicity through production of an array of soluble mediators and adhesion molecules. However, the biochemical mechanisms that control epithelial maintenance and repair are poorly understood, even though avoiding or reversing the changes in the airways which are likely to result from chronic inflammation and epithelial restitution remains a significant challenge in asthma therapy. The purpose of this review is to highlight the potential of the epidermal growth factor receptor (EGFR/c-erbB) and its ligands in restitution of the bronchial epithelium. This receptor tyrosine kinase plays a pivotal role in regulation of epithelial cell behaviour, having the capacity to elicit a broad spectrum of cellular responses ranging from migration or proliferation to differentiation and enhanced survival; it also has the ability to regulate expression of various inflammatory mediators, mucins, adhesion molecules, matrix proteins, and metalloproteinases which are relevant to the chronic disease phenotype.
Animals↗
Amino acid residues that influence Fc epsilon RI-mediated effector functions of human immunoglobulin E.
Immunoglobulin E (IgE) mediates its effector functions via the Fc region of the molecule. IgE binding to and subsequent aggregation of the high-affinity receptor (Fc epsilon RI) by allergen plays a pivotal role in type I hypersensitivity responses. Earlier studies implicated the C epsilon 2 and 3 interface and the A-B loop in C epsilon 3 in the IgE-Fc epsilon RI interaction. These regions and glycosylation sites in C epsilon 3 were now targeted by site-specific mutagenesis. IgE binding to Fc epsilon RI was compared with surface plasmon resonance (SPR) measurements, which assessed the binding of the soluble extracellular domain of Fc epsilon RI to IgE. Kinetic analysis based on a pseudo-first-order model agrees with previous determinations. A more refined SPR-based kinetic analysis suggests a biphasic interaction. A model-free empirical analysis, comparing the binding strength and kinetics of native and mutant forms of IgE, identified changes in the kinetics of IgE-Fc epsilon RI interaction. Conservative substitutions introduced into the A-B loop have a small effect on binding, suggesting that the overall conformation of the loop is important for the complementary interaction, but multiple sites across the C epsilon 3 domain may influence IgE-Fc epsilon RI interactions. Asn394 is essential for the generation of a functional IgE molecule in mammalian cells. A role of Pro333 in the maintenance of a constrained conformation at the interface between C epsilon 2-3 emerged by studying the functional consequences of replacing this residue by Ala and Gly. These substitutions cause a dramatic decrease in the ability of the ligand to mediate stimulus secretion coupling, although only small changes in the association and dissociation rates are observed. Understanding the molecular basis of this phenomenon may provide important information for the design of inhibitors of mast cell degranulation.
Amino Acids↗
Linkage of asthma to markers on chromosome 12 in a sample of 240 families using quantitative phenotype scores.
We present evidence of linkage between markers on chromosome 12 and asthma using the BETA program for nonparametric single- and multipoint linkage analysis. We have used quantitative scores as our phenotypic variables, combining data into indices for asthma and atopy and maximizing heritability. The largest single-locus LODs were achieved for asthma: D12S342 and asthma score (LOD 2.255), D12S324 and asthma affection (LOD 2.214), and D12S366 and wheeze (LOD 3.307). The region of interest identified using multipoint analysis, with a maximum LOD of 2.29, centers around D12S97 at location 173.5 cM with a standard error of 6.5 for the asthma score and close agreement for asthma affection and wheeze. Such evidence merits further investigation of this area in an attempt to define the region with greater precision with a view to identifying candidate genes. We hope that the methods presented will encourage researchers to use phenotypic information in a way that encourages meta-analysis.
Adolescent↗
The role of mast cell tryptase in regulating endothelial cell proliferation, cytokine release, and adhesion molecule expression: tryptase induces expression of mRNA for IL-1 beta and IL-8 and stimulates the selective release of IL-8 from human umbilical vein endothelial cells.
Mast cells are found frequently in close proximity to blood vessels, and endothelial cells are likely to be exposed to high concentrations of their granule mediators. We have investigated the proinflammatory actions of the major mast cell product tryptase on HUVEC. Addition of purified tryptase was found to stimulate thymidine incorporation, but induced little alteration in cell numbers, suggesting it is not a growth factor for HUVEC. Expression of ICAM-1, VCAM-1, and E-selectin was not altered following incubation with tryptase, but the potent granulocyte chemoattractant IL-8 was released in a dose-dependent fashion in response to physiologically relevant concentrations, with maximal levels in supernatants after 24 h. The actions of tryptase on HUVEC were inhibited by heat inactivation of the enzyme, or by preincubating with the protease inhibitors leupeptin or benzamidine, suggesting a requirement for an intact catalytic site. Reverse-transcription PCR analysis indicated up-regulation of mRNA for IL-8 as well as for IL-1 beta in response to tryptase or TNF-alpha. However, tryptase was a more selective stimulus than TNF-alpha and did not induce increased expression of mRNA for granulocyte-macrophage CSF or stimulate the release of this cytokine. Leukocyte accumulation in response to tryptase may be mediated in part through the selective secretion of IL-8 from endothelial cells.
Cell Adhesion Molecules↗