Genetic variants of microsomal epoxide hydrolase and glutamate-cysteine ligase in COPD.

The genetic factors that contribute to the development of chronic obstructive pulmonary disease (COPD) are poorly understood. Many candidate genes have been proposed, including enzymes that protect the lung against oxidative stress, such as microsomal epoxide hydrolase (EPHX1) and glutamate-cysteine ligase (GCL). To date, most reported findings have been for EPHX1, particularly in relation to functional variants associated with fast and slow metabolism of epoxide intermediates. The present study aimed to identify any association of variation in these genes with COPD susceptibility or severity. In total, 1,017 white COPD patients and 912 nondiseased age and sex matched smoking controls were genotyped for six single nucleotide polymorphisms (SNPs) in EPHX1 (including the fast and slow variants and associated haplotypes), and eight SNPs in the two genes encoding GCL. GCL is a rate-limiting enzyme in the synthesis of glutathione, a major contributor to anti-oxidant protection in the lung. No association of variation was found in EPHX1 or GCL with susceptibility to COPD or disease severity. This is the largest reported study to date and is well powered to detect associations that have been previously suggested. The current data indicate that these genetic variants are unlikely to be related to susceptibility or disease severity in white chronic obstructive pulmonary disease patients.

Glucocorticoid treatment reduces exhaled nitric oxide in cystic fibrosis patients.

In cystic fibrosis (CF), low concentrations of exhaled nitric oxide (NO) and reduced expression of inducible nitric oxide synthase (iNOS) in airway epithelium have been reported. However, abundant iNOS expression has been found in the subepithelial tissues and elevated concentrations of NO metabolites in breath condensate and sputum. These conflicting results may be explained by increased scavenging of NO by superoxide radicals, resulting in rapid conversion to peroxynitrite, so that only a small proportion of the NO produced in the lung tissue reaches the airway lumen. If iNOS were active in the CF lung, exhaled NO would be further reduced by glucocorticoid treatment. CF patients (n = 13) were recruited to a double-blind, placebo-controlled study with crossover. Treatment comprised prednisolone or placebo for 5 days with a 9 day washout. After each treatment, exhaled NO was measured, spirometry performed and blood collected for measurement of serum nitrogen dioxide/nitrous oxide (NO2/NO3). Ten patients (8 male) completed the study. Following prednisolone treatment (mean +/- SD) exhaled NO concentration (3.1 +/- 1.6 parts per billion (ppb)) was significantly reduced versus placebo treatment (4.9 +/- 4.2 ppb; p<0.05, Wilcoxon signed-rank test). Spirometric indices and serum NO2/NO3 concentration were unchanged. These findings support the hypothesis that glucocorticoids suppress nitric oxide production in cystic fibrosis airways by reducing inducible nitric oxide synthase expression or by inhibiting recruitment of neutrophils, cells which express inducible nitric oxide synthase.

Induced sputum : whole sample.

With the introduction of fiberoptic bronchoscopy and the ability to carry out bronchoscopic biopsy and broncho-alveolar lavage (BAL) in patients and control subjects, characterisation of inflammation in airways diseases such as asthma and chronic obstructive respiratory disease (COPD) has been possible. This has allowed emphasis to be placed on the role of inflammation in diseases such as asthma (1) and COPD (2). Bronchoscopy, being an invasive procedure, carries an associated morbidity (3,4). Although bronchoscopy is carried out in patients with moderate or even severe airflow limitation for clinical indications, it is not ethically justified to carry out research bronchoscopies on such patients, as it is essential to pursue research procedures carrying the minimum risk to volunteer subjects. Thus BAL, for the purposes of research in airways diseases, is limited to patients with mild airflow obstruction, thus requiring extrapolation of findings to a more heterogeneous group of patients. As a consequence of its invasive nature, the number of times the procedure can be repeated is limited so that it may be difficult to study the kinetics of the inflammatory response. In addition, it may be difficult to recruit volunteers for studies that necessitate bronchoscopy.

A polymorphism in the tumor necrosis factor-alpha gene promoter region may predispose to a poor prognosis in COPD.

STUDY OBJECTIVES: To determine whether the adenine (A)-guanine (G) substitution polymorphism at position - 308 on the tumor necrosis factor-alpha gene confers susceptibility to COPD or to the development of a more severe form of disease. DESIGN: A cross-sectional study was undertaken to compare the frequency of the A allele in a group of 106 patients with COPD with that in a control population (n = 99). Patients were followed up prospectively for a period of 2 years. PARTICIPANTS AND SETTING: Participants included 106 COPD patients recruited from a respiratory outpatient clinic and 99 control subjects recruited from patients admitted for cardiac catheterization. MEASUREMENTS AND RESULTS: DNA was extracted from venous blood, and each subject was genotyped for the polymorphism by polymerase chain reaction amplification and restriction digestion using Nco1. There was no increased frequency of the A allele in patients compared to control subjects. AA homozygous patients had less reversible airflow obstruction (p<0.05) and a significantly greater mortality (both all-cause and respiratory deaths) on follow-up (p<0.001), despite a shorter cigarette smoking history. CONCLUSIONS: This study suggests that homozygosity for this A allele predisposes to more severe airflow obstruction and a worse prognosis in COPD.

Total sputum nitrate plus nitrite is raised during acute pulmonary infection in cystic fibrosis.

Nitric oxide (NO) can be detected in exhaled gas in human subjects. It is produced by nitric oxide synthase (NOS) and is rapidly metabolized to nitrite and nitrate (NO2/NO3). Exhaled NO is reported to be elevated in patients with asthma, bronchiectasis, or upper respiratory tract infection. Recent reports have shown no increase of exhaled NO in stable cystic fibrosis (CF). We hypothesized that NOS activity is increased in patients with acute pulmonary exacerbation of CF. We therefore measured exhaled NO and sputum NO2/NO3 in three subject categories: patients with acute pulmonary exacerbation of CF, patients with stable CF, and healthy control subjects. Mean +/- SD exhaled NO was significantly higher in control subjects (8.8 +/- 4.9 ppb) than in both acute (3.8 +/- 3.9 ppb) and stable (5.0 +/- 2.5 ppb) patients. Sputum NO2/NO3 was significantly higher in acute patients (774 +/- 307 micromol/L) when compared with both stable patients (387 +/- 203 micromol/L) and control (421 +/- 261 micromol/L) subjects. Sputum NO2/NO3 did not return to normal in a subgroup of patients assessed after 2 wk of intensive antibiotic and glucocorticoid treatment. These results confirm that exhaled NO is not a useful measure of airway inflammation in CF. Elevated levels of sputum NO2/NO3 suggest that NOS is activated during acute pulmonary exacerbations of CF.

Neutrophil adhesion molecule surface expression and responsiveness in cystic fibrosis.

The neutrophil-dominated inflammation of the lung in cystic fibrosis (CF) has traditionally been seen as a physiological response to continuous opportunistic infection. Recent studies suggest, however, that regulation of the inflammatory response itself may be altered in CF. Neutrophil migration from the bloodstream involves alterations in surface expression of the adhesion molecules L-selectin and Mac-1 (CD11b/CD18). The aim of this study was to assess neutrophil adhesion molecule expression and responsiveness in CF. Neutrophils from chronic (n = 16) and acutely infected (n = 13) CF patients and 15 normal control subjects were directly assessed by Fluorescence-activated cell sorter (FACS) analysis for surface expression of L-selectin and CD11b before and after stimulation with interleukin 8 (IL-8) or f-Met-Leu-Phe (fMLP). Neutrophils from stable (n = 5) and acutely infected (n = 5) non-CF bronchiectasis patients were also assessed. Surface upregulation of CD11b was similar in all groups. Basal levels of L-selectin were also comparable among all groups, however, when stimulated, neutrophils from both stable and acutely infected CF patients shed significantly less L-selectin than those from control subjects (p < 0.05 and p < 0.01, respectively). This decreased responsiveness was not observed in either stable or acutely infected non-CF bronchiectasis patients. These results add to the accumulating evidence suggestive of a defective inflammatory response in CF.

Exhaled nitric oxide and bronchoalveolar lavage nitrite/nitrate in active pulmonary sarcoidosis.

Increased exhaled nitric oxide (NO) may reflect respiratory tract inflammation in untreated asthmatics. We compared exhaled NO and bronchoalveolar lavage (BAL) nitrate/nitrite (NO3-/NO2-) in 10 patients who had untreated, active pulmonary sarcoidosis with those of normal control subjects. Exhaled NO concentrations, determined by chemiluminescence, were similar in patients and control subjects (peak NO concentration of patients [mean +/- SD]: 13.6 +/- 5.9 parts per billion [ppb], peak NO concentration of control subjects: 11.2 +/- 5.7 ppb, p = 0.32; mean alveolar NO concentration of patients: 7.8 +/- 4.4 ppb, mean alveolar NO concentration of control subjects: 7.1 +/- 4.2 ppb, p = 0.70; end-tidal NO concentration of patients: 6.9 +/- 4.5 ppb, end-tidal NO concentration of control subjects: 6.6 +/- 4.0 ppb, p = 0.60). BAL NO2- was assayed using a modified Griess reaction after reduction of NO3- to NO2-. There was no significant difference in mean BAL NO2- concentrations, expressed as nanomoles per milliliter of epithelial lining fluid (patients: 544 nmol/ml, control subjects: 579 nmol/ml, p = 0.81) or as nanomoles per milliliter of BAL fluid (patients: 6.7 nmol/ml, control subjects: 5.7 nmol/ml, p = 0.41). These data suggest that excess NO generation does not accompany the respiratory tract inflammation of pulmonary sarcoidosis.

Late response to allergen is associated with increased concentrations of tumor necrosis factor-alpha and IL-5 in induced sputum.

Bronchial antigen challenge of sensitized atopic patients with asthma results in an early fall in FEV1, followed in a proportion of patients by a late (4 to 24 hours) fall. The late response is accompanied by an increase in bronchial reactivity, which is widely believed to reflect local influx and degranulation of inflammatory cells, particularly eosinophils, in association with elevated local secretion of cytokines. We hypothesized that the development of a late-phase bronchoconstrictor response and airway eosinophilia after allergen challenge of sensitized atopic patients with asthma is associated with elevated induced sputum concentrations of the eosinophil-active cytokines IL-5 and granulocyte-macrophage colony-stimulating factor and the proinflammatory cytokine tumor necrosis factor-alpha. We counted inflammatory leukocytes and measured cytokine concentrations in induced sputum at baseline and 24 hours after inhalational allergen challenge of 15 atopic patients with asthma who had previously demonstrated a late response. We observed significant increases in the numbers of eosinophils and the concentrations of their granule products, eosinophil cationic protein and eosinophil peroxidase. In contrast, the numbers of neutrophils and concentrations of two of their products, myeloperoxidase and human neutrophil lipocalin, did not significantly change. The numbers of sputum eosinophils correlated with the maximal late-phase fall in FEV1. Concentrations of IL-5 and tumor necrosis factor-alpha, but not granulocyte-macrophage colony-stimulating factor, were significantly elevated after allergen challenge. We conclude that the relatively noninvasive technique of induced sputum production can be used to monitor the effect of bronchial provocation on cytokine concentrations in asthma.

Cellular profiles in asthmatic airways: a comparison of induced sputum, bronchial washings, and bronchoalveolar lavage fluid.

BACKGROUND: Analysis of bronchoalveolar lavage fluid has improved our understanding of the pathogenesis of asthma. Safety issues and access to expert resources limit this techniques as a research tool. Induced sputum is a non-invasive method of collecting airway fluid which is applicable to subjects with a range of severity of airflow obstruction. The method of sputum collection and processing differs between groups. A study was undertaken to compare induced sputum with bronchoscopically collected fluid. METHODS: Sixteen patients with mild stable asthma underwent both sputum induction and bronchoscopic examination with bronchial washings and bronchoalveolar lavage (BAL) in random order, with each procedure being separated by an interval of 12 days. Airway fluid was processed and stained for differential cell counting. RESULTS: Induced sputum was relatively rich in neutrophils and eosinophils compared with bronchial washings and BAL fluid (mean (SE) 1.3 (0.4)%, 5.0 (2.7)%, and 36.4 (3.7)% neutrophils and 0.6 (0.1)%, 1.6 (0.6)%, and 3.3 (1.1)% eosinophils in BAL fluid, bronchial washings, and induced sputum, respectively). The proportions of cells obtained at sputum induction correlated with those in bronchial washings but not BAL fluid (r = 0.6 and 0.7 for neutrophils and eosinophils, respectively, p < 0.05). By contrast, induced sputum had a lower proportion of lymphocytes and macrophages than bronchial washings or BAL fluid, without any correlation. CONCLUSION: Induced sputum is rich in neutrophils and eosinophils and poor in lymphocytes, suggesting an origin in the larger airways. Induced sputum adequately reflects the findings in fluid collected by direct bronchoscopy.

Granulocyte activation markers in induced sputum: comparison between chronic obstructive pulmonary disease, asthma, and normal subjects.

Airway inflammation is present in asthma and is thought to play a significant part in the development of airflow obstruction. In chronic obstructive pulmonary disease (COPD), neutrophilic inflammation is present in the airway lumen, whereas the submucosa displays a lymphocytic infiltrate. Less is known about the nature and mechanisms of inflammation in COPD than in asthma. Induced sputum allows noninvasive sampling of respiratory tract secretions from patients and control subjects, allowing characterization of cells and measurement of soluble markers. We exploited this technique in order to compare the presence and quantify specific markers of eosinophil and neutrophil activation in subjects with asthma or COPD, and control subjects. Differential cell counts showed significantly higher neutrophil percentages in the patients with COPD compared with other groups, while patients with asthma had higher numbers of eosinophils. The neutrophil markers myeloperoxidase (MPO), from primary granules in neutrophils, and human neutrophil lipocalin (HNL), released from secondary granules, were elevated in patients with asthma and COPD compared with control subjects but markedly more so in COPD. The difference between COPD and asthma was more marked for HNL than for MPO suggesting that HNL may be a better marker for discriminating between these conditions. Concentrations of the eosinophil granule protein, eosinophil cationic protein (ECP), and the eosinophil granule-derived enzyme, eosinophil peroxidase (EPO) were raised in the patients with asthma and those with COPD.

Effects of inhaled and oral glucocorticoids on inflammatory indices in asthma and COPD.

The role of glucocorticoids in the treatment of chronic obstructive pulmonary disease (COPD) is controversial. We have previously described high numbers of neutrophils and high concentrations of the inflammatory cytokines interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha), and of the cell activation markers eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), myeloperoxidase (MPO), and human neutrophil lipocalin (HNL) in COPD patients as compared with controls, and have postulated that the cytokines TNF-alpha and IL-8 play a role in propagating the inflammatory response in COPD. We have now studied the effects of inhaled and oral glucocorticoids on these inflammatory indices in induced sputum. Initially, we studied the effect of a 2-wk course of inhaled budesonide (800 mg twice daily for 2 wk) in 13 patients with severe COPD (mean FDV1: 35% predicted). There was no clinical benefit in either lung function or symptom scores, and no significant change in the inflammatory indices as measured by total and differential cell counts and concentrations of TNF-alpha eosinophil activation markers ECP and EPO, and neutrophil activation markers MPO and HNL. Because the lack of anti-inflammatory effect might have been due to poor drug delivery as a result of severe airflow limitation, we undertook a study examining the antiinflammatory effect of oral prednisolone (30 mg daily for 2 wk) in patients with COPD and undertook the same measurements in 10 patients with atopic asthma. Sputum eosinophil numbers, ECP, and EPO were significantly reduced in the asthmatic patients but were not modified in COPD. This confirms the clinical impression that inhaled steroids have little antiinflammatory effect, at least in the short term in this group of patients, and suggests that the inflammatory process in COPD is resistant to the antiinflammatory effect of glucocorticoids.

Differences in interleukin-8 and tumor necrosis factor-alpha in induced sputum from patients with chronic obstructive pulmonary disease or asthma.

Asthma and chronic obstructive pulmonary disease are characterized by chronic airway inflammation. Studies using bronchoalveolar lavage (BAL) have shown an increased proportion of eosinophils in the BAL fluid from asthmatics compared with that from normal subjects, whereas studies of chronic obstructive pulmonary disease (COPD) have shown increased numbers of neutrophils. Induced sputum allows sampling of respiratory tract secretions from patients and control subjects, providing a noninvasive method of studying airway secretions and allowing characterization of cells and measurement of soluble markers. We investigated whether induced sputum was a useful method of studying airway fluid from patients with moderate to severe COPD and whether it could be used to compare inflammation in this condition with that in asthma. An initial reproducibility study was undertaken. Sputum was induced twice in 13 patients with severe COPD at a 14-d interval. Total and differential cell counts were carried out and were found to be reproducible over this period. Sputum was then induced in 14 patients with COPD, 23 patients with asthma, 12 healthy cigarette smokers, and 16 normal nonsmoking control subjects. We found a significant increase in neutrophils and increased concentrations of tumor necrosis factor-alpha (TNF alpha) and interleukin-8 (IL-8) in the patients with COPD compared with the smoking and nonsmoking control subjects. Interleukin-8, but not TNF alpha, was significantly higher in the COPD group than in the asthmatic group. We conclude that the cytokines TNF alpha and IL-8 may be involved in the inflammation in COPD.

Reduced dose salbutamol in comparison with standard dosage for symptom relief in asthma.

Regular treatment with beta 2-agonists has been reported to be associated with an increase in risk of asthma death or near death, and with a deterioration in asthma symptom control. Low-dose beta 2-agonists provide effective bronchodilatation and bronchoprotection, even though maximal bronchodilatation is not achieved, and they may offer a better safety profile. In a double-blind, randomized,, cross-over study, we evaluated the efficacy of low-dose salbutamol metered-dose inhaler (50 micrograms.puff-1), used over a period of 2 weeks, compared with a standard dose (100 micrograms.puff-1) in control of asthma symptoms in 20 moderately severe asthmatic subjects using inhaled glucocorticosteroid therapy. Asthma control was assessed by symptom scores, peak flow rates, spirometry, inhaler usage and, where possible, by bronchial responsiveness to methacholine. Despite a 46% reduction in mean weekly salbutamol dosage, mean forced expiratory volume in one second (FEV1), morning and evening peak expiratory flow (PEF), PEF variability, dose of methacholine provoking a 20% decrease in FEV1 (PC20) (n=9), and symptom scores showed no difference between low-dose and standard inhaler treatment periods. Low-dose inhaler administration resulted in a small but significant increase in number of inhaler actuations. Low-dose salbutamol metered-dose inhaler may, thus, be useful for control of symptoms in moderately severe asthma. This strategy could be used to achieve a reduction in total beta 2-agonist usage, which may minimize any potential for adverse effects.

Acute and chronic effects of cigarette smoking on exhaled nitric oxide.

Cigarette smoking is associated with an increased risk of respiratory tract infections, chronic airway disease, and cardiovascular diseases, all of which may be modulated by endogenous nitric oxide (NO). We have investigated whether cigarette smoking reduces the production of endogenous NO. We compared exhalations of 41 current cigarette smokers with normal lung function and 73 age-matched non-smoking controls. Peak exhaled NO levels were measured by a modified chemiluminescence analyzer. The effects of inhaling a single cigarette in smokers were also measured. In control subjects we also measured the effects of inhalation of NO itself and carbon monoxide, both constituents of tobacco smoke. Peak exhaled NO concentrations were significantly reduced in smokers (42 +/- 3.9 compared with 88 +/- 2.7 parts per billion in nonsmokers, p < 0.01), with a significant relation between the exhaled NO and cigarette consumption (r = 0.77, p < 0.001). Smoking a single cigarette also significantly (p < 0.02), but transiently, reduced exhaled NO. Inhalation of carbon monoxide and NO had no effect on exhaled NO in normal subjects. Cigarette smoking decreased exhaled NO, suggesting that it may inhibit the enzyme NO synthase. Since endogenous NO is important in defending the respiratory tract against infection, in counteracting bronchoconstriction and vasoconstriction, and in inhibiting platelet aggregation, this effect may contribute to the increased risks of chronic respiratory and cardiovascular disease in cigarette smokers.