Activated memory T helper cells in bronchoalveolar lavage fluid from patients with atopic asthma: relation to asthma symptoms, lung function, and bronchial responsiveness.

BACKGROUND: Bronchial mucosal inflammation and epithelial damage are characteristic features of asthma. Activation of T helper lymphocytes may contribute to this process by mechanisms including the release of cytokines promoting eosinophil infiltration and activation. METHODS: Bronchial washings and bronchoalveolar lavage fluid were obtained from 29 atopic asthmatic patients (19 with current symptoms and 10 symptom free) and 13 normal volunteers. Flow cytometry was used to assess T cell phenotype and activation status in bronchoalveolar lavage fluid and peripheral blood, and differential cell counts were made on bronchial washings and bronchoalveolar lavage fluid. Findings were related to severity of disease as reflected by symptom scores, baseline lung function, and airway responsiveness. RESULTS: CD4 T lymphocytes in bronchoalveolar lavage fluid and blood from asthmatic patients were activated by comparison with controls (CD4 CD25, median 16.8% v 8.7% for bronchoalveolar lavage fluid, and 15.3% v 8.7% in blood). Bronchoalveolar lavage fluid CD4 T cells from both asthmatic patients and controls were of memory phenotype (95.8% and 96.8% CD45RO and 1.7% and 0.4% CD45RA respectively), whereas both CD45RO and CD45RA T cells were present in blood. Patients with asthma and current symptoms showed increased bronchoalveolar T cell activation compared with patients without symptoms (CD4 CD25 18.7% v 12.3%). Within the asthmatic group there was a significant association between CD4 CD25 lymphocytes and asthma symptom scores (rs = 0.75), airway methacholine responsiveness (log PC20, rs = -0.43) and baseline FEV1 (rs = -0.39). A correlation was also found between CD4 CD25 lymphocytes and eosinophils in bronchoalveolar lavage fluid (rs = 0.48). Eosinophils in bronchoalveolar lavage fluid were increased in asthmatic patients compared with controls and the percentage of eosinophils in bronchoalveolar lavage fluid correlated with asthma symptom score. A relation was found between percentage of epithelial cells in bronchoalveolar lavage fluid and FEV1 and methacholine PC20. CONCLUSION: These results support the hypothesis that selective activation of memory CD4 T cells contributes to eosinophil accumulation, bronchial hyperresponsiveness, and symptoms in asthma.     

Cell specific markers for eosinophils and neutrophils in sputum and bronchoalveolar lavage fluid of patients with respiratory conditions and healthy subjects

BACKGROUND: Highly specific protein markers for eosinophils and neutrophils could be a valuable diagnostic aid in various respiratory disorders. The cell specificity of monoclonal antibodies against eosinophil peroxidase (EPO), eosinophil cationic protein (ECP), human neutrophil lipocalin (HNL), and myeloperoxidase (MPO) was investigated using immunocytochemical techniques. METHODS: Induced sputum and bronchoalveolar lavage fluid samples from 14 patients with respiratory conditions and four healthy individuals were studied. Antigens were detected at their intracellular sites in cells with well preserved structures using optimal techniques for fixation, permeabilisation, and immunolabelling. RESULTS: Anti-EPO antibodies reacted only with eosinophils, and anti-HNL antibodies only with neutrophils. Anti-ECP antibodies reacted with both eosinophils and neutrophils and anti-MPO antibodies with neutrophils and monocytes. Cells not stained by monoclonal anti-EPO and anti-HNL antibodies included lymphocytes, monocytes, macrophages, squamous epithelial cells, and ciliated epithelial cells. CONCLUSIONS: EPO, a unique component of eosinophils, and HNL, a unique component of neutrophils, are useful markers for the identification of eosinophils and neutrophils, respectively, in sputum and bronchoalveolar lavage fluid.     

Comparison of airway immunopathology of eosinophilic bronchitis and asthma

BACKGROUND: Eosinophilic bronchitis is a condition characterised by a corticosteroid responsive cough, sputum eosinophilia, and normal tests of variable airflow obstruction and airway responsiveness. We performed a detailed comparative immunopathological study to test the hypothesis that the different airway function in patients with eosinophilic bronchitis and asthma reflects differences in the nature of the lower airway inflammatory response. METHODS: Exhaled nitric oxide was measured and induced sputum, bronchoscopy, bronchial wash (BW), bronchoalveolar lavage (BAL), and bronchial biopsy were performed in 16 subjects with eosinophilic bronchitis, 15 with asthma, and 14 normal controls. RESULTS: Both eosinophilic bronchitis and asthma were characterised by an induced sputum, BW and BAL eosinophilia, an increased number of epithelial and subepithelial eosinophils, and increased reticular basement membrane thickness. The median concentration of exhaled nitric oxide was higher in those with eosinophilic bronchitis (12 ppb) or asthma (8.5 ppb) than normal controls (2 ppb) (95% CI of the difference 5 to 16, p<0.0001 and 2 to 11.3, p=0.004, respectively). There were no group differences in epithelial integrity or the number of subepithelial T lymphocytes, mast cells or macrophages. CONCLUSION: With the exception of our previously reported association of smooth muscle mast cell infiltration with asthma, the immunopathology of eosinophilic bronchitis and asthma are similar which suggests that eosinophilic airway inflammation, increased exhaled nitric oxide, and increased basement membrane thickening are regulated independently of airway hyperresponsiveness.     

Correlation of CD4:CD8 ratio and tumour necrosis factor (TNF)alpha levels in induced sputum with bronchoalveolar lavage fluid in pulmonary sarcoidosis

BACKGROUND: An increased CD4:CD8 lymphocyte ratio and raised cytokine levels in bronchoalveolar lavage (BAL) fluid are characteristic of pulmonary sarcoidosis. Sputum induction has been used as a non-invasive tool for investigating the airways and may be useful in investigating inflammation in patients with sarcoidosis in whom endobronchial, peribronchial, and parenchymal inflammation is present. This study aimed to correlate the total and differential cell counts, CD4:CD8 ratio, and tumour necrosis factor (TNF)alpha levels between induced sputum and BAL fluid in patients with pulmonary sarcoidosis. METHODS: Fourteen patients with newly diagnosed biopsy proven sarcoidosis and six healthy controls were investigated. Sputum induction and BAL was carried out at the initial visit and repeated following six months of treatment with oral prednisone. RESULTS: There was no correlation of differential cell counts between induced sputum and BAL fluid. The CD4:CD8 ratio in induced sputum correlated strongly with that in BAL fluid (5.5 (0. 4:1) versus 4.4 (0.2:1); r = 0.8, p<0.001) and the fall in the ratio following six months of treatment in sputum paralleled that in BAL fluid (3.4 (0.2:1) versus 2.4 (0.2:1)). The TNF alpha levels in sputum also correlated with levels in the BAL fluid (11.9 (1.5) pg/ml versus 17.6 (2.7) pg/ml; r = 0.8, p<0.001). The fall in sputum TNF alpha levels following six months of treatment paralleled the fall in BAL fluid levels (6.7 (0.9) pg/ml versus 11.6 (1.3) pg/ml). CONCLUSIONS: The CD4:CD8 ratio and TNF alpha levels in induced sputum correlated with those in BAL fluid and paralleled changes with treatment. Induced sputum may therefore be a non-invasive surrogate for certain parameters in BAL fluid in patients with sarcoidosis.     

Airway hyperresponsiveness and bronchial mucosal inflammation in T cell peptide-induced asthmatic reactions in atopic subjects

BACKGROUND: Subjects with allergic asthma develop isolated late asthmatic reactions after inhalation of allergen-derived T cell peptides. Animal experiments have shown that airway hyperresponsiveness (AHR) is CD4+ cell-dependent. It is hypothesised that peptide inhalation produces increases in non-specific AHR and a T cell-dominant bronchial mucosal inflammatory response. METHODS: Bronchoscopy, with bronchial biopsies and bronchoalveolar lavage (BAL), was performed in 24 subjects with cat allergy 6 h after aerosol inhalation of short overlapping peptides derived from Fel d 1, the major cat allergen. Biopsy specimens and BAL fluid were studied using immunohistochemistry and ELISA. RESULTS: Twelve of the 24 subjects developed an isolated late asthmatic reaction without a preceding early (mast cell/histamine-dependent) reaction characteristic of whole allergen inhalation. These responders had significant between-group differences (responders vs non-responders) in the changes (peptide vs diluent) in AHR (p = 0.007) and bronchial mucosal CD3+ (p = 0.005), CD4+ (p = 0.006) and thymus- and activation-regulated chemokine (TARC)+ (p = 0.003) but not CD8+ or CD25+ cells or eosinophils, basophils, mast cells and macrophages. The between-group difference for neutrophils was p = 0.05 but with a non-significant within-group value (peptide vs diluent, responders, p = 0.11). In BAL fluid there was a significant between-group difference in TARC (p = 0.02) but not in histamine, tryptase, basogranulin, C3a or C5a, leukotrienes C(4)/D(4)/E(4), prostaglandins D(2) or F(2alpha). CONCLUSIONS: Direct activation of allergen-specific airway T cells by peptide inhalation in patients with atopic asthma leads to increased AHR with local increases in CD3+ and CD4+ cells and TARC but no significant changes in eosinophils or basophil/mast cell products. These findings support previous animal experiments which showed a CD4+ dependence for AHR.     

Effect of exposure to swine dust on levels of IL-8 in airway lavage fluid

BACKGROUND: Inhalation of swine dust causes airway inflammation with influx of inflammatory cells, predominantly neutrophils, into the lungs. A study was undertaken to determine whether or not exposure to swine dust induces release of interleukin 8 (IL-8) into upper and lower airways and how this possible release is related to cellular influx. A further aim was to study the relationship between the inflammatory response and swine dust exposure. METHODS: Thirty one healthy, non- smoking, previously unexposed subjects were exposed to swine dust during three hours work in a swine house. Bronchoalveolar lavage (BAL) was performed two weeks before and 24 hours after the exposure (n = 16). Nasal lavage and acoustic rhinometry were carried out 1-2 hours before and seven hours after the start of the exposure (n = 31). Exposure measurements were performed with personal sampling equipment. RESULTS: The exposure led to 19-fold and 70-fold increases in the neutrophil concentrations in nasal lavage and BAL fluid, respectively (p < 0.001). In BAL, fluid macrophages, lymphocytes and eosinophils increased significantly. The IL-8 levels in BAL fluid increased from < 31.3 ng/l to 63 (43-109) ng/l (median (25-75th percentile), p < 0.001), and in nasal lavage fluid the concentrations increased from 144 (97- 227) ng/l to 1064 (864-1437) ng/l (p < 0.001). IL-8 levels showed a significant correlation with the increase in neutrophils in the nasal lavage fluid but not in the BAL fluid. Acoustic rhinometry demonstrated significant swelling of the nasal mucosa. The air concentration of inhalable dust was 23.3 (20.0-29.3) mg/m3, endotoxin 1.3 (1.1-1.4) micrograms/m3, and muramic acid 0.99 (0.78-2.1) microgram/m3. CONCLUSIONS: The concentration of IL-8 increases in BAL fluid and nasal lavage fluid following exposure to swine dust and may be one of the chemoattractants contributing to the recruitment of neutrophils to the nasal cavity and the alveolar space.


     

Sputum T lymphocytes in asthma,COPD and healthy subjects have the phenotype of activated intraepithelial T cells (CD69+ CD103+)

BACKGROUND: T cells of intraepithelial phenotype have previously been detected in bronchoalveolar lavage (BAL) fluid in a range of lung diseases; these cells express the adhesion molecule alpha(E)beta(7) integrin, CD103, the ligand for epithelial cell E-cadherin. In subjects with asthma CD4+ lymphocytes are the predominant T cell subtype found in bronchial biopsy specimens and in BAL fluid, whereas CD8+ lymphocytes have been shown to predominate in subjects with chronic obstructive pulmonary disease (COPD). The aim of this study was to analyse the expression of CD103, activation markers (CD25 and CD69), and chemokine receptors (CXCR3, CCR5 and CCR3) on CD4+ and CD8+ lymphocytes from sputum and peripheral blood of subjects with asthma, COPD, and healthy controls. METHODS: T cell surface markers were assessed by immunofluorescence labelling and flow cytometry of gated lymphocytes among CD45+ leucocytes in sputum cell suspensions. RESULTS: Sputum lymphocytes expressed higher levels of CD103 and CD69 than blood lymphocytes in all subject groups, with CD103 expressed at higher levels on CD8+ than on CD4+ cells. There were no detectable differences in numbers of CD4+ and CD8+ T cells between subjects with asthma, COPD and controls. The percentage of sputum lymphocytes expressing CXCR3 was lower in subjects with asthma or COPD than in healthy controls; CCR3 was not detectable on sputum or blood lymphocytes. CONCLUSIONS: Sputum T lymphocytes are predominantly of activated intraepithelial phenotype (CD103+ CD69+), and normal numbers of CD4+ and CD8+ T cell populations are found in the sputum of patients with asthma and COPD.     

Increased levels of the chemokines GROalpha and MCP-1 in sputum samples from patients with COPD

BACKGROUND: Patients with chronic obstructive pulmonary disease (COPD) have increased numbers of neutrophils and macrophages in their lungs. Growth related oncogene-alpha (GROalpha) attracts neutrophils, whereas monocyte chemoattractant protein-1 (MCP-1) attracts monocytes that can differentiate into macrophages. The aim of this study was to determine the concentration of GROalpha and MCP-1 in bronchoalveolar lavage (BAL) fluid and sputum from non-smokers, healthy smokers and patients with COPD, and to see if there was a correlation between the concentrations of these chemokines, lung function, and numbers of inflammatory cells. METHODS: BAL fluid and sputum from non-smokers (n=32), healthy smokers (n=36), and patients with COPD (n=40) were analysed for the presence of GROalpha and MCP-1 using ELISA. Cells counts were performed on the samples and correlations between the concentrations of these chemokines, lung function, and inflammatory cells observed. RESULTS: Median (SE) GROalpha and MCP-1 levels were significantly increased in sputum from patients with COPD compared with non-smokers and healthy smokers (GROalpha: 31 (11) v 2 (2) v 3 (0.8) ng/ml; MCP-1: 0.8 (0.4) v 0.2 (0.1) v 0.1 (0.04) ng/ml, p<0.05), but not in BAL fluid. There were significant negative correlations between both GROalpha and MCP-1 levels in sputum and forced expiratory volume in 1 second (FEV(1)) % predicted (GROalpha: r=-0.5, p<0.001; MCP-1: r=-0.5, p<0.001), together with significant positive correlations between GROalpha and MCP-1 and neutrophil numbers in sputum (GROalpha: r=0.6, p<0.001; MCP-1: r=0.4, p<0.01). CONCLUSION: These results suggest that GROalpha and MCP-1 are involved in the migration of inflammatory cells, thus contributing to the inflammatory load associated with COPD.     

Exhaled nitric oxide correlates with airway eosinophils in childhood asthma

BACKGROUND: Exhaled nitric oxide has been proposed as a marker for airway inflammation in asthma. The aim of this study was to compare exhaled nitric oxide levels with inflammatory cells and mediators in bronchoalveolar lavage fluid from asthmatic and normal children. METHODS: Children were recruited from elective surgical lists and a non-bronchoscopic bronchoalveolar lavage (BAL) was performed after induction of anaesthesia. Exhaled nitric oxide (parts per billion) was measured by two techniques: tidal breathing and restricted breath. RESULTS: Median (interquartile range) exhaled nitric oxide measured by restricted breath was increased in asthmatics compared with normal children (24.3 (10.5-66.5) v 9.7 (6.5-16.5), difference between medians 14.6 (95% CI 5.1 to 29.9), p=0.001). In asthmatic children exhaled nitric oxide correlated significantly with percentage eosinophils (r=0.78, p<0.001 (tidal breathing) and r=0.78, p<0.001 (restricted breath)) and with eosinophilic cationic protein (r=0.53, p<0.01 (restricted breath)), but not with other inflammatory cells in the BAL fluid. The area under the receiver operator characteristic curves for the prediction of the presence of eosinophilic airways inflammation by exhaled nitric oxide (tidal and restricted) was 0.80 and 0.87, respectively. CONCLUSIONS: Exhaled nitric oxide correlates closely with percentage eosinophils in BAL fluid in asthmatic children and is therefore likely to be a useful non-invasive marker of airway inflammation.     

Role of sputum differential cell count in detecting airway inflammation in patients with chronic bronchial asthma or COPD.

BACKGROUND: Sputum may provide an alternative source of bronchial cells to investigate characteristics of airway inflammation and its functional correlates in patients with asthma or chronic obstructive pulmonary disease (COPD). METHODS: Two groups of clinically stable patients were studied: a group of 43 patients with mild or moderate asthma and a group of 18 patients with COPD. Twenty normal subjects formed a control group. Sputum production was either spontaneous or induced with inhaled hypertonic saline for five minute periods for up to 20 minutes. The concentration of saline was increased at intervals of 10 minutes from 3% to 4%. Plugs from the lower respiratory tract were selected for differential counting in cytocentrifugation preparations. Bronchial provocation tests were performed by inhaling progressive concentrations of histamine from a DeVilbiss 646 nebuliser and the concentration of histamine which caused a 20% fall in the forced expiratory volume in one second (FEV1) was calculated (PC20FEV1). RESULTS: Neutrophils predominated in the sputum of subjects with COPD while eosinophils predominated in the sputum of those with chronic asthma. However, in 28% of asthmatic subjects an increased percentage of neutrophils was found. In asthmatic patients the differential count of eosinophils was inversely related to the FEV1, FEV1/VC, and bronchial hyperresponsiveness, and directly related to clinical scores. CONCLUSIONS: The cellular profile of sputum in normal subjects and in patients with asthma and COPD is different. The concentration of eosinophils in the sputum correlates with the severity of asthma.     

Tumour necrosis factor (TNFalpha) as a novel therapeutic target in symptomatic corticosteroid dependent asthma

BACKGROUND: Tumour necrosis factor alpha (TNFalpha) is a major therapeutic target in a range of chronic inflammatory disorders characterised by a Th1 type immune response in which TNFalpha is generated in excess. By contrast, asthma is regarded as a Th2 type disorder, especially when associated with atopy. However, as asthma becomes more severe and chronic, it adopts additional characteristics including corticosteroid refractoriness and involvement of neutrophils suggestive of an altered inflammatory profile towards a Th1 type response, incriminating cytokines such as TNFalpha. METHODS: TNFalpha levels in bronchoalveolar lavage (BAL) fluid of 26 healthy controls, 42 subjects with mild asthma and 20 with severe asthma were measured by immunoassay, and TNFalpha gene expression was determined in endobronchial biopsy specimens from 14 patients with mild asthma and 14 with severe asthma. The cellular localisation of TNFalpha was assessed by immunohistochemistry. An open label uncontrolled clinical study was then undertaken in 17 subjects with severe asthma to evaluate the effect of 12 weeks of treatment with the soluble TNFalpha receptor-IgG1Fc fusion protein, etanercept. RESULTS: TNFalpha levels in BAL fluid, TNFalpha gene expression and TNFalpha immunoreative cells were increased in subjects with severe corticosteroid dependent asthma. Etanercept treatment was associated with improvement in asthma symptoms, lung function, and bronchial hyperresponsiveness. CONCLUSIONS: These findings may be of clinical significance in identifying TNFalpha as a new therapeutic target in subjects with severe asthma. The effects of anti-TNF treatment now require confirmation in placebo controlled studies.     

Value of eosinophil cationic protein and tryptase levels in bronchoalveolar lavage fluid for predicting lung function impairment in anaesthetised, asthmatic children

Bronchial hyperactivity, a key feature of active asthma in children, is a risk factor for respiratory adverse events in the peri-operative period. The presence of activated eosinophils in the lungs and mast cell degranulation can contribute to bronchial hyperreactivity. Eosinophil cationic protein is released by activated eosinophils and tryptase reflects mast cell degranulation. This study focused on the relationship of respiratory mechanics, eosinophil cationic protein and tryptase levels in bronchoalveolar lavage fluid in asthmatic and healthy children under general anaesthesia. We measured eosinophil cationic protein and tryptase levels in bronchoalveolar lavage fluid from 21 asthmatic and 21 healthy children following induction of general anaesthesia. Respiratory system resistance and dynamic compliance were measured during mechanical ventilation. Eosinophil cationic protein was more common in bronchoalveolar lavage fluid from asthmatics (12/21) than from controls (4/21, p = 0.01) and was present at higher levels (p = 0.002). Tryptase was also more common in the asthmatics (8/21 vs 1/21, p = 0.01). Respiratory resistance was significantly higher in asthmatic children with detectable eosinophil cationic protein levels than in those with undetectable eosinophil cationic protein levels (p = 0.019). Furthermore, 50% of the asthmatics with detectable eosinophil cationic protein exhibited bronchospasm after sampling their bronchoalveolar lavage fluid. These findings suggested that high levels of eosinophil cationic protein in the bronchoalveolar lavage fluid are associated with irritable airways, presumably secondary to airway inflammation, and this might be a useful marker for respiratory adverse events in the peri-operative period.     

Effect of natural allergen exposure during the grass pollen season on airways inflammatory cells and asthma symptoms.

BACKGROUND: Bronchial challenge with allergen causes a specific form of airways inflammation consisting of an influx of neutrophils, eosinophils, and T cells. Because the relevance of the challenge model to clinical asthma is uncertain, the cellular changes that occur in the lungs of asthmatic subjects during natural seasonal allergen exposure were investigated. METHODS: Seventeen grass pollen sensitive asthmatic subjects with previously reported seasonal exacerbations of asthma kept records of symptoms and underwent fibreoptic bronchoscopy with bronchoalveolar lavage (BAL) and endobronchial biopsy before and during the peak of the grass pollen season. The BAL cells were analysed for differential cell counts and by flow cytometry for T cell subsets and surface activation markers. The biopsy samples were processed into glycol methacrylate resin and immunohistochemical analysis was performed for mast cells, activated eosinophils, T cells and interleukin 4 (IL-4), a cytokine with a pivotal role in allergen-induced inflammation. RESULTS: In the pollen season there was an increase in T lymphocyte activation in the BAL fluid as identified by increased expression of interleukin 2 receptor (IL-2R). In the submucosa these changes were paralleled by an increase in CD4+ T cells. By contrast, the numbers of metachromatic cells in BAL fluid staining with toluidine blue were reduced, possibly because of degranulation following allergen stimulation. In keeping with mast cell activation, the number of mucosal mast cells staining for secreted IL-4 increased during the season. In comparison with the period shortly before the onset of the season, all but two subjects experienced an asthma exacerbation which followed the rise in pollen counts but, compared with the period preceding the first bronchoscopic examination, asthma symptoms were not increased during the pollen season. CONCLUSIONS: The data suggest that natural allergen exposure, leading to a clinical exacerbation of asthma, may induce an inflammatory response involving T cells, mast cells and eosinophils. The relationship between allergen exposure, cellular infiltration and activation, and clinical symptoms appears to be complex, with factors other than allergen also contributing to asthmatic activity.     

Lymphocyte dynamics: caution in interpreting BAL numbers

In various allergic and inflammatory lung diseases the number and subset composition of lymphocytes in the bronchoalveolar lavage (BAL) fluid are taken as indicators of the state of the disease. The number of lymphocytes in the BAL fluid depends on three main parameters: (1) entry into the bronchoalveolar space from the different compartments of the lung, (2) persistence in the bronchoalveolar space which is modified by the rate of local proliferation and apoptosis, and (3) exit into the draining bronchial lymph node via the lymphatic system. In healthy individuals lymphocytes in the BAL fluid seem to be a stable pool: each day there is hardly any entry, local cell division or cell death and few lymphocytes emigrate from this compartment. In contrast, during inflammatory, toxic and allergic reactions all parameters can increase rapidly with more lymphocytes entering, proliferating and/or undergoing apoptosis locally. Very little is known about factors such as cytokines and chemokines which may regulate these parameters. When interpreting data on lymphocyte numbers in patients, lymphocyte dynamics in the bronchoalveolar space have to be considered, and in the future it may be possible to manipulate these lymphocyte fluxes for therapeutic purposes.


     

Enhanced virulence, airway inflammation and impaired lung function induced by respiratory syncytial virus deficient in secreted G protein

BACKGROUND: Respiratory syncytial virus (RSV) infection can cause bronchial hyperresponsiveness and asthma exacerbations. In mice it results in airway inflammation and airway hyperresponsiveness. Since viral factors influencing these responses are not well defined, a study was undertaken to investigate the role of secreted G protein of human RSV in determining virulence, inflammatory responses, and changes in lung function. METHODS: BALB/c mice were infected with a spontaneous mutant of RSV deficient in secreted G protein (RSV-DeltasG) or with wild type RSV (RSV-WT). Viral titres, numbers of pulmonary inflammatory cells, and concentrations of interferon (IFN)-gamma, interleukin (IL)-4, IL-5 and IL-10 in bronchoalveolar lavage (BAL) fluid were determined. Airway function was assessed at baseline and following methacholine provocation using barometric whole body plethysmography. RESULT: Following infection with RSV-DeltasG, viral titres were increased 50-fold compared with RSV-WT. Influx of eosinophils and macrophages to the lung and concentrations of IFN-gamma and IL-10 in BAL fluid were also significantly higher following infection with RSV-DeltasG. Airway function, both at baseline and after methacholine provocation, was significantly decreased following infection with RSV-DeltasG compared with RSV-WT. CONCLUSION: Secreted G protein is likely to be a regulatory factor in RSV infection limiting infectivity of the virus, inflammatory responses in the lungs, and reduction in lung function.     

支气管哮喘和慢性阻塞性肺疾病患者诱导痰中血管内皮生长因子水平的变化及意义

目的探讨血管内皮生长因子（VEGF）在支气管哮喘和慢性阻塞性肺疾病（COPD）发病中的作用。方法分别选择缓解期支气管哮喘患者（哮喘组）30例、稳定期COPD患者（COPD组）28例和健康志愿者（健康对照组）24例进行肺功能测定和用诱导痰检查方法对痰进行炎性细胞分类计数,并用EMSA法测定诱导痰上清液中VEGF水平。结果哮喘组诱导痰中嗜酸粒细胞数为0．9（0．4—1．4）×10^9/L,明显高于COPD组和健康对照组的0．1（0—0．2）×10^9/L、0．0（0～0．1）×10^9/L（P值均＜0．05）;COPD组诱导痰中中性粒细胞数为2．3（1．8～2．8）×10^9/L,明显高于哮喘组和健康对照组的1．1（0．2～1．9）×10^9/L、1．0（0．8～1．2）×10^9/L（P值均＜0．05）。哮喘组、COPD组和健康对照组诱导痰上清液中VEGF水平分别为（2．3±0．5）、（0．3±0．1）、（0．9±0．2）μg／L,三组间比较差异有统计学意义（P值均＜0．05）。哮喘组诱导痰上清液中VEGF水平与诱导痰中嗜酸粒细胞数呈正相关（r=0．62,P＜0．05）,与第1秒用力呼气容积（FEV。）占预计值百分比呈负相关（r=-0．56,P＜0．05）;COPD组诱导痰上清液中VEGF水平与FEV,占预计值百分比呈正相关（r=0．43,P＜0．05）,与诱导痰中中性粒细胞数无相关性（r=0．21,P＞0．05）。结论支气管哮喘患者诱导痰上清液中VEGF表达上调,VEGF可能参与了支气管哮喘的气道炎性反应过程;COPD患者诱导痰上清液中VEGF表达下降,VEGF可能参与了COPD的发病过程。     

Ongoing airway inflammation in patients with COPD who do not currently smoke

BACKGROUND: Inflammatory changes in the airways in chronic obstructive pulmonary disease (COPD) are largely attributed to smoking, yet they may be present even if patients do not currently smoke. The differences in inflammatory cells and the factors contributing to these differences were examined in the airways of patients with COPD who do not currently smoke. METHODS: Eighteen non-atopic subjects with COPD (14 men) of mean (SD) age 62 (8) years and forced expiratory volume in one second (FEV(1)) 59 (13)% predicted and 11 non-atopic healthy subjects (eight men) of mean (SD) age 58 (8) years, FEV(1) 104 (11)% predicted were studied. Sputum induction and bronchoscopy with bronchoalveolar lavage (BAL) and biopsies were performed. RESULTS: Patients with COPD had more mucosal EG2+ cells (eosinophils) (median (range) 40 (0-190) versus 5 (0-40) cells/mm(2), p = 0.049) and CD68+ cells (1115 (330-2920) versus 590 (450-1580) cells/mm(2), p = 0.03), and a tendency towards more CD4+ but not CD8+ lymphocytes than healthy controls. Furthermore, patients with COPD had higher percentages of sputum neutrophils (77 (29-94) versus 36 (18-60)%, p = 0.001) and eosinophils (1.2 (0-8.5) versus 0.2 (0-3.1)%, p = 0.008), BAL fluid eosinophils (0.4 (0-1.7) versus 0.2 (0-0.5)%, p = 0.03), and higher concentrations of sputum eosinophilic cationic protein (ECP) (838 (115-23 760) versus 121 (35-218) ng/ml, p<0.001). Concentrations of ECP expressed per eosinophil were not higher. Patients with COPD with high mucosal EG2+ cell numbers also had high mucosal CD4+ cell numbers. Sputum eosinophilia was associated with a decrease in FEV(1)/VC and BAL fluid eosinophilia with a decrease in mucosal NP57+ cells (neutrophils). CONCLUSIONS: Subjects with COPD who do not currently smoke have increased numbers of inflammatory cells. Eosinophils are increased in number in the airways in COPD but do not seem to be activated. The increased eosinophil numbers are probably due to recruitment as a result of ongoing inflammation. Macrophages and lymphocytes may play a part in this inflammation.     

Feasibility of sputum induction in lung transplant recipients

Sputum induction (SI) is nowadays being applied as a non-invasive and safe method to investigate airway inflammation in pulmonary diseases. We investigated the feasibility of SI after lung transplantation (LTX), and compared sputum and bronchoalveolar lavage (BAL) cellular characteristics and interleukin-8 (IL-8) levels. Results were also compared with 11 healthy subjects. SI as performed between 26 and 1947 d after LTX in 19 recipients, was successful in 16 of 22 attempts (73%). Six patients failed to produce sputum after induction, mostly just post-LTX and with having a lower forced expiratory volume in 1 s (FEV1). The success rate in clinically stable patients after the first month post-LTX was 93%. Side-effects were absent. Sputum recovery, viability and squamous cell contamination were comparable between LTX patients and healthy subjects. In the LTX group, total cell counts, neutrophil percentages and IL-8 levels were much higher in SI than BAL (1.6 x 10(6)/mL, 65.5% and 54.2 ng/mL vs. 0.1 x 10(6)/mL, 3.0% and 0.01 ng/mL; p < 0.001). Although LTX-neutrophil percentages in SI and BAL correlated properly (rho=0.72, p=0.04), both techniques are not interchangeable. We conclude that sputum induction is feasible, well tolerated, and without major side-effects in stable patients after the first month post-LTX. Induced sputum may be a useful tool to study inflammatory changes of the airways after LTX, and because of the large quantity of neutrophils sampled, especially for further studies on the pathogenesis of bronchiolitis obliterans.     

Increased matrix metalloproteinase-9 concentration and activity after stimulation with interleukin-17 in mouse airways

BACKGROUND: The proteolytic enzyme matrix metalloproteinase (MMP)-9 can degrade structural compounds such as the extracellular matrix and the basement membrane in the airways and lungs. MMP-9 has therefore been implicated in remodelling of the airways and lungs during severe asthma and chronic obstructive pulmonary disease (COPD). METHODS: The effect of the T lymphocyte derived proinflammatory cytokine interleukin (IL)-17 on MMP-9 protein release and activity in the airways was studied in vivo and in vitro. RESULTS: In vivo, intranasal stimulation of mice with IL-17 induced the release of the precursor molecule proMMP-9 in bronchoalveolar lavage (BAL) fluid, associated with a pronounced local accumulation of neutrophils that stained positive for MMP-9. Stimulation with IL-17 also increased the concentration of free soluble MMP-9 that was proteolytically active as determined by a gelatinase substrate assay. The concentration of MMP-9 in BAL fluid had a strong positive correlation with the number of neutrophils; the amount of MMP-9 per neutrophil was not increased by IL-17 stimulation. In vitro, stimulation of mouse neutrophils with IL-17 did not increase the concentration of proMMP-9 in the conditioned medium. CONCLUSION: Local stimulation with IL-17 increases the concentration of biologically active MMP-9 as well as its precursor molecule in mouse airways in vivo. This increase in proteolytic load is probably mainly due to an increased number of neutrophils and not to an increase in the release of MMP-9 from each neutrophil. These findings indicate a link between the T lymphocyte cytokine IL-17 and increased proteolytic load in the airways which may be relevant for chronic inflammatory airway diseases such as severe asthma and COPD.     

Decreased peroxynitrite inhibitory activity in induced sputum in patients with bronchial asthma

BACKGROUND: The production of peroxynitrite, an extremely potent oxidant, is increased in inflammatory lung disease. It is therefore important to measure antioxidant activity against peroxynitrite in epithelial lining fluid to examine the physiological effects of peroxynitrite in the airways of patients with asthma. This study was designed to determine whether peroxynitrite inhibitory activity in induced sputum is correlated with clinical characteristics and airway inflammatory indices in asthmatic patients. METHODS: Inflammatory indices were measured in induced sputum from 25 patients with asthma and 12 normal control subjects. Peroxynitrite inhibitory activity was also measured by monitoring rhodamine formation in sputum samples. RESULTS: Peroxynitrite inhibitory activity in induced sputum was significantly lower in asthmatic patients (52.4 (24.5)%) than in normal control subjects (92.1 (3.9)%, p<0.0001). Its activity was significantly correlated with forced expiratory volume in 1 second (FEV(1)) % predicted (r=0.774, p<0.0001) and bronchial hyperreactivity to methacholine (r=0.464, p=0.023). There was a significant negative correlation between peroxynitrite inhibitory activity and the degree of eosinophilic airway inflammation (% eosinophils, r=-0.758, p<0.0001; eosinophil cationic protein, r=-0.780, p<0.0001). CONCLUSIONS: Decreased peroxynitrite inhibitory activity occurs in induced sputum of asthmatic patients. Since even in patients with stable asthma the airway lining fluid lacks peroxynitrite inhibitory activity, large amounts of peroxynitrite, which are further increased during an acute asthma attack, would not be completely inactivated and asthmatic airways might have markedly increased susceptibility to peroxynitrite induced airway injury.