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1.  Increases in CD4+ T lymphocytes, macrophages, neutrophils and interleukin 8 positive cells in the airways of patients with bronchiectasis 
Thorax  1998;53(8):685-691.
BACKGROUND—Bronchiectasis is a chronic suppurative lung disease characterised by irreversible dilation of the bronchi and persistent purulent sputum. The immunopathology of the disease was studied using a quantitative immunostaining technique with particular reference to T lymphocytes, macrophages, and granulocytes.
METHODS—Bronchial mucosal biopsy specimens were obtained by fibreoptic bronchoscopy from 12 patients with bronchiectasis (six receiving inhaled steroids) and 11 normal healthy controls. Immunostaining (APAAP method) was performed on frozen cryostat sections with a panel of monoclonal antibodies to total leucocytes (CD45), T lymphocyte phenotypic markers (CD3, CD4, CD8), macrophages (CD68), eosinophils (EG2), and neutrophils (elastase).
RESULTS—There was a mononuclear cell infiltrate in both patients with bronchiectasis and normal controls, but an overall increase in total leucocyte cell numbers (CD45+ cells) was identified in those with bronchiectasis (median values 422 cells/mm2 versus 113 cells/mm2 in control tissue, p<0.001). Intense infiltration of CD3+ T lymphocytes was observed compared with healthy controls (292 cells/mm2 and 40 cells/mm2, respectively, p<0.001). This comprised predominantly CD4+ T cells (118 cells/mm2) rather than CD8+ T cells (47 cells/mm2). CD3+ cell counts were reduced in those subjects on inhaled steroids compared with those not receiving inhaled steroids (197cells/mm2 versus 369 cells/mm2, p<0.05), as were CD4+ cell counts (82 cells/mm2 versus 190 cells/mm2, p<0.05). Neutrophil and macrophage cell numbers were also increased in patients with bronchiectasis (114 cells/mm2 and 213 cells/mm2, respectively) compared with controls (41 neutrophils/mm2 and 40 macrophages/mm2). EG2+ (activated) eosinophil numbers were much lower than T cells, macrophages, and neutrophils in patients with bronchiectasis but were increased compared with controls (36 cells/mm2 versus 0 cells/mm2, p<0.001). In view of the markedly increased neutrophil counts in patients with bronchiectasis, biopsy specimens were immunostained for interleukin 8 (IL-8) which was highly significantly increased compared with controls (47 cells/mm2 versus 15 cells/mm2, p<0.01). IL-8+ cells were less prominent in steroid treated patients than in patients not receiving treatment (30 cells/mm2 versus 60 cells/mm2, p<0.05). A further characteristic of bronchiectasis was mucous gland hypertrophy. Gland area comprised up to 40% of the tissue in some bronchiectasis sections while no hypertrophy was noted in control biopsy specimens (p<0.05).
CONCLUSION—Airway inflammation in bronchiectasis is characterised by tissue neutrophilia, a mononuclear cell infiltrate composed mainly of CD4+ T cells and CD68+ macrophages, and increased IL-8 expression. Inhaled corticosteroid treatment in patients with bronchiectasis is associated with a less marked infiltration by T cells and IL-8+ cells within the bronchial mucosa, although this finding requires confirmation in a prospective placebo controlled trial.

PMCID: PMC1745299  PMID: 9828857
2.  Pediatric severe asthma is characterized by eosinophilia and remodeling without TH2 cytokines 
The pathology of pediatric severe therapy-resistant asthma (STRA) is little understood.
We hypothesized that STRA in children is characterized by airway eosinophilia and mast cell inflammation and is driven by the TH2 cytokines IL-4, IL-5, and IL-13.
Sixty-nine children (mean age, 11.8 years; interquartile range, 5.6-17.3 years; patients with STRA, n = 53; control subjects, n = 16) underwent fiberoptic bronchoscopy, bronchoalveolar lavage (BAL), and endobronchial biopsy. Airway inflammation, remodeling, and BAL fluid and biopsy specimen TH2 cytokines were quantified. Children with STRA also underwent symptom assessment (Asthma Control Test), spirometry, exhaled nitric oxide and induced sputum evaluation.
Children with STRA had significantly increased BAL fluid and biopsy specimen eosinophil counts compared with those found in control subjects (BAL fluid, P < .001; biopsy specimen, P < .01); within the STRA group, there was marked between-patient variability in eosinophilia. Submucosal mast cell, neutrophil, and lymphocyte counts were similar in both groups. Reticular basement membrane thickness and airway smooth muscle were increased in patients with STRA compared with those found in control subjects (P < .0001 and P < .001, respectively). There was no increase in BAL fluid IL-4, IL-5, or IL-13 levels in patients with STRA compared with control subjects, and these cytokines were rarely detected in induced sputum. Biopsy IL-5+ and IL-13+ cell counts were also not higher in patients with STRA compared with those seen in control subjects. The subgroup (n = 15) of children with STRA with detectable BAL fluid TH2 cytokines had significantly lower lung function than those with undetectable BAL fluid TH2 cytokines.
STRA in children was characterized by remodeling and variable airway eosinophil counts. However, unlike in adults, there was no neutrophilia, and despite the wide range in eosinophil counts, the TH2 mediators that are thought to drive allergic asthma were mostly absent.
PMCID: PMC3381727  PMID: 22385633
Pediatric asthma; eosinophilia; remodeling; severe therapy-resistant asthma; mediators
3.  PCR based bronchoscopic detection of common respiratory pathogens in chronic cough: a case control study 
Viral respiratory tract infection is the most frequent cause of acute cough and is reported at onset in about one third of patients with chronic cough. Persistent infection is therefore one possible explanation for the cough reflex hypersensitivity and pulmonary inflammation reported in chronic cough patients.
Bronchoscopic endobronchial biopsies and bronchoalveolar lavage cell counts were obtained from ten healthy volunteers and twenty treatment resistant chronic cough patients (10 selected for lavage lymphocytosis). A screen for known respiratory pathogens was performed on biopsy tissue. Chronic cough patients also underwent cough reflex sensitivity testing using citric acid.
There was no significant difference in incidence of infection between healthy volunteers and chronic cough patients (p = 0.115) or non-lymphocytic and lymphocytic groups (p = 0.404). BAL cell percentages were not significantly different between healthy volunteers and chronic cough patients without lymphocytosis. Lymphocytic patients however had a significantly raised percentage of lymphocytes (p < 0.01), neutrophils (p < 0.05), eosinophils (p < 0.05) and decreased macrophages (p < 0.001) verses healthy volunteers. There was no significant difference in the cough reflex sensitivity between non-lymphocytic and lymphocytic patients (p = 0.536).
This study indicates latent infection in the lung is unlikely to play an important role in chronic cough, but a role for undetected or undetectable pathogens in either the lung or a distal site could not be ruled out.
Trials registration
Current Controlled Trials ISRCTN62337037 & ISRCTN40147207
PMCID: PMC3496690  PMID: 22978556
Biopsy; Bronchoalveolar lavage; Bronchoscopy; Cough; Infection; PCR; Virus
4.  Evidence for eosinophil activation in bronchiectasis unrelated to cystic fibrosis and bronchopulmonary aspergillosis: discrepancy between blood eosinophil counts and serum eosinophil cationic protein levels 
Thorax  1998;53(6):498-500.
BACKGROUND—Increased serum levels of eosinophil cationic protein (ECP) have been detected in adolescent patients with cystic fibrosis. However, ECP concentrations in adult patients with bronchiectasis unrelated to cystic fibrosis have not been studied.
METHODS—Eosinophil numbers and serum concentrations of ECP were determined in 14 patients with known or newly diagnosed bronchiectasis and compared with age and sex matched patients with allergic bronchial asthma, chronic obstructive pulmonary disease (COPD), and controls in whom bronchiectasis or obstructive pulmonary disease could be excluded.
RESULTS—Serum ECP levels were significantly raised both in patients with bronchiectasis (median (range) 22.5 µg/l (7-85)) and allergic asthma (35.0 µg/l (7-128)) compared with the sex and age matched subjects suffering from COPD (6.7 µg/l (1.5-28); p<0.006) and non-obstructive normal controls (7.5 µg/l (3.5-19); p<0.003). In contrast, significantly increased peripheral eosinophil numbers were observed in patients with bronchial asthma (305 × 106/l; p<0.01) but not in those with bronchiectasis (102 × 106/l), COPD (117 × 106/l), and healthy controls (101 × 106/l).
CONCLUSIONS—The discrepancy between eosinophil counts and eosinophil numbers in patients with bronchiectasis suggests that serum ECP levels may be more relevant in assessing local eosinophil involvement than blood eosinophil numbers.

PMCID: PMC1745234  PMID: 9713451
5.  BAL fluid cells in newly diagnosed pulmonary sarcoidosis with different clinical activity 
Sarcoidosis is associated with an increase in the number of alveolar T cells (CD3+ cells) and an increase of the CD3+CD4+ lymphocyte subset. However, the number of lymphocytes and the CD4/CD8 ratio in bronchoalveolar lavage (BAL) fluid are highly variable in sarcoidosis. Comparative studies have demonstrated that geographic and ethnic factors are linked to the specific characteristics of patients with sarcoidosis.
Aim of the study
To investigate peculiarities of BAL fluid (BALF) cell patterns in different clinical activity of pulmonary sarcoidosis at the time of diagnosis.
Material and methods
A total of 308 non-treated patients (138 asymptomatic and 170 with sarcoidosis-related symptoms) and 40 previously empirically steroid-treated patients with newly diagnosed sarcoidosis have been prospectively examined.
Significant BAL fluid lymphocytosis and increased CD4/CD8 ratio were characteristic for all three sarcoidosis patient groups. A total of 12% of asymptomatic patients, 3% of patients with sarcoidosis-related symptoms, and 5% of previously treated symptomatic patients had normal BALF cell counts. Non-treated patients with sarcoidosis-related symptoms had significantly higher lymphocytosis (45±19% versus 39±17%, P<0.01), CD4/CD8 ratio (9.3±5.0 versus 5.7±4.5, P<0.001), and total BALF cell count (411±322 106/mL versus 334±273 106/mL, P<0.05), compared with asymptomatic patients. However, previously treated symptomatic patients had lower lymphocytosis (39±15% versus 45±19%, P=0.058), and total BALF cell count (292±166 106/mL versus 411±322 106/mL, P<0.05) compared with non-treated symptomatic patients. The same trend was noticed for CD4/CD8 ratio (8.3±4.8), although a statistically significant difference was not achieved.
Independently of clinical symptoms at the time of diagnosis sarcoid patients have significantly different BAL fluid cell patterns compared to healthy persons. BAL fluid cell changes are more prominent in corticosteroid non-treated patients with clinically active sarcoidosis. Treatment with systemic corticosteroids may modify typical BALF cellular patterns of sarcoidosis.
PMCID: PMC2852744  PMID: 19242869
Bronchoalveolar lavage; bronchoscopy; sarcoidosis
6.  Chronic pulmonary sarcoidosis: relationship between lung lavage cell counts, chest radiograph, and results of standard lung function tests. 
Thorax  1985;40(7):501-507.
Thirty three consecutive untreated patients with pulmonary sarcoidosis, confirmed histologically or by Kveim test, were investigated to correlate cell counts in bronchoalveolar lavage fluid with clinical features, the chest radiograph, and results of lung function tests. A persistently abnormal radiograph had been observed for one year or more in 26 (79%) and for two years or more in 20 (61%), but only 24% had dyspnoea. Twenty (61%) of 33 patients showed an increased percentage of lymphocytes in bronchoalveolar lavage fluid, although only eight (24%) exceeded 28%. A moderate increase of neutrophils, up to 12%, was found in 14 (42%). Lymphocyte percentage counts were higher in the group of patients without evidence of radiographic contraction suggesting fibrosis, and this contrasted with higher percentage neutrophil counts in those with contraction. There was also a correlation between the percentages of neutrophils and increasing radiographic profusion scores (p less than 0.001), suggesting that neutrophils may reflect the severity of the parenchymal legions as well as fibrotic distortion, and an inverse correlation with vital capacity (p less than 0.001) and transfer factor (TLCO) (p less than 0.1 greater than 0.05). No significant correlation was found between the lymphocyte counts and radiographic profusion scores, vital capacity or TLCO; but it was noted that all eight patients with high lymphocyte counts (greater than 28%) had radiographic profusion scores less than 12. This study shows that, especially in sarcoidosis with more extensive radiographic shadows of long duration, bronchoalveolar lavage neutrophils may be important as well as lymphocytes in clinical assessment of "activity" of disease. These observations are important because they throw doubt on whether the lavage lymphocyte count alone can be used as an indicator of the need to start corticosteroid treatment.
PMCID: PMC460121  PMID: 4035616
7.  Asbestosis: assessment by bronchoalveolar lavage and measurement of pulmonary epithelial permeability. 
Thorax  1985;40(7):508-514.
Thirty two patients with asbestosis were assessed by means of bronchoalveolar lavage (27 patients) and the half time clearance from lungs to blood (T1/2LB) of an inhaled aerosol of diethylenetriamine pentacetate (DTPA) labelled with technetium 99m (32 patients). T1/2LB was also measured in 20 non-smoking normal individuals and 17 smokers without a history of exposure to asbestos. Thirteen patients (46%) showed an increase in the percentage of neutrophils with or without an increase in the percentage of eosinophils and eight (29%) showed an increased percentage of lymphocytes. The number of neutrophils plus eosinophils expressed as a percentage of the total count was positively correlated with the length of the history of disease (r = 0.53, p less than 0.025) and greater percentages were associated with more severe impairment of lung function. Smokers had lower percentages of lymphocytes than non-smokers (p less than 0.002) and showed increased proportions of neutrophils and eosinophils more often than non-smokers (p less than 0.05). In 18 non-smokers with asbestosis the mean T1/2LB was 33.8 (range 10.0-62.0) minutes, significantly less than 57.2 (30.5-109) minutes in 20 non-smoking normal subjects (p less than 0.002). In non-smokers shorter T1/2LB correlated with a longer time since first exposure to asbestos (r = -0.65, p less than 0.005), longer duration of exposure (r = -0.70, p less than 0.001), and a shorter time since last exposure (r = 0.59, p less than 0.01). Shorter T1/2LB was also associated with increased inflammatory activity as shown by higher bronchoalveolar lavage cell counts (r = -0.53, p less than 0.025) and higher combined percentages of neutrophils, eosinophils, and lymphocytes (r = -0.47, p less than 0.05). The techniques of bronchoalveolar lavage and measurement of inhaled solute clearance may be useful in assessing inflammatory activity in asbestosis.
PMCID: PMC460122  PMID: 4035617
8.  Effect of natural allergen exposure during the grass pollen season on airways inflammatory cells and asthma symptoms. 
Thorax  1996;51(6):575-581.
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.
PMCID: PMC1090485  PMID: 8693436
9.  Predictive value of bronchoalveolar lavage cell analysis in sarcoidosis. 
Thorax  1988;43(4):284-288.
Patients with histopathologically proved sarcoidosis were studied serially by means of bronchoalveolar lavage, initially at the time of diagnosis and then six and 12 months later. Two years later they were evaluated by chest radiography and lung function tests and classified in terms of recovery or progression over the previous two years. The recovery of lymphocytes and granulocytes in lavage fluid was of limited prognostic value for persistent lung disease. In contrast, patients with increased numbers of mast cells recovered by lavage were more likely to deteriorate. Significantly increased mast cell counts (greater than or equal to 0.5% of total cells recovered) were seen in at least one lavage investigation in 15 of the 16 patients with more active and progressive disease, but in only eight of 23 patients with inactive disease (p less than 0.001). A persistent increase of mast cells on serial measurement occurred in nine of the 16 patients with active disease and in four of the 23 patients in whom the disease was inactive (p less than 0.02). The finding in the two subsequent lavages of lymphocytosis (lymphocytes greater than 30% of recovered cells) or neutrophilia (neutrophils greater than 15%) combined with mastocytosis (mast cells greater than or equal to 0.5%) occurred in nine of the 16 patients with active disease but in no patients with inactive disease.
PMCID: PMC461214  PMID: 3406915
10.  CD4/CD8 ratio and cytokine levels of the BAL fluid in patients with bronchiectasis caused by sulfur mustard gas inhalation 
To analyze cytokine levels in BAL fluid of patients with bronchiectasis due to mustard gas inhalation.
29 victims with mustard gas-induced bronchiectasis and 25 normal veterans as control group.
PFTs,, high-resolution CT scans of the chest, analyses of BAL fluids for five cytokines (IL-8, IL-1β, IL-6, TNF-α, IL-12) and analyses of BAL fluids for cellular and flow-cytometric analysis of the phenotype of bronchoalveolar cells were performed in all cases.
CD4 lymphocytes expressed as percentage or absolute number were significantly higher in patients with bronchiectasis than in controls (32.17 ± 16.00 vs 23.40 ± 6.97%, respectively; p = 0.01; and 3.31 ± 2.03 vs 1.88 ± 0.83 × 103 cells/ml, respectively; p = 0.001). The CD4/CD8 ratio was significantly higher in patients with bronchiectasis than in controls (3.08 ± 2.05 vs 1.68 ± 0.78; p = 0.002).
There were significant differences in cytokine (IL-8, IL-1β, IL-6, TNF-α, IL-12) levels of BAL fluid between patients with bronchiectasis and healthy controls.
A significant correlation was observed between the HRCT scores and both the percentage and the absolute number of CD4 lymphocytes in BAL fluid in patients with bronchiectasis (r = -0.49, p = 0.009; r = -0.50, p = 0.008; respectively). HRCT scores showed a significant correlation with CD4/CD8 ratios (r = 0.54, p = 0.004) too.
Of measured BAL cytokines, only IL-8 (r = -0.52, p = 0.005) and TNF-aα (r = 0.44, p = 0.01) showed significant correlations with the HRCT scores.
The increased levels of cytokines CD4 lymphocytes in the BAL fluid suggest the possible causative mechanism in the lung in sulfur mustard gas-induced bronchiectasis by the recruitment of neutrophils into the lung.
PMCID: PMC1781448  PMID: 17224076
11.  Correlation of CD4:CD8 ratio and tumour necrosis factor (TNF)α levels in induced sputum with bronchoalveolar lavage fluid in pulmonary sarcoidosis 
Thorax  2000;55(8):696-699.
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)α 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 TNFα 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 TNFα 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 TNFα 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.

PMCID: PMC1745837  PMID: 10899249
12.  Bronchoalveolar lavage fluid cell counts in cryptogenic fibrosing alveolitis and their relation to therapy. 
Thorax  1980;35(5):328-339.
Bronchoalveolar lavage was used to sample inflammatory cells from the lungs of 51 patients with cryptogenic fibrosing alveolitis (CFA) (24 smokers, 12 ex-smokers, and 15 non-smokers). The smokers with CFA have been compared with 15 smoking control subjects in whom there was no radiographic abnormality or clinical evidence of chronic bronchitis. Significantly lower volumes of lavage fluid were recovered from the smokers with CFA (p < 0.001) and the fluid contained lower percentages of macrophages (p < 0.01), reflecting increased percentages of eosinophils (p < 0.001) and neutrophils (p < 0.01). Similar changes were seen in the ex-smokers and non-smokers. There was also an increase in the percentages of lymphocytes when the whole group of CFA patients was compared with the control subjects (p less than or equal to 0.05). No significant differences were found when patients with "lone" CFA were compared with those having associated systemic disease. The only feature distinguishing smokers from non-smokers with CFA was the presence of pigmented cytoplasmic inclusions in the macrophages from the smokers (p < 0.001). However, there were lower numbers of pigmented macrophages in the smoking CFA patients by comparison with the control subjects suggesting either a change in phagocytic capacity or turnover rate in this disease. Profiles of differential cell counts in individual patients showed that increases of eosinophils over 3% or neutrophils over 4% or both with lymphocyte counts of less than 11% related to a poor clinical response to corticosteroids, but lymphocyte percentages greater than 11% related to improvement (p < 0.05).
PMCID: PMC471286  PMID: 7434282
13.  Desensitisation of neutrophil responses by systemic interleukin 8 in cystic fibrosis. 
Thorax  1994;49(9):867-871.
BACKGROUND--Inflammation associated with neutrophil infiltration is a commonly observed feature of children with cystic fibrosis. Production of the major neutrophil chemotactic cytokine interleukin 8 (IL-8) is potentially of great importance in the pathology of cystic fibrosis. Concentrations of IL-8 in both sputum and bronchoalveolar lavage fluid have been found to be higher in children with cystic fibrosis than in controls. The IL-8 induced chemotactic response and numbers of IL-8 receptors on peripheral neutrophils obtained from children with cystic fibrosis have been compared with a control group of children. METHODS--Cells were isolated from 18 patients with cystic fibrosis (aged 4-20 years) and 13 controls (aged 5-12 years) by dextran centrifugation followed by separation on Lymphoprep. Chemotaxis was assayed using multiwell microchemotaxis chambers and 5 microns polycarbonate filters. Filters were fixed and stained with Haema-Gurr for counting. Results were expressed as numbers of neutrophils per high power field (HPF). RESULTS--At the optimum concentration (1 x 10(-8) mol/l) the number of cells migrating were similar for controls (150 (12)/HPF) and for the cystic fibrosis group (140 (14)/HPF)). At lower concentrations the numbers of neutrophils migrating were lower for the cystic fibrosis group. Scatchard analysis of 125I-labelled IL-8 binding revealed lower numbers of receptors on neutrophils from patients with cystic fibrosis (22,000 per cell) than from controls (75,000 per cell). CONCLUSIONS--Reduced responsiveness to IL-8 of neutrophils from patients with cystic fibrosis is associated with receptor desensitisation as a result of exposure to high systemic levels of IL-8.
PMCID: PMC475175  PMID: 7940424
14.  Matrix Metalloproteinase-1 Polymorphism (-1607G) and Disease Severity in Non-Cystic Fibrosis Bronchiectasis in Taiwan 
PLoS ONE  2013;8(6):e66265.
Bronchiectasis is characterized by an irreversible dilatation of bronchi and is associated with lung fibrosis. MMP-1 polymorphism may alter its transcriptional activity, and differentially modulate bronchial destruction and lung fibrosis.
To investigate the association of MMP-1 polymorphisms with disease severity in non-cystic fibrosis (CF) bronchiectasis patients, 51 normal subjects and 113 patients with bronchiectasis were studied. The associations between MMP-1 polymorphisms, lung function, and disease severity evaluated by high resolution computed tomography (HRCT) were analyzed.
The frequency of MMP-1(-1607G) allele was significantly higher in patients with bronchiectasis than normal subjects (70.8% vs 45.1%, p<0.01). Forced expiratory volume in 1 second (FEV1) was decreased in bronchiectasis patients with 1G/1G (1.2±0.1 L, n = 14) and 1G/2G (1.3±0.1 L, n = 66) genotypes compared to the 2G/2G genotype (1.7±0.1 L, n = 33, p<0.01). Six minute walking distance was decreased in bronchiectasis patients with 1G/1G and 1G/2G compared to that of 2G/2G genotype. Disease severity evaluated by HRCT score significantly increased in bronchiectasis patients with 1G/1G and 1G/2G genotypes compared to that of 2G/2G genotype. Bronchiectasis patients with at least one MMP-1 (-1607G) allele showed increased tendency for hospitalization. Serum levels of pro-MMP-1, active MMP-1 and TGF-β1 were significantly increased in patients with bronchiectasis with 1G/1G and 1G/2G genotype compared with 2G/2G genotype or normal subjects. Under IL-1β stimulation, peripheral blood monocytes from subjects with 1G/2G or 1G/1G genotype secreted higher levels of TGF-β1compared to subjects with 2G/2G genotype.
This is the first report to address the influence of MMP-1 polymorphisms on lung function and airway destruction in non-CF bronchiectasis patients. Bronchiectasis patients with MMP-1(-1607G) polymorphism may be more vulnerable to permanent lung fibrosis or airway destruction due to the enhanced MMP-1 and TGF-β1 activity. Upregulated MMP-1 activity results in proteolytic destruction of matrix, and leads to subsequent fibrosis.
PMCID: PMC3679085  PMID: 23776649
15.  Expression of the T Helper 17-Associated Cytokines IL-17A and IL-17F in Asthma and COPD 
Chest  2010;138(5):1140-1147.
Asthma and COPD are characterized by airway dysfunction and inflammation. Neutrophilic airway inflammation is a common feature of COPD and is recognized in asthma, particularly in severe disease. The T helper (Th) 17 cytokines IL-17A and IL-17F have been implicated in the development of neutrophilic airway inflammation, but their expression in asthma and COPD is uncertain.
We assessed IL-17A and IL-17F expression in the bronchial submucosa from 30 subjects with asthma, 10 ex-smokers with mild to moderate COPD, and 27 nonsmoking and 14 smoking control subjects. Sputum IL-17 concentration was measured in 165 subjects with asthma and 27 with COPD.
The median (interquartile range) IL-17A cells/mm2 submucosa was increased in mild to moderate asthma (2.1 [2.4]) compared with healthy control subjects (0.4 [2.8]) but not in severe asthma (P = .04). In COPD, IL-17A+ cells/mm2 submucosa were increased (0.5 [3.7]) compared with nonsmoking control subjects (0 [0]) but not compared with smoking control subjects (P = .046). IL-17F+ cells/mm2 submucosa were increased in severe asthma (2.7 [3.6]) and mild to moderate asthma (1.6 [1.0]) compared with healthy controls subjects (0.7 [1.4]) (P = .001) but was not increased in subjects with COPD. IL-17A and IL-17F were not associated with increased neutrophilic inflammation, but IL-17F was correlated with the submucosal eosinophil count (rs = 0.5, P = .005). The sputum IL-17 concentration in COPD was increased compared with asthma (2 [0-7] pg/mL vs 0 [0-2] pg/mL, P < .0001) and was correlated with post-bronchodilator FEV1% predicted (r = −0.5, P = .008) and FEV1/FVC (r = −0.4, P = .04).
Our findings support a potential role for the Th17 cytokines IL-17A and IL-17F in asthma and COPD, but do not demonstrate a relationship with neutrophilic inflammation.
PMCID: PMC2972626  PMID: 20538817
16.  Persistent sputum cellularity and neutrophils may predict bronchiectasis 
The clinical utility of using quantitative cell counts in sputum for the evaluation of obstructive airway diseases has been previously demonstrated. Although neutrophil counts and activation products have been reported to increase in the sputum of patients with bronchiectasis, it is not known whether these are predictive parameters. This study aimed to assess the accuracy and measurement properties of quantitative sputum cell counts to identify bronchiectasis detected by high-resolution computed tomography scans of the thorax. If validated in larger prospective studies, these parameters may decrease the need for high-resolution scans to detect bronchiectasis and limit radiation exposure.
Quantitative cell counts in sputum provide an accurate assessment of the type and severity of bronchitis.
To examine whether sputum cell counts could identify bronchiectasis in patients with recurrent bronchitis.
A retrospective survey of a clinical database (January 2004 to January 2005) of quantitative cell counts from sputum selected from expectorate in patients with obstructive airways diseases was used to identify predictors of bronchiectasis using ROC curves. This was prospectively evaluated (February 2005 to April 2008) using high-resolution computed tomography scans of thorax that were independently scored by a radiologist who was blinded to the clinical details.
The retrospective survey identified 41 patients with bronchiectasis among 490 patients with airway diseases. Total cell count of 60×106/g or greater of the selected sputum with predominant neutrophils on two occasions had a sensitivity of 86.7%, a specificity of 87.5%, and positive and negative predictive values of 93% and 78%, respectively, to identify bronchiectasis. In the prospective study, 10 of 14 (71%) patients who met these criteria were identified to have bronchiectasis. Both total cell count and the percentage of neutrophils correlated with radiographic bronchiectasis severity.
Persistent or recurrent intense sputum cellularity with neutrophilia is suggestive of bronchiectasis.
PMCID: PMC3205103  PMID: 22059180
Bronchiectasis; Neutrophil; Sputum cell counts
17.  Effectiveness of treatment with high-frequency chest wall oscillation in patients with bronchiectasis 
High-frequency airway clearance (HFCWC) assist devices generate either positive or negative trans-respiratory pressure excursions to produce high-frequency, small-volume oscillations in the airways.
HFCWC can lead to changes in volume of 15–57 ml and in flow up to 1.6 L/s, which generate minimal coughing to mobilize secretions. The typical treatment lasts 20–30 minutes, and consists of short periods of compression at different frequencies, separated by coughing.
The aim of this study was to find the more efficacious treatment in patients with bronchiectasis: traditional techniques of chest physiotherapy (CPT) versus high frequency oscillation of the chest wall in patients with bronchiectasis.
37 patients were enrolled. Seven of them were excluded. Computer randomization divided the patients into three groups:
– 10 patients treated with HFCWO by using the Vest® Airway Clearance System;
– 10 patients treated with traditional techniques of air way clearance (PEP bottle, PEP mask, ELTGOL, vibratory positive expiratory pressure);
– 10 patients received medical therapy only (control group).
To be eligible for enrollment, participants had to be between 18 and 85 years old and have a diagnosis of bronchiectasis, confirmed on high resolution computed tomography. Exclusion criteria: lack of informed consent, signs of exacerbation, cystic fibrosis. Before the treatment, each patient had blood tests, sputum volume and cell count, pulmonary function tests and on the quality of life inventories (MMRC, CAT, BCSS). The results were processed through the covariance analysis, performed with the R-Project statistical program. It has been considered a positive result p <005.
Both treatments (traditional CPT and HFCWO) showed a significant improvement in some biochemical and functional respiratory tests as well as in the quality of life compared to the control group. The use of HFCWO compared to CPT also produced a significant improvement in blood inflammation parameter C-RP (p ≤0.019), parameters of lung functionality associated with bronchial obstruction (FVC, FEV1) (p ≤0.006 and p ≤0.001), and in the dyspnea. Improvement in quality of life scales was noted. (BCSS, CAT) (both p ≤0.001). No significant changes of total cell counts in sputum samples were observed in the two groups. In the HFCWO group a significant reduction of neutrophils percentage (p≤0.002) and a significant increase of macrophages percentage (p ≤0.012).
The HFCWO technique provides an improvement both in pulmonary function and quality of life related parameters in patients with chronic hypersecretive disease. Since those patients need daily airway clearance, this treatment should be included among the principal options in chest physiotherapy. The study was registered as ChiCTR-TRC-12002134 at
PMCID: PMC3623823  PMID: 23556995
Bronchiectasis; High frequency chest wall oscillation; Chest physiotherapy; Lung function; Sputum cell count; Dyspnea scales
18.  Number and activity of inflammatory cells in bronchoalveolar lavage fluid in asthma and their relation to airway responsiveness. 
Thorax  1988;43(9):684-692.
Bronchial responsiveness to inhaled methacholine was measured four to six days before fibreoptic bronchoscopy in 22 asthmatic patients (10 smokers) and 20 control subjects (12 smokers). The asthmatic patients had a baseline FEV1 greater than 60% predicted and a PD20FEV1 (provocative dose of methacholine causing a 20% fall in FEV1) of 0.006-3.7 mg. The 20 control subjects had normal pulmonary function and a PD20FEV1 above the maximum cumulative dose of methacholine of 6.4 mg. Bronchoalveolar lavage of a middle lobe segment (lingula in four subjects) was performed with three sequential 60 ml aliquots of sterile saline. Cellular metabolic activity was stimulated with latex in aliquots of resuspended cells, and measured by means of luminol enhanced chemiluminescence to assess neutrophil activity and lucigenin enhanced chemiluminescence to assess macrophage activity. Mean absolute total cell counts were similar in the asthmatic and control groups but there were differences in differential cell counts, with a significant increase in eosinophil (p less than 0.05) and lymphocyte (p less than 0.005) counts in asthma. PD20FEV1 was negatively correlated with percentage neutrophil counts (p less than 0.005). Luminol enhanced chemiluminescence/1000 neutrophils was increased about twofold in asthmatic subjects (p less than 0.001), but was not correlated with PD20FEV1 Lucigenin enhanced chemiluminescence/1000 macrophages was increased nearly fourfold in asthmatic patients (p less than 0.001) and showed a negative correlation with PD20FEV1 (p less than 0.01). The macrophage count was increased twofold in current smokers in both groups, but other cell numbers were not altered significantly. Smoking did not affect cellular metabolic activity in either group. This study supports the idea that an inflammatory process is present in the airways of those with asthma, and suggests a relation between bronchial responsiveness and both neutrophil numbers and macrophage activity.
PMCID: PMC461456  PMID: 3194874
19.  Comparison of exhaled and nasal nitric oxide and exhaled carbon monoxide levels in bronchiectatic patients with and without primary ciliary dyskinesia 
Thorax  2003;58(1):68-72.
Methods: The levels of exhaled nitric oxide (eNO), carbon monoxide (eCO) and nasal NO (nNO) from bronchiectatic patients with PCD (n=14) were compared with those from patients with non-PCD bronchiectasis without (n=31) and with cystic fibrosis (CF) (n=20) and from normal subjects (n=37) to assess the clinical usefulness of these measurements in discriminating between PCD and other causes of bronchiectasis.
Results: Exhaled NO levels were lower in patients with PCD than in patients with non-PCD non-CF bronchiectasis or healthy subjects (median (range) 2.1 (1.3–3.5) ppb v 8.7 (4.5–26.0) ppb, p<0.001; 6.7 (2.6–11.9) ppb, p<0.001, respectively) but not lower than bronchiectatic patients with CF (3.0 (1.5–7.5) ppb, p>0.05). Nasal levels of nNO were significantly lower in PCD patients than in any other subjects (PCD: 54.5 (5.0–269) ppb, non-PCD bronchiectasis without CF: 680 (310–1000) ppb, non-PCD bronchiectasis with CF: 343 (30–997) ppb, control: 663 (322–1343) ppb). In contrast, eCO levels were higher in all patient groups than in control subjects (PCD: 4.5 (3.0–24.0) ppm, p<0.01, other bronchiectasis without CF: 5.0 (3.0–15.0) ppm, p<0.001; CF: 5.3 (2.0–23.0) ppm, p<0.001 v 3.0 (0.5–5.0) ppm). Low values in both eNO and nNO readings (<2.4 ppb and <187 ppb, respectively) identified PCD patients from other bronchiectatic patients with a specificity of 98% and a positive predictive value of 92%.
Conclusion: The simultaneous measurement of eNO and nNO is a useful screening tool for PCD.
PMCID: PMC1746449  PMID: 12511725
20.  Airway remodelling in children with cystic fibrosis 
Thorax  2007;62(12):1074-1080.
The relationship between airway structural changes and inflammation is unclear in early cystic fibrosis (CF) lung disease. A study was undertaken to determine changes in airway remodelling in children with CF compared with appropriate disease and healthy controls.
Bronchoalveolar lavage and endobronchial biopsy were performed in a cross‐sectional study of 43 children with CF (aged 0.3–16.8 years), 7 children with primary ciliary dyskinesia (PCD), 26 with chronic respiratory symptoms (CRS) investigated for recurrent infection and/or cough and 7 control children with no lower airway symptoms. Inflammatory cells, cytokines, proteases and matrix constituents were measured in bronchoalveolar lavage fluid (BALF). Reticular basement membrane (RBM) thickness was measured on biopsy specimens using light microscopy.
Increased concentrations of elastin, glycosaminoglycans and collagen were found in BALF from children with CF compared with the CRS group and controls, each correlating positively with age, neutrophil count and proteases (elastase activity and matrix metalloproteinase‐9 (MMP‐9) concentration). There were significant negative correlations between certain of these and pulmonary function (forced expiratory volume in 1 s) in the CF group (elastin: r = −0.45, p<0.05; MMP‐9:TIMP‐1 ratio: r = −0.47, p<0.05). Median RBM thickness was greater in the CF group than in the controls (5.9 μm vs 4.0 μm, p<0.01) and correlated positively with levels of transforming growth factor‐β1 (TGF‐β1; r = 0.53, p = 0.01), although not with other inflammatory markers or pulmonary function.
This study provides evidence for two forms of airway remodelling in children with CF: (1) matrix breakdown, related to inflammation, proteolysis and impaired pulmonary function, and (2) RBM thickening, related to TGF‐β1 concentration but independent of other markers of inflammation.
PMCID: PMC2094274  PMID: 17526676
21.  Hyaluronan and type III procollagen peptide concentrations in bronchoalveolar lavage fluid in idiopathic pulmonary fibrosis. 
Thorax  1989;44(2):126-131.
The connective tissue components hyaluronan (hyaluronic acid) and type III procollagen peptide were measured in bronchoalveolar lavage fluid in 22 patients with idiopathic pulmonary fibrosis and 21 healthy control subjects. The patients with idiopathic pulmonary fibrosis had higher concentrations of hyaluronan (median 46 micrograms/l) and type III procollagen peptide (median 0.45 micrograms/l) than the healthy controls (9 and less than 0.02 micrograms/l; p less than 0.001). The patients had normal serum concentrations of hyaluronan and of the procollagen peptide, and albumin concentrations in lavage fluid similar to those of the control subjects. Neutrophil and lymphocyte counts in lavage fluid were increased on average 10 and two fold respectively in the patients with idiopathic pulmonary fibrosis and both correlated with the amount of hyaluronan recovered (p less than 0.05). An inverse correlation was seen between the transfer factor for carbon monoxide and hyaluronan concentrations in lavage fluid in the patients (p less than 0.05). Deterioration in lung function and radiographic progression were seen over six months in 12 of the patients. These patients had higher lavage fluid concentrations of hyaluronan and type III procollagen peptide than the patients whose disease was stable (p less than 0.01). Increased synthesis of hyaluronan and type III procollagen peptide in lung parenchyma may reflect activation or proliferation (or both) of pulmonary fibroblasts in idiopathic pulmonary fibrosis and seems to be linked to the severity and activity of the lung disease.
PMCID: PMC461712  PMID: 2928996
22.  Characterizing Mucous Cell Remodeling in Cystic Fibrosis 
Rationale: Relatively few studies have characterized mucous cells or mucins in detail in cystic fibrosis (CF), and the relationship between mucous cell abnormalities and neutrophilic inflammation is uncertain.
Objectives: To characterize mucous cell phenotypes and mucin profiles in CF and to determine if neutrophils accumulate around goblet cells in the epithelium and gland acini in the submucosa.
Methods: Bronchial biopsies were collected from 7 subjects with CF and 15 control subjects, and the morphology of mucous cells was measured. Immunostains for gel-forming mucins and neutrophil elastase were quantified.
Measurements and Main Results: Goblet cell size was increased in CF (p = 0.004), but the number of goblet cells was normal. The volume of submucosal glands was fourfold higher than normal (p = 0.031), but the proportion of mucous and serous cells in CF glands was normal. The patterns of expression of gel-forming mucins in epithelial and submucosal compartments in CF were similar to normal. Although neutrophil elastase immunostaining was intense in the epithelium in CF, neutrophils were largely absent around gland acini in the submucosa.
Conclusion: The most prominent pathologic feature in the CF airway is an increase in submucosal gland volume, but serous cell transdifferentiation to mucous cells does not occur, nor are gland acini inflamed with neutrophils. The mechanism for increased submucosal gland volume in CF deserves further study.
PMCID: PMC2648101  PMID: 16917116
cystic fibrosis; MUC5AC; MUC5B; neutrophil elastase; submucosal glands
23.  Boswellic acids extract attenuates pulmonary fibrosis induced by bleomycin and oxidative stress from gamma irradiation in rats 
Chinese Medicine  2011;6:36.
Interstitial pulmonary fibrosis is characterized by an altered cellular composition of the alveolar region with excessive deposition of collagen. Lung inflammation is also common in pulmonary fibrosis. This study aims to test the inhibition of 5-lipooxygenase (5-LOX) by boswellic acid (BA) extract in an experimental model of pulmonary fibrosis using bleomycin (BL).
Boswellic acid extract (1 g/kg) was force-fed to rats seven days prior to administration of BL or gamma irradiation or both. BL (0.15 U/rat) in 25 μl of 0.9% normal saline (NS) or 0.9% NS alone was administered intratracheally. Rats were exposed to two fractionated doses of gamma irradiation (0.5 Gy/dose/week) with a gamma cell-40 (Cesium-137 irradiation units, Canada) during the last two weeks of the experiment. BA was administered during BL or irradiation treatment or both. After the animals were sacrificed, bronchoalveolar lavage was performed; lungs were weighed and processed separately for biochemical and histological studies.
In rats treated with BL, levels of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) were significantly elevated (P = 0.05 and P = 0.005). Hydroxyproline was highly and extensively expressed. Immunoreactive compounds were abundantly expressed, represented in the levels of macrophages infiltrate, accumulation of eosinophils and neutrophils in the lung as well as the aggregation of fibroblasts in the fibrotic area. The levels of lipoxygenase enzyme activity were significantly increased (P = 0.005). Antioxidant activities measured in BL-treated rats deteriorated, coupled with the elevation of both levels of plasma lipid peroxide (LP) content and bronchoalveolar lavage lactate dehydrogenase activity. BA-treated rats had reduced number of macrophages, (P = 0.01), neutrophils in bronchoalveolar lavage (P = 0.01) and protein (P = 0.0001). Moreover, the hydroxyproline content was significantly lowered in BA-treated rats (P = 0.005). BA extract inhibited the TGF-ß induced fibrosis (P = 0.01) and 5-LOX activity levels (P = 0.005).
Histologically, BA reduced the number of infiltrating cells, ameliorated the destruction of lung architecture and attenuated lung fibrosis.
BA attenuates the BL-induced injury response in rats, such as collagen accumulation, airway dysfunction and injury. This study suggests that the blocking of 5-LOX may prevent the progression of fibrosis.
PMCID: PMC3199276  PMID: 21961991
24.  Relationships between radiographic change, pulmonary function, and bronchoalveolar lavage fluid lymphocytes in farmer's lung disease. 
Thorax  1986;41(1):28-33.
Ninety four dairy farmers were investigated by chest radiography, pulmonary function tests, and bronchoalveolar lavage. They were divided into five groups--1: 11 subjects with acute farmer's lung; 2: 25 subjects with previously diagnosed farmer's lung who had stayed on their farm; 3: 15 farmers with previously diagnosed farmer's lung who had left the farm; 4: 23 precipitin positive symptomless farmers; 5: 20 precipitin negative symptomless farmers. The study evaluated the relationships between radiographic changes measured with a scoring system derived from the International Labour Office (ILO) classification, the results of pulmonary function tests, and bronchoalveolar lavage fluid. Thirty eight subjects had radiographic evidence of interstitial pulmonary infiltrates. Group 1 had the highest percentage of lymphocytes recovered by bronchoalveolar lavage (mean 66.3 (SD 19.2]. For all subjects carbon monoxide transfer factor (TLCO) and total lung capacity were negatively correlated with radiographic changes (r = -0.45 and -0.30; p less than 0.001 and less than 0.01 respectively). TLCO was also negatively correlated with radiographic change in group 2 (r = -0.59, p less than 0.005). The percentage of lavage lymphocytes was correlated with radiographic changes for all subjects (r = 0.36, p less than 0.001), but this correlation was not seen within groups. This study shows good correlation between radiographic abnormalities, pulmonary function changes and the cellular composition of bronchoalveolar lavage fluid.
PMCID: PMC460248  PMID: 3704964
25.  Effect of exposure to swine dust on levels of IL-8 in airway lavage fluid 
Thorax  1997;52(7):638-642.
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. 

PMCID: PMC1758613  PMID: 9246137

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