Relationship between bronchoalveolar lavage neutrophil numbers and lavage fluid elastase and antielastase activities

Elastase and antielastase activities were measured in bronchoalveolar lavage fluid (BALF) and their relationship to bronchoalveolar lavage (BAL) neutrophil numbers was assessed in order to determine whether the elevated BAL neutrophil count can predict a shift in the elastase/antielastase balance. BAL samples were obtained from 133 randomly selected patients undergoing diagnostic bronchoscopy with BAL. Elastase and antielastase activities were determined using the synthetic substrate MeO-Suc-Ala-Ala-Pro-Val-pNA. In a random subset of 24 samples, the antioxidant capacity was measured as the inhibition of peroxyl radical-mediated oxidation of B-phycoerythrin. Only 7 of the BAL samples exhibited measurable elastase activity and all but one of these had a BAL neutrophil count greater than 100 × 103/ml. Antielastase activity was measurable in 124 samples exhibiting no free elastase activity. There was a tendency for lower antielastase activity to be associated with higher neutrophil numbers, but this did not translate into a statistically significant correlation over all samples. There was no significant correlation between antioxidant capacity and either the neutrophil number or antielastase activity. It is concluded that BAL neutrophil numbers do not, in general, predict the status of elastase/antielastase balance in the epithelial lining fluid and that the antioxidant mechanisms in the epithelial lining fluid do not appear to be related to the antielastase capacity.     

Acute effect of smoking on the functional activity of alpha1-protease inhibitor in bronchoalveolar lavage fluid

To assess the acute effect of smoking on the functional activity of alpha1-protease inhibitor (alpha-Pl) in bronchoalveolar lavage (BAL), we studied 38 smokers (mean age 25 +/- 6.5 yr), who had 2 fiberoptic bronchoscopic lavages in sequence, the first after 8 h of abstinence from smoking, and the second at varying time intervals after smoking. Twenty-two smokers were tested before, and 10 min to 3 h after, smoking 2 medium-tar filter cigarettes; 16 smokers were were tested before, and 2 min to 60 min after, smoking 4 cigarettes. Eight nonsmoking volunteers had 2 BAL performed in sequence as control subjects. Initial BAL from control subjects and from smokers after 8 h of abstinence had similar alpha-Pl activity (mean 0.495 +/- SD 0.017 micrograms of pancreatic elastase/micrograms alpha-Pl, about 90% of the activity of purified alpha-Pl). After smoking, we did not find significant inactivation of alpha-Pl except in the 6 smokers lavaged 1 h after smoking 2 cigarettes, whose alpha-Pl activity decreased slightly to 90.0 +/- SD 10.6% of their initial activity (p less than 0.05). We also obtained BAL from 7 smokers only after smoking, and did not find inactivation of alpha-Pl. We conclude that in young healthy smokers: (1) alpha-Pl in BAL after 8 h of abstinence from smoking is active similar to nonsmoking control subjects, and (2) after smoking 2 to 4 cigarettes, there is no, or very limited, inactivation of alpha-Pl.     

Decline in FEV(1) in community-based older volunteers with higher levels of neutrophil elastase in bronchoalveolar lavage fluid

BACKGROUND: Neutrophil elastase (NE) is thought to be one of the key proteinases in the development of chronic obstructive pulmonary disease (COPD). Previously, we have shown that the NE-alpha1-proteinase inhibitor (NE-alpha1PI) complex in bronchoalveolar lavage (BAL) fluid was markedly elevated in asymptomatic smokers who had subclinical emphysema on CT scans. We proposed that excessive NE-alpha1PI complex in BAL fluid was a factor which might differentiate smokers who were developing emphysema from others. OBJECTIVE: In this study, we addressed the question of whether elevated levels of the NE-alpha1PI complex in BAL fluid are linked to the accelerated decline in pulmonary functions in those subjects. METHODS: We conducted a follow-up study to analyze the decline in FEV(1) for 4.3 years on average for 26 community-based volunteers who had received pulmonary function tests, CT scans and BAL. The levels of the NE-alpha1PI complex in BAL fluid and in plasma was measured. RESULTS: Neither pulmonary function measurements nor the presence of emphysema on CT scans could predict the decline in FEV(1). The number of inflammatory cells in BAL fluid was also not an indicator of progression. By contrast, subjects with higher levels of the NE-alpha1PI complex in BAL fluid had a significantly accelerated decline in FEV(1) compared to those with lower levels. CONCLUSION: These data seem to support the hypothesis that NE in the lung is related to the onset and/or progression of COPD.     

A sensitive double-sandwich ELISA for neutrophil elastase. Assay results in unconcentrated bronchoalveolar lavage fluid

We developed a sensitive double-sandwich ELISA assay for neutrophil elastase (NE) using affinity-purified NE antibody. The assay was capable of detecting NE levels of 0.2 ng/ml and was used to determine NE in bronchoalveolar lavages (BAL) of 12 healthy subjects (6 nonsmokers and 6 smokers) with a mean age of about 27 yr. NE levels in the unconcentrated cell-free supernatant of BAL, subjected to high-speed centrifugation (17,000 x g for 30 min) to sediment subcellular debris, were similar in the smokers who abstained overnight from smoking and in the nonsmokers (24.4 +/- 13.9 versus 23.7 +/- 12.3 ng/mg [SD] albumin). NE levels were significantly higher in lavage fluid not subjected to high-speed centrifugation, reflecting the presence of NE bound to subcellular debris that was sedimented by high-speed centrifugation. Concentration by ultrafiltration through a Millipore CX-10 filter was accompanied by loss of protein with a relatively greater loss of NE than albumin, resulting in lower NE/albumin ratios in concentrated than in unconcentrated lavage. It is therefore recommended that NE levels be determined on unconcentrated BAL after high-speed centrifugation to sediment subcellular debris.     

Effects of ageing and smoking on SP-A and SP-D levels in bronchoalveolar lavage fluid.

Surfactant protein (SP)-A and SP-D are collagen-like glycoproteins that are synthesised in the distal pulmonary epithelium. This study examined the effects of ageing and long-term smoking on SP-A and SP-D in the lungs. The possible links to the development of pulmonary emphysema were also investigated. Sequential lavage was performed in young and middle-aged or elderly nonsmokers and asymptomatic current smokers with various smoking histories. Middle-aged or elderly smokers were further categorised according to the presence of emphysema by high-resolution computed tomography. Levels of SP-A and SP-D in bronchial lavage (BL) fluid and in bronchoalveolar lavage (BAL) fluid were quantified by ELISA. Significant decreases in SP-A were seen with age in nonsmokers in BL fluid, but not in BAL fluid. Middle-aged or elderly smokers with emphysema had lower levels of SP-A in both BL and BAL fluids when compared with young subjects, and in BL fluid when compared with middle-aged or elderly smokers without emphysema. SP-D did not change with age alone, however, it was decreased in middle-aged or elderly smokers when compared with similarly aged nonsmokers. In conclusion, surfactant protein-A may decrease with age alone or due to the cumulative effects of long-term smoking and development of emphysema, while surfactant protein-D decreases due to long-term smoking.     

Acute respiratory distress syndrome induced by rifampicin with high levels of neutrophil and eosinophil products in bronchoalveolar lavage fluid

We reported a case with acute respiratory distress syndrome (ARDS) caused by rifampicin during therapy for pulmonary tuberculosis. A high level of eosinophil cationic protein in bronchoalveolar lavage fluid (BALF) was detected as well as interleukin-8 and neutrophil elastase. Based on these results together with the positive result of the drug lymphocyte-stimulating test, we concluded that rifampicin was the causative drug leading to ARDS. Corticosteroid therapy resulted in clinical improvement and resolution of the pulmonary infiltrates on the chest radiograph without the recurrence of pulmonary tuberculosis.     

Cotinine levels in serum and bronchoalveolar lavage fluid

Cotinine is a major metabolite of nicotine. This study was planned to investigate the relationship between bronchoalveolar lavage (BAL) fluid cotinine levels and serum cotinine levels in smokers and nonsmokers with various pulmonary diseases and to investigate whether these levels are affected by passive smoking. Serum and BAL fluid cotinine levels were measured in 27 patients. BAL cotinine levels were measured using a sensitive ELISA kit produced to measure cotinine in saliva. Plates were read by microuant (BioTek, USA) micro plate reader. All patient serum cotinine levels were detectable except for one nonsmoker patient. However, BAL fluid cotinine levels were measurable in only 6 patients (two of them were nonsmokers). A significant positive correlation was seen between serum and BAL fluid cotinine levels (r = 0.726; p = 0.000). Serum cotinine levels were significantly higher in present smokers than non-smokers (21.0 +/- 16.01; 5.35 +/- 7.65; p = 0.004). However, there were no significant differences in BAL fluid cotinine levels between smokers and nonsmokers. Passive smoking can increase nicotine metabolites in serum and other body fluids, including BAL fluid. Since BAL fluid and serum cotinine levels were well correlated, there is no need to use invasive procedures, such as bronchoscopy and expensive, time consuming BAL fluid analyses. Serum cotinine levels can give a rough idea of smoking status. BAL fluid cotinine meaurements should be done for only scientific reasons.     

Cell population and elastase activity in bronchoalveolar lavage fluid from rats exposed to high concentration of nitrogen dioxide

The purpose of this study was to clarify what elastase acts primarily to injure the lung tissue when animals are exposed to nitrogen dioxide (NO2). Rats were exposed to 50 ppm NO2 for 5 hours a day on 7 consecutive days. Approximately six animals were sacrificed after the exposure of the 1st, 3rd, 5th and 7th days. On the 1st day, diffuse alveolar edema was seen accompanying deposits of fibrin nets, suggesting the increase of permeability. Inflammatory cell infiltration was seen in terminal bronchioles and alveolar ducts on the 3rd day, however these changes diminished gradually thereafter. The increase of total cell count was seen in bronchoalveolar lavage fluid on day 3 and thereafter. The number of neutrophils was raised markedly on the 3rd day, whereas that of macrophages showed a gradual increase thereafter. The protein concentration of bronchoalveolar lavage fluid increased markedly on the 1st day and was reduced rapidly thereafter. Elastase activity was measured in bronchoalveolar lavage fluid using succinyl-trialanyl-p-nitroanilide as substrate. Total elastase activity was significantly increased on the 1st and 3rd day. Inhibition studies using EDTA and DFP suggested that most of the elastase activity was due to a metaleoprotease. These data suggest that elastase from macrophage may be mainly related to nitrogen dioxide-induced lung injury.     

Levels of elastase activity in bronchoalveolar lavage fluids of healthy smokers and nonsmokers

Elastase activity was measured in concentrated, cell-free bronchoalveolar lavage (BAL), using the synthetic substrate butyloxycarbonyl-L-alanyl-L-alanyl-L-prolyl-L-valyl-amino-methylcoumarin. The BAL fluids obtained from young, asymptomatic smokers with normal urine desmosine concentrations 1 h after they had smoked 2 cigarettes showed significant increases in elastase levels compared with those in nonsmoking control subjects [nanomoles substrate hydrolyzed (3 h) per milligram lavage albumin = mean 2.7 +/- 1.9 SD (11 smokers) versus 0.5 +/- 0.4 (11 nonsmokers), p less than 0.01]. Repeated BAL samples were obtained at later times from one smoker with a high initial enzyme value and from one nonsmoking control subject. Elastase activity varied over time, but both subjects consistently remained within their respective group ranges. Inhibition studies on pooled BAL from smokers showed that the elastase activity present had properties of both serine and metalloenzymes, suggesting that neutrophils and/or monocytes (serine enzyme) as well as macrophages (metalloenzyme) contributed to the observed activity. Lung lavage cells obtained from 2 of the smokers and 2 of the nonsmokers were stained with both a chromogenic substrate and by indirect immunofluorescence for the serine enzyme. Positively stained neutrophils were readily found in smokers' lavages, but no, or only rare, positive mononuclear cells could be identified. By contrast, peripheral blood mononuclear cells from all 4 subjects stained positively with either method. These results show that some asymptomatic smokers have significantly more elastase activity in their bronchopulmonary secretions than do nonsmokers (as measured with a low molecular weight synthetic substrate). Furthermore, the enzyme activity recovered in smokers' BAL appears to be derived mainly from neutrophils (serine enzyme) and macrophages (metalloenzyme), rather than from monocytes.     

Proangiogenic activity in bronchoalveolar lavage fluid from patients with asthma

RATIONALE: Asthmatic airways have an increased number and size of vascular structures, which contribute to airflow obstruction and hyperresponsiveness. OBJECTIVES: We examined whether proangiogenic mediators are elevated in bronchoalveolar lavage fluid (BALF) from subjects with asthma and if this translated to induction of angiogenesis. METHODS: Angiogenic activity in BALF from 12 healthy, nonatopic subjects and 10 atopic subjects with mild asthma was evaluated by examining tubule formation at 11 days in cocultures of human endothelial cells with dermal fibroblasts. Vascular structures were visualized by anti-CD31 labeling and quantified by image analysis. Angiogenic growth factors in BALF from healthy subjects and subjects with asthma were identified using antibody arrays and by ELISA. MEASUREMENTS AND MAIN RESULTS: Angiogenic activity induced by BALF from healthy subjects was not different from basal tubule formation (p>0.05). However, induction of tubular structures by asthmatic BALF was 2.5-fold greater (p<0.001) compared with healthy samples. Similarly, levels of proangiogenic growth factors (angiogenin, vascular endothelial growth factor [VEGF], monocyte chemotactic protein-1) were increased approximately 2.5-fold (p<0.05) in BALF from subjects with asthma, whereas antiangiogenic factors (endostatin, Ang-2) were unchanged. A blocking anti-VEGF antibody abolished tubule formation induced by BALF from either healthy subjects or subjects with asthma (p<0.01). Immunodepletion of VEGF had no effect on basal tubule formation induced by healthy BALF but abrogated enhanced tubule formation by asthmatic BALF (p<0.01). CONCLUSIONS: BALF collected from subjects with asthma but not healthy subjects is functionally active in promoting angiogenesis in vitro. The proangiogenic capacity of BALF from subjects with asthma resides in elevated VEGF derived from asthmatic airways. This observation supports VEGF as a key factor in vascular remodeling in asthma.     

Acute effect of nitrogen dioxide exposure on the functional activity of alpha-1-protease inhibitor in bronchoalveolar lavage fluid of normal subjects

Nitrogen dioxide is one form of an oxidizing free radical that is sufficiently stable to exist in relatively high concentrations in ambient air and cigarette smoke. We examined the effect of NO2 exposure on the functional activity against pancreatic elastase of alpha-1-protease inhibitor (alpha 1PI) in bronchoalveolar lavage (BAL) fluid of nonsmoking subjects. Ten nonsmokers (mean age, 25 +/- 2 SE yr) were exposed to NO2 (3 or 4 ppm) for 3 h with intermittent exercise. Seven nonsmokers (mean age, 24 +/- 2 SE yr) underwent a similar protocol but were exposed to NO2-free air and served as control subjects. Bronchoalveolar lavage was performed 3.5 to 4 h after the end of exposure. Exposure to NO2 caused a 45% decrease in functional activity of alpha 1PI in BAL. There was no significant difference in immunoreactive alpha 1PI between the groups whether expressed as micrograms per 100 ml of recovered fluid or per milligram of albumin. This inactivation of alpha 1PI was not associated with any neutrophil migration into the air spaces of the lung. The "elastaselike" activity of BAL using synthetic elastinlike chromophore substrate succinyl-trialanine-nitroanilide showed no significant difference between the NO2-exposed group (221 +/- 39 SE ng/dl BAL) and the control group (196 +/- 61 SE ng/dl BAL). Assay for human leukocyte elastase (HLE) in concentrated BAL using the synthetic substrate Methoxysuc-Ala3-Pro-Val-aminomethylcoumarin did not detect any HLE activity in the BAL. These results showed that nonsmoking subjects exposed to relatively low concentrations of NO2 for a short time have a significant inactivation of alpha 1PI in the lower respiratory tract fluid than did nonsmoking control subjects.     

Cell profile and elastase activity in diffuse panbronchiolitis investigated by bronchoalveolar and bronchial lavage.

To examine the mechanism of tissue damage which causes bronchiolectasis in diffuse panbronchiolitis (DPB), the cellular components, elastase and its main inhibitor, alpha 1-protease inhibitor (alpha 1-PI) were measured in bronchoalveolar and bronchial lavage fluid (BALF and BLF) from 14 DPB patients. A predominant increase in the neutrophil count was observed in DPB. Elastase activity in BALF and BLF was about 1,000-fold higher in the DPB group than in the control group. An inhibitor study and a positive correlation between elastase activity and the neutrophil count in both lavage fluids from the DPB group indicated that the activity was mainly that of neutrophil elastase. Western blot analysis of alpha 1-PI showed that most of the alpha 1-PI in the lavage fluids from DPB group was degraded. These results indicated that neutrophil infiltration increases the level of elastase in the DPB lesions; this increase seems to be closely related to tissue damage.     

Antibody activity to Propionibacterium acnes in bronchoalveolar lavage fluid in sarcoidosis

While increased levels of circulating antibody to various microorganisms have been reported in sarcoidosis patients, the pathogenesis of the disease is still unknown. In this report, the levels of antibody activities against Propionibacterium acnes (P. acnes) were measured in bronchoalveolar lavage fluid (BALF) in patients with sarcoidosis, using an enzyme-linked immunosorbent assay method. Each immunoglobulin class of antibody activity to P. acnes was corrected by albumin concentrations in BALF. The levels of whole immunoglobulin antibody activities to P. acnes in BALF were as follows: 412.3 +/- 443.9 O.D./albumin 1 mg (M +/- SD) in 31 untreated sarcoidosis patients, 556.6 +/- 341.8 in 10 sarcoidosis patients treated with prednisolone, and 231.5 +/- 156.8 in 16 control individuals. The levels of antibody activities were significantly elevated in untreated patients (p less than 0.05) and in treated patients (p less than 0.02) compared to those of controls. However, considering the treated vs. untreated patients, there was no significant difference in levels. The serum levels of whole immunoglobulin antibody activities were 0.484 +2- 0.191 O.D. in 38 untreated patients, 0.410 +/- 0.166 in 13 treated patients and 0.571 +/- 0.254 in 52 controls. The levels of antibody activity were significantly lower in treated patients than in the controls (p less than 0.05). However, there was no significant difference between the untreated patients and controls. To assess the site of antibody production, the secretion ratio was calculated by dividing the levels in BALF to those in serum. For this purpose, each serum level of antibody activity was also corrected by serum albumin concentration as with BALF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine deaminase activity in bronchoalveolar lavage fluid of sarcoidosis patients]

We studied adenosine deaminase (ADA) activity in bronchoalveolar lavage fluid (BALF) specimens from 24 patients with sarcoidosis. Mean BALF-ADA activity was significantly (p < 0.0001) elevated in patients with sarcoidosis (1.02 +/- 1.01 IU/L, mean +/- SD) compared to the subjects in a healthy control group (0.08 +/- 0.29 IU/L). In the sarcoidosis patients with high BALF-ADA activity (> or = 1.0 IU/L), AaDO2 was significantly (p < 0.0001) elevated compared to its level in patients with normal BALF-ADA activity (< 1.0 IU/L). BALF-ADA activity was significantly (p < 0.01) higher in patients who exhibited lung-field accumulations on 67Ga scintigrams compared to those with no accumulations, and significantly (p < 0.001) higher in patients undergoing corticosteroid treatment compared to in those patients who did not receive such treatment. These findings were similar to the results of studies using data on BALF-ADA/albumin ratios. Furthermore, they suggest that the localized production of ADA may increase in sarcoidosis patients displaying 67Ga scintigram lung-field accumulations with increased AaDO2, and that BALF-ADA activity may be a useful indicator of disease activity and the need for treatment.     

Immunoglobulin levels in bronchoalveolar lavage fluid of children with chronic chest disease

The concentration and distribution of immunoglobulin isotypes (IgG, IgM, sIgA) and IgG-subclass levels (IgG-1-4) were measured in bronchoalveolar lavage fluid (BALF) in 47 children with chronic chest disease (age range 1.0-9.9 years) and 18 healthy controls (age range 1.0-6.25 years). Of these patients, 19 had nonallergic asthma (Group A), 19 suffered from recurrent pneumonia or chronic bronchitis (Group B), and 9 patients had IgG-2 deficiency (Group C). In all individuals, IgG was the predominant immunoglobulin in the lower respiratory tract, followed by IgA and IgM. In patients of Group A and B, IgG, IgM and IgA levels in BALF were significantly elevated when compared to controls. Assessment of IgG-subclass concentrations in BALF revealed that IgG-1 levels were increased in Group A and B when compared to controls (P < 0.05). Since this difference could not be explained by difference in age, it is possibly due to the inflammatory process at the mucosal level. IgG-2 levels were elevated in all patients except those with IgG-2 deficiency. IgG-2 concentration in the IgG-2 deficent group was lower compared to controls (P < 0.005) and patients in Group A (P < 0.0005) and B (P < 0.005). IgG-3 levels were elevated in asthmatics in group A compared to healthy controls (P < 0.005). IgG-4 concentrations were the same in all study groups. Since IgG-subclasses in percentage of total IgG were similar in BALF and serum, our results do not indicate a local production of any of the IgG-subclasses in the respiratory tract.     

Low neutral endopeptidase levels in bronchoalveolar lavage fluid of lung cancer patients

Neutral endopeptidase (NEP) is a cell surface enzyme found in normal human lung and which hydrolyzes small bioactive peptides, some of which act as growth factors for normal and malignant airway epithelial cells. Expression of NEP varies widely in human lung tissue from different individuals. NEP is often expressed at low or undetectable levels in both small-cell and non-small-cell lung cancer, and inhibits the growth of lung cancer cell lines. Variation in the expression of NEP could be a factor in susceptibility to lung cancer. We hypothesized that NEP could be measured in bronchoalveolar lavage fluid (BALF) and that airway levels of NEP would be low in lung cancer patients as compared with normal controls. We measured NEP and total protein in cell-free BALF supernatant, and expressed the respective concentrations as a ratio. NEP levels showed wide variation in BALF of healthy volunteers. Most patients with lung cancer had no NEP detectable in BALF. The mean NEP/total protein ratio was significantly lower in patients with lung cancer (0.87 +/- 0.7 ng NEP/mg protein) than in normal healthy subjects (14.0 +/- 4.3, p < 0.0003). We conclude that NEP levels are highly variable in BALF of normal volunteers, and are low or undetectable in most BALF specimens from patients with lung cancer. Low NEP levels in the airways may be a factor in the pathogenesis of carcinoma of the lung.     

Inflammatory cytokine levels in induced sputum and bronchoalveolar lavage fluid in pulmonary sarcoidosis

BACKGROUND: The immune inflammatory process in patients with sarcoidosis is not only compartmentalized within the alveolar walls, but also involves the bronchial airways. Analysis of induced sputum has been used as a non-invasive tool for investigating the airways and may reflect the endobronchial and parenchymal inflammation in patients with sarcoidosis. This present study was designed to measure the soluble pro-inflammatory cytokine levels interleukin-1 (IL-1), interleukin-6 (IL-6), tumuor necrosis factor-alpha (TNF-alpha) and percentage of macrophages expressing these cytokines in induced sputum and bronchoalveolar lavage (BAL) fluid in patients with pulmonary sarcoidosis. METHODS: Sputum induction and BAL was carried out in 27 patients with newly diagnosed sarcoidosis. Control group consisted of six patients with a normal chest radiograph (three patients with carcinoma esophagus and three patients with doubtful history of hemoptysis). Induced sputum was also obtained from 10 non-smoking, non-atopic healthy controls. RESULTS: Percentage of macrophages expressing pro-inflammatory cytokines and soluble cytokine levels in induced sputum were higher in patients with sarcoidosis compared to both groups of controls. There was good correlation between IL-6 and TNF-alpha levels (r = 0.49, 0.58 p < 0.05) and percentage of macrophages expressing all three cytokines (r = 0.56-0.71, p < 0.01) between induced sputum and BAL fluid. Mild positive correlation between cytokine levels in sputum and age was also noted (r = 0.33-0.38, p < 0.05). CONCLUSIONS: Induced sputum may reflect changes in cytokine milieu in BAL in sarcoidosis.