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.     

Inflammatory and functional effects of increasing asthma treatment with formoterol or double dose budesonide

Adding a long-acting beta(2)-agonist to inhaled corticosteroids (ICS) for asthma treatment is better than increasing ICS dose in improving clinical status, although there is no consensus about the impact of this regimen on inflammation. In this double-blind, randomized, parallel group study, asthmatics with moderate to severe disease used budesonide (400 mcg/day) for 5 weeks (run-in period); then they were randomized to use budesonide (800 mcg/day - BUD group) or budesonide plus formoterol (400 mcg and 24 mcg/day, respectively - FORMO group) for 9 weeks (treatment period). Home PEF measurements, symptom daily reporting, spirometry, sputum induction (for differential cell counts and sputum cell cultures), and hypertonic saline bronchial challenge test were performed before and after treatments. TNF-alpha, IL-4 and eotaxin-2 levels in the sputum and cell culture supernatants were determined. Morning and night PEF values increased in the FORMO group during the treatment period (p<0.01), from 435+/-162 to 489+/-169 and 428+/-160 to 496+/-173 L/min, respectively. The rate of exacerbations in the FORMO group was lower than in the BUD group (p<0.05). Neutrophil counts in sputum increased in both groups (p<0.05) and leukocyte viability after 48h-culture increased in the FORMO group (p<0.05). No other parameter changed significantly in either group. This study showed that adding formoterol to budesonide improved home PEF and provided protection from exacerbations, although increase of leukocyte viability in cell culture may be a matter of concern and needs further investigation.     

The bacteriology of bronchiectasis in Hong Kong investigated by protected catheter brush and bronchoalveolar lavage

Bacteria often colonize the lower respiratory tract of patients with bronchiectasis. Although the role of these bacteria in the pathogenesis of the disease is uncertain, their accurate identification is important for epidemiologic and treatment purposes. Therefore, the aims of this study were: (1) to identify these bacteria in patients with bronchiectasis without cystic fibrosis using the protected catheter brush (PCB) in order to avoid oropharyngeal contamination, and (2) to compare the results of bronchoalveolar lavage (BAL) with PCB. Quantitative culture was performed on PCB and BAL specimens obtained from the most severely affected lobes of 23 patients with bronchiectasis. Results of PCB showed no significant growth (less than 10(3) colony-forming units [cfu]/ml) in nine patients and 17 significant isolates (greater than 10(3) cfu/ml) in the rest: H. influenzae, 5; P. aeruginosa, 4; K. ozaenae, 2; S. aureus, 2; P. fluorescens, 1; S. pneumoniae, 1; Veillonella, 1; and coag.-ve Staph., 1. For BAL, the results were the same (20 isolates) regardless of whether 10(4) or 10(5) cfu/ml was chosen as the cutoff point. More organisms were cultured from BAL specimens, and these included all but one of the organisms cultured from PCB. We conclude that the bacteriology of bronchiectasis in Hong Kong is different from that reported in sputum studies in the West (mainly H. influenzae, S. pneumoniae, and S. aureus), and with 10(4) cfu/ml as the cutoff point, BAL gives comparable results to PCB.     

The effects of corticosteroid administration on the bronchoalveolar cells obtained from guinea pigs by lung lavage.

The effects of systemic administration of corticosteroids on the bronchoalveolar cell population obtained from guinea pigs by lung lavage were studied. The results of this study demonstrate that in contrast to the marked decrease in the percentage of T lymphocytes in the peripheral blood of animals treated with steroids, there was no significant decrease in the percentage of T lymphocytes in the bronchoalveolar cell population. In addition, corticosteroid administration did not significantly affect either the number of alveolar macrophages obtained by lavage or the Fc receptor activity of these cells. When considered in the context of previously reported data, these results emphasize that caution must be used in reaching conclusions about the integrity of the pulmonary immune response from the results of studies using a bronchoalveolar cell population obtained by lung lavage. This observation must be considered when evaluating the significance of studies of the human pulmonary immune response performed with cells obtained by segmental bronchopulmonary lavage.     

Plasma cells in the bronchoalveolar lavage fluid of a patient with eosinophilic pneumonia. Morphologic proof of local production of antibodies

We describe a case of eosinophilic pneumonia in which plasma cells appeared in bronchoalveolar lavage fluid (BALF). A 49-year-old man presented with wheezing, lung infiltrates, peripheral eosinophilia, and extremely high IgE levels in serum and BALF. A differential count of BALF revealed 56.6 percent lymphocytes and 1.3 percent plasma cells. The appearance of plasma cells suggests local maturation of B cells and represents a morphologic proof of local production of immunoglobulins. The increased number of lymphocytes suggests their role in B cell differentiation via the lymphokine network.     

Elevated levels of antimicrobial peptides in bronchoalveolar lavage fluid in patients with chronic eosinophilic pneumonia

BACKGROUND: Chronic eosinophilic pneumonia (CEP) is an idiopathic pulmonary disease. As the lung is in direct communication with the environment, inhaled antigen may activate immune mechanisms in the airway that may participate in the pathogenesis of idiopathic pulmonary diseases. Defensins are antimicrobial peptides that consist of alpha-defensin (HAD) in neutrophils and beta-defensin (HBD) in epithelial cells. Defensins act as innate immunity against pathogens acquired from the environment and as mediators to induce local inflammation. OBJECTIVES: The aim of the present study was to determine whether immune mechanisms in the airway are induced in CEP patients. METHODS: We measured BALF defensin levels in patients with CEP, acute EP (AEP) and drug-induced eosinophilic pneumonia (drug-EP). We also measured BALF levels of IL-5, GM-CSF, eotaxin and RANTES. These substances can recruit eosinophils. RESULTS: BALF HAD levels were higher in patients with CEP than in those with drug-EP and normal controls. HBD-2 was detected in BALF of 10 of 11 CEP patients and in 3 of 5 AEP patients while its level was below detection in drug-EP patients and normal controls. BALF HBD-2 levels correlated with the proportion of lymphocytes in CEP patients. CONCLUSION: The defensin-linked immune system is activated in CEP but not in drug-EP. This suggests that inhaled antigen(s) may be involved in the pathogenesis of CEP.     

Elevated levels of thymus- and activation-regulated chemokine in bronchoalveolar lavage fluid from patients with eosinophilic pneumonia

Thymus- and activation-regulated chemokine (TARC/CCL17) is a lymphocyte-directed CC chemokine, which plays a role in the recruitment of CC chemokine receptor-4 positive T helper 2 (Th2) cells. In this study, we measured concentrations of TARC and Th2 cell-derived cytokines in bronchoalveolar lavage (BAL) fluid, as well as TARC concentrations in serum from patients with eosinophilic pneumonia and other interstitial lung diseases. TARC was significantly elevated in BAL fluids from patients with eosinophilic pneumonia (median, 240 pg/ml), whereas TARC was undetectable (< 7 pg/ml) in most cases of hypersensitivity pneumonitis, sarcoidosis, and idiopathic pulmonary fibrosis, as well as in healthy control subjects. Also, when present, quantities were less than 20 pg/ml. Elevated concentrations of interleukin (IL)-4, IL-5, and IL-13 were also detected in BAL fluid from patients with eosinophilic pneumonia. Interestingly, TARC concentrations in BAL fluids were closely correlated with the concentrations of IL-5 and IL-13. A serial examination showed that elevated TARC in BAL fluid rapidly fell to below detectable limits preceding decreases in IL-5 concentration and eosinophil percentage. Our results, in concordance with previous studies, demonstrate the potential activity of TARC for recruiting Th2 cells to the lungs and suggest a significant role for TARC in the pathogenesis of eosinophilic pneumonia.     

Studies of bronchoalveolar lavage cells and fluids in pulmonary sarcoidosis. I. Enhanced capacity of bronchoalveolar lavage cells from patients with pulmonary sarcoidosis to induce angiogenesis in vivo

Both allogeneic immunocompetent CD4+ lymphocytes and activated macrophages of mice can induce neovascularization when inoculated intradermally into host animals. Because sarcoidosis is associated with an increase in both activated macrophages and CD4+ effector lymphocytes in the lung, we carried out experiments in which cells obtained by bronchoalveolar lavage (BAL) of patients with pulmonary sarcoidosis were tested in a murine intradermal angiogenesis assay. BAL cells from patients with pulmonary sarcoidosis induced a significantly greater degree of angiogenesis than those from normal volunteers or from patients with other lung diseases. Moreover, the degree of angiogenesis induced by BAL cells from patients with sarcoidosis correlated positively with the severity of the disease. When BAL cells were separated into macrophage and lymphocyte subpopulations by flow cytometric techniques, the observed angiogenic activity was restricted primarily or exclusively to macrophages; lymphocytes were unable to induce angiogenesis in this xenogeneic assay system. These experiments suggest that pulmonary macrophages may play a role in the pathogenesis of sarcoidosis by inducing changes in the pulmonary microvasculature. Moreover, we hypothesize that these vascular changes may be induced not only in the lung but also in other organ systems such as skin, muscle, and eye in which microangiopathies are associated with sarcoid disease.     

The reversible effect of lignocaine on the stimulated metabolic activity of bronchoalveolar lavage cells

Lignocaine concentrations were measured in the aspirate from a low volume (100 ml) bronchoalveolar lavage (BAL) in twenty patients who had received topical 4% lignocaine as required, and compared to those found in the aspirate from a 180 ml BAL in ten patients who had received 1.5% isotonic lignocaine. The median BAL supernatant lignocaine concentration was significantly lower at 0.14 mM (range 0.07-0.44 mM) in the group who received 1.5% lignocaine, compared to 1.08 mM (range 0.03-7.05 mM) in those given 4% lignocaine (p less than 0.01). The effect of increasing concentrations of lignocaine on BAL neutrophil and pulmonary macrophage metabolic activity, as assessed by latex-stimulated luminol- and lucigenin-amplified chemiluminescence (CL), respectively, in mixed BAL cell populations, were measured following preincubation of "washed" harvested BAL cells with 0.4-8.0 mM lignocaine. There was no demonstrable decline in either cell activity with lignocaine concentrations of up to 2 mM, with dose-dependent inhibition of both above this threshold. Cell viability was unaffected. In a further experiment, the inhibition induced by 8 mM lignocaine on both pulmonary macrophage (lucigenin-amplified CL of harvested BAL cells) and isolated peripheral blood neutrophil metabolic activity was completely reversed by a single "wash", following both 30 and 60 min incubations at 4 degrees C, which was equivalent to resuspending harvested BAL cells in fresh medium after separation from BAL supernatant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of lymphocytes and increased interleukin-5 levels in bronchoalveolar lavage fluid in acute eosinophilic pneumonia.

Acute eosinophilic pneumonia (AEP) is a recently described illness and the number of case reports has increased during the last few years. However, the role of interleukin (IL)-5 and activated lymphocytes in the pathogenesis or activity of AEP is still not clear. The clinical features, lymphocyte surface analysis and IL-5 concentrations in bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) of a young female patient with AEP are described before and at 2 weeks, 4 weeks and 6 months after a 3-day course of i.v. methylprednisolone. Serum and BALF concentrations of IL-5 before treatment were 5,200 and 8,400 pg x mL(-1), respectively. Activated CD4 lymphocytes bearing CD25 and human leukocyte antigen (HLA)-DR in BALF were higher than in PB. Treatment caused a rapid fall in these cells and levels of IL-5 in BALF returned to normal levels in parallel with clinical improvement. There was no evidence of recurrence after cessation of steroid therapy. In contrast, eosinophilia in BALF persisted for 4 weeks after steroid therapy in spite of normalization of the chest radiograph and arterial blood gases. The number of CD8+CD11b- (suppressor/cytotoxic) T-cells subsequently increased while the number of CD8+CD11b+ cells decreased. These results suggest that activated CD4 cells and interleukin-5 elevation contribute to the development of acute eosinophilic pneumonia rather than persistent eosinophilia in the lung and that a short course of steroid therapy may effectively control acute eosinophilic pneumonia.     

Elevated uric acid and adenosine triphosphate concentrations in bronchoalveolar lavage fluid of eosinophilic pneumonia

Background

Recent evidence has suggested that the innate immune response may play a role in the development of eosinophilic airway inflammation. We previously reported that uric acid (UA) and adenosine triphosphate (ATP), two important damage-associated molecular pattern molecules (DAMPs), activate eosinophil functions, suggesting that these molecules may be involved in the development of eosinophilic airway inflammation. The objective of this study was to measure the concentrations of DAMPs including UA and ATP in the bronchoalveolar lavage fluid (BALF) of patients with eosinophilic pneumonia (EP).

Characterization of bronchoalveolar lavage cells and macrophage-derived chemoattractant activity in pancreatic elastase-induced emphysema in hamsters

Bronchoalveolar lavage (BAL) was performed 30 days after endotracheal saline (C) or pancreatic elastase (E) administration to hamsters. E animals had twice as many cells as C animals (7.04 +/- 0.07 vs. 6.69 +/- 0.05 log10 cells, p less than 0.0001, paired t-test) and greater numbers of polymorphonuclear neutrophils (PMN) (6.61 +/- 0.05 vs. 5.27 +/- 0.04 log10 cells/animal, p less than 0.0001, t-test). On flow cytometric analysis and cell sorting, BAL cells from C animals showed a homogeneous population of cells with high light scatter and laser-excited intrinsic autofluorescence that were predominantly (greater than 99%) pulmonary alveolar macrophages (PAM). BAL cells from E animals had 80% PAM with characteristics similar to those of C animals; the remaining 20% of cells were smaller with lower forward and 90 degrees light scatter and four- to fivefold lower autofluorescence. These were found to be predominantly PMN on cytological examination of a sorted fraction. Fc receptors were expressed by greater than 95% of BAL cells from both C and E animals. To explain the altered cell population in E animals, PAM from both E and C animals were cultured in vitro, and the supernatants were assayed for neutrophil chemoattractant activity (NCA). In vitro PAM from E animals produced greater NCA than control PAM after 48 h in culture when stimulated with aggregated immunoglobulin or serum activates zymosan (p less than or equal to 0.003, ANOVA). Analyses of BAL supernatants 24 h after elastase administration indicated the presence of a heat-stable chemoattractant for PMN; such chemotactic activity, however, was not detectable 1 week or 4 weeks after the administration of elastase.     

T-cell dominated inflammatory reactions in the bronchi of asthmatics are not reflected in matched bronchoalveolar lavage specimens.

Samples of bronchoalveolar lavage (BAL) and endobronchial biopsies were obtained from five patients with clinically diagnosed asthma (ATS criteria). A comparison was made of the presence and distribution of immunocompetent lymphocytes and macrophages within each sample. Significantly raised numbers of T lymphocytes, CD45RO+ lymphocytes, RFD1+ macrophage-like cells and RFD7+ macrophages were seen in the bronchial biopsies. In contrast four out of five of the BAL specimens showed a normal differential cell count, the one exception being a patient exhibiting a degree of lymphocytosis. Further, immunocytological investigation demonstrated a normal distribution of T-cell subsets and macrophage subsets in asthmatic BAL with the exception that in four out of five of these patients a raised number of macrophage-like cells exhibiting phenotypic markers of monocytes was observed. Correlation between BAL and biopsy data was seen in the number of CD45RO+ T-cells present. No other parameters exhibited a significant correlation. Raised expression of HLA-DR was recorded in all asthmatic biopsies, yet lavage cells from the same patients failed to exhibit any increase of HLA-DR density over normal. It is concluded that the immune-associated inflammation present in endobronchial biopsies of clinically stable asthmatics is not reflected in bronchoalveolar lavage samples taken from the same patients.     

Increased interleukin 6 production by bronchoalveolar lavage cells in patients with active sarcoidosis

Alveolitis of sarcoidosis is characterized by activated alveolar macrophages (AMs) and T cells. The mediators interleukin-1 (IL-1) and interleukin 6 (IL-6) released by AMs represent essential factors for the progression of the T cells in the cell cycle. The role of IL-1 in pulmonary sarcoidosis has previously been studied; however, the relevance of other mediators (i.e. IL-6) has not yet been evaluated. We measured the spontaneous and lipopolysaccharide (LPS)-induced release of IL-6 and tumor necrosis factor a (TNF) by bronchoalveolar lavage cells (BAL) and peripheral blood mononuclear cells (PBMNC) in 6 control subjects (group A) and in 15 patients with sarcoidosis, 10 with active (group B), 5 with inactive disease (group C). IL-6 as well as TNF were spontaneously released by BAL cells of the active group in significantly greater amounts compared to both other groups; IL-6: A, 165.5 pg/ml/24 hr/10⁶ cells (range, 0–604), B, 946 (0–2467), C, 16.6 (0–83); TNF: A, 162 pg/ml/24 hr/10⁶ cells (0–523), B, 803 (100–17352), C, 100 (0–379). In all groups autologous PBMNC proved to be quiescent, releasing only baseline levels of the cytokines tested. After stimulation with LPS all these cells released great quantities of IL-6 and TNF. In active disease a positive correlation between IL-6 and TNF release was observed (r = 0.77, p < 0.02). The present study documents that in active sarcoidosis the spontaneous release of IL-6 by BAL cells parallels the spontaneous release of TNF. IL-6 is capable of initiating the proliferation and activation of T cells in the lung. Offprint requests to: J. Müller-Quernheim     

PGE2 and PGF2 alpha content in bronchoalveolar lavage fluid obtained from patients with eosinophilic pneumonia

Cell differential, prostaglandins (PGs) E2 and F2 alpha in the bronchoalveolar lavage fluid (BALF) in two patients with eosinophilic pneumonia were measured at different times in the course of the disease. Results showed markedly increased numbers of eosinophils and increased content of prostaglandin (PG) E2 in BALF obtained from the patients with eosinophilic pneumonia compared to that of normal volunteers. Interestingly, the increased content of PGE2 in BALF obtained from the patients reverted to normal range with corticosteroid treatment. This change in PGE2 content in BALF was accompanied by normalization of number and percentage of eosinophils in BALF. These findings indicated that PGE2 level in lower respiratory tract of patients with eosinophilic pneumonia may be related to an increased number of eosinophils.     

Allergic and nonallergic asthmatics have distinct patterns of T-cell activation and cytokine production in peripheral blood and bronchoalveolar lavage.

Activation of lymphocyte subpopulations was determined in conjunction with levels of cytokines in peripheral blood and bronchoalveolar lavage (BAL) of asthmatics. Allergic asthmatics had increased numbers of CD4+ IL-2R+ T cells in peripheral blood and BAL, and T-cell activation closely correlated with numbers of low-affinity IgE receptor (CD23) bearing B cells. In contrast, in nonallergic asthmatics both CD4+ and CD8+ T cells from blood and BAL had increased expression of IL-2R, HLA-DR, and VLA-1. Furthermore, in the nonallergic asthmatics CD8+ T cells were decreased in blood but increased in BAL. Cytokine levels were determined in BAL fluid and supernatants from purified peripheral blood T cells and enriched BAL lymphocyte preparations. Allergic asthmatics were characterized by increased levels of IL-4 and IL-5, and this elevated IL-4 contributed to the elevated IgE levels found in these allergic subjects. In contrast, nonallergic asthmatics had elevated levels of IL-2 and IL-5, with IL-2 contributing to T-cell activation. In both types of asthma, the close correlation of IL-5 levels with eosinophilia suggests that IL-5 is responsible for the characteristic eosinophilia of asthma. Thus, we provide evidence of distinct T-cell activation resulting in different spectra of cytokines in allergic and nonallergic asthma.     

Quantification of cells recovered by bronchoalveolar lavage. Comparison of cytocentrifuge preparations with the filter method

Controversy exists as to the appropriate methods to use in the processing of bronchoalveolar lavage (BAL) fluid for total cell numbers and cellular differential analysis. It has been shown that cell losses (primarily lymphocytes) occur by the most commonly employed methods. Therefore, we examined the total cell and differential counts obtained by several methods of cytocentrifuge preparation and by the filter preparation in 46 consecutive patients with interstitial lung disease and 29 healthy volunteers undergoing bronchoalveolar lavage. The retrieved lavage fluid was pooled, and an aliquot was used to determine the total cell count, cell viability, and the differential cell count by the filter and cytocentrifuge techniques. The remaining fluid was centrifuged (800 g for 10 min), and the cell pellet was resuspended in Hank's balanced salt solution without Ca2+ and Mg2+. An aliquot of these centrifuged and resuspended cells was used for repeat determination of the cell viability, total cell count, and cellular differential by cytocentrifuge technique. Autologous serum was added to another aliquot of these centrifuged and resuspended cells to arrive at a 10% protein solution, and the cellular differential obtained by cytocentrifuged preparation.(ABSTRACT TRUNCATED AT 250 WORDS)