Prognostic value of neutrophils and NK cells in bronchoalveolar lavage of sarcoidosis

BACKGROUND: Not all the patients with sarcoidosis need pharmacological therapy, and the decision to start therapy is based mainly on clinical conditions. The aim of this study was to evaluate the prognostic value of the leukocyte and lymphocyte subpopulations in the bronchoalveolar lavage fluid from these patients. METHODS: Thirty-three nonsmoking patients with sarcoidosis were included and classified based on the presence of L?fgren's syndrome (n = 11), the radiological stage (12 at Stage I, 17 at Stage II, and 4 at Stage III), and their follow-up. Differential leukocyte subsets and the lymphocyte subpopulations were determined by flow cytometry. RESULTS: The percentage of neutrophils was lower in patients with L?fgren's syndrome (P = 0.038) and in patients at Stage I (P = 0.002). Patients with a poor outcome had a higher percentage of neutrophils (P = 0.004) and NK cells (P = 0.023) than those with a stable disease. Finally, a higher percentage of NK cells was found in those patients who needed a steroid treatment (P = 0.012). CONCLUSIONS: Increased percentages of neutrophils and NK cells in the bronchoalveolar lavage fluid from patients with sarcoidosis are associated with a poor outcome and a higher probability to need steroids treatment. The percentage of neutrophils was also lower in patients with L?fgren's syndrome.     

Neutrophil chemotactic activity produced by normal and activated human bronchoalveolar lavage cells

Activated macrophages can secrete a number of mediators that can attract inflammatory cells and enhance secretion of phlogistic substances from these cells. The ultimate effect of activated bronchoalveolar lavage (BAL) cells may be fibrotic lung injury. Inasmuch as pulmonary sarcoidosis is a disease associated with spontaneous activation of macrophages and lymphocytes among BAL cells, cells obtained from patients with sarcoidosis were compared with normal cells. We report that adherent BAL cells in culture from patients with sarcoidosis (n = 21) release during a resting period in vitro more chemotactic activity for neutrophils (PMNs) than do BAL cells from normal individuals (n = 14). After density fractionation of the respiratory cells by albumin gradient, cells from high-density fractions in the group with sarcoidosis secrete more chemotactic activity for neutrophils than cells from less dense fractions. The PMN chemotactic activity spontaneously released in vitro by BAL cells from patients with sarcoidosis correlates with the percentage of PMNs recovered by BAL. Immunochemical bioassay and high-performance liquid chromatographic (HPLC) analysis of BAL cell supernatants revealed a complex pattern of chemotactic factors to be present. Generally, three peaks of chemotactic activity were noted on HPLC 1-60 separations at greater than 20 kd, 8 to 10 kd, and less than 1 kd apparent molecular weights. Significantly, interleukin-1 was present in these supernatants, whereas complement components and leukotriene B4 were absent. Sarcoid BAL cells, principally alveolar macrophages, are activated in vivo as manifested by spontaneous secretion of chemotactic factors for PMNs in vitro. Interleukin-1 and other less well characterized molecules were detected. The presence of PMNs among the lavage cells of some patients with sarcoidosis appears to be an in vivo biologic correlate of this activation. These data provide additional criteria of BAL cell activation in patients with pulmonary sarcoidosis and provide further evidence concerning factors that attract inflammatory cells into the lung.     

Adenosine induces histamine release from human bronchoalveolar lavage mast cells

Previous studies have shown that in vitro adenosine enhances histamine release from activated human lung mast cells obtained by enzymic dispersion of lung parenchyma. However, adenosine alone has no effect on histamine release from these cells. Given the evidence for direct activation of mast cells after endobronchial challenge with adenosine and previous studies indicating that mast cells obtained at bronchoalveolar lavage are a better model for asthma studies than those obtained by enzymic dispersion of lung tissue, the histamine-releasing effect of adenosine was examined on lavage mast cells. Bronchoalveolar lavage fluid was obtained from patients attending hospital for routine bronchoscopy (n=54). Lavage cells were challenged with adenosine or adenosine receptor agonists (20 min, 37 degrees C) and histamine release determined using an automated fluorometric assay. Endogenous adenosine levels were also measured in lavage fluid (n=9) via an HPLC method. Adenosine alone caused histamine release from lavage mast cells in 37 of 54 patients with a maximal histamine release of 20.56+/-2.52% (range 5.2-61%). The adenosine receptor agonists (R)-N6-(2-phenylisopropyl)adenosine, 5'-N-ethylcarboxamidoadenosine and CGS21680 also induced histamine release from lavage mast cells. Preincubation of lavage mast cells with the adenosine receptor antagonist xanthine amine congener caused significant inhibition of the response to adenosine (P=0.007). There was an inverse correlation between endogenous adenosine levels in the lavage fluid and the maximal response to in vitro adenosine challenge of the lavage cells. The findings of the present study indicate a means by which adenosine challenge of the airways can induce bronchoconstriction and support a role for adenosine in the pathophysiology of asthma. The results also suggest that cells obtained from bronchoalveolar lavage fluid may provide the ideal model for the testing of novel, adenosine receptor, targeted therapies for asthma.     

Bronchoalveolar lavage in sarcoidosis

A characteristic feature of BAL in patients with sarcoidosis is an increase of the total cell number, lymphocytes percent, or CD4/CD8 ratio of T lymphocytes. With respect to T lymphocytes, sarcoidosis has been recognized as a granulomatous disease characterized by dominant expression of Th1 cytokines. Recently Th1 cytokine profile in both CD4+ and CD8+ T lymphocytes in BAL fluid were demonstrated at the single-cell level. Furthermore, alveolar macrophages collected from BAL fluid were also investigated. Increased expression of 25-hydroxyvitamin D3 1 alpha-hydroxylase with mRNA level in alveolar macrophage were revealed in active sarcoidosis. Previously we showed that the B allele of the vitamin D receptor gene polymorphism may be regarded as a risk factor in the onset of sarcoidosis. The metabolism of vitamin D may be related to granuloma formation.     

Leukotriene A4 hydrolase in human bronchoalveolar lavage fluid.

We examined cell-free human bronchoalveolar lavage fluid (BALF) for enzymes of the 5-lipoxygenase pathway. BALF was obtained from six patients who were active smokers and six nonsmokers. Enzymatic activity in cell-free BALF was assessed by specific assays for leukotriene (LT) A4 hydrolase, 5-lipoxygenase, and LTC4 synthase using HPLC. Only LTA4 hydrolase enzymatic activity was found. This activity ranged from 101 to 667 when expressed as picomoles of LTB4 produced per milliliter BALF. Enzymatic activity in smokers vs nonsmokers was 484 +/- 120 vs 129 +/- 32 pmol LTB4/ml BALF (mean +/- SD, P < 0.0001). There were no leukotrienes found in BALF before assay. Immunoblot analysis revealed an immunoreactive band at a relative molecular mass of 69,000 D in all samples, consistent with LTA4 hydrolase, but no evidence of 5-lipoxygenase. BALF had greater LTA4 hydrolase activity per milligram of protein than neutrophil cytosol, epithelial cell cytosol, plasma, or serum. The synthesis of LTB4 was significantly increased when neutrophils were stimulated in BALF. These data indicate the selective presence of LTA4 hydrolase in BALF which is significantly increased in smokers. This enzyme in BALF may contribute to the inflammatory response in tobacco-related lung disease.     

Features of bronchoalveolar lavage differentiating hypersensitivity pneumonitis and pulmonary sarcoidosis at time of initial presentation

To evaluate the usefulness of bronchoalveolar lavage (BAL) as an aid in differentiating hypersensitivity pneumonitis (HP) and pulmonary sarcoidosis (S), we studied the cellular and protein components of BAL and pulmonary function tests (PFT) in 11 patients with acute HP and 23 patients with S at the time of initial presentation and compared the findings with those of normals. The only significant difference between the two groups of patients, on the basis of all the PFT performed, was an exercise-induced hypoxemia PaO2 = 75 +/- 4 torr) in patients with HP that, on average, was not found in patients with S (PaO2 = 88 +/- 3 torr) at the same 1.6 l min-1 O2 consumption. In patients with HP, the total cellular yield of BAL was, on average, 4 times that of patients with S and 6 times that of normals (p less than 0.01 for both). Macrophages, as % of total cells, were reduced in both groups of patients but increased in terms of cells ml-1. Lymphocytes in patients with HP averaged 61.4 +/- 6.5%, in patients with S, 32.6 +/- 4.2%, both values being significantly higher than normal (3.2 +/- 0.9%). However the percentages of lymphocytes in BAL did not clearly separate individual patients with HP from those with S.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of bronchoalveolar cells and fibronectin levels in bronchoalveolar lavage fluids from patients with lung disorders

Differential cell counts and fibronectin levels were recorded in bronchoalveolar lavage fluids (BALF) from patients with lung cancer, idiopathic pulmonary fibrosis (IPF), sarcoidosis, pneumonia, acquired immunodeficiency syndrome (AIDS), and chronic obstructive lung disease (COLD). In all groups fibronectin levels were significantly higher than in the control group; patients with sarcoidosis had a six-fold higher fibronectin level (mean values), AIDS 5.4-fold, pneumonia 4.4-fold, lung cancer, IPF and COLD 2.4-3.0-fold. In control smokers the fibronectin level was significantly higher compared to healthy nonsmokers (p less than 0.002). The increased fibronectin levels could not be explained by contamination of BALF with blood or leakage of plasma proteins. Thus, increased fibronectin levels probably reflect local (e.g. macrophage/fibroblast) synthesis.     

Propofol in bronchoalveolar lavage during anaesthesia

BackgroundThe lung protecting effect of propofol requires methods to measure the propofol concentration of the epithelial line fluid covering the alveolar surface.We hypothesized that (1) propofol can be determined in bronchoalveolar lavage (BAL) by reversed phase high performance liquid chromatography with fluorescence detection. (2) Positive end-expiratory pressure (PEEP) ventilation may have an effect on propofol concentration in BAL (cpB).Methods76 surgical patients were investigated after institutional review board approval. After criteria-based exclusion 45 samples were included. For group I (n = 15) BAL was performed directly after induction, for group Z (n = 15, PEEP = 0 cm H₂O) and P (n = 15, PEEP = 10 cm H₂O) at the end of anaesthesia. BAL and plasma samples were analysed for propofol by reversed phase high performance liquid chromatography with fluorescence detection. Data from all groups were compared by non-parametric Mann–Whitney U-test.ResultsPropofol can be detected in BAL. CpB varied between 23 and 167 μg l^? 1 in all groups. Patients ventilated with PEEP (group P) showed significantly higher cpB (median 74.5 μg l^? 1) compared to those immediately after induction of anaesthesia (median 42.0 μg l^? 1) (group I), but not to those ventilated without PEEP in group Z (median 52.5 μg l^? 1).ConclusionEpithelial line fluid, sampled by BAL, can be used to determine cpB by reversed phase high performance liquid chromatography with fluorescence detection. Continuous propofol infusion and PEEP ventilation may have an effect on cpB.     

Interstitial lung disease. Assessment by bronchoalveolar lavage

Bronchoalveolar lavage is a new bronchoscopic technique that permits assessment of changes in the cellular traffic in the alveolar spaces. During a 16-month period, 120 patients underwent bronchoalveolar lavage at our institution. Control subjects (N = 11) had a predominance of alveolar macrophages (94 +/- 1%) with a few lymphocytes (4 +/- 1%), whereas 35 patients with idiopathic pulmonary fibrosis had a substantial increase in the number of polymorphonuclear leukocytes (17 +/- 2%), and 32 patients with sarcoidosis had an appreciable increase in the number of lymphocytes (27 +/- 2%). Further subtyping of these lymphocytes in 13 patients with sarcoidosis revealed the cells to be predominantly from the T-helper subclass (helper/suppressor ratio of 5.3/1.0; normal 1.8/1.0). In contrast, three other patients with a lymphocytic alveolitis (51 +/- 8% lymphocytes) had a pronounced predominance of T-suppressor lymphocytes (helper/suppressor ratio of 0.1/1.0) in the lavage fluid. Two of the three patients were thought to represent an unusual subset of patients with idiopathic pulmonary fibrosis, and the third patient had pulmonary involvement secondary to angioimmunoblastic lymphadenopathy. Thus, bronchoalveolar lavage may be a useful means by which to assess the influx of inflammatory or immune effector cells into the alveolar structures in patients with interstitial lung disease, and this procedure offers promise as a quantitative means by which to assess the disease activity and the response to therapeutic intervention in these patients.     

Isolation of human alveolar macrophages and lymphocytes from bronchoalveolar lavage fluid by centrifugal elutriation

Cell populations obtained by bronchoalveolar lavage (BAL) from humans showed varying proportions of lymphocytes and macrophages, about 5% and 94%, respectively, in healthy controls (n = 12), and about 24% and 75%, respectively, in patients with sarcoidosis (n = 13). The present study was carried out to examine centrifugal elutriation as a means to obtain from human BAL fluids highly purified populations of viable cells, required for further biochemical investigations. After centrifugation of BAL fluids at 400 g for 5 min, cells were resuspended in Hank-Tris solution, loaded into a Beckman elutriator rotor (rotor speed 2800 rpm) and elutriated with Hank-Tris in approximately 10 min. Cells were collected at flow rates of 20 ml/min (fraction I), 32 ml/min (fraction II) and, after stopping the rotor, 80 ml/min (fraction III). Fraction I contained mainly lymphocytes, about 54% and 74% in controls and sarcoidosis, respectively. Fraction III consisted almost solely of macrophages, while fraction II contained mixed populations. Total cell recovery was about 74% and 55% in controls and sarcoidosis, respectively. Half of the missing cells were lost during centrifugation, the rest during elutriation. Cell viability was 85-90% after the first centrifugation. Following elutriation, the viability was found unchanged in fraction III, but decreased to about 50% in fraction I of controls. The decrease may be due to the accumulation of dead cells with decreased density in this fraction. The results showed that centrifugal elutriation is a highly suitable technique for the isolation of macrophages from human BAL fluids. Enriched lymphocyte populations may be obtained from sarcoid BAL fluids.     

Determination of desmosine in bronchoalveolar lavage fluids by time-resolved fluoroimmunoassay

BACKGROUND: Urinary excretion of desmosine has been reported to be increased in patients with pulmonary fibrosis; however, several investigators have pointed out that measuring urinary desmosine is not a very useful indicator of lung wall destruction. We developed a sensitive time resolved fluoroimmunoassay (TR-FIA) to identify trace amounts of desmosine in bronchoalveolar lavage fluid (BALF), and applied this method to analyse BALF samples from healthy subjects and patients with interstitial lung diseases. METHODS: In the proposed TR-FIA, a polystyrene strip was coated with desmosine-conjugated gelatin. The strip was then incubated with rabbit anti-desmosine antibody and the test solution. The desmosine bound to the solid phase and free desmosine in the sample or standard solution were allowed to compete to bind to the anti-desmosine. The solid-phase antibody was detected by Eu-complex conjugated anti-rabbit IgG. RESULTS: The detectable limit of desmosine was 50 fmol/ml in the TR-FIA developed in this study. TR-FIA showed low cross-reactivity against amino acids. BALF desmosine levels were significantly higher in patients with idiopathic fibrosis and sarcoidosis compared with healthy subjects. CONCLUSIONS: Desmosine levels in BALF may be useful to investigate lung disease.     

肺泡灌洗液中淋巴细胞表型的测定及临床意义

目的 建立以流式细胞仪（ＦＣＭ）检测肺泡灌洗液（ＢＡＬＦ）中淋巴细胞表型的方法并对其临床意义进行研究。方法 采用ＦＩＴＣ＿ＣＤ４５／ＰＥ－ＣＤ１４设门并结合ＰＩ染色排除非淋巴细胞,细胞碎片等对检测的干扰,以ＦＣＭ分析４３例肺部疾病患者ＢＡＬＦ和对照组外周血淋巴细胞上各怕的表达,并与免疫组化检测方法进行方法学比较。结果 ＦＣＭ法与免疫组化法相关良好,其为精密度远优于免疫组化法。４３例ＢＡＬＦ中Ｔ细胞     

Morphological and secretory properties of bronchoalveolar lavage mast cells in respiratory diseases

We have studied various functional and morphological characteristics of mast cells obtained in bronchoalveolar lavage from fifty-two patients with several lung diseases. The percentage of mast cells ranged from 0.04 to 0.6% (bronchial carcinoma), 0.05-0.3% (sarcoidosis), 0.06-0.25% (asthma), 0.04-1.8% (miscellaneous) and 0.02-0.04% (normals). There were no significant differences in the mast cell counts between the disease groups. Lung mast cells exhibited heterogeneity of size, shape and intensity of staining. Cells from thirty-seven subjects were further studied for total histamine content and histamine release using various secretagogues. There was a significant correlation (P less than 0.001) between the histamine content of the total lavage cell population and mast cell counts. The calculated mean histamine content per mast cell was 6.35 pg. Histamine was released in a dose-dependent fashion after stimulation with anti-IgE, calcium ionophore and phorbol myristate acetate with a time course of histamine release characteristic of the mast cell. Unlike peripheral blood basophils, no release was observed following incubation with f-met-leu-phe (10(-6)-10(-8) M) and neither cell type released histamine following incubation with 48/80 (10 micrograms/ml). Inhibition of anti-IgE-induced histamine release was obtained following pre-incubation with salbutamol (10(-4)-10(-6) M). These studies indicate that bronchoalveolar lavage is a suitable model for the study of human lung mast cells.     

Evidence for lipid-associated serine proteases and metalloproteases in human bronchoalveolar lavage fluid

1. We have investigated the nature of elastase activity in bronchoalveolar lavage samples from healthy cigarette smokers and subjects with emphysema. 2. Initial experiments with pure human leucocyte elastase showed this enzyme to be inhibited by high concentrations (greater than 10 mmol/l) of ethylenediaminetetra-acetate, indicating that results of previous studies of 'metalloelastase' activity in bronchoalveolar lavage were ambiguous. 3. We have nevertheless demonstrated the presence in bronchoalveolar lavage of an elastase with the characteristics of a metalloproteinase, although samples also contained a substantial amount of activity that was sensitive to serine proteinase inhibitors. 4. Fractionation of lavage fluid supernatant by size-exclusion chromatography demonstrated most of the elastase activity to be of molecular mass greater than 300 kDa. Treatment of samples with lipase or detergent caused a reduction in metalloelastase activity and the generation of lower-molecular-mass components (90-100 kDa and 40 kDa) which were predominantly serine elastases. This suggested that the enzymes were associated with lipid.     

Inhibition of adenovirus-mediated gene transfer by bronchoalveolar lavage fluid.

Human and animal trials with recombinant adenovirus have been discouraging, since the level of recombinant gene expression was low. Nonspecific and specific immune response mediated by, for example, macrophages, T cells and immunoglobulins may prevent infection or cause death of infected cells. We analyzed the effect of bronchoalveolar lavage fluid (BAL) on the efficiency of adenoviral infection in vitro. A total of 26 BAL samples of randomly selected patients was examined. Adenovirus-mediated gene transfer was quantified using AdCMV. Null, a recombinant adenovirus, in a modified titer assay based on immunocytochemical detection of infected 293 cells. In addition, the concentration of anti-adenovirus-type 5 IgA, IgG and IgM antibodies in BAL was determined by ELISA. 53.8% of the BAL samples (14 out of 26) reduced adenoviral infectivity by at least 50% (factor of inhibition > or = 2). All BAL samples effecting, a reduction in adenoviral infectivity contained detectable amounts of anti-adenovirus-type 5-IgA antibodies. However, the correlation between the concentration of IgA antibody and the strength of inhibition was weak (r = 0.336). Even high levels of anti-adenovirus-type 5-IgM, IgG or IgA antibodies did not influence adenoviral infectivity consistently. This observation indicates that BAL contains (a) anti-adenovirus-type 5 antibodies which are not directed against adenoviral epitopes responsible for the viral adherence and uptake process; and/or (b) inhibitors of viral infectivity different from antibodies.