Mechanisms of neutrophil accumulation in the lungs of patients with idiopathic pulmonary fibrosis.

Neutrophils are a characteristic feature of the alveolitis of idiopathic pulmonary fibrosis (IPF). a chronic disorder limited to lung. One mechanism by which neutrophils may be selectively attracted to lung and not other tissues is via the secretion of the neutrophil-specific chemotactic factor by alveolar macrophages. To evaluate the role of alveolar macrophages in modulating the migration of neutrophils to he lung in IPF, alveolar macrophages, obtained by bronchoalveolar lavage of patients with IPF, were evaluated for their ability to release a chemotactic factor for neutrophils. Unstimulated alveolar macrophages from normal individuals did not release the factor. In patients with IPF, there was a significant correlation between the proportions of neutrophils in lavage fluid and the release of a chemotactic factor for neutrophils by alveolar macrophages (p less than 0.001). The chemotactic factor released by IPF alveolar macrophages was of low molecular weight (400-600), at least partially lipid in nature, and preferentially attracted neutrophils compared with monocytes. Several lines of evidence suggested that immune complexes in the lung stimulated alveolar macrophages of patients with IPF to release the chemotactic factor. First, immune complexes stimulated normal macrophages to release the factor.Second, there was a significant correlation between the release of the chemotactic factor by IPF alveolar macrophages and the levels of immune complexes in bronchoalveolar lavage fluid. Third, bronchoalveolar lavage fluid containing immune complexes stimulated normal macrophages to release the factor. Fourth, IPF alveolar macrophages that released large amounts of the chemotactic factor had an apparent suppression of their immunoglobulin (Ig)G Fc receptor function, suggesting that immune complexes were bound to their surface. In contrast, the IgG Fc receptor function of IPF alveolar macrophages that released only small amounts of the factor was similar to that of normal macrophages. These studies suggest that neutrophils are attracted to the lung in patients with IPF by a potent chemotactic factor released by alveolar macrophages that have been stimulated, in vivo, via their IgG Fc receptor by immune complexes.     

Cellular analysis in bronchoalveolar lavage fluids in infiltrative and fibrotic stages of idiopathic pulmonary fibrosis

We performed cellular analysis of bronchoalveolar lavage fluids (BALF) from 20 patients in different stages of idiopathic pulmonary fibrosis (IPF) and compared the results with those from 16 controls and 10 sarcoidosis patients. Patients with IPF were divided into infiltrative and fibrotic groups as diagnosed by transbronchial lung biopsy and chest x-rays. Total cell numbers counted from BALF in IPF patients were not different from those from controls and were lower than in sarcoidosis patients (p less than 0.01). Lymphocytes in BALF were significantly higher in the infiltrative type IPF than in the fibrotic type (p less than 0.05), but there was no significant difference in fibrotic type IPF and control subjects. Numbers of neutrophils and eosinophils counts were not significantly different between infiltrative type and fibrotic type. The neutrophil count in fibrotic type IPF was higher than in control subjects (p less than 0.01) but not in infiltrative type IPF. 67Ga scintiscanning uptake correlated with the lymphocyte population (r = 0.65, p less than 0.01) but with neutrophils. These findings suggest that lymphocytes play a major role in the pathogenesis of the infiltrative type of IPF and that neutrophils are related to the development of fibrotic changes in IPF.     

两种方法支气管肺泡灌洗患者低氧血症对比分析

目的观察远端导管方法与普通方法支气管肺泡灌洗患者低氧血症的程度。方法随机把呼吸科病房60例肺部感染患者分为远端导管方法支气管肺泡灌洗组(试验组)、普通方法支气管肺泡灌洗组(对照组)各30例,对两种方法患者低氧血症的发生、发生时间及发生程度进行对比分析。结果试验组低氧血症的发生率、发生程度均明显低于对照组,而发生时间和低氧程度差异无统计学意义。结论远端导管方法支气管肺泡灌洗较普通方法支气管肺泡灌洗,可以降低患者低氧血症的发生率和发生程度。     

Local cellular protection in patients with various forms of pulmonary tuberculosis

To study the cellular component of local immune defence, bronchoalveolar lavage fluid (BALF) was examined in 69 patients: 24 children and adolescents with primary tuberculosis and 45 patients with secondary patterns of tuberculosis complicated by catarrhal and purulent endobronchitis. With no appreciable lesions in the bronchial mucosa the cellular composition of BALF and functions of alveolar macrophages (AM) in patients with primary tuberculosis are determined by the phase of specific process. In secondary tuberculosis associated with endobronchitis, the cellular composition of BALF is mainly determined by the character of bronchial pathology whereas AM function by the patterns of both specific and nonspecific processes.     

Morphological characterization of basophilic cells in bronchoalveolar lavage fluids from patients with bronchial asthma and idiopathic pulmonary fibrosis

A marked increase in the number of basophilic cells (BCs) was found in the bronchoalveolar lavage (BAL) fluids from the patients with bronchial asthma and idiopathic pulmonary fibrosis (IPF). Histochemical analysis revealed that basophils were the major components of BCs in asthmatic patients, while formalin-insensitive BCs, which are presumed to be connective tissue mast cells, were observed in BAL fluids from IPF patients. In control subjects, almost all of BCs were mucosal mast cells.     

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.     

Cellular composition and T-lymphocyte subpopulations in the bronchoalveolar lavage fluid in patients with fibrosing alveolitis and pulmonary granulomatosis

Cytological and immunological investigations of bronchoalveolar lavage fluid (BLF) have been performed in 87, 27, 32 and 94 patients with fibrosing alveolitis (FA), exogenous allergic alveolitis (EAA), histiocytosis and pulmonary sarcoidosis (PS), respectively. The diseases appeared to demonstrate significant differences in BLF cell composition and relations between T-lymphocyte theophyllin-resistent and theophyllin-sensitive subpopulations. The findings can be applied in differential diagnosis of disseminated processes in the lungs. The number of neutrophils and the subpopulations correlation in FA and EAA may serve criteria of the disease activity and adequacy of the treatment administered.     

Vascular endothelial growth factor in bronchoalveolar lavage from normal subjects and patients with diffuse parenchymal lung disease

Vascular endothelial growth factor (VEGF) is a potent angiogenic agent that is expressed by epithelial cells in the mature lung of various animal species. We hypothesized that VEGF levels in lower respiratory tract secretions may vary with age or with lung inflammation in human beings. We measured VEGF165 in bronchoalveolar lavage fluid (BALF) from normal volunteers (NVs) of varying age and from patients with cystic fibrosis (CF), sarcoidosis, or idiopathic pulmonary fibrosis (IPF). A considerable gradient in VEGF levels was found with relatively high VEGF concentrations in BALF as compared with serum VEGF. VEGF levels were 303 +/- 34 pg/mL (mean +/- SEM) in serum samples from patients with CF (N = 9) versus 122 +/- 16 pg/mL for the comparable, youngest group of NVs (P < .01). BALF VEGF concentrations were 165 +/- 17 pg/mL for CF upper lobe BALF (N = 9), 140 +/- 17 pg/mL for CF lower lobe BALF (N = 9), and 235 +/- 24 pg/mL for young adult NVs (N = 29). Serum VEGF levels did not differ significantly between NVs and patients with interstitial lung disease, but mean BALF VEGF levels declined significantly with advancing age in NVs and were significantly depressed in patients with IPF (32 +/- 6 pg/mL) as compared with all other groups, including the oldest group of NVs (134 +/- 13 pg/mL, P < .0001). We conclude that a considerable gradient in VEGF concentration exists from epithelial bronchoalveolar surface fluid to serum. Concentrations of VEGF in lower respiratory tract secretions vary with age and are significantly depressed in IPF.     

儿童闭塞性细支气管炎特征及支气管肺泡灌洗

目的 探讨儿童感染后闭塞性细支气管炎(bronchiolitis obliterans,BO)临床特征、支气管肺泡灌洗(bronchoalveolar lavage,BAL)疗效及灌洗液(BAL fluid,BALF)细胞学特点.方法 浙江大学医学院附属儿童医院10例诊断为BO的患儿,均行纤维支气管镜检查.分析其病原、临床特征、肺高分辨CT(HRCT)、血T细胞亚群、BALF细胞学特点及BAL疗效.结果 10例均为重症肺炎后BO,病原为腺病毒4例,肺炎支原体4例,麻疹1例.所有患者均表现反复或持续喘息、咳嗽、气促,两肺广泛哮鸣音和湿啰音.肺HRCT提示马赛克征6例(6/10),两肺炎症伴间质变4例(4/10),伴气体潴留3例,支气管扩张、肺不张、支气管壁增厚各1例.血CD8+T细胞增高9例(9/10)、CD4+/CD8+比值下降10例(10/10).BALF中性粒细胞分类均明显增高(10/10).BAL治疗后,9例临床症状及体征均明显改善而出院.结论 腺病毒和肺炎支原体是儿童感染后BO的主要病原,细胞免疫功能昆乱导致气道炎症损伤可能参与BO的发病机制;血CD8+T细胞及BAL中性粒细胞明显增高预示BO的发生;BAL治疗可明显改善BO症状.     

儿童闭塞性细支气管炎特征及支气管肺泡灌洗

目的 探讨儿童感染后闭塞性细支气管炎(bronchiolitis obliterans,BO)临床特征、支气管肺泡灌洗(bronchoalveolar lavage,BAL)疗效及灌洗液(BAL fluid,BALF)细胞学特点.方法 浙江大学医学院附属儿童医院10例诊断为BO的患儿,均行纤维支气管镜检查.分析其病原、临床特征、肺高分辨CT(HRCT)、血T细胞亚群、BALF细胞学特点及BAL疗效.结果 10例均为重症肺炎后BO,病原为腺病毒4例,肺炎支原体4例,麻疹1例.所有患者均表现反复或持续喘息、咳嗽、气促,两肺广泛哮鸣音和湿啰音.肺HRCT提示马赛克征6例(6/10),两肺炎症伴间质变4例(4/10),伴气体潴留3例,支气管扩张、肺不张、支气管壁增厚各1例.血CD8+T细胞增高9例(9/10)、CD4+/CD8+比值下降10例(10/10).BALF中性粒细胞分类均明显增高(10/10).BAL治疗后,9例临床症状及体征均明显改善而出院.结论 腺病毒和肺炎支原体是儿童感染后BO的主要病原,细胞免疫功能昆乱导致气道炎症损伤可能参与BO的发病机制;血CD8+T细胞及BAL中性粒细胞明显增高预示BO的发生;BAL治疗可明显改善BO症状.     

肺泡巨噬细胞弹性蛋白酶在COPD患者气道炎症和重塑中的作用

[目的]探讨肺泡巨噬细胞(AM)弹性蛋白酶(MMP-12)在慢性阻塞肺疾病(COPD)发生发展过程中的作用.[方法]以24例COPD患者和25名健康者为研究对象,收集其支气管肺泡灌洗液(BALF)和支气管黏膜,对BALF细胞成分进行瑞氏染色、分类计数,用考马斯亮蓝法测定BALF中蛋白的含量,用双抗体夹心ELISA法测定BALF中MMP-12的浓度,用免疫组织化学方法计算气道黏膜中MMP-12阳性AM数和气道黏膜厚度 .[结果]COPD组BALF中细胞总数、AM数及中性粒细胞(PMN)数均高于正常对照组(均P＜0.05);COPD组 BALF和AM培养上清液中MMP-12浓度、气道黏膜中MMP-12阳性AM数及气道黏膜厚度均高正常对照组(均P＜0.01);BALF中MMP-12浓度与 AM培养上清液中MMP-12浓度呈正相关(r=0.678,P＜0.01);COPD组 BALF中MMP-12的浓度及黏膜内MMP-12阳性AM数均与黏膜厚度均呈正相关(r=0.613,P＜0.01; r=0.479,P＜0.05);COPD组BALF中的AM数、MMP-12浓度、黏膜内MMP-12阳性AM数及黏膜厚度均与FEV1%Pre呈负相关(r=-0.488,P＜0.05; r=-0.619,P＜0.01; r=-0.561,P＜0.01;r=-0.642,P＜0.01).[结论]COPD稳定期患者气道内存在以AM和PMN增高为主的慢性非特异性炎症,MMP-12可能参与了其气道炎症和气道重塑的发生发展过程.     

Evaluation of the functional activity of alveolar macrophages based on the results of a study of blood cells]

NBT test basal values and those after cell stimulation with killed BCG culture were measured by spectrophotometry in alveolar macrophages isolated from bronchoalveolar lavage fluid, in peripheral blood neutrophils and monocytes of 49 patients with newly detected pulmonary tuberculosis. Stimulation coefficient was estimated as the ratio of induced to spontaneous NBT-test values. The findings evidence that spontaneous NBT-test values are regularly growing in all three cell types, and the stimulation coefficients lowers as the process grows in severity at the expense of pulmonary tissue disintegration and bronchial obstruction; these parameters adequately reflect the status of the body specific reactivity. Significant correlations were revealed between spontaneous NBT-test parameters in all 3 cell types and their stimulation coefficients. This permits a conclusion that spontaneous and induced NBT-test of the peripheral blood cells may help assess alveolar macrophage function.     

重度烟雾吸入致大鼠急性肺损伤的免疫应答及其机制探讨

目的分析重度烟雾吸入致吸人性急性肺损伤(ALI)对大鼠肺自然免疫及特异性免疫反应。方法分别复制一氧化碳(CO)浓度为2×10~(-3)(低浓度)和4×10~(-3)(高浓度)重度烟雾吸入致大鼠吸人性ALI模型。观察染毒后0～24 h大鼠肺组织病理学变化;检测支气管肺泡灌洗液(BALF)中致炎及抗炎细胞因子的浓度;用流式细胞仪检测外周血及BALF中淋巴细胞亚群数,BALF中CD45~+淋巴细胞和非淋巴细胞数量以及CD4~+/CD8~+变化。结果肺组织病理学检查证实染毒后可致明显肺损伤。染毒2 h BALF中肿瘤坏死因子-α(TNF-α)呈一过性升高,高浓度组较低浓度组更明显,之后下降;4 h白细胞介素-6(IL-6)、γ-干扰素(IFN-γ)开始升高,其中IL-6低浓度组较高浓度组明显,IFN-γ高浓度组较低浓度组明显,至12 h达高峰,24 h开始下降,但仍高于正常对照组水平(P＜0．05或P＜0．01);6～24 h IL-10与正常对照组比较均显著升高,尤以24 h明显(P＜0．05或P＜0．01)。外周血及BALF中CD4~+、CD8~+、自然杀伤细胞、B细胞及总T细胞均较正常对照组明显下降(P＜0．05或P＜0．01)。BALF中CD45~+淋巴细胞数和CD4~+/CD8~+均较正常对照组明显减少,非淋巴细胞数较正常对照组明显增多,且高浓度组较低浓度组变化趋势明显(P＜0．05或P＜0．01)。结论重度烟雾吸入致吸入性ALI的过程中伴有持续且过度的肺自然免疫反应,这种自然免疫反应部分由活化的中性粒细胞及巨噬细胞所介导;而肺特异性免疫反应受到明显的抑制。     

Flow cytometric characterization of cell populations in bronchoalveolar lavage and bronchial brushings from patients with chronic obstructive pulmonary disease

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a serious, chronic inflammatory disease of the airway associated with cigarette smoking. Leucocytes are involved in the inflammatory process in the airways in COPD. There is a need for accurate characterization of cellular populations in bronchoalveolar lavage (BAL) due to variation in the predominant cell types reported, which were investigated mostly with manual counting techniques. METHODS: Bronchial brushings and BAL were obtained from human subjects undergoing fiber optic bronchoscopy. Flow cytometry was applied to identify various cell types. Quenching of autofluorescence of BAL-derived alveolar macrophages was achieved with eta-octyl beta-D-galactopyranoside and crystal violet. Comparisons of cell counts obtained with flow cytometric and manual counting methods were performed. RESULTS: Correlation analysis showed that manual cell counting methods overestimated the percentage of macrophages when compared with flow cytometric methods (R2 = 0.54). There was also a small tendency by manual counting to underestimate the percentage of lymphocytes and neutrophils. Using flow cytometry, the percentage and absolute numbers of alveolar macrophages and lymphocytes in BAL were not significantly different between patients with COPD and control subjects. The percentage and absolute numbers of neutrophils were higher in BAL from patients with moderate to severe COPD. CONCLUSIONS: This novel flow cytometric assay for identification of various cell types from heterogenous samples of BAL and bronchial brushing will allow further investigation of cell characteristics, such as cytokine production and receptor expression, and an accurate evaluation of apoptosis for different cell types and provide a rationale for urgently required effective treatments for COPD.     

Bronchoalveolar lavage in diagnosis of chronic obstructive pulmonary disease]

In addition to understanding the pathophysiology, bronchoalveolar lavage (BAL) and bronchial lavage (BL) are used for the diagnosis of chronic obstructive pulmonary disease (COPD). Abundant neutrophils and mononuclear cells (lymphocytes) with a few of eosinophils are observed in BAL or BL fluids from chronic bronchitis patients, while abundant neutrophils and macrophages are seen in BAL fluids from emphysema patients. COPD patients combined with bronchial asthma show an increase in eosinophil number in BAL or BL fluid. In BAL fluids from diffuse panbronchiolitis (DPB) patients, increased neutrophils alone are observed without any increases in eosinophils or mononuclear cells.     

特发性肺纤维化患者支气管肺泡灌洗液中特异性蛋白标记物研究

目的 分析特发性肺纤维化(IPF)患者支气管肺泡灌洗液(BALF)中蛋白质谱变化,寻找特异性蛋白标记物,评价这些标记物对IPF的诊断价值及临床应用前景.方法 收集IPF患者及对照组BALF标本,应用表面增强激光解析/电离飞行时间质谱(SELDI-TOF-MS)技术筛选差异蛋白.通过非线性支持向量机方法建立诊断模型,并留一交叉验证法评估各模型的预测效果.结果 IPF患者BALF中筛选出8个表达有显著差异的蛋白质峰,其中检测效率最高的4个低表达蛋白质峰质荷比(m/z)分别为1947.634、1975.519、2488.535及4950.230.以这4个蛋白质峰组合建立诊断模型,其特异性和敏感性均为100%.结论 应用SELDI-TOF-MS技术分析获得的低分子量蛋白质峰在诊断中具有较高的灵敏度和特异性,这些小分子可能在IPF发病中起重要作用.     

Bronchoalveolar lavage fluid and plasma proteins, chemiluminescence response and protein contents of polymorphonuclear leukocytes from blood and lavage fluid in traumatized patients

The technique of bronchoalveolar lavage was used to obtain serial samples of lavage every two days from non-contused lung areas of seven traumatized patients and four normals; blood was drawn simultaneously. Urea, total protein, albumin, alpha 1-proteinase inhibitor, alpha 2-macroglobulin, lactate dehydrogenase, beta-N-acetyl-glucosaminidase, myeloperoxidase, and elastase enzyme activity, as well as complexed and total elastase concentrations were determined in bronchoalveolar lavage fluids and plasma samples. Lavage fluid cell pattern was counted. Polymorphonuclear leukocytes were isolated from lavage fluids and blood samples. Granulocyte contents of elastase enzyme activity, complexed and total elastase concentrations, and myeloperoxidase and lactate dehydrogenase activity were determined. Polymorphonuclear leukocyte stimulatory functions were measured by luminol-enhanced chemiluminescence. The following results were obtained for the patient group: Patterns of lavage fluid cells were shifted in favour of polymorphonuclear leukocytes and lymphocytes. The protein determinations of bronchoalveolar lavage fluids and plasma samples gave information about the extent of alterations of permeability of the capillary-interstitial-alveolar space (albumin/urea and alpha 1-proteinase inhibitor/urea ratios) as well as about the amounts of cytoplasmic and lysosomal enzymes released by phagocytes (lactate dehydrogenase/urea, beta-N-acetylglucosaminidase/urea, elastase/urea ratios). Polymorphonuclear leukocytes isolated from bronchoalveolar lavage fluids contained a decreased content of myeloperoxidase and elastase enzyme activities and total elastase concentration; the content of complexed elastase was found to be increased more than 100 fold. From chemiluminescence measurements there was evidence for decreased zymosan-induced stimulatory function, while the photon emission rate of polymorphonuclear leukocytes after passage into the alveolar space was increased.     

Association of reactive nitrogen species metabolites, myeloperoxidase, and airway inflammation in lung transplants.

BACKGROUND: We have previously reported that patients who had single or double lung transplants had higher concentrations than controls of nitrite and nitrate, which are metabolites of reactive nitrogen species (RNS), in bronchoalveolar lavage fluid (BALF) and serum. METHODS: This study investigates implications of RNS metabolites as markers of airway inflammation in a distinct group of lung transplant patients (n = 40). All patients underwent spirometry, routine surveillance transbronchial lung biopsies, and bronchoalveolar lavage as required by clinical protocol. Four normal controls also had bronchoscopy for measurement of BALF nitrite (NO2-) and nitrate (NO3-). BALF NO2- and NO3-, myeloperoxidase (MPO), protein, and urea were assayed. Total nitrite (NO2- plus enzymatically reduced NO3-) and urea were measured in serum. RESULTS: BALF RNS metabolites were mainly NO3-. Forced expiratory volume in 1 s (FEV1) obtained near bronchoscopy was compared with best postoperative FEV1. Total nitrite in transplant patients' BALF and serum were 3.8 +/- 0.2 and 49 +/- 5 microM, respectively. Total nitrite in controls' BALF and serum were 2.2 +/- 0.7 and 19 +/- 2 microM, respectively (P < 0.05 compared with transplant values). Serum total nitrite correlated (Pearson product moment) with percentage of neutrophils in BALF (R = 0.650, P < 0.0001), MPO (R = 0.431, P = 0.0055), change in FEV1 from baseline (deltaFEV1) (R = -0348, P = 0.0298), and days after transplantation (R = 0.345, P = 0.0294). None of the associated variables, airway inflanmmation (quantified as a score, "B"), deltaFEV1, serum, or BALF total nitrite, were explained by infection. Univariate analysis of airway inflammation in patients showed that it was associated with BALF neutrophils, deltaFEV1, and serum total nitrite. CONCLUSIONS: Serum nitrite appears to reflect the degree of airway inflammation in this lung-transplant study group.     

Exhaled markers of oxidative stress in idiopathic pulmonary fibrosis

BACKGROUND: Expired breath condensate (EBC) has never been used to explore the level of oxidative stress in idiopathic pulmonary fibrosis (IPF). Therefore, the aim of this study was to measure the levels of H2O2 and 8-isoprostane, as biomarkers of oxidative stress, in the EBC of patients with IPF. MATERIALS AND METHODS: We investigated 16 patients with IPF and 15 healthy subjects as the control group. The levels of H2O2 and 8-isoprostane were measured in the EBC of all subjects and were compared between the IPF and control groups. In patients with IPF, H2O2 and 8-isoprostane were further correlated with pulmonary function tests (PFTs), the resting pO2 and the differential cell count from the bronchoalveolar lavage fluid (BALF). RESULTS: The mean (95%CI) concentration of H2O2 was increased in the patients with IPF compared with the normal subjects (0.36, 0.24-0.47 microM vs. 0.16, 0.10-0.23 microM, P=0.003). The mean (95%CI) concentration of 8-isoprostane was also increased in the patients with IPF compared with the controls (74, 38-110 pg mL-1 vs. 33, 28-39 pg mL-1, P=0.02). In the patients with IPF, the diffusing capacity of the lung for carbon monoxide was negatively correlated with the levels of H2O2 in EBC (P=0.03, r=-0.58). No other correlation was found between the oxidative stress markers in the EBC and PFT values, pO2 or BALF cell count. CONCLUSIONS: Our data suggest that H2O2 and 8-isoprostane are increased in the EBC of patients with IPF. H2O2 may be correlated with the severity of the disease in IPF.