Down modulation of L-Selectin expression on eosinophils recovered from bronchoalveolar lavage fluid after allergen provocation

In allergic asthma eosinophils infiltrate into the lung after allergen challenge. The mechanism of this cellular infiltration is not fully understood. L-Selectin is involved in leucocyte-endothelial cell recognition and participates in homing of leucocytes into sites of inflammation. To find indications for a role of L-Selectin in the migration of eosinophils to the bronchoalveolar space we measured L-Selectin expression on eosinophils in peripheral blood and bronchoalveolar lavage fluid (BAL) 4 hr after the early allergic reaction after allergen challenge. Nine patients with allergic asthma participated in the study. An eosinophil specific high depolarization signal enabled us to measure L-Selectin expression on eosinophils in a FACS analysis without isolation of these cells. Eosinophils recovered from BAL showed a strong decrease of L-Selectin expression compared to blood eosinophils. This decrease in L-Selectin expression can be induced in vitro by activation of eosinophils with PMA or FMLP whereas priming of eosinophils during several hours with GM-CSF did not influence L-Selectin expression. Our results are a first indication that L-Selectin may play a role during homing of eosinophils in the lung in asthma after allergen challenge. Moreover, the low expression of L-Selectin on eosinophils in the lung is a further indication that these cells exhibit an activated phenotype.     

Increased CD69 expression on peripheral blood eosinophils after specific inhalation challenge

BACKGROUND: CD69 is a molecule expressed on human eosinophils after cytokine-activation. Different studies have described the eosinophil activation, evaluated by CD69 expression, at the site of an allergic inflammation. In this study we evaluated the expression of CD69 on peripheral blood eosinophils after a specific inhalation challenge (SIC), in order to better define the state of activation of peripheral blood eosinophils after exposure to sensitizers. METHODS: CD69 expression was evaluated by flow cytometry in nine asthmatic patients before and after a positive SIC with high or low molecular weight agents (pollens, house dust mites, Penicillia, isocyanates) and in 11 asthmatic patients who underwent an inhalation challenge with placebo. CD69 expression was evaluated at baseline, 120 min, and 240 min after the SIC or the placebo. RESULTS: Baseline (before challenge) CD69 expression was comparable between the group of SIC positive patients and the placebo group. CD69 expression on peripheral eosinophils significantly increased 240 min after the challenge in positive SIC patients compared to placebo. In patients with a positive SIC the percentage of peripheral blood eosinophils significantly decreased at 120 and 240 min after the inhalation challenge with respect to the baseline. CONCLUSION: CD69 expression on peripheral blood eosinophils is significantly increased in asthmatic patients after exposure to the sensitizing agent. These data show that the effects of a bronchial stimulation are also detectable on peripheral blood eosinophils.     

Lysophosphatidic acid is detectable in human bronchoalveolar lavage fluids at baseline and increased after segmental allergen challenge

BACKGROUND: Lysophosphatidic acid (LPA) is a biologically active lysophospholipid and a component of normal plasma. LPA binds to receptors expressed on circulating and structural lung cells and affects cell growth and activation. Whether LPA is present in the lung has not been previously reported. OBJECTIVE: To develop an assay to measure LPA in bronchoalveolar lavage (BAL) fluids, and to study the association between LPA and allergic airway inflammation. METHODS: Seventeen allergic subjects underwent bronchoscopy and segmental allergen challenge, followed 18 h later by BAL. Supernatants were analysed for LPA content using liquid chromatography and mass spectroscopy. Expression of LPA receptors on primary bronchial epithelial cells was analysed by immunolabelling, and the effects of LPA on epithelial cell barrier function was investigated by measuring transepithelial resistance. RESULTS: LPA was detectable in BAL from control lung segments, and significantly increased 18 h after allergen challenge. Polyunsaturated species of LPA were especially increased following segmental allergen challenge. LPA levels did not strongly correlate with the number or percentages of eosinophils, neutrophils of lymphocytes, whereas MIP-3alpha (CCL20) levels correlated significantly with the allergen-driven influx of lymphocytes. The levels of LPA from control sites correlated inversely with BAL protein content, suggesting that LPA promoted epithelial barrier integrity at baseline. Experiments using primary human bronchial epithelial cells confirmed that LPA tightened the epithelial cell barrier. CONCLUSION: Lysophosphatidic acid is detectable in human BAL fluids at baseline and its expression increases during allergic inflammation. LPA does not appear to be a dominant chemoattractant for eosinophils or lymphocytes during allergic airway inflammation. In the absence of ongoing inflammation, LPA may promote epithelial barrier integrity.     

Interleukin-5 receptors on human lung eosinophils after segmental allergen challenge

BACKGROUND: IL-5 is a specific cytokine for eosinophil accumulation, activation and prolongation of survival and can be recovered in elevated concentrations from the bronchoalveolar compartment in atopic asthma following allergen challenge. OBJECTIVE: The action of IL-5 is mediated via the specific IL-5 receptor-alpha (IL-5Ralpha). Although in vitro data suggest that IL-5R expression is regulated by cytokines such as IL-3, IL-5 and GM-CSF, IL-5R regulation in vivo and its kinetics following allergen provocation are incompletely understood. METHODS: We investigated IL-5R regulation in vivo following segmental allergen provocation (SAP) with an individually standardized dose of allergen in 12 patients with atopic asthma. Lavage was performed 10 min and 18 h (eight patients) and 10 min and 42 h (eight patients) after allergen challenge. In addition to differential cell counts, IL-5Ralpha was measured by flow cytometry and IL-5 concentrations in bronchoalveolar lavage (BAL) fluid were determined by ELISA. RESULTS: IL-5Ralpha expression decreased significantly on peripheral blood and on BAL eosinophils 18 and 42 h after SAP. In contrast, IL-5 concentrations increased significantly in BAL fluid 18 and 42 h after SAP. In four and two patients, respectively, there were detectable IL-5 concentrations in serum 18 or 42 h after allergen exposure. CONCLUSIONS: Although there was no correlation between IL-5 concentrations and IL-5Ralpha expression on eosinophils in BAL, our data support previous in vitro and in vivo findings of a negative feedback mechanism between IL-5 concentrations and IL-5Ralpha expression on eosinophils.     

Surfactant protein levels in bronchoalveolar lavage after segmental allergen challenge in patients with asthma

BACKGROUND: Allergic asthma is associated with airway inflammation and dysfunction of pulmonary surfactant. Because surfactant proteins (SP) account for immunomodulatory functions as well as biophysical functions, we hypothesized that the allergic response in asthma might be accompanied by a dysregulation of SPs. METHODS: We measured levels of SP-A, SP-B, SP-C and SP-D by enzyme-linked immunosorbent assay in bronchoalveolar lavage (BAL) fluid of 23 asthma patients and 10 healthy control subjects under well-controlled conditions before and 24 h after segmental allergen provocation. These data were related to surfactant function, Th(2) cytokine levels in BAL fluid and to the degree of eosinophilic inflammation. RESULTS: In patients with asthma, allergen challenge increased BAL levels of SP-B, SP-C and SP-D while SP-A was decreased. For SP-B and SP-D, a moderate increase was also observed after saline challenge. In contrast, no alterations were observed in healthy control subjects. Levels of SP-B and SP-C in asthmatics correlated with the ratio of small to large surfactant aggregates (SA/LA ratio) and correlated negatively with BAL surface activity. Furthermore, increased SP-C but not SP-B levels after allergen challenge correlated with eosinophil numbers, interleukin (IL)-5, and IL-13 in BAL while increased SP-D levels only correlated with eosinophil numbers. CONCLUSIONS: This study demonstrates significant alterations of all SPs in BAL fluid after allergen challenge of which SP-C was most closely related to surfactant dysfunction and the degree of the allergic inflammation.     

T-cell subtypes in bronchoalveolar lavage fluid and in peripheral blood from patients with primary lung cancer

The changes in local immunology play an important role in lung cancer development. We used bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) for the analysis of cell profiles in patients with primary lung cancer. Twenty-one patients with confirmed primary lung cancer and 13 healthy volunteers were investigated. All persons were smokers. The analysis of T-cell subsets was performed with a flow cytometry method and with the following antibodies: anti CD3, CD4, CD8, CD16, CD25, CD45, CD56, and HLA-DR. We found differences in the proportion of lymphocytes between BALF and PB, and a higher proportion of T cells and a lower proportion of B and natural-killer (NK) cells in BALF. There was a significant difference in the proportion of T-cytotoxic/suppressor lymphocytes, which was elevated in the BALF of patients and decreased in patients' PB. The T-helper:T-cytotoxic/suppressor (Th:Tc/s) ratio was significantly lower in the BALF of patients. These changes were visible in patients with a small cell type. The percentage of T cells with the alpha chain of receptor to IL-2 (IL -R) was lower in the BALF of patients than in the control group. Our observations reflect local changes in lung environment in patients affected with lung cancer.     

Secretion of interleukin-1 receptor antagonist from human mast cells after immunoglobulin E-mediated activation and after segmental antigen challenge.

Mast cells produce substances with antiinflammatory properties in addition to their capacity to release proinflammatory mediators. To further probe the antiinflammatory aspect of mast-cell function we investigated the ability of human mast cells (huMCs) to produce interleukin (IL)-1 receptor antagonist (IL-1ra) in response to high-affinity Fc receptor for immunoglobulin E (Fcalpha RI) aggregation, and examined IL-1ra in bronchoalveolar lavage fluid (BALF) to determine whether it might be of mast-cell origin. Using a ribonuclease protection assay, flow cytometry, and enzyme-linked immunosorbent assay (ELISA), IL-1ra message and protein were found to be constitutively expressed in cultured huMCs. Upon stimulation through Fcalpha RI, IL-1ra message was upregulated in huMCs and IL-1ra protein secreted from cultured huMCs and isolated human lung mast cells. By immunoblot analysis, huMCs were found to produce the 17-kD form of IL-1ra and the presence of IL-1ra in human lung mast cells was confirmed by immunohistochemistry. In BALF obtained from allergic asthmatic subjects, IL-1ra production increased after specific antigen challenge, with the 17-kD isoform of IL-1ra predominating. These findings demonstrate that huMCs produce and release IL-1ra after Fcalpha RI aggregation, which may contribute to a local inhibition of IL-1-dependent effects on inflammation in the lung.     

选择性扩增外周血和支气管肺泡灌洗液中γδT细胞的研究

目的 :探讨选择性扩增外周血和支气管肺泡灌洗液 (BALF)中γδT细胞的方法 ,以获得高纯度的γδT细胞亚群。方法 :应用梯度离心法分离大鼠 (n =10 )外周血和BALF中的单个核细胞 ,经贴壁除去单核 /巨噬细胞后 ,用补体攻击αβT细胞 ,再用抗TCRγδ单克隆抗体 (mAb)通过固相法加IL 2刺激选择性培养扩增γδT细胞 (简称“攻击洗淘法”) ;通过细胞生长曲线观察细胞增殖变化 ,采用免疫组化法和流式细胞仪检测鉴定γδT细胞纯度。结果 :PBMC和BALF中的γδT细胞在抗TCRγδmAb和IL 2存在下 ,无须再用抗原刺激 ,能维持较长时间增殖 ;经“攻击洗淘法”纯化后扩增的γδT细胞纯度达到 81%～99%。结论 :“攻击洗淘法”能高度纯化扩增外周血和BALF中的γδT细胞     

Down-regulated IL-5 receptor expression on peripheral blood eosinophils from budesonide-treated children with asthma

BACKGROUND: The expression and function of cytokine receptors on peripheral blood eosinophils (PBE) from healthy and asthmatic children are poorly characterized. METHODS: The PBE count and expression of IL-5 receptor (R) and GM-CSFR positive PBE was analyzed in nonsteroid-treated asthmatic children (n = 13), budesonide-treated asthmatic children (n = 24) and healthy children (n = 16) by flow cytometry. Alterations in intracellular EG2-epitope expression were used to measure the in vitro responsiveness of PBE to recombinant IL-5 and GM-CSF. RESULTS: The PBE count was increased (P < 0.05) in both asthmatic groups, independent of treatment, as compared to healthy children. The IL-5R expression on PBE, as well as the in vitro responsiveness of PBE to recombinant IL-5, was reduced (P < 0.05), in budesonide-treated asthmatic children compared to nonsteroid-treated asthmatic children and healthy children. The proportion of GM-CSFR positive PBE and in vitro responsiveness of PBE to recombinant GM-CSF were not different between the groups. In vitro treatment with budesonide did not down-regulate the proportion of IL-5R positive PBE. CONCLUSIONS: Budesonide-treatment of asthmatic children induces a selectively reduced IL-5R expression on PBE, concomitant with a reduced in vitro responsiveness of PBE to IL-5. We suggest that this budesonide-related down-regulation of the IL-5R might be a mechanism by which steroid treatment inhibits the action of IL-5 on eosinophil accumulation and activation in vivo.     

Cytokine profile of t lymphocytes from peripheral blood and bronchoalveolar lavage fluid in patients with active pulmonary tuberculosis

The possible immunological relationship between the pattern of Th1/Th2 cytokine production and tuberculin reactivity was assessed in patients with active Mycobacterium tuberculosis infection. The production of the intracellular cytokines interferon (IFN)-gamma and interleukin-4 (IL-4) was measured in CD4(+) and CD8(+) T cells obtained from peripheral blood and bronchoalveolar lavage fluid (BALF) of 20 tuberculin skin-positive patients and compared with the findings recorded in nine tuberculin skin-negative patients with active pulmonary tuberculosis. Upon stimulation with phorbol 12-myristate acetate/ionomycin for 6 h, tuberculin-negative patients had a significantly higher proportion of IFN-gamma-producing CD4(+) T lymphocytes in BALF than in peripheral blood, while both CD4(+) and CD8(+) T-lymphocyte subsets in BALF of tuberculin-positive patients secreted more IFN-gamma than their peripheral blood counterparts. Tuberculin-negative patients had a significantly higher proportion of IFN-gamma-producing CD4(+) T lymphocytes in peripheral blood than tuberculin-positive patients. There was no significant difference in the production of IFN-gamma by BALF CD4(+) T lymphocytes, or by either peripheral blood or BALF CD8(+) T lymphocytes. In two tuberculin-negative patients, peripheral blood CD4(+) T lymphocytes produced IL-4. Study results suggested a higher immune activity in the blood of tuberculin-negative patients, with an increased lymphocyte activity in BALF versus peripheral blood in both patient groups.     

Phenotypic analysis of cells in bronchoalveolar lavage fluid and peripheral blood of maedi visna-infected sheep

A phenolypic analysis of bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) cells in maedi visna virus (MVVVinlecled sheep has been performed. The differential cell eount in BALF from MVV-infeeted animals was characterized by a significant increase (P < 0±05) i n lymphocytes and neutrophils. Lymphocyte phenotyping in BALF from MVV-infeeted sheep showed a significant decrease (P < 0·05) or CD4⁺ cells, a significant increase (P < 0·05) of CD8⁺ eells and a significant inversion (P<0·001) of the CD4⁺/CD8⁺ ratio. CD5⁺ lymphocytes were also significantly decreased (P < 0·05). γδ T cells and B cells did not differ significantly when compared with the controls. No correlation was observed between BALF and PB lymphocyte phenotypes. BALF macrophages from MVV-infeeted animals showed inereased MHC class II expression and BALF lymphocytes from the same animals demonstrated up-regulation of LFA-1 and LFA-3 expression. These findings and their relationship with lentiviral pathogenesis are discussed.     

The effect of immunotherapy on T-cell subsets in peripheral blood and bronchoalveolar lavage fluid in pollen-allergic patients

The effect of immunotherapy (IT) on T-cell subsets in peripheral blood and bronchoalveolar lavage fluid (BAL) was examined in 15 patients with rhinoconjunctivitis and asthma caused by sensitivity to birch pollen. They were treated with IT for 3 years. Seven patients were treated with highly standardized birch-pollen extract (Pharmacia, Sweden). Eight untreated patients served as controls. Histamine challenge, blood sampling, and BAL were performed before (January, February), and at the peak of, the birch-pollen season (May). The subpopulations of T cells in peripheral blood and BAL fluid were investigated by immunocytochemistry and flow cytometry. During the birch-pollen season, the percentage of CD3+ and CD4+ cells of blood mononuclear cells in the IT patients increased significantly ( P <0.03 and P <0.02, respectively). The percentage of CD8 + cells remained unaltered. In control patients, no changes of T-cell subsets in the peripheral blood were observed. T-cell subsets in BAL did not change during the season in relation to preseasonal values for either IT-treated or non-IT-treated patients.     

Microfluidics-based capture of human neutrophils for expression analysis in blood and bronchoalveolar lavage

Gene expression analysis can be a powerful tool in predicting patient outcomes and identifying patients who may benefit from targeted therapies. However, isolating human blood polymorphonuclear cells (PMNs) for genomic analysis has been challenging. We used a novel microfluidic technique that isolates PMNs by capturing CD66b(+) cells and compared it with dextran-Ficoll gradient isolation. We also used microfluidic isolation techniques for blood and bronchoalveolar lavage (BAL) samples of patients with acute respiratory distress syndrome (ARDS) to evaluate PMN genomic alterations secondary to pulmonary sequestration. PMNs obtained from ex vivo lipopolysaccharide (LPS)-stimulated or -unstimulated whole blood from five healthy volunteers were isolated by either dextran-Ficoll gradient, microfluidics capture, or a combination of the two techniques. Blood and BAL fluid PMNs were also isolated using microfluidics from seven hospitalized patients with ARDS. Gene expression was inferred from extracted RNA using Affymetrix U133 Plus 2.0 GeneChips. All methods of PMN isolation produced similar quantities of high-quality RNA, when adjusted for recovered cell number. Unsupervised analysis and hierarchical clustering indicated that LPS stimulation was the primary factor affecting gene expression patterns among all ex vivo samples. Patterns of gene expression from blood and BAL PMNs differed significantly from each other in the patients with ARDS. Isolation of PMNs by microfluidics can be applied to both blood and BAL specimens from critically ill, hospitalized patients. Unique genomic expression patterns are obtained from the blood and BAL fluid of critically ill patients with ARDS, and these differ significantly from genomic patterns seen after ex vivo LPS stimulation.     

Effects of dexamethasone on apoptosis of eosinophils infiltrated into bronchoalveolar lavage fluid after Sephadex bead treatment in rat]

Eosinophilic inflammation of the airways is a key characteristic of asthma. Glucocorticoids can suppress the inflammatory response in part by promotion of eosinophilic apoptosis. We investigated the effects of glucocorticoids on leukocyte infiltration and apoptotic resolution of eosinophils and neutrophils in Sephadex-treated rat lung. Sephadex beads were injected intravenously, followed 24 h later by i.p. administration of dexamethasone (DEX, 0.1 mg/kg) or its vehicle. At 24 h post-DEX treatment, the bronchoalveolar lavage fluid (BALF) was collected. Differential leukocyte counts and the numbers of apoptotic eosinophils and neutrophils, and macrophages with engulfed eosinophils or neutrophils in BALF were determined microscopically from Diff-Quik stained cytospin preparations. Sephadex beads markedly increased cell counts of eosinophils and neutrophils in BALF. Compared with a vehicle-treated group, the DEX treatment significantly decreased the number of eosinophils, but not neutrophils, in BALF. Dexamethasone in BALF also significantly increased eosinophilic apoptosis and engulfment of apoptotic eosinophils by macrophages, but had no effect on neutrophilic apoptosis and engulfment of apoptotic neutrophils by macrophages. These results suggest that the increased clearance of eosinophils from airways by glucocorticoids may be partly due to the promotion of eosinophilic apoptosis.     

IL-9 and IL-9 receptor expression in lymphocytes from bronchoalveolar lavage fluid of patients with interstitial lung disease

IntroductionIL-9, mainly produced by T helper 9 (Th9) cells, promotes allergic airway inflammation and remodeling through the interaction with its receptor (IL-9R). Th9 cells and IL-9 have also been implicated in tissue fibrosis and autoimmunity pathways. However, the role of IL-9/IL-9R in the pathogenesis of interstitial lung disease (ILD) is unknown.AimTo evaluate IL-9/IL-9R expression in bronchoalveolar lavage fluid (BALF) lymphocytes of patients with various ILDs.MethodsConsecutive patients with ILD, who underwent BAL for diagnostic purposes, were studied. As control group, consecutive patients without evidence of ILD were included. Immunocytochemical staining of BALF lymphocytes for IL-9 and IL-9R was performed and evaluated by two independent readers.Results45 patients, of them 8 had idiopathic pulmonary fibrosis (IPF), 12 nonspecific interstitial pneumonia (NSIP), 10 sarcoidosis, 9 hypersensitivity pneumonitis (HP), 6 cryptogenic organizing pneumonia (COP), and 24 controls were studied. In the ILD group, the highest BALF lymphocyte count was seen in HP followed by NSIP, COP, sarcoidosis, and IPF (p < 0.05 for HP vs IPF). The highest percentages of IL-9 and IL-9R positive lymphocytes were seen in COP. Conversely, NSIP showed the lowest rate of IL-9, and sarcoidosis the lowest rate of IL-9R positive lymphocytes. Only in NSIP, a direct correlation between IL and 9 and IL-9R positive lymphocytes was seen (r = 0.639, p = 0.025).ConclusionBALF lymphocytes IL-9 and IL-9R expression differs between various ILDs and could reflect different pathogenetic mechanisms.     

Effect of transportation stress on blood and bronchoalveolar lavage fluid components in calves

The effect of transportation stress on the content of bronchoalveolar lavage (BAL) fluid of 20 male Holstein–Friesian calves, 4–10 months old (mean weight 160 kg) was studied. The calves were healthy and had no previous history of respiratory tract diseases. During a period of 42 days experiment, the calves were kept indoors and were fed alfalfa hay and corn silage ad libitum. After a period of adaptation, on day 21, BAL fluid, blood samples, and nasal swabs were taken from all calves; then, the calves were divided into three groups: experimental (ten calves), which were transported and were deprived of food and water during transportation; control 1 (five calves), which were not transported and had free access to food and water during the 12 h of transportation of the experimental group; and control 2 (five calves), which were not transported but were deprived of food and water for the same time as the experimental group. On day 26, BAL fluid samples and nasal swabs were taken from control group 1. Blood samples were collected simultaneously from all groups at 0, 1, 3, 6, and 12 h of transportation. On days 27, 31, and 42, all previous samplings (BAL fluid, blood, and nasal swabs) were conducted on the experimental group and control group 2. Cytological, biochemical, and bacteriologic examination of BAL fluid and hematological and biochemical examination of blood samples revealed that the number of red blood cells, white blood cells, neutrophils, and the levels of cortisol, packed cell volume, total protein, and fibrinogen significantly increased, but lymphocytes significantly decreased in the experimental group compared with control groups 1 and 2 on the day of transportation (p < 0.05). In addition, regarding BAL fluid content, total cell count, macrophages, neutrophils, and total protein increased in the experimental group (p < 0.05). Pasteurella multocida was isolated from BAL fluid of three calves in the experimental group after transportation. Alteration in BAL fluid components in this study may be due to a depressed efficiency of mucociliary system and/or decreased amount of alveolar spatial surfactant either or both of which may predispose affected livestock to show the presence of P. multocida in bronchoalveolar fluid.     

Rapid isolation of homogeneous murine bronchoalveolar lavage fluid eosinophils by differential lectin affinity interaction and negative selection

Murine models have advanced our understanding of the immune regulation of eosinophilic inflammation but there are few methods for the reliable isolation of viable populations of eosinophils from the inflamed lung. Here we describe a method to isolate murine eosinophils in high yield and purity from lung lavage fluid after induction of eosinophilic inflammation by inhalation of ovalbumin antigen in presensitized BALB/c mice. Thirteen biotinylated plant lectins were screened for their ability to bind selectively alveolar macrophages/monocytes thus permitting the purification of eosinophils by negative selection with streptavidin-conjugated magnetic beads. Bandierea (Griffonia) simplifora isolectin I and, to a lesser extent, Jacalin, provided selective enrichment of viable eosinophils which could be further purified with biotinylated anti-lymphocyte antibodies (up to 98.5% pure). FACS analysis revealed a surface marker phenotype consistent with active effector function (Fas/CD95(+), B7-1/CD80(+), L-selectin/CD62L(Lo), ICAM-1/CD54(+), CD51(+)). Eosinophils retained functional responsiveness, responding to PMA by producing superoxide, as detected by the reduction of dihydrorhodamine-123 to rhodamine. The eosinophils were also able to undergo active apoptosis, as detected by propidium iodide DNA staining, when exposed to a cross-linking anti-Fas antibody, Jo-2. The method may be of general use in studies of murine eosinophil biology.