Inflammatory mediators in bronchoalveolar lavage samples from children with and without asthma

BACKGROUND: We investigated whether eosinophils and mast cells, found in the airways of children with wheeze, were activated during relatively asymptomatic periods. METHODS: A nonbronchoscopic bronchoalveolar lavage (BAL) procedure was performed on children presenting for an elective surgical procedure. Eosinophil-derived (eosinophil cationic protein, ECP) and mast cell-derived (histamine/tryptase) mediator concentrations were measured in the BAL fluid. A detailed history and serum immunoglobulin E were used to classify the children into four groups: atopic with and without asthma, viral-associated wheeze and normal controls. RESULTS: The ECP concentrations in BAL from atopic asthmatic subjects were significantly higher than those measured in BAL from normal controls (P < 0.01), no other groups differed significantly. Histamine concentrations were elevated in both the atopic asthmatic and viral-associated wheeze groups compared with controls (P < 0.02) and additionally higher concentrations were obtained in atopics with asthma compared with atopics without asthma (P < 0.03). Tryptase concentrations did not differ between groups, although the tryptase and histamine concentrations correlated significantly (r = 0.78, P < 0.0001). CONCLUSIONS: Elevated histamine concentrations were found in children with wheeze regardless of the aetiology, whereas ECP was only elevated in those asthmatics with atopy. This suggests that even in relatively quiescent periods, there is some on going activation of airway eosinophils in children with atopic asthma.     

Pathological changes according to the severity of asthma

Background There have been many studies concerning pathological changes in bronchial mucosa from asthmatics; however, few studies has been carried out to evaluate pathological changes according to the severity of asthma. Objective This study was designed to evaluate the cellular components in bronchoalveolar lavage fluid (BALF) and histologicai abnormalities in asthmatics according to the severity ot asthma. Methods Bronchoalveolar lavages, bronchoscopic biopsies and ultrastructural examinations were performed in 13 asthmatics and 11 (BAL) or four (biopsies) non-asthmatic controls. The proportions of epithelial cells and eosinophils in BALF were significantly increased in asthmatics and showed significant correlations with PC_20Meth which reflects bronchial hyperresponsiveness. Light microscopic examination revealed loss of epithelium, inllammatory cell infiltrations and thickening of the basement membrane which also showed significant correlation with PC_20Meth. Hypertrophy of airway smooth muscles and hyperplasia of mucous glands were prominent in asthmatics but there was no difference according to the severity of asthma. Ultra-structural examination revealed that basement membrane thickening on light microscopic examination is due to the increased subepithelial collagen deposition with normal thickeness of basal lamina. Conclusion These data suggest that loss of epithelial cells, infiltration of inflammatory cells, especially eosinophils, and increased deposition of subepithelial collagen play major roles in determining the severity of asthma and non-specific bronchial hyperresponsiveness.     

Inflammatory cells and eicosanoid mediators in subjects with late asthmatic responses and increases in airway responsiveness

To determine the relationship of inflammatory cells and eicosanoid mediators to the pathogenesis of the late asthmatic response (LAR) and increases in nonspecific airway responsiveness, we studied bronchoalveolar lavage (BAL) cells and fluid in 27 subjects 12 hours after inhaled antigen challenge. Methacholine challenge was performed before antigen challenge and 24 hours later (12 hours after BAL). Eight subjects had no LAR (-LAR, less than or equal to 10% fall in FEV1), nine subjects had an equivocal LAR (+/- LAR, 11% 25% fall in FEV1), and 10 subjects had a definite LAR (+LAR, greater than 25% fall in FEV1). Subjects developing +LAR had increased airway responsiveness at baseline compared with that of subjects developing an +/- LAR, but not with subjects having -LAR. If airway responsiveness was markedly increased at baseline, further increases after antigen challenge were often not observed. We found that both percent neutrophils and eosinophils increased in BAL as the severity of the LAR increased, but significant differences between the groups with -LAR and +LAR were only observed when both cell types were considered together. In addition, there was a significant correlation between the combined cell percentages and the severity of the LAR as determined by fall in FEV1. Likewise, increases in airway responsiveness were associated with significant increases in both neutrophil and eosinophil numbers, but only neutrophils correlated with the change in airway responsiveness after antigen challenge. However, despite the significant physiologic and cellular differences that we found between our groups, no significant differences could be found in BAL eicosanoid-mediator concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of immunotherapy on the inflammation in pollen asthma

Seasonal asthma is a natural model of airway inflammation where amount of airborne antigen have direct impact and correlates to the clinical disease and degree of inflammation. Bronchial hyperresponsiveness (BHR) is an indirect measure of the degree of airway inflammation and the seasonal increase paralleling clinical disease has been shown in a number of studies. Using a model of seasonal asthma in birch pollen sensitive individuals we intended to prove the anti-inflammatory potential of immunotherapy (IT). Forty birch pollen-allergic patients, 20 IT-treated and 20 untreated participated in a series of studies. BHR expressed as PCzo histamine increased in all patients during the season although most in untreated controls; the difference between the groups was p <0.07. The level of eosinophil cationic protein (ECP) rose in untreated, but not IT-treated, patients during the season (p <0.05). Significant increase in eosinophil chemotactic activity (ECA) in serum of untreated patients during the season was noted. IT-treatment abrogated this response. After 2 years of treatment bronchoalveolar lavage (BAL) was performed on 12 patients (6 in each group). IT-treatment prevented seasonal rise of eosinophil numbers in peripheral blood (PB) and the lung while in the untreated group the increase was significant in both compartments (p <0.05 and p<0.01, respectively). At the same timc, significant increment in ECP and ECA levels in BAL wcre rccordcd in untreated patients comparcd with IT-treated (p <0.05. p <0.01, respectively). It is concluded that IT proved its anti-inflammatory effect by diminishing degree of BHR, prcventing attraction. accumulation and activation of eosinophils in the lung of allergic I asthmatics.     

Lidocaine in bronchoalveolar lavage fluid (BALF) is an inhibitor of eosinophil-active cytokines

Eosinophils and eosinophil granule proteins may play an important role in the pathogenesis of asthma. BALF from 40 patients with symptomatic asthma were analysed for cytokine activity by the eosinophil survival assay. BALF from 15 patients showed increased survival activity. Survival activities in BALF from four of these patients were almost completely blocked by anti-IL-5 MoAb, and the remaining activities were blocked by anti-granulocyte-macrophage colony-stimulating factor (GM-CSF), anti-IL-3 antibody, or both. Surprisingly, BALF samples from the other 25 patients decreased eosinophil viabilities below the levels of medium control. The inhibitory factor in these BALF was of low molecular weight, was heat-stable, was largely overcome by excess exogenously added cytokines, and was positively correlated with the concentrations of lidocaine in the BALF. Lidocaine itself inhibited eosinophil survival at concentrations less than those present in the BALF. These findings indicate that lidocaine is an inhibitor of cytokines in the eosinophil survival assay, and they suggest the need for caution in analyses of BALF containing lidocaine or other local anaesthetics.     

The 3111 Clock gene polymorphism is not associated with sleep and circadian rhythmicity in phenotypically characterized human subjects

Mutations in clock genes are associated with abnormal circadian parameters, including sleep. An association has been reported previously between a polymorphism (3111C), situated in the 3'-untranslated region (3'-UTR) of the circadian gene Clock and evening preference. In the present study, this polymorphism was assessed in: (1) 105 control subjects with defined diurnal preference, (2) 26 blind subjects with free-running circadian rhythms and characterized with regard to circadian period (tau) and (3) 16 delayed sleep phase syndrome patients. The control group was chosen from a larger population (n = 484) by Horne-Ostberg questionnaire analysis, from which three subgroups were selected (evening, intermediate and morning preference). Data from sleep diaries completed by 90% of these subjects showed a strong correlation between preferred and estimated timings of sleep and wake. The mean timings of activities for the evening group were at least 2 h later than the morning group. Genetic analysis showed that, in contrast with the previously published finding, there was no association between 3111C and eveningness. Neither was there an association between 3111C and tau, nor a significant difference in 3111C frequency between the normal and delayed sleep phase syndrome groups. To assess the effect of this polymorphism on messenger RNA (mRNA) translatability, luciferase reporter gene constructs containing the two Clock polymorphic variants in their 3'-UTR were transfected into COS-1 cells and luciferase activity measured. No significant difference was observed between the two variants. These results do not support Clock 3111C as a marker for diurnal preference, tau, or delayed sleep phase syndrome in humans.     

Inflammatory response in bronchoalveolar lavage fluid after inhaling histamine

To examine the influence of the histamine chloride challenge test on the bronchoalveolar lavage cell population, lavage fluid from 15 subjects was collected 24 h after the histamine test, and was compared with the lavage fluid from a reference group of 25 subjects. Inhaled histamine is commonly used to quantitate non-specific bronchial responsiveness. Increase in airway responsiveness after exposure to ozone or allergen is associated with airway inflammation. Bronchoalveolar lavage, has therefore become a valuable tool in the study of bronchoalveolar cells and mediators in subjects with asthma and bronchial hyperresponsiveness. The total cell number and differential cell counts in bronchoalveolar lavage fluid 24 h after inhalation challenge test with histamine-chloride were studied. There was a significant increase in lymphocytes, mast cells and neutrophils after histamine test. The conclusion was that inhaled histamine-chloride can induce an inflammatory cell response in the lung. Thus the histamine-chloride test should not be performed before bronchoalveolar lavage.     

Interleukin-4 and -13 expression is co-localized to mast cells within the airway smooth muscle in asthma

BACKGROUND: Airway smooth muscle infiltration by mast cells is a feature of asthma and not eosinophilic bronchitis. In asthma, Th2 cytokines have been implicated as playing a critical role in the development of airway inflammation and hyper-responsiveness. Whether inflammatory cells within the airway smooth muscle release these cytokines is unknown. METHODS: We have undertaken a comparative immunohistochemical study in bronchial biopsies from 14 subjects with asthma, 10 with eosinophilic bronchitis and eight normal controls recruited from two centres. RESULTS: The median number of IL-4+ cells/mm2 smooth muscle was significantly higher in subjects with asthma than eosinophilic bronchitis and normal controls for both the anti-IL-4 mAb 3H4 (2.4, 0, 0, respectively; P=0.001) and anti-IL-4 mAb 4D9 (1.6, 0, 0, respectively; P=0.02). There were no group differences in the number of IL-5+ cells (P=0.31). In six subjects with asthma, IL-13 expression by cells within the airway smooth muscle was studied. The median (range) of IL-13+cells was 2 (0.9-2.7). Ninety-four percent of the cells expressing IL-4 (3H4), 92% of those expressing IL-4 (4D9) and 100% expressing IL-13 in the airway smooth muscle were mast cells. Fifty-five percent of the mast cells within the airway smooth muscle co-localized to IL-4 (3H4), 29% to IL-4 (4D9) and 17% to IL-13. CONCLUSIONS: In asthma, IL-4+ and IL-13+ cells were present within the airway smooth muscle and were expressed predominantly by mast cells, suggesting that IL-4 and IL-13 may play an important role in mast cell-airway smooth muscle interactions.     

Inflammatory biomarkers in airways of patients with severe asthma compared with non-severe asthma

Background About 5–10% of patients with asthma suffer from poorly-controlled disease despite corticosteroid (CS) therapy.
Objective We determined whether there were any differences in inflammatory biomarkers between severe and non-severe asthma patients.
Methods Nineteen severe and 20 non-severe asthma patients were recruited and underwent collection of induced sputum, bronchoalveolar lavage (BAL) fluid and bronchial biopsies.
Results Biopsy results showed no differences in eosinophils (major basic protein positive), neutrophils, macrophages, T cells and mast cells in the bronchial submucosa. However, subbasement membrane (SBM) thickness and smooth muscle area were increased in the biopsies. No significant differences were observed in the induced sputum inflammatory cells. In BAL fluid, there was a significant increase in neutrophils but a significant decrease in macrophages. Eosinophil counts were non-significantly increased threefold in both sputum and BAL in severe asthma. Levels of IL-8 and IL-13 in sputum supernatants were similar in both groups of asthma patients. There was a significant inverse correlation between post-bronchodilator forced expiratory volume in 1 s and provocative concentration of methacholine causing a 20% fall in FEV₁ with SBM thickness.
Conclusion Differences in inflammatory cells were observed mainly in terms of increased neutrophils and reduction in macrophage numbers in BAL fluid with a trend towards increased eosinophils in severe asthma compared with non-severe asthma. However, the most notable features are the increase in features of airway wall remodelling of SBM thickness and smooth muscle area.     

Cyclosporin A treatment and airways inflammation in corticosteroid-dependent asthma

Cyclosporin A is a potent immunosuppressive agent which inhibits activation of T cells and other inflammatory ceils. It has been shown to be of clinical benefit in patients with corticosteroid dependent asthma, but there are no data on its in vivo effects on airways inflammation. In this report, we describe the case of a 47-year-old man with chronic severe corticosteroid-dependent asthma who made a dramatic clinical response to therapy with cyclosporin
A. Fibreoptic bronchoscopy with bronchoalveolar lavage and endobronchial biopsy were performed before and after a 12-month period of treatment with cyclosporin A and demonstrated a concomitant reduction in airway inflammatory indices.     

Increased IL-6 and IL-8 in bronchoalveolar lavage fluids (BALF) from patients with sarcoidosis: correlation with the clinical parameters

A variety of cytokines have been implicated in the pathogenesis of pulmonary sarcoidosis, but the exact roles of IL-6 and IL-8 are not yet clear. We studied these cytokine levels in BALF from patients with pulmonary sarcoidosis, idiopathic pulmonary fibrosis (IPF), systemic screlosis (SSc) with interstitial lung disease and control subjects. IL-6 and IL-8 levels were significantly elevated in sarcoidosis, IPF and SSc with interstitial lung disease compared with control subjects. Subjects with sarcoidosis had significantly increased levels of both cytokines compared with controls when the cytokine values were corrected by the total albumin content and the two cytokine levels correlated with each other (r=0.876). BALF IL-6 levels correlated with percent lymphocytes and percent CD3⁺ cells. Moreover, when sarcoidosis patients were divided into three groups, those who needed steroid therapy or had progressive disease showed increased cytokine levels in BALF over stable or improved patients. These observations suggest that locally derived IL-6 and IL-8 were increased in sarcoidosis and correlated with activity of this granulomatous lung disease.     

支气管肺泡灌洗联合长期小剂量阿奇霉素治疗支气管扩张患者的效果及肺功能的影响

目的：探讨支气管肺泡灌洗(bronchoalveolar lavage,BAL)联合长期小剂量阿奇霉素治疗支气管扩张症患者的临床效果及对患者肺功能的影响作用。方法：回顾性分析本院2012年2月至2014年12月治疗的83例支气管扩张症患者,其中44例患者采用常规治疗联合BAL+阿奇霉素(250 mg/次,1周2次,连续治疗6个月)作为观察组,对照组39例患者仅采取常规治疗+BAL疗法,对比2组的临床效果及肺功能变化。结果：治疗前,急性期结束时观察组和对照组的hs-CRP、WBC水平差异无统计学意义(P>0.05);经过6个月治疗,观察组患者血清hs-CRP水较对照组低(P<0.05);治疗6个月后,观察组的痰液MD、MP评分及痰液量显著低于对照组(P<0.05);急性期结束时,两组患者的PEF、FEV1、FVE1/pred测定结果差异不具有统计学差异(P>0.05);经过6个月治疗,观察组的FEV1、FVE1/pred测定值明显的优于对照组患者(P<0.05)。结论：BAL联合长期小剂量阿奇霉素治疗支气管扩张症患者临床效果更好,对患者的肺功能改善效果更佳。     

Exposure to systemic prednisolone for 4 hours reduces ex vivo synthesis of GM-CSF by bronchoalveolar lavage cells and blood mononuclear cells of mild allergic asthmatics

BACKGROUND: In acute severe asthma, the earliest clinical effects of glucocorticosteroids occur from 4 to 5 h after systemic administration, but the mechanisms are unclear. In persistent asthma, corticosteroids are thought to suppress airway inflammation by modulating the expression of adhesion molecules, enzymes, and leucotactic cytokines, including granulocyte-macrophage colony stimulating factor (GM-CSF). GM-CSF is also overexpressed in the airways of symptomatic asthmatics. OBJECTIVES: To examine the early effects of systemic corticosteroids on cytokine expression, we investigated whether ex vivo synthesis of GM-CSF is suppressed in the bronchoalveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMCs) of normal and mild allergic asthmatic subjects obtained 4 h after a single intravenous dose of prednisolone. METHODS: In a randomized, double-blind, placebo-controlled study, BAL cells and PBMCs were obtained from mild atopic asthmatic patients (n = 9) and normal subjects (n = 9) 4 h after an intravenous bolus dose of 80 mg prednisolone, and cultured for 0-18 h in the presence or absence of lipopolysaccharide (LPS; 10 microg/mL). Enzyme immunoassay was used to assess GM-CSF levels in BAL cell and PBMC culture supernatants, and in BAL fluid. RESULTS: After placebo, GM-CSF synthesis tended to be higher in BAL cells from asthmatics than in normals. LPS stimulation significantly increased median (interquartile range) GM-CSF synthesis by BAL cells ex vivo from 16.4 (23 to 74) to 35.8 (3-148) pg/106 cells in normals (P < 0.05), and from 59 (9 to 204) to 134 (24-288) pg/106 cells in asthmatics (P < 0.01). After intravenous prednisolone, the rise in GM-CSF production induced in BAL cells by LPS was completely abolished in both subject groups. In PBMCs of placebo-treated asthmatics (but not normals), LPS stimulated median GM-CSF synthesis from 164 (110 to 300) to 314 (235-485) pg/106 cells (P = 0.02), and this was blocked by intravenous prednisolone. CONCLUSIONS: LPS-stimulated GM-CSF synthesis ex vivo is abolished in BAL cells of mild asthmatic and normal subjects, and in PBMCs of asthmatics, obtained 4 h after a single intravenous dose of prednisolone. Suppression of GM-CSF synthesis in airway and blood leucocytes may contribute to the early clinical efficacy of systemic glucocorticoids in acute allergic asthma.     

IL-12p40 is essential for the down-regulation of airway hyperresponsiveness in a mouse model of bronchial asthma with prolonged antigen exposure

Background We previously reported a mouse model of bronchial asthma showing eosinophilic inflammation, but not airway hyperresponsiveness (AHR), after prolonged antigen exposure. This model showed an increase of IL-12 in the lung.
Objective The aim of this study was to investigate the role of IL-12p40 in a murine asthma model with prolonged antigen exposures.
Methods An ovalbumin (OVA)-induced asthma model was first established in wild-type (WT) and IL-12p40-deficient (IL-12p40^−/−) mice. Both strains of mice were further exposed to either OVA (prolonged exposure group) or phosphate-buffered saline (positive control group) 3 days per week for 3 weeks. During week 4, both groups of mice were given a final challenge with OVA.
Results Prolonged antigen exposures resulted in marked suppression of airway eosinophilia in both WT and IL-12p40^−/− mice. However, AHR persisted in IL-12p40^−/− but not in WT mice. There were no significant differences of IL-5, IL-13 or IFN-γ levels in bronchoalveolar lavage fluid between WT and IL-12p40^−/− mice. The hydroxyproline content of the lung and peribronchial fibrosis were, however, significantly increased in IL-12p40^−/− mice.
Conclusion The results suggest that endogenous IL-12p40 is essential for inhibition of AHR and peribronchial fibrosis, but not eosinophilic inflammation, in a murine asthma model with prolonged antigen exposures.