IL-5, IL-8 and GM-CSF immunostaining of sputum cells in bronchial asthma and chronic bronchitis

Background: Granulocyte-macrophage colony stimulating factor (GM-CSF) and inlerleukin (IL)-5 or IL-8 have been .suggested to play an important role in the pathogenesis of eosinophilic airway inflammation in bronchial asthma or neutrophilic airway inflammation in chronic bronchitis, respectively, However, GM-CSF and IL-8 have biological activities to either eosinophils or neutrophils. Objective: To investigate the contribution of these cytokines to airway inflammation, we compared the cellular differential and immunolocalization of GM-CSF, IL-5 and IL-8 in sputum cells from patients with bronchial asthma and chronic bronchitis. Methods: Cytospins of sputum cells from 12 patients with bronchial asthma and 12 with chronic bronchitis were subjected to cellular differential counting and immunocytochemistry with antihuman GM-CSF, IL-5 and IL-8 antibody. Results: The predominant cells in bronchial asthma were eosinophils and lymphocytes, while those in chronic bronchitis were neutrophils. All cytokines examined were detected in either bronchial asthma or chronic bronchitis, although the percentage of GM-CSF and IL-5 positive cells in bronchial asthma (53.4 ± 6.0 [mean±sfm ]% and 9.7 ± 2.8%, respectively) was significantly higher than that in chronic bronchitis (11.4±2.5%; P < 0.001 and 1.7plusmn;0.3%; P < 0.007. respectively). In contrast, the percentage of IL-8 positive cells in chronic bronchitis (23.8 ± 7.0%) was significantly higher than that in bronchial asthma (7.7 ± 1.9%; P < 0.04). The cells positive for IL-5 were lymphocytes in bronchial asthma and chronic bronchitis. The cells positive for GM-CSF in bronchial asthma were predominantly eosinophils. while those in chronic bronchitis were monocytes/macrophages and neutrophils. In contrast, neutrophils are mainly positive for IL-8 in chronic bronchitis, while monocytes/macrophages and bronchial epithelial cells are positive for IL-8 in bronchial asthma. Conclusion: The immunochcmical comparison of GM-CSF and IL-8 localization in sputum cells between bronchial asthma chronic bronchitis suggests the differential regulation and roles of these cytokines in eosinophilic vs neutrophilic airway inflammation, resulting in the development of different types of airway inflammation.     

Serum interleukin-5 in aspirin-induced asthma

BACKGROUND: Eosinophilopoetic cytokine IL-5 enhances cysteinyl-leukotriene (cys-LT) synthesis in eosinophils in vitro. In patients with aspirin-induced asthma (AIA) bronchial biopsies revealed eosinophil infiltration and a marked increase in IL-5 positive cells. OBJECTIVE: We wondered whether in AIA patients the bronchial IL-5 increase is reflected in peripheral blood, and if so, whether it is related to overproduction of cys-LT. METHODS: In 11 stable patients with AIA, 32 with ATA (aspirin-tolerant asthma) and in 16 controls we measured serum IL-5 concentrations and urinary LTE4, believed to reflect global cys-LT production. RESULTS: Serum IL-5 was detectable in 12 of 43 asthmatics, but in none of the control subjects. It was highest in the ATA group and differed significantly from the controls. There was no significant difference in IL-5 levels between: (i) the asthmatic groups studied, and (ii) AIA patients and controls. No relationship was found between serum IL-5 and urinary cys-LT. CONCLUSION: Overexpression of IL-5 reported in the airways of aspirin-sensitive patients with asthma was not reflected in their blood. If IL-5 affects cys-LT production, it is rather in the bronchi of the patients than in the blood.     

Detection of transforming growth factor-β in sputum from patients with bronchial asthma by eosinophil survival assay and enzyme-linked immunosorbent assay

Background We have shown that interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are present in sputum from patients experiencing acute asthma attacks, by eosinophil survival assay. The viability of guinea-pig eosinophils was significantly increased in the presence of such sputum extracts after 3 days' culture, and it was inhibited by the addition of anti-IL-5 and anti-GM-CSF antibodies. However, the contribution of IL-5 to the increase in eosinophil viabihty was less than expected from the values of IL-5 measured by enzyme-linked immunosorbent assay (ELISA). Therefore, we speculated that something in sputum inhibited the function of IL-5. Objective Tratnsforming growth factor-β (TGF-β) was the only cytokine we tested that inhibited the prolongation of survival of guinea-pig eosinophils induced by IL-5. The objective of this study is to detect TGF-β in the same sputum. Methods Guinea-pig eosinophils were cultured with or without anti-TGF-β antibody in the presence of sputum extracts, and the eosinophil viability was counted after 3 days. Measurement of TGF-β_l in sputum was performed by ELISA. Results Eosinophil viabilities with and without anti-TGF-β antibody were 79.7 ± 2.9% and 69.0 ± 2.7%, respectively, and the difference between them was statistically significant (P < 0.05, n = 9). The concentration of TGF-β₁ in the sputum was 21.7 ± 3.3 ng/mL (n = 9). Conclusion These observations suggest that TGF-β is present in sputum from patients with bronchial asthma.     

吸入糖皮质激素对哮喘儿童外周血Foxp3~+调节性T细胞及IL-2、IL-6的影响及其机制研究

研究吸入丙酸氟替卡松(FP)对哮喘患儿外周血单个核细胞(PBMC)中CD4+Foxp3+调节T细胞、细胞因子IL-2、IL-6以及转录因子STAT5的影响。以30例确诊为支气管哮喘的患儿为研究对象,随机分为未治疗哮喘组(15例)、吸入FP缓解组(15例),10例同期正常儿童为对照组。流式细胞仪检测外周血PBMC中的CD4+Foxp3+调节T细胞比率,ELISA检测血浆或培养上清中IL-2、IL-6细胞因子水平,Western blot检测PBMC中磷酸化及非磷酸化STAT5的水平。结果1.未治疗哮喘组PBMC中CD4+Foxp3+T细胞百分率在PHA刺激培养前后均明显低于正常对照组,吸入FP缓解组明显升高,与正常对照组没有差异;各组刺激后CD4+Foxp3+T细胞百分率均有升高,吸入FP缓解组、正常对照组分别升高约1.89、2.01倍,而未治疗哮喘组升高仅1.56倍;2.未治疗哮喘组血浆中IL-6水平明显高于正常组及吸入FP缓解组,而IL-2水平没有明显差异;3.PHA刺激24 h后未治疗哮喘组磷酸化STAT5(p-STAT)表达水平明显低于吸入FP组及正常对照组,而各组STAT5表达水平没有明显差异,结论吸入FP能增加哮喘患儿外周血PBMC中CD4+Foxp3+调节T细胞数量,其机制可能与降低血浆IL-6,上调STAT5磷酸化水平有关。     

Allergen challenge in asthma: association of eosinophils and lymphocytes with interleukin-5

To test whether eosinophil recruitment after pulmonary allergen challenge is associated with interleukin (IL)-5 in patients with asthma, we performed segmental bronchoprovocation (SBP) with saline, and with low and high dosages of ragweed extract in six patients with allergic asthma. Bronchoalveolar lavage (BAL) of the challenged segments was performed 5 min after challenge (immediate BAL fluid) and repeated 24 h later (late BAL fluid). Allergen challenge resulted in recruitment of eosinophils, and increased levels of eosinophil-active cytokines. A bioassay showed the predominant eosinophil-active cytokine in the late BAL fluids to be IL-5. Analysis of the late BAL fluids revealed that IL-5 levels correlated with the numbers of eosinophils and lymphocytes. This study provides evidence that IL-5 is a critical cytokine associated with eosinophil and lymphocyte recruitment into the airways of patients with asthma following exposure to allergen.     

IL-5 deficiency abolishes aspects of airway remodelling in a murine model of lung inflammation

BACKGROUND AND OBJECTIVES: Lung remodelling is a recognized feature of chronic asthma. In the present study, we have used IL-5-deficient mice to evaluate the role of this cytokine and eosinophilic inflammation in the initial stages of the structural changes occurring in the lung after antigen challenge. METHODS: Ovalbumin-sensitized wild type and IL-5-deficient mice were daily challenged for 5 consecutive days and killed 3 or 7 days after the last challenge to study the inflammatory and remodelling events, respectively. RESULTS: Wild type mice challenged with ovalbumin exhibited an accumulation of eosinophils in the bronchoalveolar lavage (BAL) fluid, associated with a production of BAL cellular fibronectin. Histological analysis also revealed an antigen-specific increase in epithelial and alveolar cell proliferation together with an increase in mucus producing epithelial cells. Eosinophilic infiltration and the associated lung remodelling were totally abrogated in IL-5-deficient mice. In wild type mice, treated intranasally with 1 microg of murine IL-5 for 5 consecutive days, no BAL eosinophilia and structural changes of the lungs could be observed. CONCLUSION: Our results demonstrate that eosinophil accumulation, but not IL-5 alone, plays a central role in the initial stages of the lung remodelling process and suggests that therapies directed at inhibiting eosinophilic inflammation may be beneficial in treating chronic asthma.     

Cysteinyl-leukotriene levels in sputum differentiate asthma from rhinitis patients with or without bronchial hyperresponsiveness

BACKGROUND: We have previously reported that asthma differs from rhinitis with or without bronchial hyperresponsiveness in the perception and degree of lower airway inflammation. OBJECTIVE: The aim of the present study was to investigate whether sputum levels of inflammatory markers could further distinguish these patient groups. METHODS: Patients with seasonal allergic rhinitis with or without asthma or bronchial hyperresponsiveness to methacholine were investigated. Induced sputum was performed during as well as off season, and analysed for cysteinyl-leukotrienes, hyaluronan, eosinophilic cationic protein (ECP) and other inflammatory markers. RESULTS: Asthmatic patients differentiated from those with rhinitis with or without bronchial hyperresponsiveness in levels of cysteinyl-leukotrienes [geometric mean: 3.3 (lower 95%-upper 95% confidence interval (CI) of geometric mean: 1.9-5.1) vs. 1.4 (0.9-2.2) and 0.7 (0.3-1.6) pg/microg total protein] and hyaluronan [0.30 (0.22-0.43) vs. 0.15 (0.10-0.20) and 0.20 (0.12-0.35) ng/microg total protein] in sputum. The levels of cysteinyl-leukotrienes decreased in sputum from the asthmatic patients, while the levels of hyaluronan remained elevated off-season. Furthermore, elevated levels of ECP were noticed among both the asthmatic and rhinitis patients with hyperresponsiveness compared with controls [0.022 (0.014-0.033) and 0.015 (0.011-0.021) compared with 0.010 (0.007-0.014) ng/microg total protein]. The level of ECP remained elevated off season. CONCLUSION: Cysteinyl-leukotrienes are possibly more related to mast cell-mediated inflammation and remodelling, also indicated by increased levels of hyaluronan during and off season. This inflammation may be partly different from the eosinophil-driven inflammation.     

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.     

Appearance of basophils in the sputum of patients with bronchial asthma

A total of 108 samples of sputum obtained from twenty patients with bronchial asthma were examined for appearance of basophils and eosinophils. Both cell types are present in sputum during an asthmatic attack and disappear at the conclusion of the attack. Their presence correlates with the severity of the disease. It has previously been demonstrated that the blood basophils count falls during attacks of bronchial asthma, and the present study suggests that basophils move from the blood stream into bronchial tissue during the acute phase of an asthmatic attack.     

Interleukin-18 promoter polymorphism in patients with atopic asthma

Allergic asthma is a chronic inflammatory disease in which interleukin-18 (IL-18) plays an important role. However, there are controversial reports on IL-18 promoter polymorphism as an independent marker of asthma susceptibility. The aim of the present study was to examine the IL-18 promoter polymorphism in patients with allergic asthma. Two hundred and thirty-one patients with allergic asthma from a Polish population diagnosed according to the National Heart, Lung, and Blood Institute (NHLBI)/WHO guidelines were examined. An allele-specific polymerase chain reaction was used to analyse polymorphisms at positions -137 and -607 in the promoter region of the IL-18 gene. Neither in the -607 C>A nor in the -137 G>C promoter polymorphism were there any differences observed between the total group of asthmatic patients and the controls in the frequencies of genotypes, alleles, diplotypes or haplotypes. In patients with severe asthma, the -607 CC and -137 GG genotypes were observed significantly more frequently (P = 0.03 for both), whereas in patients with mild and moderate asthma, the -137 CC genotype was more prevalent than in the former group. The strongest difference between mild to moderate and severe asthma was observed in -137 allele frequencies (P = 0.006). The results of the present study suggest that the -137 G allele and the C-G/C-G diplotype seem to be involved in the pathogenesis of the severe form of asthma.     

Immuno-regulatory cytokines in asthma: IL-15 and IL-13 in induced sputum

BACKGROUND: The importance of Th2-type lymphocyte function in asthmatic airway inflammation is well recognized, but less is known about the factors which regulate the function of these lymphocytes in asthma. The macrophage-derived cytokine, interleukin (IL)-15 has a number of T cell regulatory properties which might be of relevance to asthma and its treatment. OBJECTIVE: The aims were to identify and quantify the T cell regulatory cytokine IL-15 in induced sputum samples from asthmatic patients, in comparison with IL-13, and to relate the levels of these cytokines to treatment with inhaled steroids. METHODS: Induced sputum was collected from 16 asthmatics (eight steroid and eight non-steroid treated) and eight normal controls. IL-15 and IL-13 levels were measured by enzyme-linked immunoassay (ELISA) in sputum. IL-15 levels were also measured in sputum cell culture supernatants and localized to specific sputum cells by immuno-cytochemistry. RESULTS: IL-15 levels were increased and IL-13 levels were decreased in sputum fluid from steroid-treated compared with non-steroid-treated asthmatics. IL-15 was localized specifically to macrophages and the proportion of these cells expressing IL-15 correlated with sputum fluid IL-15 and IL-15 levels in cell culture supernatants, and all were higher in the steroid-treated asthmatics. CONCLUSION: IL-15 and IL-13 production appears to be reciprocally regulated by steroid therapy in asthma patients. The steroid-associated increase in IL-15 may regulate a fundamental shift away from an inflammatory Th2-type environment in asthma and may be an essential component of the cytokine modulation underlying the therapeutic benefit of corticosteroids in this condition.     

Allergen inhalation decreases adenosine receptor expression in sputum and blood of asthma patients

Background: Adenosine is a signalling nucleoside that has been proposed to contribute to the pathogenesis of asthma. Adenosine is produced in inflammatory environments and acts via adenosine receptors (A₁R, A_2AR, A_2BR, and A₃R) expressed by a wide variety of cells, resulting in pro‐ and anti‐inflammatory effects. Objective: To compare AR expression in asthma patients and healthy subjects, and to assess the effect of allergen challenge on AR expression of inflammatory cells and on cytokines in peripheral blood and sputum in asthma. Methods: Asthma patients underwent an allergen challenge, and blood and induced sputum samples were taken before and 24 h after allergen challenge to study inflammatory cells numbers, AR expression and cytokine production. Blood and sputum were investigated at one time point in healthy subjects. AR expression was measured by flow cytometry (blood) or on cytospins using immunocytochemistry (sputum). Cytokines (luminex, ELISA) and adenosine (HPLC) were measured in sputum supernatant. Results: The percentage of A_2BR expressing neutrophils in sputum was lower in asthma patients than in healthy subjects (P = 0.016). Allergen challenge decreased A₁R and A_2AR expression on neutrophils and A₁R expression on T cells in peripheral blood (all P < 0.05). Allergen challenge increased IL‐8 levels and eosinophil numbers (P < 0.05), whereas it decreased thymic stromal lymphopoietin levels and the percentage of A₁R expressing macrophages in induced sputum (P < 0.05). Conclusions: Allergen challenge has a down‐regulatory effect on AR expression in asthma, suggesting a contribution of adenosine‐related effector mechanisms in the pathophysiology.     

Neutrophil activation following TDI bronchial challenges to the airway secretion from subjects with TDI-induced asthma

BACKGROUND: The immunopathological mechanism for occupational asthma induced by toluene diisocyanate (TDI) remains to be further clarified. There have been few reports suggesting involvement of neutrophils in inducing bronchoconstriction after TDI inhalation. OBJECTIVES: To further understand the role of neutrophils in the pathogenesis of TDI-induced asthma. MATERIALS AND METHODS: Eight TDI-induced asthmatic subjects were classified as group I, and five exposed workers who had complained of work-related symptoms and worked in the same workplace, but showed negative bronchial challenges were enrolled as controls (group II). Serum neutrophil chemotactic activity during TDI bronchial challenge test was measured by the Boyden chamber method. Induced sputum was collected before and after the TDI bronchial challenge test. The myeloperoxidase (MPO) and interleukin (IL) -8 levels in the sputum were measured using RIA and ELISA. RESULT: Serum neutrophil chemotactic activity significantly increased at 10 min (P = 0.01), then decreased at 60 min (P = 0.02) and remained unchanged for up to 420 min (P = 0.07) in group I subjects, while no significant changes were found in group II subjects (P > 0.05). MPO and IL-8 were abundantly present in the sputum of all the TDI-induced asthmatic subjects and they increased significantly at 420 min after the bronchial challenges (P = 0.02, P = 0.03, respectively), while no significant changes were noted in group II subjects (P > 0.05). CONCLUSION: These findings support the view that activated neutrophils may contribute to bronchoconstriction induced by TDI which may be associated with IL-8 release.     

Genetic factors in the treatment of bronchial asthma

Owing to the recent vast progress in analytical tools and procedures to elucidate the relationship between genes and diseases, many candidate genes leading to the development of bronchial asthma have been reported. However, the quantitative phenotypes of asthma, such as decrease in forced expiratory volume in the first second, serum hyper-IgE, bronchial hyperresponsiveness and blood hyper-eosinophilia, do not represent this disease completely. On the other hand, eosinophilic inflammation of the bronchial mucosa represents accurately the feature of bronchial asthma, although accurate quantification of its status is difficult. While the production of interleukin (IL)-5 in peripheral CD4⁺ T cells probably correlates with eosinophilic inflammation of the airway, the effectiveness of anti-IL-5 antibody for the treatment of bronchial asthma is controversial. Since intervention with asthma-causing gene products may not be sufficient for the treatment of this disease, identification of therapy-responsive genes should become more important in the near future.     

雷公藤对致敏小鼠T细胞IL-5 mRNA表达及转录因子GATA-3活性的抑制作用

探讨雷公藤内酯醇 (TP )对致敏小鼠T淋巴细胞IL 5mRNA表达的影响及其机制。采用卵蛋白 (OVA )致敏的方法建立模型 ;运用原位杂交染色法 (ISH )观察TP对T淋巴细胞IL 5mRNA表达的影响 ;通过凝胶电泳迁移率实验 (EMSA )对CD4+T淋巴细胞核转录因子GATA 3的DNA结合活性进行检测 ,同时就TP的作用与地塞米松 (DM )相比较。结果表明致敏小鼠T淋巴细胞IL 5mRNA表达显著高于正常对照组 (P <0 0 1) ,经TP、DM处理后 ,其IL 5mRNA表达显著低于致敏组(P <0 0 1)。致敏小鼠CD4+ T淋巴细胞体外经伴刀豆蛋白A (ConA )刺激后 ,GATA 3的DNA结合活性与正常对照组比较显著增强 ,并呈时间依赖关系 ,经TP、DM处理后 ,GATA 3的DNA结合活性显著减弱。TP抑制IL 5基因转录的分子机制可能与其抑制GATA 3的DNA结合活性有关。     

Effect of pranlukast on bronchial inflammation in patients with asthma

BACKGROUND: Pranlukast (8-[p-(4-phenylbutyloxy) benzol] amino-2-[tetrazol-5-yl]-4-oxo-4H-1-benzopyran hemihydrate), a selective cysteinyl leukotriene receptor antagonist, has been reported to exhibit not only antileukotrine activity but also pharmacological activity including antieosinophilic effects. OBJECTIVE: This study was designed to investigate whether the antiasthmatic activity of pranlukast is associated with a reduction in eosinophilic inflammation. METHODS: A double-blind, randomized, crossover design was used. Subjects received 225 mg of pranlukast or placebo orally twice daily for 4 weeks and then, after a washout period of at least 4 weeks, crossed over to receive the alternative treatment. We assessed the effects of pretreatment with pranlukast on bronchoconstriction precipitated by inhalation of methacholine in 32 adult patients with mild or moderate bronchial asthma; those who were in stable clinical condition were allocated to this study. Blood and sputum samples were taken the morning of the methacholine provocation test. Eosinophil counts and measurement of eosinophilic cationic protein (ECP) were performed. RESULTS: After the 4 weeks of treatment with pranlukast, patients' symptoms, blood eosinophils, serum ECP, sputum eosinophils, and sputum ECP were significantly decreased. Furthermore, values of PC20-methacholine significantly improved in the treatment with pranlukast. CONCLUSION: Our results suggest that pranlukast has an anti-inflammatory effect on bronchial eosinophilic infiltration. This study raises further interesting therapeutic possibilities and argues for further trials of new approaches to the treatment of bronchial asthma.