Expression of 5-lipoxygenase and cyclooxygenase pathway enzymes in nasal polyps of patients with aspirin-intolerant asthma

In aspirin-intolerant subjects, adverse bronchial and nasal reactions to cyclooxygenase (COX) inhibitors are associated with over-production of cysteinyl-leukotrienes (cys-LTs) generated by the 5-lipoxygenase (5-LO) pathway. In the bronchi of patients with aspirin-intolerant asthma, we previously linked cys-LT over-production and aspirin hyper-reactivity with elevated immunoexpression in eosinophils of the terminal enzyme for cys-LT production, LTC4 synthase. We investigated whether this anomaly also occurs in the nasal airways of these patients. Immunohistochemical expression of 5-LO and COX pathway proteins was quantified in nasal polyps from 12 patients with aspirin-intolerant asthma and 13 with aspirin-tolerant asthma. In the mucosa of polyps from aspirin-intolerant asthmatic patients, cells immunopositive for LTC4 synthase were four-fold more numerous than in aspirin-tolerant asthmatic patients (p=0.04). There were also three-fold more cells expressing 5-LO (p=0.037), with no differences in 5-LO activating protein (FLAP), COX-1 or COX-2. LTC4 synthase-positive cell counts correlated exclusively with mucosal eosinophils (r=0.94, p<0.001, n=25). Co-localisation confirmed that five-fold higher eosinophil counts (p=0.007) accounted for the increased LTC4 synthase expression in polyps from aspirin-intolerant asthmatic patients, with no alterations in mast cells or macrophages. Within the epithelium, increased counts of eosinophils (p=0.006), macrophages (p=0.097), and mast cells (p=0.034) in aspirin-intolerant asthmatic polyps were associated only with 2.5-fold increased 5-LO-positive cells (p<0.05), while the other enzymes were not different. Our results indicate that a marked over-representation of LTC4 synthase in mucosal eosinophils is closely linked to aspirin intolerance in the nasal airway, as in the bronchial airways.     

Leukotriene C4 synthase promoter polymorphism in Japanese patients with aspirin-induced asthma

BACKGROUND: The A to C transversion in the promoter region of the gene encoding leukotriene C4 synthase (LTC4S) is proposed to be associated with the development of aspirin-induced asthma (AIA). OBJECTIVE: We investigated the frequency of the polymorphism in Japanese population and its association with clinical characteristics and cysteinyl leukotriene production. METHODS: Genotyping of LTC4S gene promoter was performed on 60 patients with AIA, 100 patients with aspirin-tolerant asthma (ATA), and 110 control subjects. We assessed the basal levels of urinary LTE4, the increment of urinary LTE4 on venous aspirin challenge, and LTC4S activity in peripheral blood eosinophils. RESULTS: The frequency of the variant C allele was significantly higher in patients with AIA (frequency of allele [q] = 0.192) than in patients with ATA (q = 0.110, P =.042). Variant C-allelic carriers experienced asthma at a significantly younger age (31.8 +/- 2.9 years [mean +/- SEM]) than wild-type A homozygotes (41.3 +/- 2.2 years, P =.007). Basal levels of LTE4 and the increment of urinary LTE4 on venous aspirin challenge did not show a difference between wild-type A homozygotes and variant C-allelic carriers. There was no relationship between the polymorphism and the LTC4S activity in eosinophils, although LTC4S activities were significantly higher in patients with AIA than in patients with ATA. CONCLUSION: Our findings reveal the lack of functionality of the polymorphism in the LTC4S gene, whereas this polymorphism might have some effect on the development of AIA, probably in linkage disequilibrium with another causatively important mutation.     

Up-regulation of human eosinophil leukotriene C4 generation through contact with bronchial epithelial cells

OBJECTIVE AND DESIGN: We studied the effect of contact with bronchial epithelial cells on the functional activity of human eosinophils, measured as the production of a bronchoconstrictor lipid mediator, leukotriene C4 (LTC4) to determine the role of cell-cell interaction in activation of airway eosinophils. MATERIALS AND METHODS: Eosinophils were isolated from peripheral blood of atopic donors. Epithelial cells were obtained from the bronchi of surgically resected lung lobes and cultured to confluence on collagen-coated plates. Eosinophils were stimulated with platelet activating factor (PAF) or serum opsonized zymosan (SOZ) after incubation with or without epithelial cells. Leukotriene C4 (LTC4) was assayed in supernatants by enzyme immunoassay. RESULTS: Bronchial epithelial cells did not produce LTC4 in response to PAF or SOZ. Eosinophils pre-incubated in collagen-coated plates for 1 h produced LTC4 in response to both PAF (130 +/- 53 fmol/10(6) eosinophils at 10 micromol/l PAF, 5 min) and SOZ (1,900 +/- 550 fmol/10(6) eosinophils at 2 mg/ml SOZ, 15 min). Eosinophils co-incubated with bronchial epithelial cells for 1 h produced significantly higher quantities of LTC4 in response to both PAF (310 +/- 94 fmol/10(6) eosinophils; P<0.01) and SOZ (5,500 +/- 1,500 fmol/10(6) eosinophils; P<0.001). Ligation of the common beta2 integrin subunit (CD18) with a monoclonal antibody inhibited PAF-stimulated and augmented SOZ-stimulated LTC4 generation by eosinophils alone but had marginal effects on the epithelium-dependent up-regulation. CONCLUSIONS: Contact with bronchial epithelial cells up-regulates the responsiveness of human eosinophils, a finding that has significant implications in the pathology of asthma.     

Asthma, inflammation, eosinophils and bronchial hyperresponsiveness

Asthmatics can have a blood eosinophilia which in some studies correlates with the severity of the disease. However, an increased number and percentage of activated eosinophils can be present in the blood without asthma. The eosinophils that contribute to asthma will be those in the lung. In the BAL fluids collected from asthmatics there is usually no change in total cell number, but there are changes in the differential cell count. A consistent finding is an increase in percentage of mast cells and eosinophils with a tendency for an increase in lymphocytes and epithelial cells and a decrease in percentage of macrophages. As with the blood eosinophilia, an increase in number of eosinophils can be present in BAL fluids without asthma. The site of localization and activation of the eosinophils in the lung may be critical. In bronchial biopsies, taken from asthmatics, increased number of mast cells, eosinophils and lymphocytes have been demonstrated in the bronchial mucosa together with shedding of columnar epithelial cells. However these changes have not been found, or have not reached significance, in all studies. An increase in number of activated eosinophils and T-lymphocytes has been demonstrated but an increase in number of degranulating mast cells has been disputed. A consistent finding has been thickening below the basement membrane. Attempts to correlate the changes in the BAL or lung biopsies with the severity of asthma, lung airways function or bronchial responsiveness have given inconsistent results. Treatment of asthmatics with inhaled steroids can reduce the cellular infiltration in the bronchial biopsies to normal levels but this produces a trivial reduction in bronchial responsiveness. It is possible that infiltration of inflammatory cells into the bronchial mucosa is intermittent, at least in mild asthma, but this produces changes leading to a long lasting bronchial hyperresponsiveness.     

An immunohistochemical study on cellular infiltration of bronchial walls in bronchial asthma]

Inflammatory cell reactions in bronchial asthma have been considered to contribute directly to asthmatic attacks. Therefore, attention has been focused on the role of inflammatory cells in the airway wall. In this study, we investigated inflammatory cells in the airway wall of bronchial asthma histologically and immunohistologically using 20 autopsy cases who died of the asthma attacks (Group A) and 11 autopsy cases who died of other causes (Group B). Six autopsy cases were treated as controls (Group C). A significantly larger number of eosinophils in the bronchi and bronchioles were found in Group A. The majority of eosinophils were found to be stained with EG2. Parts of the bronchial epithelium with marked infiltration of EG2 positive eosinophils were detached from the basal layer. Immunoelectron microscopically, the matrix of EG2 positive areas within the eosinophils corresponded to that of eosinophil granules. A significantly larger number of lymphocytes in the bronchi and bronchioles was observed in Groups A and B. Almost all of these lymphocytes were determined to be activated T lymphocytes. There were significantly more IgE positive cells in cases with bronchial asthma, especially in Group A. The majority of IgE positive cells were determined to be mast cells. These results suggest that eosinophils, lymphocytes and mast cells play an important role in the pathogenesis of bronchial asthma.     

Relationship between LTC4 generation of hypodense eosinophils and bronchial hyperreactivity in asthmatic children

In 19 asthmatic children aged 6-16 years, the degree of bronchial hyperreactivity was determined in relationship to the concentration of inhaled histamine which caused a fall of the specific conductance (sGaw) to 60% of the baseline value PC60sGaw. At the time of lung function testing, a sample of heparinized blood was obtained from each patient. Eosinophils were purified and separated into a normodense and hypodense fraction by Percoll gradient centrifugation. After in vitro stimulation by ionophore A 23187, the leukotriene C4 (LTC4) content was determined in the culture supernatants. Hypodense eosinophils of the 13 children with a histamine threshold lower than 1 mg/ml generated significantly (p less than 0.01) larger amounts of LTC4 (0.8-36.3 ng/10(6) cells) when compared to 6 children with a histamine threshold higher than 1 mg/ml (0.7-12.1 ng/10(6) cells) and 12 healthy controls (0.4-8.2 ng/10(6) cells). Preincubation of eosinophils with platelet activating factor (PAF) induced an enhanced LTC4 production, not only in hypodense cells from both asthmatic groups but also in normodense cells from patients with severe hyperresponsiveness. These results are consistent with other results which suggest an important role of eosinophils, their activation by PAF and enhanced release of spasmogenic LTC4 in the pathogenesis of asthma.     

Cellular communication in leukotriene C4 production between eosinophils and neutrophils

Eosinophilic granulocytes as well as neutrophilic granulocytes produced leukotriene C4 (LTC4) on stimulation with 1 microM A23187 (Ca2+ ionophore). In healthy volunteers, the LTC4 production in eosinophils was about 3 times the production in neutrophils. Within 15 min greater than 90% of the LTC4 was released into the supernatant. Stimulation of eosinophils and neutrophils together resulted in a synergistic increase in LTC4 production of 306 +/- 40%. LTC4 synthesis by hypodense eosinophils was also enhanced if stimulated in the presence of neutrophils. These findings suggest a communication between eosinophils and neutrophils, which may play a role in bronchial asthma.     

Asthma and COPD

The two obstructive airway diseases bronchial asthma and chronic obstructive pulmonary disease (COPD) represent major global causes of disability and death, and COPD is estimated to become the third most common cause of death by 2020. The structural and pathophysiologic findings in both diseases appear to be easily differentiated in the extremes of clinical presentation. However, a significant overlap may exist in individual patients regarding features such as airway wall thickening on computer tomography or reversibility and airway hyperresponsiveness in lung function tests. Airway inflammation differs between the two diseases. In bronchial asthma, airway inflammation is characterized in most cases by an increased number of activated T-lymphocytes, particularly CD4+ Th2 cells, and sometimes eosinophils and mast cells. The most notable difference of chronic severe asthma compared with mild to moderate asthma is an increased number of neutrophils. In stable COPD, airway inflammation is characterized by an increased number of T-lymphocytes, particularly CD8+ T cells, macrophages and neutrophils. With the progression of the disease severity, macrophage and neutrophil numbers increase. Although there may be a partial overlap between asthma and COPD in some patients, the differences in functional, structural and pharmacological features clearly demonstrate the consensus that asthma and COPD are different diseases along all their stages of severity.     

Expression of cysteinyl leukotriene synthetic and signalling proteins in inflammatory cells in active seasonal allergic rhinitis

BACKGROUND: Cysteinyl leukotrienes (CysLTs) are bioactive lipids that have been shown to contribute to allergic and inflammatory diseases. Eosinophils and mast cells have the capacity to produce large amounts of CysLTs after allergic or non-allergic stimulation. Molecular identification of both the synthetic and signalling proteins in the CysLT pathway allows the investigation of expression of the CysLT enzymes and receptors in active allergic rhinitis. OBJECTIVE: We examined the expression of the proteins involved in the synthesis of CysLTs and the cysteinyl leukotriene-1 (CysLT1) and cysteinyl leukotriene-2 (CysLT2) receptors in inflammatory cells from patients with active seasonal allergic rhinitis. METHODS: Nasal lavage samples were obtained from patients during active seasonal allergic rhinitis. Specific cellular immunocytochemical techniques were used to detect the cysteinyl leukotriene synthetic proteins, namely 5-lipoxygenase (5-LO), 5-lipoxygenase-activating protein (FLAP) and leukotriene C4 synthase (LTC4S). In situ hybridization and immunocytochemical techniques were used to identify the mRNA and proteins for the CysLT1 and CysLT2 receptors. RESULTS: 5-LO, FLAP and LTC4S, and the CysLT1 and CysLT2 receptors were expressed in the majority of eosinophils and in subsets of mast cells and mononuclear cells. 5-LO, FLAP and the CysLT1 receptor, but not LTC4S or the CysLT2 receptor, were expressed in a subset of nasal neutrophils. CONCLUSIONS: Our study demonstrates the presence of CysLT pathway proteins in key allergic and inflammatory cells from the upper airway of patients with active seasonal allergic rhinitis. Our expression data highlight the potential of CysLT-modifying agents to treat both upper and lower airway symptoms in patients suffering from allergic rhinitis and asthma.     

Leucocytes in asthma

A diagrammatic representation of the interactions between mediators of hypersensitivity and leucocytes in early-, late-phase, and ongoing asthma is shown in Fig. 1. Early-phase or immediate reactions are largely the result of bronchoconstriction consequent to the release of mediators such as histamine, PGD2, LTC4/D4 and PAF. The principal mediator cell (MC) is the mast cell (although other IgE receptor-bearing cells such as the macrophage, eosinophil and platelet might also be involved in this immediate response). The stimulus for mediator cell activation may be either immunologic (IgE-dependent) or non-immunologic (i.e. changes in osmolarity as a result of the respiratory water loss associated with exercise-induced asthma). Late-phase reactions appear to be a consequence of infiltration with neutrophils (N), eosinophils (E) and macrophages (MO). These cells are recruited and activated either by mast cell-associated chemotactic factors [such as LTB4, PAF, the eosinophil chemotactic factor of anaphylaxis (ECF-A) or high molecular weight neutrophil chemotactic activity (NCA (HMW]] and/or "lymphokines" derived from T helper cells (TH) which have been stimulated by antigen processed by the antigen processing cells (APC). These mononuclear cell interactions are under the control of regulatory T cells [T suppressor (TS) cells] and it is speculated that the availability of these subsets may determine the magnitude of the late-phase response. Lymphokines and monokines which selectively activate neutrophils, eosinophils and monocytes include LIF, EAF and INF-gamma respectively. Macrophage-derived tumour necrosis factor (TNF) also amplifies the inflammatory response by its capacity to enhance eosinophil cytotoxicity. Eosinophil-derived agents such as PAF, LTC4, MBP and ECP might be responsible for submucosal oedema and non-specific bronchial hyperreactivity which are characteristic features of late-phase reactions. T cell-derived lymphokines such as EDF (IL-5), together with GM-CSF, might lead to eosinophilopoiesis and account for the prolonged eosinophilia of ongoing chronic asthma. The T cell is prominent in the pathology of chronic asthma and is possibly "chronically activated". Thus lymphocytes, driven by as yet undetermined "antigens" (possibly viral), may perpetuate the inflammatory response in and around the bronchi. IL-5-like products from these putative activated lymphocytes might perpetuate (a) eosinophil production by the bone marrow, (b) its release into the circulation, (c) its migration into bronchial tissue and (d) activation to release PAF, LTC4, MBP, etc.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preventive effect of a novel leukotrienes antagonist ONO-1078 on leukotriene C4- and D4-induced human bronchial smooth muscle contraction

To study whether a novel leukotrienes antagonist ONO-1078 (4-oxo-8-[4-phenylbutyloxy) benzoylamino]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate) prevents leukotrienes C4- and D4-(LTC4, LTD4) induced human bronchial smooth muscle contraction, we examined the bronchial contractile response to LTC4 and LTD4 in the presence or in the absence of ONO-1078 in human bronchial strips. We prepared 4 strips from each of 5 patients undergoing lobectomy of the lung because of lung cancer. We mounted the strips in organ baths and measured the contractile response to LTC4 and LTD4 from 10(-11) M to 10(-7) M after incubation with ONO-1078 (10(-8) M, 10(-7) M and 10(-6) M) and without ONO-1078. ONO-1078 shifted the dose response curve to LTC4 to higher concentration in dose dependent fashion, and it significantly inhibited the contractile response to LTC4 and LTD4. In the presence of L-serine borate complex (45 mM), an inhibitor of gamma-glutamyl transpeptidase, ONO-1078 significantly inhibited the LTC4-induced contraction. On the other hand, ONO-1078 had no effect on the contractile response to acetylcholine. These results suggest that ONO-1078 is a specific antagonist of LTC4 and LTD4 receptors in human bronchial smooth muscle. Because the effect of LTC4 and LTD4 is thought to be an important part of the pathogenesis of bronchial asthma, our results also suggest that ONO-1078 may be a useful prophylactic drug for bronchial asthma.     

Mast cells regulate procollagen I (alpha 1) production by bronchial fibroblasts derived from subjects with asthma through IL-4/IL-4 delta 2 ratio

BACKGROUND: Asthma is characterized by inflammation and remodeling. Mast cells are generally increased in bronchial mucosa of subjects with asthma. These cells release a wide variety of cytokines and mediators that have the capacity to stimulate other resident cells such as smooth muscle cells and fibroblasts. OBJECTIVE: This study was designed to evaluate whether mast cells modulate collagen production by bronchial fibroblasts isolated from subjects with asthma and normal subjects through cytokine production. METHODS: Human mast cells were cocultured for 72 hours with primary bronchial fibroblasts isolated from bronchial biopsies of subjects with mild asthma and normal controls. Procollagen I (alpha1), IL-4Ralpha, IL-13Ralpha1, and IL-13Ralpha2 gene expression by bronchial fibroblasts and IL-4 and IL-4delta2 gene expression by mast cells were quantified by real-time RT-PCR. IL-4 production was also measured by ELISA in culture supernatants. RESULTS: Procollagen I (alpha1) gene expression by fibroblasts from subjects with asthma was significantly higher compared with cells from normal controls when cocultured with mast cells. Mast cells expressed IL-4 isoform and IL-4delta2, an alternative splice variant of IL-4. Coculture significantly increased the expression of IL-4 but not IL-4delta2 by mast cells when they were cultured with fibroblasts from subjects with asthma compared with cells from normal controls. Neutralization of IL-4 abrogated collagen mRNA expression. There was no significant change in IL-4Ralpha or IL-13Ralpha1. However, IL-13Ralpha2 gene expression was significantly reduced in fibroblasts from subjects with asthma. CONCLUSION: These results suggest that inflammatory process may regulate airway remodelling through crosstalk between inflammatory and structural cells. Targeting this crosstalk may have therapeutic application. CLINICAL IMPLICATIONS: Understanding mechanisms that govern airway remodeling and collagen deposition in asthma is a step toward therapeutic management of this disease. In this work, we found that mast cell-fibroblast crosstalk may be a potential future target to control some aspects of airway remodeling.     

Release of prostaglandin D2 and leukotriene C4 in response to hyperosmolar stimulation of mast cells

BACKGROUND: Mannitol-induced bronchoconstriction in subjects with exercise-induced asthma is associated with increased urinary excretion of 9alpha, 11beta-PGF(2), a metabolite of prostaglandin D(2) (PGD(2)) serving as a mast cell marker. It has however been questioned whether or not human mast cells release PGD(2) and leukotriene C(4) (LTC(4)) after osmotic challenge with mannitol in vitro. METHODS: Cord blood-derived human mast cells were stimulated osmotically, immunologically or with a combination of both. Supernatants were analysed for PGD(2), LTC(4) and histamine contents with enzyme immunoassays. RESULTS: Significant release of de novo synthesized eicosanoids, predominantly PGD(2) [12 (8.8, 14) pmol/10(6)cells; median (25th, 75th percentile) but also LTC(4) (0.1 (0.08, 0.15) pmol/10(6) cells] were found in mast cells in vitro in response to 0.7 M mannitol stimulation. A massive release of histamine [70 (5.3)% of total; mean (SEM)] was also found. There were no correlations between the levels of released mediators after mannitol stimulation. In contrast, there was a correlation between release of PGD(2) and LTC(4), following immunological stimulation. CONCLUSION: The findings support that hyperosmolar challenge activates mast cells, but different than antigen stimulation.     

Identification of basophils by immunohistochemistry in the airways of post-mortem cases of fatal asthma

There is increasing evidence for the role of basophils in the pathogenesis of bronchial asthma. To examine the presence of basophils in the airways of patients with fatal asthma by immunohistochemistry, we stained lung tissues from four post-mortem cases who had died from severe asthmatic attacks and four controls with a monoclonal antibody raised against tryptase (AA-1) and anti-IgE. Mast cells and basophils were identified in the bronchioles as AA-1- and anti-IgE-positive cells, and anti-IgE-positive cells, respectively. Airway mast cells were found beneath the basement membrane, near blood vessels in the submucosa, and adjacent to the submucosal glands, and scattered throughout the muscle bundles. There was a significant increase of mast cells in the asthma group compared with the control group (203.5+/-84.6/mm2, mean+/-s.d. vs 37.7+/-8.7/mm2, P<0.05, n=4). In contrast, basophils were observed in the airway lumen, in the bronchial epithelium and in the submucosa. The number of basophils in the bronchioles was 81.8+/-55.5/mm2 (n = 4); however, basophils were not found at all in the airways of the control group. Although eosinophils, B lymphocytes and macrophages bear low affinity IgE receptors and could react with anti-IgE, the location of these cells in the close sections did not correspond closely with basophils. The presence of basophils in lung tissues obtained from fatal asthma patients supports the view that basophils play a role in the pathogenesis of bronchial asthma.     

嗜酸粒细胞性支气管炎气道炎症病理特征的探讨

目的： 观察嗜酸粒细胞性支气管炎（EB）气道粘膜炎症的病理特征，并与咳嗽变异型哮喘（CVA）进行比较。 方法： 对11例EB患者行纤支镜支气管粘膜活检，并以10例正常对照、10例CVA和14例典型支气管哮喘的支气管粘膜标本作对照。光镜下测量各组气道粘膜上皮的基底膜厚度，并通过免疫组化和特殊染色技术，计算EB和CVA组气道粘膜固有层中炎症细胞（嗜酸粒细胞、肥大细胞、T淋巴细胞）的浸润密度。 结果： EB组支气管粘膜基底膜厚度［2.92 μm(2.10-6.50)μm］显著高于对照组［2.08 μm(1.62-3.40 μm)］, P<0.05，同时显著低于CVA组［5.64 μm (3.23-8.48 μm)］, P<0.05，而CVA组的基底膜厚度又显著低于典型哮喘组［9.08 μm (6.61-11.99 μm)］, P<0.01；EB组气道粘膜固有层可见肥大细胞和嗜酸粒细胞散在分布，浸润密度分别为［75 cells/mm2(35-112 cells/mm2)］和［7 cells/mm2(0-31 cells/mm2)］，显著低于CVA组［148 cells/mm2(34-200 cells/mm2)，114 cells/mm2(1-768 cells/mm2)］, P<0.05，淋巴细胞浸润密度无显著差异。 结论： EB是以嗜酸细胞浸润为特征，涉及多种炎症细胞的慢性气道炎症性疾病，但气道粘膜基底膜厚度显著低于CVA和典型哮喘，炎症细胞浸润程度低于CVA，均可能是EB缺乏气道高反应性的重要机制。     

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.     

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.     

Circadian variation of sputum inflammatory cells in mild asthma

BACKGROUND: Asthma, like many conditions, demonstrates a circadian rhythm with a worsening of lung function in the early morning hours compared with in the late afternoon. OBJECTIVE: Because eosinophilic airway inflammation is a proposed mechanism for worsening asthma, we characterized circadian variation in airway eosinophils and determined its relationship to variability in airway function. METHODS: Pulmonary function testing, sputum induction, and phlebotomy were performed at 7 am and 4 pm in 11 allergic subjects with mild asthma. Sputum was analyzed for cell viability, differential, and eosinophil-derived neurotoxin levels. IL-5 levels in serum were measured by means of ELISA. RESULTS: Subjects had a significant decrease in FEV(1) (median [interquartile range] = 80% [70%-86%] vs 85% [82%-94%], P =.009) and a greater beta-agonist reversibility (median [interquartile range] = 13% [7%-32%] vs 8% [5%-14%], P =.024) in the early morning compared with in the late afternoon. Sputum analysis showed an increase in early morning total sputum leukocytes (median [interquartile range] = 4.3 x 10(6) [2.3 x 10(6) to 6.1 x 10(6)] vs 2.6 x 10(6) [1.7 x 10(6) to 3.6 x 10(6)], P =.044) and eosinophils (median [interquartile range] = 7.0 x 10(4) [2.7 x 10(4) to 18.7 x 10(4)] vs 3.6 x 10(4) [1.0 x 10(4) to 8.2 x 10(4)], P =.024). Furthermore, sputum eosinophils correlated with beta-agonist reversibility (R (s) = 0.665, P =.019). Finally, levels of IL-5 in serum and eosinophil-derived neurotoxin in sputum were significantly increased at 7 am. CONCLUSION: These data suggest that circadian variability in pulmonary function in asthma could be related to changes in airway eosinophil recruitment and activation.