Intracellular expression and serum levels of eosinophil peroxidase (EPO) and eosinophil cationic protein in asthmatic children

BACKGROUND: Eosinophils are involved in the chronic inflammatory response in asthma and their basic proteins are thought to play a major pathophysiological role in this process. While serum levels of basic proteins have been used to monitor the ongoing allergic disease, little is known about the intracellular expression of these proteins in clinical situations. OBJECTIVE: The aim of the study was to determine the intracellular expression of eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) in asthmatic children and control subjects and relate it to serum levels of both proteins, lung function tests and immunoglobulin (Ig)E levels. METHODS: Serum ECP and EPO concentrations were determined by immunoassays in 13 asthmatic children (mean age: 9 +/- 1 years, mean FEV1: 92 +/- 10% predicted, geometric mean PC20 histamine 0.5 mg/mL) and 10 age-matched, healthy control subjects. A flow cytometric single cell assay was employed to detect intracellular ECP and EPO in peripheral blood eosinophils. RESULTS: While serum concentrations of both ECP (asthma: median 15.0 microg/L [range 3.6-57.7] vs control: 5.9 microg/L [2.7-9.1]; P = 0.02) and EPO (22.9 microg/L [5.2-82.5] vs 7. 2 microg/L [2.5-12.7]; P = 0.008) were significantly elevated in asthmatics, the intracellular expression of ECP and EPO (measured as mean fluorescence intensity) was decreased (EG1: 55.3 [17.7-120.8] vs 100.3 [46.5-264.4]; P = 0.01; EG2: 80.2 [24.1-135.3] vs 133.7 [32. 1-244.9]; P = 0.04 and EPO: 49.7 [23.1-155.8] vs 94.9 [28.8-115.2]; P = 0.03). In asthmatics there was a significant correlation of FEV1 with intracellular ECP and of bronchial hyperresponsiveness with serum EPO and ECP. Furthermore, total IgE levels were positively correlated with serum EPO only. CONCLUSION: We conclude that in asthmatics the intracellular content of ECP and EPO in peripheral eosinophils is reduced possibly due to degranulation. Epitope masking in activated eosinophils or a shift to early bone marrow-derived progenitors with less granule proteins are further possible explanations.     

Serum eosinophil peroxidase (EPO) levels in asthmatic patients

Eosinophil granular proteins are a useful eosinophilic activation marker in asthmatic patients. In this study, the eosinophil peroxidase (EPO) levels were assessed in different stages of bronchial asthma, in 123 patients suffering from asthma, classified as mild (n=49), moderate (n=49), and severe (n=25), according to the International Consensus Report on Diagnosis and Treatment of Asthma, as well as in 27 healthy controls, with the aim of evaluating the importance of this protein as a severity marker in bronchial asthma, and its possible correlation with parameters such as anamnesis, respiratory function tests, and peripheral blood eosinophil count, and also with some allergologic diagnostic tests, both in vivo and in vitro. The geometric mean serum level of EPO was 9.3±11.3 ng/ml (median±SD) in controls, and 28±37.8 ng/ml in the asthmatic patients. Depending on the asthma severity, the EPO levels were 25±30.5; 29±37.1, and 41 ±47.3 ng/ml in mild, moderate, and severe asthmatics, respectively, being the significant differences between the group of patients with mild and severe asthma (P<0.001). The number of eosinophils (eos) in peripheral blood was 157±20 eos/mm³ in the controls, 334+35 eos/mm³ in mild asthmatics, 510 ±87 eos/mm³ in moderate asthmatics, and 658±72 eos/mm³ in severe asthmatics, with significant differences between all the groups (from P<0.05 to P<0.001). Both the serum levels of EPO and the number of eosinophils were greater in patients with active asthma than in patients with inactive asthma (P<0.001). Significant negative correlations (P < 0.001) were found between serum levels of EPO and FEY, (r_s= 0.30), MEF_25-75 (r_s= -0.33), and MEF₅₀ (r_s= -0.34), and a good positive correlation (r_s= 0.80, P<0.001) was found between EPO levels and the number of eosinophils in peripheral blood. We also found a significant positive correlation between eosinophil number and clinical score (r_s= 0.54, P<0.001) and between EPO levels and the mentioned score (r_s= 0.46, P<0.001).     

Serum eosinophil granule proteins predict asthma risk in allergic rhinitis

Background: Allergic rhinitis is a common disease, in which some patients will deteriorate or develop asthma. It is important to characterize these patients, thereby offering the possibility for prevention. This study evaluated eosinophil parameters as potential indicators of deteriorating allergic airway disease. Methods: The subjects of the study included all patients who suffered seasonal allergic rhinitis and had participated in a study 6 years earlier, in which blood eosinophils, serum eosinophil cationic protein (ECP) serum eosinophil peroxidase (EPO), nasal lavage ECP and nasal lavage EPO levels were measured. Patients in the present study were interviewed on occurrence of rhinitis symptoms during the last season, rhinitis outside season, asthma‐like symptoms and asthma diagnosis, and were skin‐prick tested for common aeroallergens. Eosinophil parameters from the study 6 years earlier were then tested for the ability to predict occurrence of new allergies, worsening of rhinitis and occurrence of asthma. Results: Forty‐four patients participated in the study. In four patients seasonal rhinitis symptoms had deteriorated, 10 had experienced perennial rhinitis symptoms, 14 reported asthma‐like symptoms and seven had been diagnosed with asthma. Thirteen had developed additional sensitization. Patients developing asthma‐like symptoms compared with patients with no such symptoms had significantly higher serum ECP (16.7 μg/l vs 8.2 μg/l; P ≤ 0.01) and serum EPO (17.9 μg/l vs 8.8 μg/l; P ≤ 0.05). Results were similar, considering patients diagnosed with asthma. Blood eosinophils and nasal lavage parameters were not related to development of asthma and asthma‐like symptoms. No eosinophil parameter was related to deterioration of rhinitis or additional sensitization. Conclusion: Serum ECP and EPO in patients with seasonal rhinitis demonstrated a high predictive ability for later development of asthma.     

A novel specificity of anticytoplasmic autoantibodies directed against eosinophil peroxidase.

Vasculitis associated anticytoplasmic autoantibodies (ANCA) are directed against enzymes in the granules of both neutrophils and monocytes. These autoantibodies can be detected by indirect immunofluorescence technique (IIFT) using ethanol-fixed cytospins. We here report the identification of a novel specificity of autoantibodies, present in the sera of eight patients, that reacted only with eosinophils in the IIFT. By immunoprecipitation and ELISA experiments it was shown that the autoantibodies in these sera were directed against eosinophil peroxidase (EPO). There was no apparent influence on initial substrate conversion rate, but reduced plateau levels suggested increased inactivation of the enzyme in the course of the peroxidase reaction. Flow cytometry studies demonstrated the presence of EPO on the surface of primed eosinophils. Anti-EPO sera and purified anti-EPO immunoglobulins significantly increased the release of reactive oxygen species from primed eosinophils. The patients with anti-EPO antibodies suffered from clinically diverse disorders, with more or less generalized manifestations involving the kidneys, blood vessels, lungs and/or joints.     

嗜酸性粒细胞募集在支气管哮喘发病中的作用

支气管哮喘是一种气道慢性炎症性疾病,伴有嗜酸性粒细胞增多、杯状细胞肥大、黏液分泌增多、可逆性的气道阻塞及对吸入变应原和非特异性刺激的高反应性等特点,其中嗜酸性粒细胞募集和随后的激活在支气管哮喘发病中起着十分重要的作用。本文就嗜酸性粒细胞募集在支气管哮喘发病机制中的研究进展作一综述,为支气管哮喘的治疗提供一线新的曙光。     

Nasal and pharyngeal eosinophil peroxidase levels in adults with poorly controlled asthma correlate with sputum eosinophilia

The objective of the study was to compare nasal, pharyngeal, and sputum eosinophil peroxidase (EPX) levels with induced sputum eosinophil percentage in 10 adults with poorly controlled asthma and 10 normal controls. EPX was measured using an ELISA and normalized for grams of protein for nasal and pharynx specimens and for mL‐gram of protein for sputum. Sputum EPX levels were statistically different between asthma and control subjects (P = 0.024). EPX levels measured in the nasal and pharyngeal swab samples derived from the same patients were also different between asthma and control subjects, each displaying a high degree of significance (P = 0.002). Spearman's correlation coefficients for nasal EPX and pharyngeal EPX levels compared to induced sputum eosinophil percentage were 0.81 (P = 0.0007) and 0.78 (P = 0.0017), respectively. Thus, there is a strong association in a given patient between both nasal and pharyngeal EPX levels and the eosinophil percentage of induced sputum.     

Wheat flour peroxidase is a prominent allergen associated with baker's asthma

Background Putative wheat allergens with molecular sizes around 35kDa have been repeatedly detected in immunoblots using sera from patients with baker's asthma. However, none of these allergens had been previously isolated. Objective To purify and characterize a major component of the 35 kDa aliergenic band which seems to be present in flour from diploid. tetraploid (pasta) and hexaploid (bread) wheats. Methods Gel-filtration chromatography followed by RP-HPLC have allowed the purification of a new wheat allergen. Immunodetection with sera from allergic patients and with a specific serum for asparagine-linked complex glycans, as well as amino acid sequencing, were used to identify the isolated protein. Results A 36 kDa allergen has been purified from wheat flour. Its N-terminal and internal sequences, N-linked glycans, and enzymatic activity indicated that this allergen is a seed-specific peroxidase. Sera from six out of 10 patients hypersensitive to wheat flour displayed positive reactions when assayed against the purified dot-blotted allergen. Conclusion Seed-specific peroxidases from cereals (flours) seem to be important allergens in hypersensitive diseases associated with the manipulation of these plant materials. A new enzyme has thus been involved in IgE-mediated diseases.     

Increased eosinophil transmigration after nasal allergen challenge in children with allergic asthma and rhinitis

BACKGROUND: Eotaxin and interleukin-5 together provide the signal essential for eosinophil transmigration to airway tissue in allergic reactions. However, it is not known whether peripheral blood eosinophils (PBE) possess an increased transmigration capacity in vitro after allergen challenge in vivo before they leave the circulation. We aimed to determine whether PBE in cat-sensitized children have increased spontaneous and/or eotaxin-induced transmigration capacity in vitro, and to what extent allergen challenge alters this feature. METHODS: Fourteen cat-allergic children and four healthy controls underwent nasal challenge with cat-allergen. Blood samples were drawn prechallenge and at 2 h and 24 h postchallenge. We analyzed the in vitro transmigration of PBE, with and without eotaxin as a chemoattractant. We used a transmigration assay with fibronectin-coated membranes. Eosinophil cationic protein (ECP) and PBE counts were run in parallel. RESULTS: The spontaneous transmigration capacity of eosinophils in vitro was significantly higher at 2 h after allergen challenge (P < 0.01 vs. prechallenge) and returned to prechallenge levels at 24 h postchallenge (P < 0.02 vs. 2 h postchallenge). Addition of eotaxin further augmented the increased transmigration. In concordance, no accompanying changes were measured in the levels of eosinophils in blood or ECP in serum. Furthermore no spontaneous or eotaxin-induced eosinophil transmigration was detected in healthy controls. CONCLUSION: PBE possess increased spontaneous (and eotaxin-induced) capacity to transmigrate as early as 2 h after allergen challenge in allergic children, without accompanying signs of eosinophil activation in terms of increased PBE count or ECP level. This is probably due to the increased stage of activation of the eosinophil, often referred to as "priming".     

Detection of eosinophils using an eosinophil peroxidase assay. Its use as an assay for eosinophil differentiation factors

A colorimetric assay for peroxidase has been applied to the detection of eosinophils in bone marrow cultures and tissue cell suspensions. The substrate solution consists of 0.1 mM o-phenylenediamine in 0.05 M Tris-HCl buffer pH 8.0 containing 0.1% Triton and 1 mM hydrogen peroxide. The method is shown to be an easy and reproducible method of detecting eosinophils, with insignificant interference from neutrophils.     

Evaluation of serum eosinophil cationic protein as a predictive marker for asthma exacerbation in patients with persistent disease

BACKGROUND: Eosinophilic inflammation is a feature of asthma. However, serological markers to indicate eosinophil activation in this process are not fully defined. OBJECTIVE: To evaluate the relationship of serum eosinophil cationic protein (ECP) to asthma worsening and a marker for treatment effectiveness, 26 adult patients with an asthma exacerbation were identified. METHODS: Identified asthma subjects were treated with oral corticosteroids (prednisone) for 14 days. The lung function variables, forced expiratory volume in one second (FEV1) and peak expiratory flow (PEF), were determined as percentage of predicted and the blood total eosinophil count and serum ECP levels were measured. Patients were re-evaluated after 14 days of corticosteroid treatment and then every 3 months thereafter during a 12-month period. RESULTS: Eighteen patients responded to prednisone treatment, whereas eight did not, assessed as improvement of their lung function parameters. Different serum ECP patterns could be seen in the responders compared with the non-responders. All 18 responders had considerably increased serum ECP at the time of exacerbation, whereas the non-responders had lower serum ECP levels. The serum ECP levels decreased to a greater extent in the responder patient group than in the non-responder patients following prednisone treatment. This difference in patterns was not seen with total blood eosinophil counts. CONCLUSION: Our findings suggest that serum ECP may be used to predict a response to corticosteroid therapy in adult patients with asthma.     

Serum eosinophil cationic protein in the evaluation of asthma severity in children

To assess the sensitivity and specificity of serum eosinophil cationic protein (ECP) in the diagnosis of asthma and evaluation of asthma severity, we conducted a prospective study to compare parameters of asthma severity, peripheral blood eosinophilia, and serum ECP concentrations in 88 children presenting to a university hospital outpatient clinic with suspected ( n =59) or recently diagnosed asthma ( n =29). Serum ECP correlated significantly (r²=0.676. P=0.0001) with peripheral eosinophil counts, but only weakly with asthma severity (r,=0.21, P=0.046). Serum ECP was significantly higher in atopic children (25±11 μg/l) than in nonatopic children (16±15 μg/l) iP=0.01). Bronchial hyperresponsiveness had no significant correlation (r_s=-0.21, P=0.30) with serum ECP Lung function test results had no (peak flow) or only a weak (FEV) correlation with serum ECP. In distinguishing between children with and without asthma or in assessing asthma severity, serum ECP is not superior to the peripheral blood eosinophil count. The diagnostic sensitivity and specificity of ECP in serum for detecting symptomatically active asthma, evaluated against the cutoff level of ECP in serum of 16 mg/1, were 54% and 71%, respectively.     

Peripheral eosinophil counts as a marker of disease activity in intrinsic and extrinsic asthma

Background: Recently it has been suggested that the bronchospasm and hyperresponsiveness phenomena observed in asthma are secondary to the actions of the eosinophils; the purpose of this study was to evaluate the relationship between the peripheral number of eosinophils and various markers of disease activity in a group of asthmatics examined in childhood (mean age 10 years) and early adulthood (mean age 21 years). Methods: The relationship between eosinophil count and pulmonary function (FEV₁), respiratory symptoms, bronchial responsiveness to histamine and diurnal variation in peak expiratory flow rate (PEF) was studied in 70 subjects with bronchial asthma, of whom 24 had intrinsic and 46 extrinsic asthma. Self-reported symptoms of asthma were graded on a scale from 0 to 5, where 0 = no symptoms within the preceding 12 months and 5 = daily including nocturnal symptoms, and histamine responsiveness was analysed by means of the dose-response slope (DRS). Results: In both childhood and adulthood, a direct correlation was found between blood eosinophil count and symptom score (r= 0.69, P< 0.001 and r= 0.58, P < 0.001, respectively), whereas inverse correlations were observed between number of eosinophils and FEV, % predicted (r= .0.75, P < 0.001 and r= 0.80, P < 0.001. respectively). Furthermore, in adulthood, eosinophil count was found to be significantly correlated to hisiamine responsiveness (log DRS) (r= 0.65, P < 0.001) and diurnal PEF variation (r= 0.81, P < 0.001); these correlations were also noted after dividing the subjects into intrinsic and extrinic asthmatics. In both groups of subjects a significant inverse correlation was also found between histamine responsiveness and pre challenge FEV₁% predicted. The eosinophil count in childhood was weakly correlated to the symptom score in adulthood (r= 0.29, P < 0.02). Conlusions: This study showed a relationship between eosinophil count and seventy of asthmatic symptoms, level of pulmonary function, histamine responsiveness and diurnal variation in PEF in both intrinsic and extrinsic asthma; suggesting that the peripheral eosinophil count reflects asthmatic activity, and possibly the degree of inflammation in the airways, in both children and adults. Furthermore, a low number of eosinophils in childhood might be related to a relatively favourable prognosis with regard to symptoms of asthma in early adulthood.     

支气管哮喘患者血清嗜酸细胞阳离子蛋白的变化

探讨检测血清嗜酸细胞阳离子蛋白在支气管哮喘的诊断１病情监测及疗效判断中的价值。方法采用荧光酶免疫法测定了发作期和缓解哮喘患者各２０例,慢性阻塞性肺疾病患者１６例和正常人２０例的血清ＥＣＰ,同时进行了肺功能测定,并对发作期哮喘患者在给予吸入皮质激素治疗３ｍｏ后复测其ＥＣＰ及肺。     

Lack of relationship between eosinophil cationic protein and eosinophil protein X in nasal lavage and urine and the severity of childhood asthma in a 6-month follow-up study.

BACKGROUND: Recent studies suggest that eosinophil cationic protein (ECP) and eosinophil protein X (EPX) may be valuable markers of airway inflammation in various body fluids of asthmatic children. Most of these studies have relied on a single measure of inflammatory markers. OBJECTIVE: We measured ECP and EPX in nasal lavage fluids (NALF) and urine samples in children with asthma over a 6-month period to study the relationship between inflammatory markers and clinical severity. METHODS: Fourteen children with mild persisting asthma (mean age 11.7 years, SD 2.2) were recruited. All patients were on therapy including inhaled steroids. For a 6-month period asthma severity was monitored by at least monthly physical examination and pulmonary function tests. Daily morning and evening PEF, asthma symptoms and medication were recorded in diaries for the whole study period. Telephone interviews were performed between visits and additional visits were done in case of an increase in asthmatic symptoms or drop of PEF values under 80% of best value. An exacerbation was defined by a fall of FEV1 > 10% and an increase in asthma symptoms and additional need of beta2-agonist. NALF and urine samples were obtained at each visit and analysed for ECP (NALF only) and EPX. RESULTS: Mean observation time was 186.4 days (SD 19.8). Thirteen patients completed the study. During the study period 11 exacerbations were observed in six patients. No significant associations between PEF, PEF variability (amplitude % of mean), daily symptoms, additional beta2-agonist, FEV1 and MEF50 and nasal ECP, nasal EPX and urinary EPX were found. However, at exacerbations an average increase of nasal ECP (9.3 vs 50.3 microg/L) and EPX (nasal EPX 36.4 vs 141.7 microg/L, urinary EPX 46.4 vs 74.1 microg/mmol creatinine) was observed. CONCLUSION: Serial measurements of ECP and EPX in NALF and urine samples do not provide additional information for the practical management in monitoring childhood asthma.     

An evaluation of peripheral blood eosinophil/basophil progenitors following nasal allergen challenge in patients with allergic rhinitis

OBJECTIVE: To evaluate the effect of a single nasal allergen challenge on peripheral blood eosinophil/basophil (Eo/B) progenitor cells and induced sputum eosinophil counts in subjects with allergic rhinitis. METHODS: Sixteen adults entered a sequential nasal control and allergen challenge study, outside the pollen season. Blind assessment of peripheral blood Eo/B progenitor colony forming units (CFU), induced sputum and nasal lavage cell counts was made before and 24 h after both challenges. Subjects recorded their rhinitis symptoms and nasal peak inspiratory flow, hourly at home, following both challenges. RESULTS: When comparing the values 24 h after the control vs. the allergen challenge, there were no significant differences in Eo/B progenitor CFU (control (mean, SD): 3.6 (1.0)/10(6) cells; allergen: 4.4 (1.1)/10(6) cells) or sputum eosinophils (control (median, inter-quartile range): 1.0 (0.3-1.7)%; allergen: 0.7 (0.0-1.3)%) despite a significant increase in the percentage (median (inter-quartile range) of eosinophils in nasal lavage (control: 0.6 (0.1-0.9)%; allergen; 1.9 (0.9-8.1)% and significant worsening of nasal peak inspiratory flow and rhinitis symptoms. CONCLUSIONS: Despite a significant increase in nasal symptoms and lavage eosinophil counts, a single nasal allergen challenge was not sufficient to elicit a measurable haemopoietic response in circulation, or an increase in sputum eosinophil counts.     

抗白介素5单克隆抗体抑制哮喘小鼠嗜酸性粒细胞迁移

目的 观察抗白介素5(IL-5)单克隆抗体对哮喘小鼠嗜酸性粒细胞(Eos)从骨髓到气道迁移的影响.方法 15只雄性C57BL/6小鼠.常规法复制哮喘,并在每次卵白蛋白(Ova)激发前2 h鼻腔内滴入抗IL-5单克隆抗体,同时设立阴性对照组,即以生理盐水代替Ova 和抗IL-5抗体.在末次抗原激发后12 h测定支气管肺泡灌洗液(BALF)、外周血(PB)和骨髓(BM)中炎症细胞数以及肺组织内Eos数.结果 Ova抗原激发后12 h小鼠的BALF、PB和BM及细支气管和肺组织中的Eos数显著增加(P＜0.01, P＜0.05).抗IL-5单克隆抗体则使上述变化显著减轻(P＜0.05,P＜0.01).结论 抗IL-5单克隆抗体能显著抑制哮喘小鼠嗜酸性粒细胞的迁移.     

Sustained eosinophil cationic protein release into tears after a single high-dose conjunctival allergen challenge

Background The appearance of eosinophils is a hallmark sign of the allergic late-phase response (LPR). Eosinophil cationic protein (ECP), a readily measurable product released from activated eosinophils, has so far not been evaluated in the ocular LPR. Objective Two sets of trials were performed in order to investigate changes of local and systemic eosinophil activity and their possible link with symptoms and hyper-reactivity in the allergic LPR in the eye. Methods In the first experiment, ECP was analysed in tears and serum and the clinical reaction was evaluated during a 72-h time–course after a single, high-dose allergen challenge out of season in one eye of 15 pollen-sensitized volunteers. In a second experiment, the hypothesis of an increased clinical response to an allergen challenge in an eye that had been provoked with allergen 48h previously was tested in nine sensitized individuals. Results In the first experiment, symptoms at 10 min and 2, 4, 6, 8 and 24 h significantly exceeded base line scores of the challenged eyes. Tear ECP was significantly elevated in challenged eyes compared to contralateral eyes at 6, 8 and 24 h. In addition, symptoms and ECP release correlated significantly at the 24-h evaluation. Serum ECP remained unchanged throughout the study period. In the second experiment, conjunctival hyperreactivity 48h after an allergen challenge was not confirmed. Conclusion ECP secretion occurs in the experimental ocular LPR and is in part associated with the magnitude of the clinical reaction, which suggests a truly pathogenic role of the activated eosinophil in pollen-induced allergic conjunctivitis.