Local release of eosinophil peroxidase following segmental allergen provocation in asthma

BACKGROUND: Eosinophil peroxidase (EPO) is an eosinophilic basic protein, which leads to increased permeability and damage of bronchial epithelial cells in asthma. OBJECTIVE: As little is known about its local expression and release in humans the intracellular expression in lung and peripheral eosinophils and the concentrations of EPO in bronchoalveolar lavage (BAL) fluid and serum was investigated in patients with asthma. METHODS: Twelve mild atopic asthmatic and nine control subjects underwent segmental sham and allergen challenge. EPO concentrations in BAL fluid and serum were determined by immunoassay and flow cytometry was used to determine the intracellular expression of EPO in BAL-derived and peripheral eosinophils. RESULTS: In asthmatic patients a large increase in BAL eosinophils--total cells: median 9.5 x 10(6) (range: 0.5 to 455.0 x 10(6)); relative: 38% (1 to 91%)--was detectable 24 h following allergen challenge, but peripheral blood eosinophil counts did not change. Concentrations of EPO in BAL fluid increased from 1 microg/L (1.0 to 6.8 microg/L) to 42 microg/L (5.6 to 379.6 microg/L; P < 0.01) after allergen but not after saline challenge (1.5 microg/L; 1.0 to 21.9 microg/L), whereas in control subjects all measurements were below the detection limit. Serum concentrations of EPO increased slightly from 18.3 microg/L (3.0 to 56.8 microg/L) to 27 microg/L (3.8 to 133.9 microg/L; P < 0.05) 24 h after allergen challenge in asthmatic patients. Furthermore, the intracellular expression of EPO (measured as mean fluorescence intensity) was decreased in BAL eosinophils compared with blood eosinophils (mean fluorescence intensity 29 (7 to 71) vs. 48 (20 to 85); P < 0.01) after allergen challenge. CONCLUSION: The finding of increased EPO concentrations in the BAL fluid and decreased intracellular EPO expression in pulmonary eosinophils of asthmatic patients reflects the allergen-triggered release of EPO into the bronchial space.     

A simple method to obtain pure granule-rich eosinophil fragments (cytosomes) from normal human blood

This paper describes a simple, rapid and reproducible method to obtain pure granule-rich eosinophil fragments (cytosomes) with a high yield from normal human blood. The method is based on the treatment of whole blood with saponin and subsequent purification of the cytosomes on Percoll gradient. The enzymatic analysis of the cytosomes shows that the content of 3 granular enzymes is of the same order of magnitude already reported by others for intact eosinophils. This finding suggests that the cytosomes can be employed as starting material for studying the content of the granules or for the isolation of the granular components. The advantages offered by this method over those currently used to obtain eosinophil granules are discussed.     

Eosinophil cationic protein and eosinophil protein X in the nasal lavage of children during the first 4 weeks of life. SPACE Collaborative Study Team

Eosinophil cationic protein (ECP) and eosinophil protein X (EPX) are well established as markers of eosinophil activation. We analyzed ECP and EPX concentrations in nasal lavage fluids (NALF) of 378 neonates during their first 4 weeks of life. Inclusion criteria were a positive history of parental allergy and a positive skin prick test or specific IgE (RAST class > or = 2) against at least one out of a panel of common aeroallergens in one or both parents. Twenty-four infants with no history of parental allergy were used as controls. A volume of 2 ml of 0.9% saline was instilled into each nostril and immediately recovered by a suction device. ECP and EPX were analyzed by radioimmunoassay. In 65 samples of three consecutive lavages, EPX was detected in nine samples (13.8%) in the control group, whereas it was detected in 197/360 samples (54.7%) in the study population. The corresponding figures for ECP were 17/65 (26.2%) in the control group and 173/365 (47.4%) in the study group. Both proteins showed a skewed distribution (median/5-95th percentiles for ECP: 0 microg/l [0-69.4] and EPX: 6.6 microg/l [0-73.2]). The differences between the control group and the study group were statistically significant, regardless of the allergic disease of the parents. In children of allergic parents, activation proteins of the eosinophil granulocyte are released on the nasal mucosal surface in about 50% of the studied population at the age of 4 weeks. This early onset of eosinophil activation in the nasal respiratory epithelium may reflect a genetic predisposition to allergy or early exposure to allergens.     

The Content of Eosinophil Cationic Protein in Eosinophil Leucocytes

Eosinophil cationic protein consitutes a major pan of eosinophil Leucocyte granule protein. Low serum concentrations have previously been found in patients with bronchial asthma. As this might reflect a low intracellular content, eosinophils were isolated from normal controls and patients with bronchial asthma. Eosinophil cationic protein was estimated after extraction of the cells and a similar content was found in cells from both groups.     

Eosinophil cationic protein as a possible marker of active human Toxocara infection

BACKGROUND: Human toxocariasis is a common, worldwide helminthozoonosis that may elicit syndromes including various allergy symptoms. The diagnosis relies upon specific serology. However, this parasitosis is often self-limiting, and many subjects have residual antibodies, thus making differential diagnosis quite difficult when blood eosinophilia, a commonly accepted criterion of active helminthiasis due to tissue-dwelling parasites, is lacking. METHODS AND RESULTS: We present a patient with chronic irritant cough displaying negative allergologic screening, normal blood eosinophilia, but positive toxocariasis immunodiagnosis. Therefore, this case presented the fortuitous association of an unexplained allergic picture with residual anti-Toxocara antibodies. In an attempt to distinguish between active and past toxocaral infection, the subject's level of eosinophil cationic protein (ECP) was assessed and then compared to those of four control groups, namely, healthy volunteers, subjects presenting anti-Toxocara residual antibodies, patients with various helminthiases, and patients with active toxocaral disease. Since the patient's ECP level was found to be sharply elevated, we hypothesized that viable Toxocara larvae were still present in the tissues, and the patient was given anthelmintic therapy. At the control checkup, the cough had waned and the ECP level had decreased to below the mean value observed in both healthy subjects and in subjects with past toxocaral infections. CONCLUSIONS: These data suggest, first, that patients presenting unexplained allergic syndromes should be checked for helminthiases, even if blood eosinophilia is lacking, and, second, in such subjects displaying positive toxocariasis immunodiagnosis, ECP assessment would be a useful marker to distinguish between active and past toxocaral disease.     

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.     

A microtiter assay for activation of eosinophils

A simple microtiter assay for eosinophil activation is described. The assay used 1000–4000 eosinophils/microtiter well, and the design allows for a separate or simultaneous quantitative assessment of eosinophil adhension to protein-coated microtiter wells and degranulation after stimulation with eosinophil-activating factors. The number of adherent eosinophils is quantified indirectly by expressing the amount of eosinophil cationic protein (ECP) extracted from the adherent fraction of cells in percentage of the total amount of ECP extracted from the adherent fraction of cells in percentage of the total amount of ECP extracted from the cells added to the wells. Degranulation is quantified in the same way by expressing the amount of ECP or eosinophil protein X (EPX) released to the supernatant in percentage of the total amount of ECP or EPX. Known eosinophil-activating agents such as PMA, interleukin (IL)-3, IL-5, GM-CSF, and platelet-activating factor (PAF) all induced a time- and does-dependent adhension to albumin-coated wells, whereas L_PAF did not. KInetic experiments showed that most adhesion occurred within the first 15–30 min, reaching a plateau around 60 min. After prolonged incubation, a decline in adhesion was detected. GM-CSF-induced adhesion was completely inhibited by incubation with monoclonal antibodies directed against the common β₂-chain (CD18) of the LFA-1, Mac-1, p150,95 complexes. Monoclonal antibodies against CD11a, CD11b, CD11c, VLA-4 ALFA, ICAM-1, VCAM-1, Sialyl-Le^m, ELAM-1, and LECAM had no inhibitory effect. Simultaneous monitoring of adhesion and degranulation after stimulation of eosinophils in albumincoated wells with either PMA or GM-CSF showed that adhesion always preceded degranulation. Replacing the albumin coating of the microtiter wells with IgG or secretory IgA augmented both the spontaneous and the PMA- or GM-CSF-stimulated responses. In conclusion, the assay allows dynamic evaluation of eosinophil activationa dn can be used to assess soluble eosinophil-activating factors as well as to study eosinophil activation by solidphase-bound proteins.     

Eosinophil markers in seasonal allergic rhinitis

Background The purpose was to study activation markers of the eosinophil granulocytes in seasonal allergic rhinitis, and the impact of topical steroid therapy thereupon.
Methods Sixty-three rhinitis patients with monoallergy to grass were examined before and at peak pollen season. Blood eosinophil count, eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO) in serum and nasal lavage fluid were measured. During the season, patients were randomized to treatment with intranasal fluticasone propionate 0.1 mg o.d. ( n =26), 0.2 mg o.d. ( n =25), or placebo (n = 12). Six healthy persons served as controls.
Results During the season, all parameters, except nasal lavage ECP, increased in the placebo group (P<0.001 – P<0.05). Significant differences were seen between the steroid grotips and the placebo group for all parameters (P<0.001–F<0.05). Higher eosinophil count (P<0.05), serum EPO (F<0.02), and nasal lavage EPO (P<0.05) were found in patients before season than in controls. The following winter, 44 patients returned for repeated measurement. Lower levels of nasal lavage EPO were observed for patients than levels at the beginning of the season (P<0.0001).
Conclusions Intranasal fluticasone propionate reduced inflammation of the nasal mucosa, demonstrated locally by nasal lavage ECP and EPO, and systemically by blood eosinophils, serum ECP, and serum EPO. EPO seemed more sensitive than ECP as indicator of allergic inflammation. EPO demonstrated some perennial eosinophil activity in hay fever patients, increasing locally during spring.     

Degranulation patterns of eosinophils in advanced gastric carcinoma: an electron microscopic study

Recruitment and activation of eosinophils have been studied intensely in asthma and other allergic diseases. Less is known about the infiltration and degranulation patterns of eosinophils in the tumor stroma. Seven cases of advanced gastric carcinomas were found to be massively infiltrated by eosinophils and studied by light and electron microscopy. Gastric carcinomas, despite having similar numbers of tissue eosinophils, exhibited markedly different degranulation patterns. In 2 cases, resting nondegranulating eosinophils were found. Piecemeal degranulation was the predominant mode of secretion from eosinophils localized within the tumor stroma in 4 cases. Eosinophil exocytosis and cytolysis were rarely observed. In 1 case, crystals morphologically similar to Charcot-Leyden crystals were observed at the extracellular level as well as in phagosomes of tissue macrophages, confirming active sequestrations of eosinophil Charcot-Leyden protein by macrophages in vivo. In the same case, eosinophils showed characteristic features of early and late apoptotic changes, such as condensed chromatin, focal dilatation of nuclear envelope, and preserved plasma membrane. Morphological association between apoptotic eosinophils and deposition of granules in the tumor stroma was found. Extracellular deposition of intact granules from apoptotic eosinophils was distinct from eosinophilic (necrotic) cytolysis, and has reported previously in experimental studies in vitro. To the knowledge of the authors, this case represents the first report of late apoptotic eosinophils that release their granules within the tumor stroma in a human gastric carcinoma.     

Urine eosinophil protein X (EPX) is an in vitro parameter of inflammation in atopic dermatitis of the adult age

BACKGROUND: Eosinophils are important effector cells in several atopic diseases. The levels of eosinophil granule-derived mediators (ECP, EPX) in serum and body fluids have been proven to be correlated with disease activity in atopic respiratory diseases and atopic dermatitis. The study aimed to demonstrate an interrelationship between urine EPX and disease activity in adult patients with atopic dermatitis. METHODS: We determined urine EPX concentration, serum ECP concentration, and peripheral blood eosinophil count in 40 adult patients with mild to severe atopic dermatitis and compared it with the disease activity as assessed with the SCORAD index. RESULTS: Urine EPX and serum ECP concentrations were significantly higher in patients with severe atopic dermatitis than in patients with mild or moderate disease (median values 123.5 vs 78.3 microg/mmol creatinine, P<0.0001; 25.4 vs 14.9 microg/l, P<0.0001, respectively). We found a significant correlation between urine EPX levels, serum ECP levels, and the SCORAD (r=0.36, P<0.0001 and 0.34, P<0.0001, respectively). CONCLUSION: Urine EPX is a useful in vitro parameter of inflammation in atopic dermatitis of the adult age.     

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.     

Variations of Blood Eosinophils and Eosinophil Cationic Protein in Serum in Patients with Bronchial Asthma

Inhalation challenge test was performed in 12 patients with bronchial asthma. The subsequent variation in blood eosinophils and serum-eosinophil canonic protein was followed up. Uniform patterns in both parameters were seen suggesting active participation of the eosinophil leucocyte in allergic: inflammation.     

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.     

Major basic protein, but not eosinophil cationic protein or eosinophil protein X, is related to atopy in cystic fibrosis.

Increased eosinophil granule proteins have been described in serum and sputum samples of patients with cystic fibrosis (CF). It has been assumed that eosinophil degranulation is enhanced in atopic subjects - as in asthmatics. Since in CF no differences in eosinophil cationic protein (ECP), eosinophil protein X (EPX), and eosinophil peroxidase between atopic and nonatopic subjects have been detected, we investigated whether major basic protein (MBP) is increased in serum and sputum samples derived from atopic (n = 14) compared with nonatopic CF subjects (n = 26). In CF patients, high mean serum (sputum) levels of ECP 29.7 microg/l (2.7 mg/l), EPX 53.7 microg/l (7.9 mg/l), and MBP 984.6 microg/l but low sputum MBP levels (57.4 microg/l) were measured. In addition, in serum and in sputum samples, a significant correlation between MBP and ECP (P<0.03 and P<0.0001, respectively) or EPX (P<0.05 and P<0.0004, respectively) was detected. By subdivision of the patients into allergic and nonallergic subjects, significant differences were found for serum MBP values only(mean 1382.2 microg/l vs. 770.5 microg/l; P<0.0001), but not for ECP or EPX serum levels or for eosinophil proteins in sputum. Although no differences between atopic and nonatopic CF patients in ECP and EPX were found, serum MBP levels were higher in patients sensitized to inhalant allergens than in nonsensitized subjects. These results indicate differential release of eosinophil granule proteins in peripheral blood from eosinophils, and they also indicate that MBP in serum likely is to be a better discriminator of atopy in CF.     

Disodium Cromoglycate and Food Allergy

In a food allergic patient challenge evoked a dual asthmatic response. These reactions could be partly or completely blocked by pretreatment with disodium cromoglycate (Intal, Lomudal) orally, depending on the doses given. Pretreatment with inhalations of disodium cromoglycate gave no protection.     

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.     

Eosinophil protein X and eosinophil cationic protein as indicators of intestinal inflammation in infants with atopic eczema and food allergy

BACKGROUND: The aim of this study was to evaluate the presence of allergic intestinal inflammation in infants with food allergy and atopic eczema before and after elimination diet, and to evaluate the use of eosinophil protein X (EPX) and eosinophil cationic protein (ECP) in the monitoring of inflammatory activity. METHODS: The study material comprised 25 infants with atopic dermatitis and food allergy. Thirteen healthy infants served as controls. Faecal and serum samples were collected before an elimination diet (on the first visit to the hospital) and approximately 3 months later for the determination of EPX and ECP. RESULTS: Before the elimination diet, infants with atopic dermatitis demonstrated markedly higher faecal concentrations of EPX and ECP than healthy controls (P = 0. 0003, P < 0.0001, respectively). The faecal concentrations of EPX and ECP showed a distinct decrease as a result of an adequate elimination diet in patients with favourable clinical response (P = 0.0027, P = 0.004, respectively). CONCLUSIONS: The results indicate the presence of marked intestinal inflammation in patients with atopic dermatitis and food allergy. The determination of faecal ECP and especially of faecal EPX provides a promising noninvasive tool in monitoring intestinal inflammation and disease activity in infants with atopic eczema and food allergy.     

实验性哮喘大鼠肺组织及血管中溶血磷脂酸的变化及地塞米松的抑制作用

研究哮喘大鼠肺组织及血浆中溶血磷脂酸 (lysophosphatidicacid ,LPA)水平的变化及地塞米松治疗对其变化的影响。将 30只健康大鼠随机分为对照组、哮喘组及激素治疗组 ,每组 10只 ,以卵蛋白致敏激发制作大鼠哮喘模型。用有机溶剂提取 ,采用薄层层析的方法进行分离 ,最终用定磷的方法测定溶血磷脂酸。结果显示哮喘组肺组织的LPA(15 8 5 9± 78 80 )nmol g显著高于正常组 (6 8 94± 32 5 7)nmol g(P <0 0 1) ;激素治疗组肺组织的LPA(75 2 7±37 94 )nmol g显著低于哮喘组 (P <0 0 5 ) ,但仍高于对照组。哮喘组大鼠肺组织的LPA水平与BALF中的嗜酸细胞呈显著正相关 (P <0 0 1)。各组血浆LPA含量无显著差异。实验结果提示LPA参与哮喘的发病 ,但主要参与气道的局部炎症反应。