Nitric oxide metabolites in induced sputum: a marker of airway inflammation in asthmatic subjects

BACKGROUND AND OBJECTIVE: The role of nitric oxide (NO) needs to be further clarified in allergic inflammation. This study was designed to investigate the relationships between NO metabolites and eosinophil count, eosinophil cationic protein (ECP), interleukin (IL)-5 in induced sputum from asthmatics. METHODS: Hypertonic saline-induced sputum was obtained in 25 asthmatic subjects, among which 13 patients were examined before and after anti-asthmatic medications including steroid preparations. Ten normal subjects were enrolled as controls. Fresh expectorated sputum separated from saliva was treated with equal volume of dithiothreitol 0.1%, cytospinned for cell count, and the supernatant was collected for biochemical assay. NO metabolites were assayed by using modified Griess reaction. ECP was measured by fluoroimmunoassay, and detected IL-5 by a sandwich ELISA. RESULTS: Asthmatic subjects, compared with controls, had significantly higher concentration of NO metabolites (1035.4 +/- 125.3 vs 557.2 +/- 101.5 micromol/L, P < 0.01), higher percentage of eosinophils (25.6 +/- 4.6 vs 1.7 +/- 0.2%, P < 0.01), and higher levels of ECP (1117.8 +/- 213.9 vs 154.6 +/- 47.4 microg/L, P < 0.01) in the induced sputum. IL-5 was detected more frequently in asthmatic subjects than in control subjects (11/25 [44%] vs 1/10 [10%], P < 0.05). According to asthma severity, moderate to severe asthmatic subjects (n = 18) had higher level of NO metabolites (1143.8 +/- 156.3 vs 575.5 +/- 89.5 micromol/L, P < 0. 01), higher levels of ECP and IL-5 (P < 0.01, respectively) in the induced sputum than in those of mild asthmatic subjects (n = 7). NO metabolites, the percentage of eosinophils, the levels of ECP, and IL-5 were reduced following treatment with anti-asthmatic drugs (P < 0.01, respectively). There were significant positive correlations between NO metabolites and percentage of eosinophils or ECP (r = 0. 34, P < 0.05; r = 0.28, P < 0.05). Negative correlations were noted between FEV1, FEV1/FVC and proportion of eosinophils, ECP, or IL-5 levels. CONCLUSION: These findings confirmed that the level of NO metabolites was increased in the tracheobronchial secretion of asthmatic subjects and was paralleled with severity of asthma. Measurement of NO metabolites in induced sputum may be used for monitoring the degree of airway inflammation in asthmatics.     

Corticosteroid resistance in mild asthma: markers of persistent inflammation.

BACKGROUND: Bronchial hyperresponsiveness (BHR) is an important feature of asthma. Glucocorticosteroids (GCS) reduce BHR, probably by suppressing allergic inflammation. There are, however, two groups of asthmatics with either GCS-responsive or non-responsive BHR to methacholine. We investigated the mechanism of non-GCS-responsive BHR in mild asthma. METHODS: Non-GCS-responsive BHR asthma was defined as failure of reduction of BHR to methacholine after a 2-week course of oral prednisolone (30 mg/day). The expression of interleukin (IL)-4, IL-5, IFN-gamma mRNA in peripheral blood mononuclear cells, eosinophil count, serum cortisol, eosinophilic cationic protein (ECP), and spirometry were measured in five non-GCS-responsive BHR asthmatics and six patients with GCS-responsive BHR asthma before and after prednisolone therapy. RESULTS: With the exception of serum ECP and expression of IL-5 mRNA, no significant differences were observed between GCS-responsive BHR and non-GCS-responsive BHR asthma. The mean ECP level was significantly higher in non-GCS-responsive BHR than in GCS-responsive BHR asthma before and after prednisolone therapy. Interleukin-5 mRNA was detected in all asthmatics before prednisolone therapy; however, after prednisolone therapy, IL-5 mRNA was only detected in non-GCS-responsive BHR asthmatics. CONCLUSIONS: Our findings suggest that activation of eosinophils appears to persist in some asthmatics with non-GCS-responsive BHR due to continuous IL-5 production by lymphocytes.     

Nitric oxide metabolites,eosinophils, and eosinophilic cationic protein in patients with asthma: sputum versus blood

The monitoring of airway inflammation has assessed in bronchial asthma directly by sputum examination, and indirectly by measurements in peripheral blood. To investigate the diagnostic value of these two methods, we compared nitric oxide (NO) metabolites, eosinophils, and eosinophil cationic protein (ECP) in sputum and blood in patients with asthma and control subjects. Sputum and serum were obtained from fifteen patients with asthma, and then were examined before anti-asthma treatment, including steroid preparations. ECP was measured by fluoroimmunoassay. NO metabolites were assayed by using modified Griess reaction. Asthmatic patients, compared with control subjects, had significantly higher level of NO metabolites, higher proportion of eosinophils, and higher levels of ECP in sputum. Asthmatic patients, compared with control subjects, however, had significantly higher number of eosinophils, and were at higher levels of ECP in blood. FEV1, FEV1/FVC was negatively correlated with sputum eosinophils. The area under receiver operating characteristic (ROC) curve showed that eosinophils in sputum are significantly accurate markers than NO metabolites in sputum and blood. These findings suggest that the proportion of eosinophils in sputum have more accurate diagnostic marker of asthmatic airway inflammation than NO metabolites in sputum in differentiating asthmatic patients from control subjects.     

Relationship between sputum inflammatory markers and osmotic airway hyperresponsiveness during induction of sputum in asthmatic patients

Hypertonic saline aerosols are being used increasingly for bronchial provocation testing and induction of sputum. The aims of this study were to assess the response to challenge with 3% hypertonic saline administered via a ultrasonic nebulizer in patients with asthma, and to evaluate relationship between % fall of FEV1 during induction of sputum (osmotic airway hyperresponsiveness; osmotic AHR) and biochemical markers of induced sputum. We investigated changes in FEV1 in response to inhaling ultrasonically nebulized 3% saline in 25 patients with asthma and 10 control subjects. FEV1 was measured before, during, and after induction of sputum. We used fluoroimmunoassay to detect eosinophil cationic protein (ECP), immunohistochemical staining to detect EG2+ (secretory form of ECP) eosinophils, and a sandwich ELISA to detect interleukin (IL)-5. Protein concentration was determined by using bicinchoninic acid protein assay reagent. Asthmatics, compared with controls, had significantly higher osmotic AHR. Moderate to severe asthmatics had significantly higher osmotic AHR compared to mild asthmatics. Osmotic AHR was significantly correlated with the proportion of eosinophils, the levels of ECP, EG2+ eosinophils, IL-5, and proteins. These data suggest that osmotic AHR is closely related to the clinical status and biochemical markers of sputum supernatant in asthmatic patients.     

Relationship between dendritic cells and activated eosinophils in induced sputum of asthmatics

It has been suggested that dendritic cells (DCs) are critical antigen presenting cells for eosinophilic airway inflammation in a mouse model of asthma, and cysteinyl leukotrienes may play a role in DC trafficking in asthmatics. We investigated whether the number of DCs is increased in the induced sputum of both atopic and nonatopic asthmatics and is related to activated eosinophil count in the sputum. Sputum was induced by inhalation of hypertonic saline in 9 atopic and 12 nonatopic asthmatics and 10 nonatopic normal controls, and differential cell counts were performed. DCs and activated eosinophils were identified by immunocytochemistry with monoclonal antibodies (anti-CD1a and EG2, respectively). There were significantly higher percentages of eosinophils, EG2+ cells, and CD1a+ DC in the sputum of atopic and nonatopic asthmatics compared with normal controls, respectively. In asthmatics, the percentage of CD1a+ DC was significantly correlated with that of EG2+ cells (Rs=0.62, p=0.004). We demonstrated that the increased number of DCs was evident in the induced sputum of both atopic and nonatopic asthmatics, and the DC number was related to the activated eosinophil count, which suggests that DCs may contribute to the ongoing eosinophilic inflammation in asthmatic airways, and vice versa.     

Application of monoclonal antibodies against major basic protein (BMK-13) and eosinophil cationic protein (EG1 and EG2) for quantifying eosinophils in bronchial biopsies from atopic asthma

A monoclonal antibody prepared against the eosinophil major basis protein (MBP) was compared with the anti-eosinophil cationic protein (ECP) antibodies (EG1 and EG2) in immunostaining of bronchial biopsies from atopic asthma and controls. Anti-MBP (designated BMK-13) did not cross-react with other eosinophil basic proteins (i.e. ECP, eosinophil peroxidase [EPO] or eosinophil-derived neurotoxin [EDN]) and stained more than 98% of peripheral blood eosinophils irrespective of their degree of activation. EG2 stained 15% of resting and 75% of activated eosinophils; EG1 recognized 74% and 78% of resting and activated cells, respectively. The numbers of BMK-13, EG1 or EG2-positive staining cells in bronchial biopsies from asthma were significantly greater than atopic non-asthmatics (P less than 0.02, P less than 0.01 and P less than 0.05, respectively) and normal non-atopic controls (P less than 0.001). For each of the various groups studied, the rank order for the number of eosinophils stained was BMK-13 greater than EG1 greater than EG2. BMK-13 stained significantly more cells from bronchial biopsies of atopic asthma and atopic non asthma when compared to EG2 (P less than 0.001 and P less than 0.05, respectively). Since only a proportion of BMK-13+ cells were EG2+, these results suggest that not all tissue eosinophils are actively secreting. Thus, BMK-13 can serve as a useful pan-eosinophil marker in tissue sections since it appears to stain most eosinophils.     

Characterization of eosinophils and detection of eotaxin in skin chamber fluid after challenge with relevant allergen in patients with mild asthma

BACKGROUND: A selective recruitment of eosinophils to sites of allergic inflammation is suggested to be controlled by regulation of cytokines, chemokines and adhesion molecules. OBJECTIVE: The aim of this study was to examine whether allergen challenge in skin chambers, applied on patients with allergic rhinitis and mild asthma, results in a selective influx of activated eosinophils and detectable levels of cytokines/chemokines related to eosinophil recruitment, such as interleukin (IL)-5 and eotaxin. METHODS: A skin blister was induced on the volar aspect of each forearm; one contained PBS-heparin buffer (control) and the other was challenged with relevant allergen. Peripheral blood was drawn before the allergen was applied to the skin chamber, and the expression of CD9, CD11b and EG2-epitope on intracellular eosinophil cationic protein (ECP) was analysed in eosinophils. Chamber fluid was collected 8 h after allergen application and analysed for differential cell counts, expression of eosinophil activity markers, the presence of ECP, eotaxin, and IL-5. RESULTS: The number of recruited leucocytes was equal in the allergen-challenged chambers and in controls. However, the number of eosinophils was significantly increased in the allergen-challenged chambers, and elevated levels of released ECP were measured. Moreover, the eosinophils recruited were activated, as shown by increased expression of EG2 and CD11b, and decreased expression of CD9, in comparison with blood eosinophils. In the skin chamber fluids, higher levels of eotaxin were detected in the allergen-challenged chambers than in controls, but there were no detectable levels of IL-5. CONCLUSION: We have demonstrated a selective recruitment of eosinophils, and higher levels of released ECP and eotaxin, in skin chambers stimulated with allergen, as compared with control chambers. Allergen challenge in skin chambers is a useful tool for studies of eosinophil recruitment, their state of activation, and their involvement in the allergic inflammatory response.     

Eosinophil activity markers in peripheral blood have high predictive value for bronchial hyperreactivity in patients with suspected mild asthma

The present study aimed to evaluate the predictive value of eosinophils and markers of their activity for bronchial hyperreactivity (BHR) in a population of patients with recently developed clinical symptoms of asthma. The activation of eosinophils was estimated by measuring eosinophil cationic protein (ECP) in serum. In addition, flow cytometry was used to measure the expression of the EG2-epitope on intracellular ECP in eosinophils from peripheral blood. Twenty-eight consecutive patients with clinical history of asthma were studied. Of the 28 patients, 18 had a positive bronchial challenge test measured as PD₂₀≤ 1600 μg histamine. A significantly higher concentration of eosinophils and a trend to higher ECP in the peripheral blood was found in the hyperreactive group than in the nonreactive group. However, the intracellular expression of ECP did not correlate with the PD₂₀ value, and no significant difference between the groups was found. With one eosinophil activity marker, either serum ECP or EG2, BHR could be predicted in 70% of the patients. If we combined any two of the activity markers (serum ECP, EG2, or the percentage of eosinophils), the predictive value increased to 100%. We conclude that the blood eosinophil concentration, as well as, to some extent, serum ECP, has a high specificity for BHR in patients with recently developed clinical symptoms of asthma. Despite normal bronchial reactivity, some patients had signs of activated eosinophils, i.e., high serum ECP and increased EG2 expression. Thus, these markers may reflect early stages in the development of BHR. Our results also indicate that a combined evaluation of percentage of eosinophils and of eosinophil activity markers is of clinical value to predict BHR.     

Eotaxin in induced sputum of asthmatics: relationship with eosinophils and eosinophil cationic protein in sputum

BACKGROUND: Eosinophilic inflammation is a crucial aspect of allergic diseases such as bronchial asthma. An eosinophil-active chemokine, eotaxin, may play a role in the pathogenesis of the tissue eosinophilia accompanying asthma. METHODS: Induced sputa were obtained from 53 patients with atopic asthma and six healthy subjects, and the concentration of eotaxin in the sputum was measured by ELISA. We investigated whether the sputum content of eotaxin is related to 1) asthma status or corticosteroid therapy, and 2) other sputum indices, including percentage of eosinophils and concentration of eosinophil cationic protein (ECP). RESULTS: The patients with stable or unstable asthma showed significantly higher concentrations of sputum eotaxin than the normal controls. The level of sputum eotaxin demonstrated a positive correlation with the percentage of eosinophils in stable asthmatics not receiving corticosteroid therapy, but not in stable patients treated with corticosteroids, or in unstable patients. Sputum eotaxin demonstrated a positive correlation with ECP in asthmatic patients who were either in a stable state or not receiving steroid therapy. CONCLUSIONS: The elevated level of eotaxin detected in association with increased eosinophils and ECP in the sputum of asthmatics suggests that eotaxin is involved in the pathogenesis of eosinophilic airway inflammation. The relationship of eotaxin to airway eosinophilia may be modified by the stability status of asthma and corticosteroid therapy.     

Analysis of induced sputum to examine the effects of inhaled corticosteroid on airway inflammation in children with asthma.

BACKGROUND: Analysis of induced sputum can be performed safely in children with asthma and is useful for both cellular and biochemical markers of inflammation. Glucocorticosteroid inhalation has become the first line therapy for chronic asthma by suppressing airway inflammation, which produces the decrease of bronchial hyperreactivity and reduces the number of eosinophil in bronchial submucosa. OBJECTIVE: To determine the characteristics of the inflammatory cells and their markers in sputum and to examine the pharmacokinetic effects of glucocorticoid within 3 hours after inhalation therapy on FEV1 and sputum inflammatory indices in children with clinically defined chronic asthma. METHODS: Thirty subjects with asthma included 14 current symptomatic asthmatics and 14 normal controls inhaled 4.5% hypertonic saline for 10 minutes by nebulizer. The expectorated sputum were collected from all asthmatics before and 3 hours after corticosteroid inhalation for children with asthma and were reduced by dithiotreitol. Total cell counts and differentials were determined. ECP was measured by CAP system. Interleukin-5, GM-CSF and albumin were measured by double sandwich ELISA. RESULTS: The mean eosinophil percentage and ECP in induced sputum of asthmatics were significantly higher than that of controls. The induced sputum samples obtained after glucocorticoid inhalation showed a significant reduction in mean eosinophil percentage, but FEV1, IL-5, GM-CSF, albumin, and ECP values were not significantly decreased. CONCLUSION: The present results in induced sputum may be interpreted to reflect direct steroid action on airways and lack of effect on bone marrow effectors at 3 hours after glucocorticoid inhalation.     

Reactivity of monoclonal antibodies EG1 and EG2 with eosinophils and their granule proteins.

Use of the murine monoclonal antibodies EG1 and EG2 has been based on the assumption that EG2 recognizes activated eosinophils. We examined the reactivity of EG1 and EG2 with eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), and stimulated and nonstimulated eosinophils from normal donors. By radioimmunoassay, EG1 recognized only ECP, whereas EG2 recognized both ECP and EDN. By Western blot, EG1 reacted with ECP, EG2 reacted with both ECP and EDN, but EG2 could not distinguish between lysates of stimulated and nonstimulated eosinophils. By immunofluorescence, EG1 and EG2 at 20 microg/mL stained 95-100% of nonstimulated eosinophils, regardless of fixative; EG1 and EG2 at 0.1 microg/mL stained 61-90% of acetone- and paraformaldehyde-fixed and only 5-21% of methanol-fixed nonstimulated eosinophils. Thus, the reactivity of EG1 and EG2 with eosinophils depends on the method of fixation and antibody concentration; and EG2, in contrast to previous reports, cannot reliably discriminate between resting and activated eosinophils.     

Effect of inhaled interleukin-5 on number and activity of eosinophils in circulation from asthmatics

The aim of the present study was to examine the effects of interleukin-5 (IL-5) inhalation on changes in the activity and number of circulating eosinophils, as well as concentrations of serum total IgE, in allergic asthmatics. A randomized double-blind, placebo-controlled study design was employed in which each subject acted as his or her own control. Eight nonsmoking patients with allergic asthma were administered recombinant human IL-5 by nebulization. Total white blood cell counts and differentials, as well as concentrations of ECP and total IgE in serum, were determined before and at 2, 24, and 48 h after inhalation. Our results demonstrated that eosinophil numbers increased from baseline (3.6 +/- 1.1 x 10(5)/ml) to 6.3 +/- 1.2 x 10(5)/ml (P < 0.01) at 24 h and to 5.7 +/- 0.9 x 10(5)/ml (P < 0.01) at 48 h after IL-5 inhalation in asthmatics. Accompanying this significantly increased blood eosinophilia were significantly elevated serum ECP levels. Compared with baseline (6.3 +/- 1.1 ng/ml), ECP levels increased with time following IL-5 inhalation, reaching 17.6 +/- 2.8 ng/ml (P < 0.01) at 24 h and remaining elevated at 48 h (18.1 +/- 2.9 ng/ml, P < 0.01). IL-5 inhalation had no significant effect on levels of serum total IgE, however. These findings provide direct evidence that nebulized IL-5 not only induces a significant blood eosinophilia but also results in the activation of circulating eosinophils. Our data further support the importance of IL-5 in the pathogenesis of bronchial asthma in humans.     

Activation of beta1 integrins on blood eosinophils by P-selectin

Activation of β(1) integrins of blood eosinophils, assessed by mAb N29, correlates inversely with FEV(1) in two paradigms for studying control of human asthma. We asked whether P-selectin causes eosinophil β(1) integrin activation and results in increased adhesivity. By dual-label flow cytometry, eosinophils with high levels of surface-associated P-selectin had higher reactivity with the activation-sensitive anti-β(1) mAbs N29, 8E3, and 9EG7 than eosinophils with no or with a low-level of surface-associated P-selectin. Among patients with nonsevere asthma, surface P-selectin correlated with N29, 8E3, and 9EG7 signals. By immunofluorescence microscopy, surface-associated P-selectin was present in patches on eosinophils, some of which stained for the platelet marker thrombospondin-1. Activated β(1) and P-selectin partially colocalized on eosinophils. Soluble P-selectin added to whole blood enhanced activation of eosinophil β(1), but not β(2), integrins. In contrast, IL-5 activated eosinophil β(2), but not β(1), integrins. Eosinophils that did not attach to vascular cell adhesion molecule-1 (VCAM-1) in a static adhesion assay had a lower N29 signal than the original population. Soluble P-selectin added to whole blood enhanced eosinophil adhesion to VCAM-1. These findings are compatible with a scenario whereby P-selectin, on eosinophil-associated activated platelets or acquired from plasma or from prior interactions with endothelial cells or platelets, activates eosinophil α(4)β(1) integrin and stimulates eosinophils to adhere to VCAM-1 and move to the airway in asthma.     

Eosinophil chemoattractants in the middle ear of patients with eosinophilic otitis media

BACKGROUND: Patients with intractable otitis media associated with bronchial asthma have an extensive accumulation of eosinophils in the effusion and mucosa of the middle ear; this condition is called eosinophilic otitis media (EOM). It remained to be determined how eosinophils accumulate in the middle ear. OBJECTIVES: To clarify the pathogenesis of middle ear diseases, we measured the concentration of eosinophil chemoattractants in middle ear effusion (MEE), and carried out immunohistochemical studies of middle ear mucosa specimens to demonstrate the expression of eosinophil chemoattractants. METHODS: Middle ear effusion samples were obtained from 15 EOM patients with bronchial asthma and from six controls for the measurement of eosinophil cationic protein (ECP), IL-5, eotaxin and regulated on activation, normal T expressed and secreted concentrations. Middle ear mucosa samples were also taken from 14 EOM patients and 16 controls for immunohistochemical study. In 10 EOM patients, the numbers of immunoreactive cells as well as apoptotic cells were determined before and after the topical application of triamcinolone acetonide into the middle ear. RESULTS: In EOM, significantly higher ECP and IL-5 concentrations were detected in MEE than in serum, and ECP, IL-5 and eotaxin concentrations in MEE were higher in the EOM patients than in the controls. ECP concentration positively correlated with that of IL-5. Immunohistochemically, the numbers of cells positive for EG2 and ecalectin were significantly higher in the EOM patients than in the controls. After the topical application of triamcinolone acetonide, the numbers of infiltrating cells and immunoreactive cells distinctly decreased, whereas the number of apoptotic cells significantly increased. CONCLUSION: In EOM, locally produced IL-5 may play a crucial role in the accumulation of eosinophils in the middle ear. Chemokines such as ecalectin and eotaxin are also produced in the middle ear, and help activate and enhance the survival of eosinophils to induce the intractable condition in the middle ear. The topical application of triamcinolone acetonide induces the apoptosis of not only eosinophils but also eosinophil chemoattractant-producing cells, thereby improving the middle ear condition.     

Asthma in children less than 5 years of age: eosinophils and serum levels of the eosinophil proteins ECP and EPX in relation to atopy and symptoms

Children less than 5 years of age with asthma were assessed for total eosinophil counts and scrum levels of the eosinophil proteins, eosinophil cationic protein (ECP) and eosinophil protein X (EPX). to determine whether these measurements would reflect eosinophilic inflammation in the airways. Initially 27 symptomatic patients, 14 atopic and 13 non-atopic were investigated. They had a mean age of 1.8 years and had never been treated with inhaled steroid and had not received Intal for 2 weeks prior to the assessment. The 14 atopic patients proved to have higher mean total eosinophil counts and serum levels of ECP and EPX than the 13 non-atopic patients (eosinophil counts 0.63 10⁹/1 vs 0.26 × 10⁹/1, P < 0.001; ECP 36.9 μg/1 vs 10.8 μg/1, P < 0.001; EPX 69.0 μg/1 v.s 19.6 μg/l, P < 0.01. Thirteen of these patients required treatment with daily doses of inhaled steroid and 11 had a repeal assessment (seven atopic and four non-atopic). The mean serum EC'P of the seven atopic patients had fallen significantly (40.6 to 22.9. P < 0.05) while the total eosinophil counts did not. These results suggest a difference in numbers and activity of eosinophils in a topic compared with non-atopic asthma in young children. To determine whether the results were influenced by treatment with inhaled steroids. 31 patients who were being treated with daily inhaled steroid underwent assessment when they were symptomatic (22 samples) or asymptomatic (19 samples). Of the 31 patients. 11 were atopic and 20 non-atopic, Atopic asthmatics had higher levels of eosinophils and serum ECP than non-atopic patients when symptomatic patients were compared, despite treatment with inhaled steroid. Finally, in order to determine whether the ECP correlates with atopy rather than asthma, 19 patients who were seen for assessment of a reaction to a food (usually peanut or egg) and who had a positive skin lest to the appropriate food were examined. Twelve of these patients had a history of intermittent asthma and a mean ECP of 31 9μg/I while seven patients had no asthma and a mean ECP of 13.4 μg/l (P < 0.05), The total eosinophil counts showed the same difference. This suggests that atopy in the absence of asthma may not be associated with an elevated eosinophil count or ECP level. The data suggest that atopy contributes lo childhood asthma, even in infancy, by mobilization and activation of eosinophils. Scrum ECP might be a useful measure of eosinophil activation in asthma of early childhood.     

Macrophage migration inhibitory factor (MIF) in bronchial asthma

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine favouring the secretion of TNFalpha and IL-8 and counteracts anti-inflammatory effects of corticosteroids. Airways inflammation is a central feature of bronchial asthma and is characterized by the accumulation of eosinophils. OBJECTIVE: The aim of this study was to investigate whether MIF is related to asthma symptoms and eosinophil accumulation in the airways. METHODS: Serum MIF levels were measured by an enzyme-linked immunosorbent assay in 44 healthy subjects and 44 asthmatics. Levels of MIF in induced sputum were measured in 10 healthy subjects and 15 asthmatics. Levels of eosinophil cationic protein (ECP) in induced sputum were measured by a radioimmunosorbent assay. Fluorescence double immunostaining was conducted to examine cellular source and localization of MIF. RESULTS: Serum MIF levels were significantly increased in asthmatic patients compared with age and sex-matched control subjects. Symptomatic patients had a higher MIF level than asymptomatic patients. Induced sputum obtained from asthmatics contained higher levels of MIF than those from control subjects. MIF levels in induced sputum were correlated with ECP levels in induced sputum. MIF was colocalized with eosinophil peroxidase staining in the cytoplasm of sputum cells. CONCLUSION: Increased MIF levels are associated with asthma symptoms and one of the cellular sources of MIF in the airways are eosinophils.     

Release of granule proteins from human eosinophils stimulated with mast-cell mediators

Background It has been suggested that mast cells and eosinophils are major effector cells in the pathogenesis of allergic diseases. However, the interaction of these cells has not been thoroughly elucidated. We examined eosinophil cationic protein (ECP) release and cytosolic free calcium concentration ([Ca²⁺]) in human eosinophils induced by the major mast-cell mediators including cytokines. Methods Eosinophils from healthy donors were stimulated with the major mast-cell mediators for 20 min after preincubation with cytochalasin B for 10 min. ECP in supernatants was measured by radioimmunoassay. Moreover, t o examine changes of [Ca²⁺]i in eosinophils, Fura-2-loaded eosinophils were monitored for fluorescence changes after stimulus addition. Results Of the tested mediators (prostaglandin [PG]D₂, leukotriene (LT)B₄, platelet-activating factor (PAF), histamine, LTQ, and eosinophil chemotactic factor of anaphylaxis [ECF-A]), LTB₄ and PAF induced ECP release from eosinophils. Any cytokines produced by human mast cells, i.e., interleukin (IL)-4, IL-5, IL-8, tumor necrosis factor (TNF), or granulocyte-macrophage colony-stimulating factor (GM-CSF), did not induce ECP release in our system. ECP release triggered with LTB₄ and PAF occurred at concentrations of 10^?8-10^?6 M concentration-dependently. LTB4 and PAF also elicited a rise in [Ca²⁺]_i in eosinophils. Neither PGDj, histamine, nor LTC₄ induced ECP release, although they increased cytosolic calcium in eosinophils. Conclusions Of mast-cell mediators, LTB₄ and PAF induced eosinophil degranulation. The contribution of LTB4 and PAF from mast cells to eosinophil degranulation may be important in the pathogenesis of allergic inflammatory diseases.     

Interleukin-5 mRNA in mucosal bronchial biopsies from asthmatic subjects

Using in situ hybridization, we have investigated the expression of interleukin-5 (IL-5) mRNA in bronchial biopsies from asthmatics (n = 10) and controls (n = 9). The number of IL-5-nRNA-positive cells were compared with the number of CD25+ and EG2+ cells and total eosinophil counts. Specific hybridization signals for IL-5 mRNA were demonstrated in 6 out of the 10 asthmatic subjects but in none of the controls. The 6 IL-5-mRNA-positive asthmatics tended to have more severe disease and showed a significant increase in the degree of infiltration of the bronchial mucosa by activated T lymphocytes and eosinophils.     

Markers for eosinophils and T-lymphocytes as predictors of late asthmatic response

Bronchial allergen challenge was performed on 12 allergic asthmatics during a stable phase of their disease. After resolution of the immediate bronchial response, fractional lung lavage was performed twice, two and 24 h post-challenge. The recovery of eosinophils, eosinophil cationic protein (ECP), eosinophil chemotactic activity (ECA) and immunohistochemical staining of the phenotypically distinct population stainable by the monoclonal antibody CD45R0, agreed to indicate T-memory cells, were assessed in the two lavages. Serial measurements of lung function, and serum concentration of ECP were also done. We found that although the recoveries in bronchial washes of eosinophil cells and ECP tended to increase during the trial, none of these variables predicted the emergence of late phase bronchial response (LPR). Instead, the proportion in the 2-h lavages, of memory-cells or ECA predicted the LPR. These two variables were inversely correlated to each other in the first lavage, suggesting the T-cells to be potential major sources of ECA. The fact that T-cells and ECA, but not markers for eosinophil activation in lavage, predicted the LPR, may suggest T-cell activation to preceed the activation of the eosinophils within the lung after a bronchial allergen challenge. There was a close correlation between LPR and serum concentration of ECP obtained at the end of the trial, 24 h post-challenge, suggesting either a delayed or a continuous activation of circulating eosinophils after bronchial allergen challenge.     

T cell and eosinophil activation in mild and moderate atopic and nonatopic children's asthma in remission

BACKGROUND: Inflammation in the pathogenesis of asthma is associated with products of activated T cells and eosinophils. The aim of this study was to determine whether ongoing inflammation persists in children with different phenotypes of asthma despite the disease in remission. METHODS: Serum samples were collected from 68 children with atopic or nonatopic asthma in remission and from 15 healthy children. Soluble interleukin-2 receptor (sIL-2R), IL-2 and IL-4 were examined by using an enzyme-linked immunosorbent assay. Total and specific immunoglobulin E, and eosinophil cationic protein (ECP) were analysed by fluoroimmunoassay (Pharmacia CAP System). RESULTS: In patients with moderate persistent atopic asthma, sIL-2R was increased significantly when compared with mild persistent atopic asthma (P < 0.05). No changes of sIL-2R were seen in nonatopic asthmatics compared with atopics and controls. The level of IL-2 was elevated in moderate persistent atopic and nonatopic asthmatic children compared with controls (P < 0.05 and P < 0.05 respectively) and compared with mild persistent atopic asthmatics and mild persistent nonatopic asthmatics (P < 0.05 in both cases). The levels of IL-4 in most patients and controls remained below the sensitivity of the assay. Eosinophil cationic protein levels in moderate persistent atopic and nonatopic asthmatics were significantly higher than in mild persistent asthma severity cases (P < 0.001 and P < 0.01 respectively) and in healthy children (P < 0.01 in both cases). CONCLUSION: Changes in the concentration of sIL-2R, IL-2 and ECP reflect increased T cell and eosinophil activity in relation to the level of severity of asthma in atopic and nonatopic children, thereby proving the presence of persistent inflammation despite the absence of disease symptoms.