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.     

Evidence of eosinophil, neutrophil, and mast-cell mediators in the effusion of OME patients with and without atopy

BACKGROUND: This study was designed to measure the involvement of eosinophils, neutrophils, and mast cells in the middle ear of patients with OME and to see whether that inflammatory response differed depending on whether or not the patient was atopic. METHODS: Levels of ECP, MPO, and tryptase were measured in effusion from 97 patients whose atopic status was determined by in vitro testing with 12 inhalants and five foods. RESULTS: Seventy-nine of 97 OME patients (81%) were atopic, among whom 86% (68/79) had elevated levels of effusion ECP, and 64% (23/36) had elevated tryptase. Mean ECP was 166 microg/l vs 3.38 microg/l, mean MPO was 6231 microg/l vs 116 microg/l, and mean tryptase was 4.8 microg/l vs 1.34 microg/l in atopic vs nonatopic ears. Total serum IgE did not differ between atopics and nonatopics (P=0.28). CONCLUSIONS: The inflammatory response by eosinophils, neutrophils, and mast cells in the middle ear was distinctly different between atopic and nonatopic patients (P < 0.001). This study provides evidence that eosinophils and mast cells, both essential to a Th-2 driven immune response, are present in the majority of ears with chronic OME, and supports the hypothesis that middle-ear mucosa, like that of the rest of the upper respiratory tract, is capable of an allergic response.     

Interleukin-4 and interleukin-4 soluble receptor alpha levels in bronchoalveolar lavage from children with asthma.

BACKGROUND: In asthma there is increased expression of the Th2-type cytokine interleukin-4 (IL-4). IL-4 is important in immunoglobulin isotype switching to immunoglobulin E and adhesion of eosinophils to endothelium. OBJECTIVE: We hypothesized that levels of IL-4 in bronchoalveolar lavage (BAL) fluid would be increased in stable, atopic asthmatic children compared with controls and that levels of its physiologic inhibitor IL-4 soluble receptor alpha (IL-4sR alpha) would be correspondingly decreased. METHODS: One hundred sixteen children attending a children's hospital for elective surgery were recruited. A nonbronchoscopic BAL was performed, and IL-4 and IL-4sR alpha were measured in the BAL supernatants. RESULTS: There was no significant difference in IL-4 concentrations between atopic asthmatic children, atopic normal controls, and nonatopic normal controls [0.13 pg/mL (0.13 to 0.87) vs 0.13 pg/mL (0.13 to 0.41) vs 0.13 pg/mL (0.13 to 0.5), P = 0.65]. IL-4sR alpha levels were significantly increased in asthmatic patients compared with atopic controls [6.4 pg/mL (5.0 to 25.5) vs 5.0 pg/mL (5.0 to 9.9), P = 0.018], but not when compared with the nonatopic controls [5.2 pg/mL (5.0 to 10.6), P = 0.19]. CONCLUSIONS: Contrary to expectation, IL-4sR alpha levels are increased in BAL from stable asthmatic children compared with nonatopic controls, and we speculate that IL-4sR alpha is released by inflammatory cells in the airways to limit the proinflammatory effects of IL-4.     

Bronchial hyper-responsiveness to hypertonic saline and blood eosinophilic markers in 8–13-year-old schoolchildren

BACKGROUND: In adult asthma, bronchial hyper-responsiveness (BHR) to indirect stimuli reflects eosinophilic activation more closely than BHR to stimuli that directly cause smooth muscle contraction. AIM: To assess the relationship between BHR to the indirect stimulus hypertonic saline (HS), blood eosinophil numbers, and serum eosinophilic cationic protein (ECP) in children with and without current wheeze. METHODS: A cross-sectional survey among 8-13-year-old schoolchildren, using the International Study of Asthma and Allergic disease in Childhood questionnaire, bronchial challenge with HS, skin prick tests, serum IgE, blood eosinophil counts and ECP (in a subset). Based upon the presence of current wheeze (WHE) and BHR, we defined three case groups (WHE+BHR+, WHE-BHR+, WHE+BHR-) and the reference group (WHE-BHR-). By regression analyses, each case group was compared with the reference group for differences in atopic sensitization, blood eosinophil counts and serum ECP. RESULTS: Complete data were obtained for 470 children. BHR was present in 103 children (22%), 66 being asymptomatic and 37 symptomatic. Children of all three case groups were more often atopic. Sensitization to indoor allergens particularly occurred in children with BHR, irrespective of symptoms (P < 0.05). Children with WHE+BHR+ had highest values for blood eosinophils and serum ECP (P < 0.05). Children with WHE-BHR+ had less severe responsiveness. In atopic children with WHE-BHR+, serum ECP was higher than in children with WHE-BHR-(P < 0.05). CONCLUSIONS: BHR to HS is associated with blood markers of eosinophilic activation, particularly in atopic children.     

Bronchoalveolar lavage findings suggest two different forms of childhood asthma

Background It seems plausible that children with atopy and persistent asthma symptoms will, like their adult counterparts, have chronic airways inflammation. However, many young children with no other atopic features have episodic wheezing that is triggered solely by viral respiratory infections. Little is known as to whether airways inflammation occurs in these two asthma patterns during relatively asymptomatic periods. Methods Using a non-bronchoscopic bronchoalveolar lavage (BAL) procedure on children presenting for an elective surgical procedure, this study has investigated the cellular constituents of BAL fluid in children with a history of atopic asthma (AA) non-asthmatic atopic children (NAA) or viral associated wheeze (VAW). Results A total of 95 children was studied: 52 with atopic asthma (8.0 years, range 1.1–15.3, 36 male), 23 with non-asthmatic atopy (median age 8.3 years, range 1.7–13.6, 11 male) and 20 with VAW (3.1 years, range 1.0–8.2, 13 male). No complications were observed during the lavage procedure and no adverse events were noted post-operatively. Total lavage fluid recovered was similar in all groups and the total cell numbers were higher in the VAW group. Eosinophil (P≤ 0.005) and mast cell (P≤ 0.05) numbers were significantly elevated in the group with atopic asthma. Conclusions During relatively asymptomatic periods there is on-going airways inflammation, as demonstrated by eosinophil and mast cell recruitment, in children with asthma and atopy but not in children with viral associated wheeze or atopy alone. This strongly suggests that there are different underlying pathophysiological mechanisms in these two groups of children who wheeze.     

Mucosal and systemic inflammatory changes in allergic rhinitis and asthma: a comparison between upper and lower airways

BACKGROUND: Local airway inflammation and airway remodelling are considered important in the clinical expression of allergic asthma. OBJECTIVE: The aim of this study was to compare airway inflammation and remodelling in nasal and bronchial mucosa of subjects with allergic rhinitis with or without asthma. METHODS: Four experimental groups were formed: allergic asthma and rhinitis (n = 19); allergic rhinitis, no asthma (n = 18); atopic subjects, no asthma, no rhinitis (n = 8) and non-allergic healthy control subjects (n = 16). Blood samples, nasal and bronchial biopsy specimens were collected during stable disease. Immunohistochemistry was performed for eosinophils (MBP), mast cells (CD117) and vascular endothelium (CD31). Epithelial loss, reticular basement membrane (RBM) thickness and subepithelial vascularity was assessed with a computer-assisted image analysis system. RESULTS: In nasal and bronchial mucosa, numbers of eosinophils were significantly higher in rhinitis patients with and without asthma than in asymptomatic atopics (P < 0.05) and controls (P < or = 0.01). In bronchial mucosa, the RBM was significantly thickened in rhinitis patients with and without asthma compared to asymptomatic atopics (P < 0.05) and controls (P < 0.01), while in nasal mucosa no differences were seen. Patients with asthma and rhinitis had increased numbers of blood eosinophils (P = 0.05) and skin test reactivity (P = 0.01) compared to patients with rhinitis only. No significant differences could be found between the investigated groups with respect to serum IL-5 and eotaxin levels, the number of mucosal mast cells and the degree of epithelial loss and subepithelial vascularity. Epithelial desquamation was significantly increased in the bronchial mucosa compared to nasal mucosa, not only in asthmatics (P < 0.001), but also in atopics without asthma and rhinitis (P = 0.02). CONCLUSIONS: This study shows that allergic inflammation, increased basement membrane thickness and epithelial desquamation are present in the lower airways of atopic subjects, even before the onset of clinical symptoms. Despite the presence of inflammatory cells, no structural changes could be assessed in nasal mucosa of allergic patients.     

Expression of eotaxin in induced sputum of atopic and nonatopic asthmatics

BACKGROUND: The chemokine eotaxin has been implicated in airway eosinophilia in atopic asthma. We have compared airway eosinophils and eotaxin expression in induced sputum from well-matched atopic and nonatopic asthmatics. METHODS: Eosinophil numbers, eosinophil cationic protein (ECP), and the expression of eotaxin were examined in induced sputum from atopic asthmatics (AA = 11), nonatopic asthmatics (NAA = 11), and atopic (AC = 12) and normal (NC = 10) controls. Slides were prepared for differential cell counts by Romanowsky stain, and ECP levels were measured by RIA. Eotaxin expression was detected by in situ hybridization, with 35S-labelled riboprobes and immunocytochemistry. RESULTS: The numbers of eosinophils and ECP concentration were increased in the sputum of AA and NAA compared with AC and NC (P < 0.05). The numbers of eotaxin mRNA+ and immunoreactive cells were increased in NAA, but not AA, when compared with controls (P < 0.05). Eotaxin immunoreactive cells in NAA were significantly higher than in AA (P < 0.05). Eotaxin was expressed predominantly by macrophages, eosinophils, and epithelial cells. In NAA, but not AA, the numbers of eotaxin mRNA+ cells were correlated with histamine PC20 (r = -0.81, P < 0.01) and eosinophil numbers in sputum (r = 0.7, P < 0.05). CONCLUSIONS: Eotaxin production by macrophages, eosinophils, and epithelial cells may play a more pronounced role in airway eosinophilia in nonatopic than in atopic asthma.     

Airway eosinophilic inflammation, epithelial damage, and bronchial hyperresponsiveness in patients with mild–moderate, stable asthma

Allergic asthma is characterized by chronic recruitment of eosinophils in the airways. Once activated, eosinophils release toxic products, including eosinophil cationic protein (ECP), able to damage airway epithelial cells. To test the hypothesis that also in mild-moderate stable asthma, a significant eosinophil activation could occur, we studied 25 asthmatic patients (34 ± 19 years old), of whom 18 were allergic (27 ± 12 years) and seven nonallergic (42±10 years), with FEV₁ values ±70% of predicted, and eight normal volunteers (controls, 33 ±11 years). All subjects underwent methacholine (MCh) challenge on the first visit, and bronchoalveolar lavage (BAL) on the second visit (approximately 3–4 days later). BAL cells were counted and albumin (Alb) (as index of protein dilution in BAL fluid) and ECP levels (as index of eosinophil activation) in BAL fluid were measured. As compared to controls, a significant increase in BAL eosinophil and in BAL epithelial cell numbers was observed in asthmatic patients (P>0.05, each comparison), with no differences between the two asthmatic patient subgroups. Detectable ECP levels (>2 μg/1) were found in BAL of 18 asthmatic patients (14 allergic and four nonallergic asthmatic patients), while Alb levels were measurable in 25 BAL fluids and found to be similar in controls and asthmatic patients, and in the two asthmatic patient subgroups (P>0.05, each comparison). In BAL of asthmatic patients, positive correlations were found between eosinophil numbers and 1) ECP/Alb levels (r= 0.50, P = 0.020); 2) epithelial cell numbers (r = 0.S0, P = 0.014). In asthmatic patients, a significant negative correlation was found between bronchial reactivity to MCh (log Pd₁₅) and ECP/Alb levels in BAL fluid (r=-0.6, P= 0.005), whereas no correlation was found between log Pd₁₅ MCh and BAL eosinophil or epithelial cell number (P>0.1, each correlation). These data suggest that bronchial eosinophil recruitment and activation may occur also in mild-moderate stable asthma and that bronchial epithelium damage and airway responsiveness may be partially associated with the eosinophilic inflammatory reaction.     

Twenty-four-hour time course of eosinophil granule proteins ECP and EPX during bronchial allergen challenges in serum of asthmatic children

To study in vivo monitoring variables for bronchial allergen challenges, we investigated the time course of the eosinophil granule proteins, eosinophil cationic protein (ECP) and eosinophil protein × (EPX) after allergen provocation in serum. Thirty-two asthmatic children sensitive to house-dust mites and six healthy young adult controls were challenged by bronchial allergen provocations with Dermatophagoides pteronyssinus and D. farinae. Blood samples were taken at regular intervals up to 24 h. Base-line concentrations of ECP ( P <0.004), EPX ( P <0.002), and eosinophils ( P <0.001) were found to be increased in asthmatic children, as compared with healthy controls. ECP and EPX concentrations showed a uniform pattern with two characteristic features: 1) a rapid increase for both mediators up to 30 min after provocation over base-line values ( P <0.0001 and P <0.001), followed by a rapid decrease nearly to base-line values in the next 30 min; and 2) a steady increase for ECP and EPX up to 10 h ( P <0.02 and P <0.01), and even higher levels at 24 h, after challenge ( P <0.002 and P <0.003). We conclude that although eosinophils are activated in asthmatic children after bronchial allergen challenge, ECP and EPX concentrations are not suitable monitoring variables. Base-line eosinophils seem to predict the occurrence of a late-phase asthmatic reaction after allergen provocation.     

Eosinophils are activated in middle ear mucosa and middle ear effusion of patients with intractable otitis media associated with bronchial asthma

BACKGROUND: Although patients with intractable otitis media associated with bronchial asthma have extensive accumulation of eosinophils in the middle ear mucosa and middle ear effusion, systematic histological and immunohistochemical studies have not been performed. OBJECTIVES: To clarify the pathogenesis of middle ear diseases, we carried out immunohistochemical studies on middle ear specimens, particularly focusing on the characteristics of accumulated eosinophils. METHODS: Middle ear specimens obtained from eight adult patients and from 17 controls were immunohistochemically stained using monoclonal antibodies against EG1, EG2, mast cell tryptase, IgA and IgE. The concentration of eosinophil cationic protein (ECP) in middle ear effusion samples was also measured. RESULTS: In the asthmatic patients, severe round-cell infiltration was observed in the submucosa and most of the EG1-positive cells were also EG2-positive. In the control patients, the mucosa showed a fibrotic change with a few inflammatory cells, and EG1- or EG2-positive cells were quite few. The expression of IgE was found not only on the surface of mast cells but also within the plasma cells in the asthmatic patients, and the number of IgE-positive cells was about twice as high as that of mast cells. A significantly higher concentration of ECP was noted in middle ear effusion obtained from the asthmatic patients than that from the control patients. CONCLUSION: Most of the eosinophils in the middle ear mucosa and middle ear effusion were activated, resulting in degranulation and release of ECP, and local IgE production occurs in the middle ear mucosa, indicating that the intractable inflammation is closely associated with IgE-mediated late phase response with eosinophil accumulation.     

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.     

Specific immunoglobulin E for staphylococcal enterotoxins in nasal polyps from patients with aspirin-intolerant asthma

BACKGROUND: Nasal polyps infiltrated with eosinophils are commonly found in chronic asthmatic patients, more frequently in those with aspirin-intolerant asthma (AIA) than aspirin-tolerant asthma (ATA). Some studies have suggested a contribution of superantigens derived from Staphylococcus sp to nasal polyposis and eosinophilia, but their relative importance in AIA and ATA subjects is unknown. OBJECTIVE: We investigated whether local production of specific IgE to staphylococcal enterotoxins A and B (SEA and SEB) and relationships with markers of eosinophilic inflammation differ in the nasal polyps of AIA and ATA subjects. METHODS: Fifteen AIA subjects with positive responses to lysine-aspirin bronchoprovocation and 15 ATA subjects underwent polypectomy. Immunoassays were used to quantify eosinophil cationic protein (ECP), IL-5, mast cell tryptase, soluble IL-2 receptors (sIL-2R), total IgE, and specific IgE for SEA and SEB. RESULTS: ECP levels in nasal polyp homogenates were higher in AIA subjects than in ATA subjects (P < 0.02), with no significant differences in tryptase, IL-5 or sIL-2R. Total IgE, and specific IgE to both SEA and SEB, were detectable in some nasal polyps from both subject groups, but median levels were markedly higher in AIA subjects than in ATA subjects (P = 0.04, 0.01, 0.05, respectively). Levels of specific IgE to SEA and SEB correlated significantly with levels of ECP and IL-5, but not those of tryptase or sIL-2R. CONCLUSION: These findings suggest that staphylococcal superantigens may drive local eosinophilic inflammation in nasal polyp tissue, and that this is exacerbated in subjects with AIA.     

Identification of IL-16 as the lymphocyte chemotactic activity in the bronchoalveolar lavage fluid of histamine-challenged asthmatic patients

Objective: We have previously demonstrated that the earliest lymphocyte chemotactic factors present in bronchoalveolar lavage fluid (BALF) of subjects with atopic asthma after subsegmental antigen challenge are IL-16 and MIP-1α, of which IL-16 appears to contribute a majority of the chemotactic activity. Because IL-16 is released in vitro after histamine stimulation of CD8⁺ T cells and epithelial cells, we evaluated the potential role of histamine in the release of IL-16 into the airways of allergic asthmatics in vivo. Methods: Eight allergic asthmatic subjects, six normal subjects, and six atopic nonasthmatic subjects were challenged with saline in the lingula and with serial concentrations of histamine (1 × 10^-7 to 5 × 10^-5 mol/L) in the right middle lobe followed by bronchoalveolar lavage (BAL) 15 minutes and 6 hours later. Results: The BALF from saline- and histamine-challenged lobes of normal subjects and atopic nonasthmatic subjects contained no significant lymphocyte chemoattractant activity. In six of the eight atopic asthmatic subjects, the histamine-challenged but not saline-challenged segment contained IL-16 chemotactic activity but no other identifiable lymphocyte chemoattractant activities at 6 hours. Conclusions: IL-16 appears in the airways after histamine challenge and therefore could contribute to the earliest infiltration of CD4⁺T cells and eosinophils observed after antigen challenge due to histamine release from mast cells. (J Allergy Clin Immunol 1998;101:786-792.)     

Relationship between childhood atopy and wheeze: what mediates wheezing in atopic phenotypes?

BACKGROUND: The nature of the relationship between childhood wheeze and atopy remains uncertain. OBJECTIVE: To characterize childhood wheeze among atopic phenotypes in a longitudinal birth cohort study. METHODS: A whole population birth cohort (N = 1,456) was recruited in 1989. Children were seen at birth and at 1, 2, 4, and 10 years of age to obtain information on asthma and allergic disease development and relevant risk factors for these states. Skin prick testing at ages 4 (n = 980) and 10 (n = 1,036) years was used to define atopic phenotypes. Wheezing in these states was characterized, and logistic regression was used to identify independent risk factors for wheeze onset in different atopic phenotypes. RESULTS: Wheeze ever occurred in 37% of never atopics, 38% of early childhood atopics, 65% of chronic childhood atopics, and 52% of delayed childhood atopics. Chronic childhood atopics had significant wheezing morbidity and bronchial hyperresponsiveness. Their wheezing was associated with male sex, early eczema, family history of eczema, and early tobacco exposure. Never atopic wheeze was related to maternal asthma, parental smoking, and respiratory tract infections. Exclusive breastfeeding protected against early childhood atopic wheeze. Maternal asthma, family history of urticaria, and dog ownership increased delayed childhood atopic wheeze. CONCLUSIONS: In many respects, chronic childhood atopy is the atopic phenotype associated with the most significant forms of childhood wheezing. In such children, heritable drive, allergens, and synergy with other environmental triggers seem to be crucial determinants of wheeze onset. Where such sensitization is absent, numerous environmental factors plus genetic predisposition may assume importance for wheezing.     

Serum eosinophilic cationic protein and blood eosinophil counts for the prediction of the presence of airways inflammation in children with wheezing

BACKGROUND: Serum eosinophilic cationic protein (ECP) concentrations may be useful noninvasive markers of airways inflammation in atopic asthma. However, the usefulness of serum ECP measurement for the prediction of airways inflammation in children with a history of wheezing is unknown. OBJECTIVE: To determine the test accuracy of serum ECP and blood eosinophil percentage as noninvasive markers of eosinophilic airways inflammation. METHODS: Bronchoalveolar lavage (BAL) fluid and peripheral blood samples for eosinophil percentages and serum ECP were obtained from children undergoing elective surgery and who gave a history of wheezing in the previous year. Sensitivity, specificity and likelihood ratios (LH) and the area under the curve (AUC) for the receiver operator characteristic (ROC) curve were calculated for each blood marker for the prediction of airways inflammation defined by a BAL eosinophil percentage > 0.86. Data were analysed on the basis of how recently symptoms had occurred. RESULTS: Seventy-seven children (median age 6.75 years) were studied. An AUC of 0.75 (log serum ECP concentration) and 0.76 (log blood eosinophil percentage) was obtained for predicting airways inflammation. A serum ECP > 13 microg/L yielded a LH of 4.4, whereas using a cutoff blood eosinophils > 4% yielded a LH of 1.9, for the prediction of elevated eosinophils in BAL. Serum ECP and eosinophil percentages in BAL and blood were lowest (not statistically significant) when last symptoms had occurred more than 12 weeks previously. CONCLUSIONS: Serum ECP and blood eosinophil percentages are useful markers for predicting eosinophilic airways inflammation in wheezing children.     

Phenotype of IL-16-producing cells in bronchial mucosa: evidence for the human eosinophil and mast cell as cellular sources of IL-16 in asthma.

BACKGROUND: We have previously shown increased expression of the CD4+ cell chemoattractant interleukin (IL)-16 in bronchial biopsies of atopic asthmatic subjects compared to normal controls. IL-16 immunoreactive cells were identified as both epithelial cells and non-epithelial inflammatory cells. The aim of this study was to characterize and compare the phenotype of non-epithelial inflammatory cells that express IL-16 immunoreactivity in bronchial biopsies from non-atopic normal controls and atopic asthmatic subjects. METHODS: Sections from endobronchial biopsies obtained from non-atopic normal controls and atopic asthmatics were processed for double immunocytochemistry. IL-16 immunoreactivity was assessed using a polyclonal anti-IL-16 antibody and the avidin-biotin complex-diaminobenzidine method. The phenotype of IL-16 immunoreactive cells was assessed using anti-CD3, anti-MBP, anti-tryptase and anti-CD68 mAbs and the alkaline phosphatase complex-Fast Red method. RESULTS: In normal subjects, the majority of IL-16 immunoreactive cells were CD3+ T cells (71.1+/-10.3%) and CD68+ macrophages (22.4+/-8.1%). IL-16 immunoreactivity coexpressed with tryptase+ mast cells in 4 of 7 normal subjects whereas IL-16 immunoreactivity coexpressed with MBP+ eosinophils in only 1 normal subject. In atopic asthmatic subjects, IL-16 immunoreactive cells were mainly CD3+ T cells (60.8+/-8.7%) and MPB+ eosinophils (16.8+/-8.2%). IL-16 immunoreactivity also coexpressed with tryptase+ mast cells (10.6+/-4.0%) in all asthmatic subjects. The number of IL-16 immunoreactive cells that coexpressed MBP was higher in asthmatic subjects compared to normal controls (p = 0.003). CONCLUSION: Our data show that T cells are the major non-epithelial cellular source of IL-16 in normal and asthmatic airways. Eosinophils and mast cells comprised other potential cellular sources of IL-16 in asthmatic airways.     

Indirect evidence of bronchial inflammation assessed by titration of inflammatory mediators in BAL fluid of patients with asthma

Bronchial inflammation is a characteristic of asthma that may be examined indirectly by bronchoalveolar lavage (BAL). Nine normal individuals were compared with 38 age-matched adults with asthma of variable severity to appreciate the importance of cell activation in the severity of asthma. The severity of asthma was appreciated by the clinical score of Aas and the pulmonary function of the patients. FEV1 ranged between 35% and 130% of predicted. The indirect activation of eosinophils (EOSs), mast cells, fibroblasts, and neutrophils was examined by the titration of eosinophil cationic protein (ECP), tryptase, hyaluronan (HA), and myeloperoxidase (MPO) by radioimmunoassay in BAL fluid (BALF) and cytology of BALF. In the adults with asthma, there was a significantly increased number of EOSs and a significantly increased level of all mediators but MPO. MPO levels were increased in seven patients only; three of these patients were previous smokers. Only ECP and HA levels were significantly correlated with the severity of asthma. These results demonstrate EOSs, mast cells, and fibroblasts are activated in asthma, whereas the involvement of neutrophils is less clear. There was a significant correlation between ECP and HA levels, suggesting a common activation of EOSs and fibroblasts.     

Occupational asthma and rhinitis due to glutaraldehyde: changes in nasal lavage fluid after specific inhalatory challenge test

BACKGROUNDd: Glutaraldehyde (GA) is a known respiratory sensitizers, and some studies have reported occupational asthma in exposed workers. Specific changes in nasal lavage fluid (NLF) induced by high-molecular-weight allergen provocation in sensitized subjects were described previously. The purpose of this study was to evaluate the changes in cytogram, protein content, eosinophil cationic protein (ECP), and mast-cell tryptase concentrations in NLF after GA inhalation challenge in patients with a positive history of GA-induced asthma and late or dual asthmatic response due to exposure to low-level GA. METHODS: A single-blind, placebo-controlled study was performed on 11 health workers with occupational asthma and rhinitis due to GA. The control groups comprised 10 atopic subjects with perennial asthma and rhinitis and 10 healthy ones. A "nasal pool" technique was used to evaluate the examined parameters in nasal washings before and 30 min, 4 h, and 24 h after the inhalatory provocation with GA and placebo. RESULTS: There was a significant increase in eosinophil number and percentage, and albumin, ECP, and tryptase concentrations in NLF from patients with occupational asthma and rhinitis when compared to controls. CONCLUSIONS: The results indicate the immunologic mechanism of GA-induced asthma and the applicability of the "nasal pool" technique as the diagnostic procedure in GA-induced airway allergy.     

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.     

Interrelationships among asthma, atopy, rhinitis and exhaled nitric oxide in a population-based sample of children

BACKGROUND: Exhaled nitric oxide (eNO) has attracted increasing interest as a non-invasive marker of airway inflammation in asthma. However, little evidence exists on the influences exerted on eNO by the interrelations among atopic status, asthma and rhinitis. METHODS: Among the 1156 children who participated in a large-scale epidemiological survey on asthma and allergies (ISAAC II: International Study of Asthma and Allergies in Childhood Phase II) in the city of Clermont-Ferrand, 53 asthmatics without corticosteroid treatment and 96 non-asthmatics were invited to perform eNO and skin prick tests (SPTs) to 12 common allergens. RESULTS: Atopic asthmatic children had higher eNO than non-atopic asthmatic children (28.9+/-9.1 vs. 17.1+/-13.1 p.p.b.; P=0.0004) with a significant increase when one SPT or more are positive (26.5+/-7.8 vs. 17.1+/-13.1 p.p.b.; P=0.03). Similarly, non-asthmatic, atopic subjects had higher eNO than non-atopic subjects with a significant increase when two SPTs or more are positive (19.4+/-9.8 vs. 11.7 +/-6.7 p.p.b.; P=0.003). In the case of equal levels of positive SPTs (0, 1, >/=2), asthmatic children always had higher eNO than non-asthmatic ones. Furthermore, among non-asthmatic children, the eNO level increased only in atopics who had rhinitis (20.7+/-13 vs. 12.5+/-6.4 p.p.b. in atopic controls (subjects without rhinitis and asthma) and 12.3+/-6.6 p.p.b. in non-atopic controls; P=0.001), whereas among asthmatic children, eNO level increased in atopics independently of rhinitis (28.2+/-9.5 p.p.b. in those with rhinitis and 30.9+/-8.1 p.p.b. in those without) as well as in non-atopics with rhinitis (22.5+/-17.2 p.p.b.). CONCLUSIONS: Our data suggest that besides atopy and asthma, allergic rhinitis should also be taken into account in the assessment of eNO.