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.     

Granulocyte function in the airways of allergen-challenged pigs: effects of inhaled and systemic budesonide

Backgroumd Late airways obstruction and eosinophil infiltration after allergen challenge are often seen in human asthma and animal models of allergy. This inflammatory reaction, which may be a link between acute and chronic asthma, is blocked by glucocorticoid pretreatment. However, the role of eosinophils in late airways obstruction and the primary site of action of glucocorticoids, i.e. locally or systemically, have not been fully determined. Objectives This study was initiated to find out the role of eosinophils and neutrophils in allergen-induced late airways obstruction in the pig. The effect of pretreatment with budesonide (BUD) given locally or systemically on cellular responses seen within 8 h after allergen challenge was also studied. Methods Twenty-five minipigs were actively sensitized with Ascaris suum antigen and challenged under anaesthesia with antigen in the lower airways. Pigs were given BUD as an aerosol (l0μg/kg) or an intravenous infusion (5μg/kg) 1 h before allergen challenge. In one group, high doses of BUD (50μg/kg) were infused twice with a 3-h interval before allergen challenge. As a positive control, one group was given the BUD vehicle as an infusion and as a negative control, one group not treated with BUD was given the irrelevant antigen ovalbumin. Eosinophils and neutrophils in lung tissue specimens were detected and levels of eosinophil peroxidase (EPO) and myeloperoxidase (MPO) in bronchoalveolar lavage (BAL) fluid were measured using specific antibodies against porcine EPO and MPO. Results The number of eosinophils in lung tissue and BAL fluid and the level of EPO in BAL fluid were significantly increased 8 h after A. suum challenge in pigs not treated with BUD. With regard to possible recruitment and activation of neutrophils the only significant finding was an increase in the number of cells in BAL fluid. The eosinophil numbers and the level of EPO in BAL fluid were shown to be decreased by all BUD treatments in all the compartments studied compared to the positive control. However, the number of eosinophils in lung tissue and EPO levels in BAL fluid did not correlate with the magnitude of the late airways obstruction. Conclusion Although eosinophils are present in the bronchial wall and lumen and are apparently activated, a causative relationship between this granulocyte and the late bronchial obstruction could not be established in this model.     

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.     

The importance of eosinophil activation for the development of allergen-induced bronchial hyperreactivity in conscious,unrestrained guinea-pigs

Using a newly developed guinea-pig model of asthma, characterized by allergen-induced early and late phase asthmatic reactions, bronchial hyperreactivity (BHR) and airway inflammation, the importance of eosinophil activation for the development of BHR to inhaled histamine was investigated at 6 h (after the early reaction) and 24 h (after the late reaction) after allergen provocation. Eosinophil activation was assessed by a sensitive kinetic assay for eosinophil peroxidase (EPO) activity, suitable for bronchoalveolar lavage (BAL) analysis. A significant 2±9-fold (P < 0±01) increase in bronchial reactivity to histamine was observed at 6 h after allergen exposure, which was associated with a 2±9-fold increase in the number of eosinophils (P < 0±05) and a 6±7-fold increase in EPO activity (P < 0±01) in the BAL fluid. At 24 h after allergen exposure the bronchial reactivity to histamine was lower (1±7-fold), but still significantly enhanced (P < 0±01). By contrast, the number of eosinophils was further increased compared with 6 h after provocation (3±8-fold, P < 0±05), while the EPO activity remained stable at 6 h levels. The number of eosinophils was significantly correlated with EPO activity at 6 h (r = 0±62; P < 0±05), but not at 24 h after provocation. No significant correlation was observed between the number of eosinophils in the BAL fluid and BHR to histamine at either time point. Remarkably, EPO activity was significantly correlated to BHR at 24 h (r = 0±66; P < 0±004), but not at 6 h after provocation. These results indicate that: newly infiltrated eosinophils have the highest activation state during the early asthmatic reaction; EPO may play a role in the development of BHR to inhaled histamine after the late reaction and that besides EPO, additional mechanisms may contribute to BHR to histamine after the early asthmatic reaction.     

The effect of salmeterol on the early- and late-phase reaction to bronchial allergen and postchallenge variation in bronchial reactivity, blood eosinophils, serum eosinophil cationic protein, and serum eosinophil protein X

The late asthmatic response to bronchial allergen challenge and the associated increase in nonspecific bronchial reactivity provides a model for studying the acute inflammatory mechanisms in asthmatic airways. In 12 asthmatic patients (aged 22–37 years) with known dual reaction to allergen challenge, salmeterol 50 μg, 100 μg, or placebo was administered as a single dose 10 min before allergen challenge in a double-blind, randomized order on three different study days 2 weeks apart. The bronchial reactivity (BH) to histamine was measured the day before and 24 and 48 h after allergen challenge. Salmeterol significantly inhibited the early ( P <0.02) and the late ( P <0.05) asthmatic reactions. After placebo, mean BH was significantly increased above base line at 24 and 48 h ( P <0.02). After 50 and 100 μg salmeterol, BH was less than base line at 24 h and returned to prechallenge values at 48 h. Blood eosinophils increased significantly ( P <0.05) 24 and 48 h after allergen challenge, and no difference was found between treatments. After pretreatment with placebo, serum eosinophil cationic protein (s-ECP) and serum eosinophil protein X (s-EPX) increased significantly ( P <0.05) 24 and 48 h after allergen challenge. After treatment with salmeterol 50 μg, s-EPX, but not s-ECP, increased significantly 24 h after challenge, but was normal at 48 h. After salmeterol 100 μg, no change in s-EPX or s-ECP was found. The results showed that salmeterol eliminated the allergen-induced dual asthmatic reaction and gave protection against increased BH. Measurement of blood eosinophils, s-ECP, and s-EPX indicated that salmeterol may modify this inflammatory cell activity, because raised s-levels of ECP and EPX may be linked to eosinophil activation and granule secretion. In addition to inducing a long-lasting bronchodilation, salmeterol may influence acute inflammation.     

Interleukin-5 receptors on human lung eosinophils after segmental allergen challenge

BACKGROUND: IL-5 is a specific cytokine for eosinophil accumulation, activation and prolongation of survival and can be recovered in elevated concentrations from the bronchoalveolar compartment in atopic asthma following allergen challenge. OBJECTIVE: The action of IL-5 is mediated via the specific IL-5 receptor-alpha (IL-5Ralpha). Although in vitro data suggest that IL-5R expression is regulated by cytokines such as IL-3, IL-5 and GM-CSF, IL-5R regulation in vivo and its kinetics following allergen provocation are incompletely understood. METHODS: We investigated IL-5R regulation in vivo following segmental allergen provocation (SAP) with an individually standardized dose of allergen in 12 patients with atopic asthma. Lavage was performed 10 min and 18 h (eight patients) and 10 min and 42 h (eight patients) after allergen challenge. In addition to differential cell counts, IL-5Ralpha was measured by flow cytometry and IL-5 concentrations in bronchoalveolar lavage (BAL) fluid were determined by ELISA. RESULTS: IL-5Ralpha expression decreased significantly on peripheral blood and on BAL eosinophils 18 and 42 h after SAP. In contrast, IL-5 concentrations increased significantly in BAL fluid 18 and 42 h after SAP. In four and two patients, respectively, there were detectable IL-5 concentrations in serum 18 or 42 h after allergen exposure. CONCLUSIONS: Although there was no correlation between IL-5 concentrations and IL-5Ralpha expression on eosinophils in BAL, our data support previous in vitro and in vivo findings of a negative feedback mechanism between IL-5 concentrations and IL-5Ralpha expression on eosinophils.     

Increased expression of IL-16 immunoreactivity in bronchial mucosa after segmental allergen challenge in patients with asthma

BACKGROUND: We have previously shown increased expression of the CD4(+) cell chemoattractant IL-16 in bronchial mucosa of patients with asthma. We investigated the effects of allergen challenge on airway IL-16 expression. METHODS: We investigated the expression of IL-16 immunoreactivity in bronchial biopsy samples obtained from atopic asthmatic subjects (n = 19) and normal subjects (n = 6) 24 hours after segmental allergen challenge. Control biopsy samples were obtained either at baseline or after diluent challenge. IL-16 expression was correlated to numbers of CD4(+) cells, CD25(+) cells, and activated eosinophils. IL-16 bioactivity was assessed in bronchoalveolar fluid obtained from patients with asthma. RESULTS: IL-16 expression was higher in control biopsy specimens obtained from subjects with asthma compared with normal subjects (P<.05). In patients with asthma, numbers of IL-16 immunoreactive cells were significantly higher in biopsy specimens obtained after allergen challenge compared with control biopsy specimens (P<.001). Allergen provocation was associated with release of IL-16 in bronchoalveolar fluid in patients with asthma. In normal subjects, there was no difference in the number of IL-16-immunoreactive cells in biopsy specimens obtained after allergen challenge compared with biopsy specimens obtained after diluent challenge. Allergen challenge was associated with an increase in the numbers of EG2(+) eosinophils in patients with asthma but not in normal subjects. IL-16 expression correlated with the numbers of CD4(+) cells and CD25(+) cells after allergen challenge in asthmatic subjects with a provocative concentration required to decrease the FEV(1) by 20% of its baseline value (PC(20)FEV(1)) < 4 mg/mL. IL-16-immunoreactive cells were identified mainly as T cells and eosinophils in asthmatic subjects after allergen challenge. CONCLUSION: Endobronchial allergen provocation in atopic asthmatic patients resulted in increased airway expression of IL-16 and release of bioactive IL-16 in airways. IL-16 may contribute to the immunoregulation of the inflammatory infiltrate in the airways in response to antigen.     

Increases in eotaxin‐positive cells in induced sputum from atopic asthmatic subjects after inhalational allergen challenge

BACKGROUND: Eosinophils are believed to be critical proinflammatory cells in airway mucosal damage in asthma. Eotaxin is a C-C chemokine with selective activity for eosinophils and basophils. Previous studies have shown increased expression of eotaxin in the airways of asthmatics at baseline. We aimed to investigate eotaxin expression during the late-phase reaction to allergen inhalation in atopic asthmatics. METHODS: Sputum induction was performed before and 24 h after inhalational allergen challenge in atopic asthmatics, and eotaxin protein was detected immunocytochemically. RESULTS: Thirteen patients with a mean decrease in forced expiratory volume in 1 s of 28% (+/-1.5) during the early asthmatic reaction, and 39% (+/-4.7) during the late asthmatic reaction produced sufficient sputum for study. The percentage of eosinophils in sputum was increased 24 h after allergen challenge (P<0.004), and eosinophil percentages in sputum after challenge correlated with the magnitude of the late-phase reaction (r=0.56, P=0.05). The percentage of eotaxin-positive cells increased from 12.6% (range 2-43.8) to 24.3% (8.1-47.1, P<0.005). Allergen-induced increases in eotaxin-positive cells correlated with increases in eosinophils (r=0.63, P<0.01). CONCLUSIONS: These findings suggest that eotaxin may contribute to allergen-induced recruitment of eosinophils to the airway in asthmatic subjects.     

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.     

Eotaxin-3 but not eotaxin gene expression is upregulated in asthmatics 24 hours after allergen challenge.

Eotaxin is an important mediator of eosinophil recruitment and activation in the airways of asthmatics. Eotaxin-2 and eotaxin-3 are two recently identified chemokines with activity similar to that of eotaxin. Using quantitative polymerase chain reaction analysis, we determined the messenger RNA (mRNA) expression of eotaxin, eotaxin-2, and eotaxin-3 relative to GAPDH mRNA expression in bronchial biopsies and bronchoalveolar lavage fluid (BALF) cells obtained from subjects with mild asthma, asthmatic subjects 24 h after allergen challenge, and normal control subjects. In bronchial biopsies, gene expression was upregulated in asthmatic subjects as compared with control subjects for eotaxin (log median values 3.18 pg/microg, 95% confidence interval [CI]; 2.27 to 3.79 versus 4.37 pg/microg, 95% CI; 3.97 to 4.65, P = 0.003) and for eotaxin-2 (0.82 pg/microg, 95% CI; 0.08 to 1.72 versus 2.97 pg/microg, 95% CI; 1.97 to 3.45, P = 0.006), but no further increase was observed after allergen challenge. In contrast, eotaxin-3 mRNA expression was not increased in asthmatic compared with control subjects, but was dramatically enhanced 24 h after challenge (median log value 1.93 pg/microg, 95% CI; 0.74 to 3.92 versus 4.62 pg/microg, 95% CI; 3.05 to 6.23, P = 0.036). No significant difference between groups was observed in BALF cell gene expression for any of the chemokines examined. These data suggest that eotaxin-3 rather than eotaxin or eotaxin-2 may account for the ongoing eosinophil recruitment to asthmatic airways in the later stages (24 h) following allergen challenge.     

The changes in cell population and chemical mediators in BALF following local allergen challenge]

In eight subjects who showed dual asthmatic response (DAR) in bronchial provocation tests (BPT) with specific allergens, local allergen challenge (LAC) was conducted using a flexible bronchofiberscope. We examined the concentrations of histamine, leukotriene B4 and C4 (LTB4 and LTC4) and cell populations in bronchoalveolar lavage fluid (BALF) collected before LAC (control), during the immediate response phase (IR), and the late response phase (LR). Control BALF was collected from the left lingula (B4 or B5), and BALF in the IR or LR phase from the right middle lobe (B4 or B5). Each BAL was conducted with 50 ml of saline at 37 degrees C and repeated three times in succession. It was noted that histamine increased significantly (p less than 0.05) in IR-BALF from the control level. In addition, the concentrations of LTC4 and the numbers of eosinophils increased in IR-BALF. In LR-BALF, the numbers of eosinophils (p less than 0.01), and the concentrations of histamine (p less than 0.05), LTC4 (p less than 0.05) and LTB4 increased. From these results, it was suggested that infiltration of eosinophils and various chemical mediators in the bronchial mucosa play important roles in the development of late asthmatic response.     

Expression of prostaglandin E(2) receptor subtypes on cells in sputum from patients with asthma and controls: effect of allergen inhalational challenge

BACKGROUND: Prostaglandin (PG) E 2 binds to 4 G-protein-coupled receptors designated EP 1 through EP 4 . Although PGE 2 plays an immunomodulatory role in asthma, there is little information on the expression of PGE 2 receptors in this disease. OBJECTIVE: We hypothesized that profiles of E-prostanoid (EP) receptor expression are altered on asthmatic bronchial inflammatory cells in vivo and further altered by allergen challenge in vivo and proinflammatory mediators in vitro. METHODS: The numbers and phenotypes of EP 1-4 immunoreactive induced sputum cells from atopic asthmatics (n = 13; before and 24 hours after allergen inhalational challenge) and normal controls (n = 9; 3 after saline challenge) and EP 1-4 expression on purified blood eosinophils from both groups (n = 4 for each) before and after stimulation with LPS and/or IL-5 in vitro were measured by using single and double immunocytochemistry. RESULTS: Subsets of sputum cells of all phenotypes expressed all 4 EP receptors in both patients with asthma and controls. There were significantly greater numbers of macrophages expressing all 4 EP receptors and increased percentages of macrophages expressing EP 2 and EP 4 in patients with asthma compared with controls. Allergen bronchial challenge of patients with asthma was associated with a selective influx of eosinophils, but the percentages of these and other leukocytes expressing all 4 EP receptors were unchanged. Compared with sputum, only small percentages of peripheral blood eosinophils expressed each receptor, but this was increased by culture with exogenous IL-5 or LPS. CONCLUSION: E-prostanoid receptor expression is increased on airway macrophages of patients with asthma at baseline and may be altered on eosinophils after allergen challenge in vivo in response to inflammatory stimuli.     

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.     

Induction of allergic inflammation in the lungs of sensitized sheep after local challenge with house dust mite

Background Previous sheep models of asthma are based on sheep sensitized to nematode (Ascaris) allergens and these have been used to evaluate the physiological and pharmacological effects of potential anti‐asthma agents. The immunological mechanisms associated with the allergic response in sheep lungs has not been examined in detail. Objective To develop an experimental sheep model of allergic lung inflammation based on a relevant major human allergen, house dust mite, and to define the immunological features of the allergic response in this model. Methods Sheep immunized subcutaneously with solubilized house dust mite extract were given a single bronchial challenge with house dust mite. Bronchoalveolar lavage (BAL) and peripheral blood leucocytes were collected before and after challenge for flow cytometry, and tissue samples were taken post‐mortem (48 h post‐challenge) for histology and immunohistochemical analyses. Results Immunizations with 50 μg house dust mite induced an allergen‐specific IgE response in 50 to 60% of sheep (allergic sheep), with higher antigen doses increasing specific IgG₁ but not IgE. Lung challenge of allergic sheep with house dust mite led to the initial recruitment of neutrophils (at 6 h post‐challenge) followed by eosinophils and activated lymphocytes into the lung tissue and BAL, similar to the late‐phase allergic response seen in human asthma. Eosinophil recruitment peaked at 48 h post‐challenge, representing 10 to 33% of BAL leucocytes in allergen‐challenged allergic sheep compared to 0 to 3% in allergen‐challenged control (naïve) sheep. Lymphocytes recovered from the lung after allergen challenge were enriched for CD4⁺ T cells and were more activated than lymphocytes in blood. There was significant down‐regulation of CD62L (L‐selectin) and CD49d (VLA‐4) expression after allergen challenge on BAL eosinophils and lymphocytes compared to blood. In addition, VCAM‐1 (ligand for VLA‐4) was up‐regulated on blood vessels of allergen‐challenged lungs. Eosinophils, CD4⁺ T cells and CD45R⁺ B cells were the most prominent leucocytes found in lung tissue 48 h after allergen challenge. Conclusion This study demonstrates, for the first time, the ability of house dust mite to induce allergic responses in sheep lungs. This novel sheep model of allergic lung inflammation using relevant human allergens, exhibits similarities to human asthmatic disease and will be a useful tool for studies of the immunological and physiological mechanisms of allergic asthma.     

Down modulation of L-Selectin expression on eosinophils recovered from bronchoalveolar lavage fluid after allergen provocation

In allergic asthma eosinophils infiltrate into the lung after allergen challenge. The mechanism of this cellular infiltration is not fully understood. L-Selectin is involved in leucocyte-endothelial cell recognition and participates in homing of leucocytes into sites of inflammation. To find indications for a role of L-Selectin in the migration of eosinophils to the bronchoalveolar space we measured L-Selectin expression on eosinophils in peripheral blood and bronchoalveolar lavage fluid (BAL) 4 hr after the early allergic reaction after allergen challenge. Nine patients with allergic asthma participated in the study. An eosinophil specific high depolarization signal enabled us to measure L-Selectin expression on eosinophils in a FACS analysis without isolation of these cells. Eosinophils recovered from BAL showed a strong decrease of L-Selectin expression compared to blood eosinophils. This decrease in L-Selectin expression can be induced in vitro by activation of eosinophils with PMA or FMLP whereas priming of eosinophils during several hours with GM-CSF did not influence L-Selectin expression. Our results are a first indication that L-Selectin may play a role during homing of eosinophils in the lung in asthma after allergen challenge. Moreover, the low expression of L-Selectin on eosinophils in the lung is a further indication that these cells exhibit an activated phenotype.     

Repeated inhalation of low doses of cat allergen that do not induce clinical symptoms increases bronchial hyperresponsiveness and eosinophil cationic protein levels.

The aim of this study was to investigate whether repeated exposure to subclinical doses of cat allergens, not inducing asthma symptoms, could affect eosinophil cationic protein (ECP) levels in bronchoalveolar lavage (BAL) or in peripheral blood, without the appearance of clinical symptoms. Twelve patients with mild asthma, all sensitized to cats and not exposed to cat allergen at home, underwent a series of inhalations of cat allergen or placebo for 8 days over 2 weeks. A methacholine challenge was performed before and after the allergen and saline exposures, and BAL and blood were sampled for ECP measurements and eosinophil counts. No patients experienced asthma symptoms. However, PD20 methacholine (geometric mean) decreased significantly from 263 microg before to 126 microg after inhalation of allergen. Inhalation of saline did not induce any significant change in PD20. The change in log PD20 before and after cat allergen exposure was statistically different from the change in log PD20 before and after saline. Median ECP levels in BAL and serum increased significantly after allergen exposure, from 0.8 to 3.1 microg/l (p<0.02) and from 15.9 to 31.4 microg/l (p<0.05), respectively. No change was observed after saline inhalations. The change in BAL and serum ECP levels was statistically significant compared to that in the control group. The number of eosinophils did not change, however, nor did IL-5 and RANTES levels in BAL and serum. In conclusion, our results show that (1) exposure of asthma patients to repeated low doses of allergen, which did not provoke any clinical symptoms, is capable of inducing a local eosinophil activation associated with an increase in nonspecific bronchial hyperresponsiveness and (2) the increase in serum ECP levels due to eosinophil activation precedes the occurrence of asthma symptoms and may thus be a marker of allergen exposure in allergic asthma.     

Resolution of allergic airways inflammation but persistence of airway smooth muscle proliferation after repeated allergen exposures

BACKGROUND: Chronic inflammation in asthmatic airways can lead to characteristic airway smooth muscle (ASM) thickening and pathological changes within the airway wall. OBJECTIVE: We investigated the long-term effects of repeated allergen exposure. METHODS: Brown-Norway (BN) rats sensitized to ovalbumin (OVA) were exposed to OVA or saline aerosol every third day on six occasions and studied 24 h, 7 days and 35 days after the final exposure. We measured airway inflammation, ASM cell proliferation (by incorporation of bromodeoxyuridine; BrdU) and bronchial responsiveness to acetylcholine. RESULTS: At 24 h, in OVA-exposed rats, we detected elevated OVA-specific serum IgE, increased numbers of macrophages, eosinophils, lymphocytes and neutrophils in the bronchoalveolar lavage (BAL) fluid and increased numbers of MBP+ (major basic protein) eosinophils and CD2+ T cells within the bronchial submucosa. This coincided with increased numbers of ASM cells expressing BrdU and with bronchial hyper-responsiveness (BHR). At 7 days, BHR was detected in OVA-exposed rats, coincident with increased numbers of macrophages and lymphocytes in BAL fluid together with increased numbers of CD2+ T cells within the bronchial submucosa. This coincided with increased numbers of ASM cells expressing BrdU. By day 35, the number of ASM cells expressing BrdU remained elevated in the absence of cellular infiltration and BHR. CONCLUSION: Repeated OVA-challenge results in persistent ASM cell proliferation in the absence of bronchial inflammation and BHR, which lasts for at least 1 week following cessation of exposure.