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

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

Monitoring of seasonal variability in bronchial hyper-responsiveness and sputum cell counts in non-asthmatic subjects with rhinitis and effect of specific immunotherapy

Bronchial hyper-responsiveness (BHR) is documented in a proportion of non-asthmatic individuals with allergic rhinitis (NAAR) and reflects inflammatory events in the lower airways. Natural exposure to allergens is known to modulate BHR and the level of airway inflammation in asthma, but less consistently in NAAR. Specific immunotherapy (SIT) attenuates symptoms possibly by reducing BHR and airway inflammation. The influence of natural exposure to Parietaria pollen on BHR and sputum cell counts of NAAR was investigated and the effect of Parietaria SIT examined. Thirty NAAR, monosensitized to Parietaria judaica, participated in a randomized, double-blind, placebo-controlled, parallel group study of the effects of a Parietaria pollen vaccine on symptoms/medication score, BHR to inhaled methacholine and adenosine 5'-monophosphate (AMP), and cell counts in the sputum collected out of and during the pollen seasons for 36 months. Seasonal variation in BHR to inhaled methacholine and AMP and changes in sputum cell counts were documented. Changes were consistent for AMP, but not methacholine, and invariably associated with modifications in sputum eosinophils and epithelial cells. The clinical efficacy of Parietaria SIT was associated with a decline in the seasonal deterioration of BHR to AMP, whereas no significant effect was observed on BHR to methacholine or sputum cell differentials. Between-groups comparison of the seasonal changes in PC15 methacholine values and sputum cell differentials calculated as the AUC were not statistically significant, whereas a significant difference in PC15 AMP was demonstrated throughout the study (P=0.029), the median (inter-quartile range) AUC values being 2478.5 (1153.3-3600.0) and 1545.5 (755.3-1797.9) for the SIT- and placebo-treated group, respectively. Bronchial airways of NAAR exhibit features of active inflammation that deteriorate during natural allergen exposure, particularly with regard to BHR to AMP. The clinical efficacy of Parietaria SIT was exclusively associated with attenuation in seasonal worsening of PC15 AMP, suggesting that AMP may be useful in monitoring changes in allergic inflammation of the airways.     

Effects of once daily dosing with inhaled budesonide on airway hyperresponsiveness and airway inflammation following repeated low-dose allergen challenge in atopic asthmatics

BACKGROUND: Repeated low-dose allergen challenge increases airway hyperresponsiveness and sputum eosinophils in atopic asthmatics. Inhaled corticosteroids attenuate the airway responses to high-dose allergen challenge, but have not been evaluated against repeated low dose challenge. OBJECTIVE: This study evaluates the effects of once daily treatments of two doses of inhaled budesonide on airway responses to repeated low-dose allergen challenge. METHODS: Eight atopic asthmatics with a dual airway responses to inhaled allergen were recruited into a randomized, double-blind crossover, placebo-controlled study. In the mornings of four consecutive days (day 1-day 4), subjects inhaled budesonide 100 microg, 400 microg, or placebo, 30 min before inhaling a concentration of allergen causing a 5% early fall in FEV1. Airway hyperresponsiveness to methacholine and sputum eosinophils were measured at baseline, on the afternoon of day 2, day 4, and 24 h after the last challenge. There was a 1-week washout between each of the three treatment periods. RESULTS: The repeated low-dose allergen challenge induced increases in the percentage sputum eosinophils from 2.0 +/- 0.7% at baseline to 16.6 +/- 7.1% on day 4 (P = 0.002), and this effect was reduced by once daily budesonide 100 microg to 5.6 +/- 1.8% (P = 0. 01) and by once daily budesonide 400 microg to 3.1 +/- 0.9% (P = 0. 004). Also, the allergen-induced methacholine airway hyperresponsiveness which occurred by day 4 (P = 0.03) of the repeated low dose challenge was inhibited by budesonide 400 microg (P = 0.017). CONCLUSION: Both budesonide 100 microg and 400 microg inhaled once daily significantly reduces allergen-induced sputum eosinophilia after repeated low dose challenge; however, only the higher dose also attenuates the allergen-induced airway hyperresponsiveness.     

Cytokine production from sputum cells after allergenic challenge in IgE-mediated asthma

BACKGROUND: Th2 cytokine production from airway cells is thought to govern the eosinophilic airways inflammation in allergic asthma. Induced sputum has become a widely used technique to assess airways inflammation. METHODS: By applying the technique of induced sputum to collect airways cells, we have assessed the spontaneous production of a set of cytokines, including interleukin-4, 6, 10, interferon-gamma and tumour necrosis factor-alpha, 6 h after a bronchial allergenic challenge with Dermatophagoides pteronyssinus (Dpt) in 12 sensitized asthmatics and compared the results obtained after inhalation of saline as control. A group of eight healthy non-allergic subjects was enrolled to control for any non-specific effect of Dpt. Cytokines were measured by a dynamic immunoassay during a 24-h sputum cell culture. RESULTS: Allergen challenge in sensitized asthmatics caused an acute and a late bronchospasm together with a rise in sputum eosinophil counts. Afterwards allergen sputum cells from allergic asthmatics displayed a rise in their production of IL-4 (P < 0.01), IL-6 (P < 0.05) and IL-10 (P < 0.05) when compared to saline. By this time sputum generation of IL-4 in atopic asthmatics was greater than in healthy subjects (P < 0.001). Furthermore, in allergic asthmatics there was a strong correlation between the rise in interleukin-4 production from sputum cells and the rise in sputum eosinophils (r = 0.87, P < 0.001). CONCLUSIONS: Sputum cell culture is a useful model to assess cytokine production in allergic asthmatics who show a marked up-regulation of Th2 cytokines following acute allergen exposure. The rise in sputum eosinophil count following allergen challenge strongly correlates with the rise in IL-4 generation from sputum cells.     

Airway inflammation in asthma and perennial allergic rhinitis. Relationship with nonspecific bronchial responsiveness and maximal airway narrowing

BACKGROUND: Eosinophilic airway inflammation is the hallmark of asthma, but it has also been reported in other conditions such as allergic rhinitis. We have tested whether the analysis of cells and chemicals in sputum can distinguish between patients with mild allergic asthma, those with allergic rhinitis, and healthy controls. The relationship between inflammation markers in sputum and nonspecific bronchial hyperresponsiveness to methacholine (BHR) (PD20 and maximal response plateau [MRP] values) was also evaluated. METHODS: We selected 31 mild asthmatics and 15 rhinitis patients sensitized to house-dust mite. As a control group, we studied 10 healthy subjects. Every subject underwent the methacholine bronchial provocation test (M-BPT) and sputum induction. Blood eosinophils and serum ECP levels were measured. Sputum cell differentials were assessed, and eosinophil cationic protein (ECP), tryptase, albumin, and interleukin (IL)-5 levels were measured in the entire sputum supernatant. RESULTS: Blood eosinophils and serum ECP levels were higher in asthma patients and rhinitis than in healthy controls, but no difference between asthma patients and rhinitis patients was found. Asthmatics had higher eosinophil counts and higher ECP and tryptase levels in sputum than rhinitis patients or control subjects. Sputum albumin levels were higher in asthmatics than in controls. Rhinitis patients exhibited higher sputum eosinophils than healthy controls. An association between sputum eosinophil numbers and MPR values (r= -0.57) was detected, and a trend toward correlation between sputum ECP levels and PD20 values (r= -0.47) was found in the rhinitis group, but not in asthmatics. No correlation between blood eosinophilic inflammation and lung functional indices was found. CONCLUSIONS: Induced sputum is an accurate method to study bronchial inflammation, allowing one to distinguish between rhinitis patients and mildly asthmatic patients. The fact that no relationship was detected between sputum inflammation and BHR suggests that other factors, such as airway remodeling, may be at least partly responsible for BHR in asthma.     

Eosinophils and eosinophilic cationic protein in induced sputum and blood: effects of budesonide and terbutaline treatment.

BACKGROUND: There is a need for easily measurable markers of airway inflammation to guide the use of anti-inflammatory treatment in asthma. Eosinophilic cationic protein (ECP) levels in sputum and blood correlate with clinical severity, and serial measurements of ECP have been proposed as a suitable candidate. AIMS AND METHODS: Our aim was to confirm that sputum and serum ECP measurements would provide a more sensitive indicator of responses to asthma treatment than eosinophil counts per se, in a randomized, placebo-controlled, crossover study of terbutaline, budesonide, and their combination in patients with chronic persistent asthma. We compared the changes in eosinophil counts and ECP in induced sputum and blood during each treatment period. RESULTS: Budesonide and combined treatment caused a significant reduction in sputum eosinophils (-2.7% and -2.3%, respectively, P < 0.05). Sputum eosinophils increased with terbutaline (+3.9%, P = 0.049). In contrast, the changes for sputum ECP were not significant. There was a similar treatment effect on blood eosinophils, but not for serum ECP. Correlations between sputum and blood eosinophils were significant with and without budesonide, but were nonsignificant between sputum and blood ECP during the active treatments. Correlations between sputum eosinophils and ECP, and between blood eosinophils and serum ECP were greatest during treatment with placebo or terbutaline alone: budesonide weakened or abolished these relationships. CONCLUSIONS: Compared with eosinophil counts, ECP measurements in either induced sputum or serum failed to reflect treatment-related changes in chronic asthma. We conclude that ECP is not a sensitive or reliable means of evaluating airway inflammation, and can not be recommended for assessing responses to anti-inflammatory therapy.     

Comparison of allergen-induced changes in bronchial hyperresponsiveness and airway inflammation between mildly allergic asthma patients and allergic rhinitis patients

Bronchial eosinophilic inflammation and bronchial hyperresponsiveness (BHR) are the main features of allergic asthma (AA), but they have also been demonstrated in allergic rhinitis (AR), suggesting a continuity between both diseases. In spite of not fully reproducing natural allergenic exposure, the allergen bronchial provocation test (A-BPT) has provided important knowledge of the pathophysiology of AA. Our aim was to verify the existence of a behavior of AA and AR airways different from the allergen bronchial challenge-induced airway eosinophilic inflammation and BHR changes. We studied a group of 31 mild and short-evolution AA and 15 AR patients, sensitized to Dermatophagoides pteronyssinus. The A-BPT was performed with a partially biologically standardized D. pteronyssinus extract, and known quantities of Der p 1 were inhaled. Peripheral blood (eosinophils and ECP) and induced sputum (percentage cell counts, ECP, albumin, tryptase, and interleukin [IL]-5) were analyzed, before and 24 h after A-BPT. Methacholine BHR, assessed before and 32 h after the A-BPT, was defined by M-PD20 values and, when possible, by maximal response plateau (MRP). The A-BPT was well tolerated by all the patients. AA presented a lower Der p 1 PD20 and a higher occurrence of late-phase responses (LPR). M-PD20 values decreased in AA, but not in AR, patients. MRP values increased in both groups. Eosinophils numbers and ECP levels increased in blood and sputum from both AA and AR, but only the absolute increment of sputum ECP levels was higher in AA than AR patients (P = 0.025). The A-BPT induced no change in sputum albumin, tryptase, or IL-5 values. We conclude as follows: 1) In spite of presenting a lower degree of bronchial sensitivity to allergen, AR patients responded to allergen inhalation with an eosinophilic inflammation enhancement very similar to that observed among AA. 2) MRP levels increased in both AA and AR patients after allergen challenge; however, M-PD20 values significantly changed only in the AA group, suggesting that the components of the airway response to methacholine were controlled by different mechanisms. 3) It is possible that the differences between AR and AA lie only in the quantitative bronchial response to allergen inhalation.     

Relationship between cutaneous allergen response and airway allergen-induced eosinophilia

BACKGROUND: Determinants of changes in airway caliber after allergen challenge include nonallergic airway responsiveness, immune response and dose of allergen given. However, determinants of the airway inflammatory response to allergens remain to be determined. AIM: To assess the relationship between skin reactivity to airborne allergens and lower airway eosinophilic response to allergen exposure in asthma and allergic rhinitis. METHODS: Forty-two subjects with mild allergic asthma (mean age 24 years) and 14 nonasthmatic subjects with allergic rhinitis (mean age 25 years) had allergen skin prick tests and titration with the allergen chosen for subsequent challenge. On a second visit, 31 asthmatic subjects had a conventional challenge while 11 asthmatic subjects and all rhinitic subjects had a low-dose allergen challenge over four subsequent days. Induced sputum samples were obtained at 6 and 24 h after the conventional challenge and at days 2 and 4 of the low-dose challenge. RESULTS: In the asthmatic group, there was a weak correlation between wheal diameter induced by the concentration used for challenge and increase in eosinophils 6 h postconventional challenge (r = 0.372, P = 0.05), but no correlation was observed following the low-dose challenge. Rhinitic subjects showed a correlation between wheal diameter with the allergen dose used for bronchoprovocation and increase in eosinophils at day 2 of low dose (r = 0.608, P = 0.02). CONCLUSION: This study suggests that immediate immune responsiveness to allergen, assessed by the magnitude of the skin response, is a significant determinant of allergen-induced airway eosinophilia and can help to predict the airway inflammatory response.     

Gradual increase in priming of human eosinophils during extravasation from peripheral blood to the airways in response to allergen challenge

BACKGROUND: Eosinophils isolated from the blood of patients with allergic asthma exhibit enhanced responsiveness to multiple stimuli compared with cells from normal controls, a phenomenon generally referred to as priming . This priming response is essential for optimal activation with augmented responses including chemotaxis, cytotoxicity, respiratory burst, and the release of proinflammatory lipid mediators. OBJECTIVE: To monitor the kinetics of priming of eosinophils in the peripheral blood and in the bronchoalveolar lavage fluid of patients with allergic asthma before and after allergen challenge. METHODS: Priming of blood eosinophils obtained from patients with allergy and donors without allergy was measured by labeling with monoclonal phage antibodies A17 and A27 recognizing priming-associated epitopes on phagocytes. In addition, blood and bronchoalveolar lavage fluid eosinophils from subjects with allergy after segmental and whole lung allergen challenge were similarly analyzed. RESULTS: A dose-dependent cytokine-induced upregulation of priming-associated epitopes on blood eosinophils was found. Patients with allergic asthma exhibited an in vivo partially primed eosinophil phenotype, which is further primed in vitro after cytokine or chemokine incubation. Priming was increased in peripheral blood 6 hours after whole lung challenge as well as after segmental allergen challenge. Interestingly, eosinophils obtained from the bronchoalveolar lavage fluid 48 hours after segmental allergen challenge exhibited a higher primed phenotype. CONCLUSION: These data are consistent with a model in which local allergic inflammatory reactions induce partial systemic eosinophil priming in the peripheral blood. Eosinophils found in the airway are highly primed, consistent with the markedly upregulated inflammatory capacity observed in these cells.     

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

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

The relationship of sputum eosinophilia and sputum cell generation of IL-5

BACKGROUND: Eosinophil recruitment to the airway after antigen challenge is regulated by many factors, including airway cell generation of cytokines. OBJECTIVES: The purpose of this study was to determine the relationship between sputum cell generation of IL-5 and the appearance of eosinophils in the sputum after antigen challenge. METHODS: Sputum samples from 11 allergic subjects were collected before and again 4 and 24 hours after antigen challenge. In 6 of these subjects, induced sputum samples were also obtained 48 hours and 7 days after challenge. Sputum leukocyte differential and cell counts and eosinophil-derived neurotoxin levels were determined. Sputum cells were then cultured with PHA (10 microg/mL) to stimulate IL-5 and IFN-gamma, which were measured in culture supernatants. RESULTS: An increase in sputum eosinophils and eosinophil-derived neurotoxin levels was detected at 4 hours after antigen challenge, with peak values at 24 hours. In contrast, significant increases in ex vivo generation of IL-5 by sputum cells was not seen until 24 hours after challenge. At 24 hours, PHA-induced IL-5 correlated with airspace eosinophil values (r (s) = 0.78, P <.01). In addition, the ratio of IFN-gamma/IL-5 decreased at 24 hours (P <.05) and had an inverse correlation with sputum eosinophils (r (s)= -0.68, P <.05). CONCLUSION: Although eosinophils are increased in the airway lumen as early as 4 hours, the ex vivo generation of IL-5 by sputum cells is first noted in samples obtained 24 hours after antigen challenge. This suggests that the early (4 hours) recruitment of eosinophils to the airway lumen may be regulated by factors other than IL-5 or that mucosal cells (rather than airspace cells) contribute to the IL-5 generation at this time point. Furthermore, IL-5 generation by airspace cells may be more responsible for either eosinophil recruitment or retention at later time points.     

IL-12 regulates bone marrow eosinophilia and airway eotaxin levels induced by airway allergen exposure

BACKGROUND: Airway allergen exposure causes local eosinophilic cell infiltration. This cellular inflammatory response is likely to involve the release of eosinophils from peripheral storage pools, and possibly also regeneration of eosinophils in the bone marrow. IL-12 is an inhibitory cytokine in allergic inflammation, shown to reduce eosinophilic cell infiltration. The aim of the present study was to determine whether airway allergen exposure increases bone marrow eosinophil production, and, if so, whether IL-12 modulates this effect. METHODS: Ovalbumin-sensitized C57BL/6 mice and IL-12 knockout (KO) mice were exposed to allergen via the airway route, and the inflammatory cell response was evaluated in bronchoalveolar lavage fluid, blood, and bone marrow. RESULTS: Allergen instillation intranasally produced a dose-dependent inflammatory response in the lower airways of sensitized mice. This inflammatory response was dominated by eosinophils, but there were also increases of both lymphocytes and neutrophils. Sensitization and airway allergen exposure also increased the IL-5-dependent growth of bone marrow cells in vitro. The enhanced bone marrow responsiveness in vitro was paralleled by an increased number of bone marrow eosinophils in vivo. After sensitization and repeated allergen exposure, IL-12 KO mice showed higher eosinophil levels in both BAL and bone marrow than parallel wild-type control mice. Furthermore, BAL-eotaxin levels were increased in IL-12 KO mice as opposed to parallel wild-type controls after allergen exposure. CONCLUSIONS: Airway allergen exposure induced systemic immunologic responses, including increased eosinophil numbers in both airways and bone marrow, and also enhanced IL-5 responsiveness in bone marrow cells. IL-12 may regulate airway eosinophilia at both the level of eosinophilopoiesis and the level of local recruitment of eosinophils into the airways.     

Characterization of airway inflammation after repeated exposures to occupational agents

BACKGROUND: Little is known about the comparative kinetics of eosinophil recruitment after exposure to low- and high-molecular-weight sensitizers in subjects with occupational asthma (OA). OBJECTIVES: The aims of the study were to investigate the kinetics of changes in inflammatory mediators associated with eosinophil infiltration (IL-5 and eotaxin) and to examine the nature of the airway inflammation induced in response to different types of occupational agents. METHODS: We investigated 15 subjects with OA caused by high- and low-molecular-weight agents. The subjects were exposed to increasing doses of the relevant occupational agent over 3 to 4 days until a 20% fall in FEV(1) occurred. Methacholine challenge and sputum induction were performed at the end of each day of exposure. Sputum samples were assessed for differential cell counts, including eosinophils, IL-5, and eotaxin messenger RNA. RESULTS: There was an increase in sputum eosinophils, eotaxin, and IL-5 on the day preceding the occurrence of asthmatic reaction, although there was no change in functional parameters (FEV(1) and PC(20)). Increase in sputum eosinophils was more prominent in subjects exposed to low-molecular-weight agents than to high-molecular-weight agents. CONCLUSION: Changes in eosinophils, IL-5, and eotaxin precede functional changes after exposure to occupational agents in subjects with OA. Eosinophil inflammation is a feature of exposure to both high- and low-molecular-weight agents. Induced sputum may be a useful tool in the early diagnosis of OA.     

Increased prostaglandin E2 concentrations and cyclooxygenase-2 expression in asthmatic subjects with sputum eosinophilia

BACKGROUND: Prostaglandin E2 (PGE2) is known to be produced within human airways, but it is not clear whether in airway diseases it can play a deleterious or a beneficial role. Recently it has been reported that PGE2 can enhance eosinophil survival in vitro. OBJECTIVE: To evaluate whether the concentrations of PGE2 in asthmatic airways correlate with the number of eosinophils and can be responsible for eosinophil-enhanced survival and to identify the cyclooxygenase isoform contributing to the synthesis of PGE2 by cells present in asthmatic airways. METHODS: Reversed-phase high-performance liquid chromatography and/or specific radioimmunoassay was used to measure PGE2 concentrations in induced sputum supernatants from 14 control and 30 asthmatic subjects. Correlations between concentrations of PGE2 and the number of eosinophils in induced sputum were evaluated. Expression of cyclooxygenase-2 (COX-2) in induced sputum cells was determined by immunocytochemistry, and the effect of COX-2 inhibition on PGE2 production was evaluated with the use of radiolabeled arachidonic acid. The effects on eosinophil apoptosis by PGE2 or induced sputum supernatants were studied by using peripheral blood eosinophils obtained by negative immunomagnetic selection. RESULTS: PGE2 concentrations resulted in elevated samples from asthmatic subjects and directly correlated with the percentage of eosinophils and the concentrations of eosinophilic cationic protein. Immunostaining for COX-2 showed enhanced expression in macrophages of asthmatic subjects when compared with control subjects, and the use of a specific COX-2 inhibitor provided evidence that PGE2 synthesis was the result of COX-2 enzymatic activity in asthma-induced sputum cells. Supernatant from induced sputum of asthmatic subjects with high eosinophil counts caused a decreased apoptosis of peripheral blood eosinophils when compared with control subjects, and immunoprecipitation of PGE2 significantly reverted this phenomenon, suggesting that PGE2 was present in biologically relevant concentrations in induced sputum. CONCLUSIONS: The results obtained suggest that COX-2 expression in alveolar macrophages from asthmatic subjects may contribute to enhanced eosinophil survival through an increased PGE2 production.     

Eosinophils, bronchial hyperreactivity and late-phase asthmatic reactions

We have measured airways reactivity (methacholine PC₂₀) and blood eosinophils in eleven asthmatics with allergen-induced late-phase reactions. Before challenge there was a significant (P<0.05) inverse correlation between blood eosinophils and the methacholine PC₂₀. After allergen inhalation eosinophil counts were significantly (P<0.01) elevated at 24 hr. These increases in blood eosinophil counts were not observed in patients who developed single early responses. The magnitude of the changes in eosinophil counts (24 hr minus pre-challenge values) also correlated with the baseline methacholine PC₂₀ (P<0.01) in the late-phase responders. These observations suggest that there may be a direct association between eosinophils and airway reactivity in subjects who develop late-phase asthmatic reactions.     

Airway eosinophilia is not a requirement for allergen-induced airway hyperresponsiveness

BACKGROUND: House dust mites (HDMs) are the major source of perennial allergens causing human allergic asthma. Animal models mimicking as closely as possible the allergic features observed in human asthma are therefore interesting tools for studying the immunological and pathophysiological mechanisms involved. Especially the role of eosinophils and allergen-specific immunoglobulin (Ig) E in the pathophysiology of airway hyperresponsiveness (AHR) remains a subject of intense debate. OBJECTIVE: To develop a mouse model of allergic airway inflammation and hyperresponsiveness based on the use of purified house dust mite allergen (Der p 1) as clinical relevant allergen. Furthermore, we studied the effects of low dose allergen exposure on the airway eosinophilia and AHR. METHODS: On day 0, C57Bl/6 mice were immunized with purified Der p 1 intraperitoneally. From day 14-20, the mice were exposed daily to a 30-min aerosol of different concentrations of house dust mite extract. RESULTS: Mice, actively immunized with Der p 1 and subsequently exposed to HDM aerosols, developed AHR, eosinophil infiltration of the airways and allergen-specific IgE. Moreover, lowering the concentration of the HDM aerosol also induced AHR and IgE without apparent eosinophil influx into the airways. Der p 1-sensitized mice exposed to PBS produced IgE, but did not show AHR or eosinophil influx. CONCLUSION: This in vivo model of HDM-induced allergic airway changes suggests that AHR is not related to either eosinophil influx or allergen-specific serum IgE, thereby reducing the importance of these factors as essential elements for allergic AHR.     

Repeatability of allergen-induced airway inflammation.

BACKGROUND: Allergen inhalation challenge is a useful clinical model to investigate the effects of asthma therapies on allergen-induced airway responses; however, the repeatability of allergen-induced airway inflammation is not known. OBJECTIVE: The purpose of this study was to investigate the repeatability of allergen-induced increases in sputum eosinophils. This information will allow the prediction of the number of subjects required in studies evaluating asthma therapies. METHODS: Seventeen subjects completed 2 allergen challenges using the same dose of allergen, at least 3 weeks apart. Allergen-induced airway responses were measured for 7 hours after challenge. Differential cell counts from induced sputum were determined the day before and 7 and 24 hours after challenge; methacholine PC20 was measured the day before and 24 hours after challenge. RESULTS: The intraclass correlation coefficient for maximum percent late fall in FEV1 was 0.32 and for the area of the late response was 0.61. The sample size predicted to be necessary to observe 50% attenuation of the maximum percent late fall in FEV1 and the late area under the curve with a power of 0.95 was 9 subjects. The intraclass correlation coefficient for percent of allergen-induced sputum eosinophils was 0.60 at 7 hours and 0.53 at 24 hours after challenge. With a randomized cross-over study design, the sample size predicted to be necessary to observe 50% attenuation of allergen-induced percent of eosinophils with a power of 0.95 was 5 subjects. CONCLUSION: Allergen inhalation challenge with measurements of sputum eosinophils is a noninvasive and reliable method for evaluating the anti-inflammatory effects of asthma therapies.     

Migration and accumulation of eosinophils toward regional lymph nodes after airway allergen challenge

BACKGROUND: Eosinophils play a major role in allergic airway inflammation because of their ability to release toxic mediators. In addition, they are able to migrate toward draining thoracic lymph nodes (TLNs) after intratracheal administration, where they can function as antigen-presenting cells. OBJECTIVE: In this study, we evaluated in vivo eosinophil migration toward the TLN after allergen sensitization and analyzed expression of molecules involved in antigen presentation. METHODS: Mice were sensitized by intraperitoneal injection of ovalbumin on days 1 and 10 and challenged once intranasally with ovalbumin on day 20. The kinetics of eosinophilia was evaluated in blood, lung tissue homogenate, bronchoalveolar lavage fluid, and TLN. Cell surface staining was analyzed by flow cytometry. RESULTS: The kinetics of eosinophil recruitment was similar in TLN, lung tissue, and blood, beginning at 12 hours and peaking at 48 hours after allergen challenge. Approximately 70% of TLN eosinophils expressed MHC class II molecules, compared with less than 25% in blood and lungs. Moreover, TLN eosinophils expressed higher levels of MHC class II and CD86 compared with blood and lung eosinophils. Most eosinophils expressed CD80 and CD54, whereas only a few eosinophils expressed CD40. Eosinophils in lungs and TLN appeared to be activated with lower CD62-ligand expression compared with blood eosinophils. CONCLUSION: The presence of eosinophils with a different phenotype in the TLN at early time points after allergen challenge of sensitized mice supports their capacity to serve as antigen-presenting cells, sustaining allergic/inflammatory responses in the airways.     

Sputum eosinophilia: an early marker of bronchial response to occupational agents

Background: False‐negative responses to specific inhalation challenge (SIC) with occupational agents may occur. We explored whether assessing changes in sputum cell counts would help improve the identification of bronchial reactivity to occupational agents during SICs. Methods: The predictive value of the changes in sputum cell counts after a negative FEV₁ response to a first challenge exposure to an occupational agent was determined using the changes in airway calibre observed during repeated challenges as the ‘gold standard’. The study included 68 subjects investigated for work‐related asthma in a tertiary centre. After a control day, the subjects were challenged with the suspected occupational agent(s) for up to 2 h. All subjects who did not show an asthmatic reaction were re‐challenged on the following day. Additional challenges were proposed to those who demonstrated a ≥ 2% increase in sputum eosinophils or an increase in nonspecific bronchial hyperresponsiveness to histamine after the second challenge day. Results: Six of the 35 subjects without changes in FEV₁ on the first challenge developed an asthmatic reaction on subsequent challenges. ROC analysis revealed that a >3% increase in sputum eosinophils at the end of the first challenge day was the most accurate parameter for predicting the development of an asthmatic response on subsequent challenges with a sensitivity of 67% and a specificity of 97%. Conclusions: An increase in sputum eosinophils is an early marker of specific bronchial reactivity to occupational agents, which may help to identify subjects who will develop an asthmatic reaction only after repeated exposure.     

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.