Blood eosinophils from asymptomatic allergies have a reduced capacity to produce oxygen-free radicals

Background The eosinophil granulocyte is an inflammatory cell that plays an active part in diseases such as asthma and rhinitis. This study aimed to investigate oxidative metabolism by blood eosinophils taken from allergic rhinitis patients, asthmatics, and nonallergic controls before and during the birch-pollen season.
Methods Twenty patients with allergy to birch pollen and seasonal symptoms of rhinitis, some of whom were also asthmatic, were followed before and during the birch-pollen season in Sweden. The cells were purified using a Percoll gradient and the MACS system. Eosinophil purity in all samples was >95%. Oxidative metabolism was measured by a chemiluminescence (CL) assay, with luminol and lucigenin acting as enhancers, and PMA, serum-treated zymosan (STZ), interleukin (IL)-5. or RANTES as stimuli. Results The allergic subjects showed reduced luminol CL when activated before the season with PMA (P = 0.40) or STZ (P = 0.0055). This was not seen during pollen exposure. STZ-activated lucigenin CL was also reduced before the season (P = 0.0027). The reduction was most evident in the group with asymptomatic rhinitis. In terms of eosinophil stimulation. IL-5 and RANTES were equally effective in allergic and nonallergic subjects, both before and during the pollen season.
Conclusions Blood eosinophils from asymptomatic allergies may have a lower capacity to produce oxygen-free radicals than eosinophils from nonallergics.     

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

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

Increases in collagen type I synthesis in asthma: the role of eosinophils and transforming growth factor-bβ

BACKGROUND: Collagen type I is one of the major deposits in thickening of the reticular basement membrane of asthma. OBJECTIVE AND METHODS: In this study, we assessed turnover of collagen type I in asthma by measuring procollagen type I C-terminal peptide (PICP) and collagen type I C-terminal telopeptide (ICTP) in induced sputum. RESULTS: PICP but not ICTP was found to be significantly higher in asthma subjects than in normal volunteers (P < 0.05). In asthma, PICP was inversely correlated with %FEV(1.0) (r = -0.539), and its levels significantly increased upon exacerbation (P < 0.05), indicating that collagen synthesis increases during asthma exacerbation. Additionally, PICP was found to significantly correlate with eosinophil counts in sputum (r = 0.539), indicating that eosinophils stimulate collagen turnover. Because eosinophils can produce TGF-beta, a potent stimulator of collagen synthesis, we immunocytochemically examined TGF-beta-positive cells in sputum. TGF-beta-positive cells significantly correlated with eosinophil counts (r = 0.811) and PICP (r = 0.569), suggesting that TGF-beta released from eosinophils is involved in collagen synthesis. CONCLUSIONS: The results of the present study suggest that collagen synthesis is stimulated in asthmatic airways by eosinophils through TGF-beta, while collagen degradation is not, and that PICP in sputum can act as a new marker for airway inflammation in asthma.     

Increased levels of IL-5 positive peripheral blood eosinophils and lymphocytes in mild asthmatics after allergen inhalation provocation

BACKGROUND: In allergic inflammation eosinophils and TH2-like lymphocytes are supposed to be the major effector cells and considered to contribute as cellular source of the key cytokine interleukin (IL)-5. OBJECTIVE: The purpose of this study was to enable detection of IL-5 containing leucocytes and to investigate whether the number of these cells in the blood circulation differed between healthy and asthmatics before and after allergen provocation. METHODS: The distribution of intracellular IL-5 in human peripheral blood eosinophils (PBE) and lymphocytes (PBL) has been investigated using fixation and cell membrane permeabilization with octyl-glucopyranoside, the FOG-method, and flow cytometry. The intracellular staining was performed on leucocytes without any prior purification and in vitro stimulation. The specificity of IL-5 binding to intracellular compartment of both PBE and PBL was confirmed by complete inhibition with human recombinant IL-5. RESULTS: Preformed intracellular IL-5 was detected in the main population of PBE (> 70%) in both healthy individuals and asymptomatic patients. Moreover, preformed intracellular IL-5 was also detected in 4.8% and 2.4% of PBL from healthy individuals and asymptomatic patients, respectively. There was a correlation between the absolute number of PBE and IL-5 positive PBE. In patients with pollen-related asthma, the number of IL-5 positive PBE and PBL increased significantly 24 h after an allergen inhalation provocation (P < 0.05). In the healthy control group no differences regarding IL-5 positive PBE and PBL were obtained pre- and post-allergen challenge. CONCLUSIONS: In patients with mild allergic asthma, but not in healthy individuals, allergen provocation induces an increased absolute number of IL-5 positive PBE and PBL. The reason for the relatively high number of IL-5 positive PBL is unclear, but a plausible explanation might be that other lymphocyte subsets besides CD4+ TH2 can produce IL-5. However, enumeration of IL-5 positive leucocytes may be used as an activity marker and also be a useful tool in monitoring the inflammation in asthma.     

Airway mast-cell activation in asthmatics is associated with selective sputum eosinophilia

BACKGROUND: Tryptase is a serine endoprotease selectively released from mast cells. Although mast cells are known to be activated after experimental allergic provocation, their role in naturally occurring asthma is still debated. METHODS: We have investigated the levels of tryptase in the whole induced sputum collected from 51 asthmatics (31 atopic and 20 intrinsic) seen in our outpatient clinic and 22 normal nonatopic healthy volunteers. Tryptase was measured by a new immunoassay based on B12 monoclonal antibody recognition of total tryptase (UniCAP System, Pharmacia) with a sensitivity of 1 ng/ml. RESULTS: While being below the threshold of detection in all normal volunteers, tryptase was detectable in the sputum from 9/51 asthmatics (18%) including five atopic and four intrinsic asthma cases. In these patients, among whom three were asymptomatic asthmatics, the values ranged between 1 and 6.1 ng/ml. The asthmatics with detectable sputum tryptase had greater sputum eosinophil counts (P<0.05) but lower neutrophil counts (P<0.05) than those in whom tryptase was undetectable. When compared to control subjects, asthmatics without tryptase had still greater eosinophil counts (P<0.0001) but also raised neutrophil counts (P<0.05). No significant difference could be found between asthmatics with tryptase and those without tryptase with respect to the age, the baseline lung function, the methacholine bronchial responsiveness, and the frequency of treatment with inhaled steroids. CONCLUSIONS: With the UniCAP System, tryptase was detectable in the sputum from 18% of asthmatics irrespective of atopy and current symptoms. Asthmatics with tryptase appeared to have a selective increase in sputum eosinophil counts while those without tryptase displayed a mixed sputum granulocyte infiltration with raised eosinophil and neutrophil counts.     

Blood eosinophils,eosinophil-derived proteins,and leukotriene C4 generation in relation to bronchial hyperreactivity in children with atopic dermatitis

To assess the relation among eosinophil-related variables in the peripheral blood, bronchial hyperreactivity, and the presence of atopic dermatitis in children aged 5–14 years, we studied 11 patients with atopic dermatitis alone, six with asthma and atopic dermatitis, 12 with asthma alone, and 12 healthy controls. Eosinophil counts, levels of eosinophil cationic protein, and the capacity of eosinophils to generate leukotriene (LT) C₄, as well as bronchial hyperreactivity and a severity score for atopic dermatitis, were determined. Eosinophil variables were significantly higher in both patient groups with atopic dermatitis than in normal controls. In particular, ionophore A 23187 LTC₄ generation was higher in patients with atopic dermatitis alone (median 82, range 25–273 ng/10⁶ cells) and patients with combined asthma and atopic dermatitis (median 68, range 32–583 ng/10⁶ cells) than in normal controls (median 9, range 1–67 ng/10⁶ cells). However, there was no difference between the group of atopic dermatitis patients with asthma and without asthma. We conclude that eosinophil variables in the peripheral blood are mainly influenced by the presence of atopic dermatitis, and not the presence and the severity of asthma in patients with both asthma and atopic dermatitis.     

The role of interleukin-5, interleukin-8 and RANTES in the chemotactic attraction of eosinophils to the allergic lung

BACKGROUND: Bronchoalveolar lavage (BAL) fluid from patients with birch-pollen allergy lavaged during the season showed an elevated chemotactic activity for eosinophils compared with BAL fluid from the same patients before the start of the season. AIM: The aim of this study was to identify the eosinophil chemotactic agents in the BAL fluid, to compare these findings with in vitro studies on selected cytokines, and to investigate the interactions between these cytokines. METHODS: Neutralizing antibodies for interleukins (IL) -2, -5 and -8, RANTES and leukaemia inhibitory factor (LIF) were added to the BAL fluid, and the chemotactic activity was tested with eosinophils from allergic donors. Eosinophils from healthy donors were preincubated with IL-5 in order to mimic the primed state of eosinophils from allergics, and the migration towards recombinant IL-5, IL-8, and RANTES in different combinations was measured. Eosinophils from allergic donors were also used. RESULTS: Anti-IL-5, anti-IL-8 and anti-RANTES inhibited the chemotactic activity in the BAL fluid. Recombinant RANTES induced migration, which was enhanced by preincubation of the cells with IL-5. Only eosinophils from symptomatic allergics responded to IL-8, and IL-5 was not sufficient to prime normal eosinophils in vitro to an IL-8 response. A negative correlation was found between the level of in vivo activation of the cells and their response to IL-5, and a positive correlation with the response to RANTES. CONCLUSION: IL-8 and RANTES are important for eosinophil accumulation to the lung of pollen-allergic asthmatics. IL-5 alone may not be responsible for the priming of eosinophils in vivo, but is an essential cofactor for the other chemoattractants.     

Allergen challenge in asthma: association of eosinophils and lymphocytes with interleukin-5

To test whether eosinophil recruitment after pulmonary allergen challenge is associated with interleukin (IL)-5 in patients with asthma, we performed segmental bronchoprovocation (SBP) with saline, and with low and high dosages of ragweed extract in six patients with allergic asthma. Bronchoalveolar lavage (BAL) of the challenged segments was performed 5 min after challenge (immediate BAL fluid) and repeated 24 h later (late BAL fluid). Allergen challenge resulted in recruitment of eosinophils, and increased levels of eosinophil-active cytokines. A bioassay showed the predominant eosinophil-active cytokine in the late BAL fluids to be IL-5. Analysis of the late BAL fluids revealed that IL-5 levels correlated with the numbers of eosinophils and lymphocytes. This study provides evidence that IL-5 is a critical cytokine associated with eosinophil and lymphocyte recruitment into the airways of patients with asthma following exposure to allergen.     

Cytokine production from sputum cells in eosinophilic versus non-eosinophilic asthmatics

The inflammatory pathways involved in asthma are more complex than the sole Th2-mediated eosinophilic airway inflammation. Different phenotypes of asthma have been recently highlighted and are probably underlied by different immunological profiles. The aim of the study was to assess cytokine production from sputum cells in eosinophilic versus non-eosinophilic asthmatics. Induced sputum was obtained from 48 consecutive stable mild to moderate asthmatics (20 eosinophilic asthmatics, 28 non-eosinophilic asthmatics) and 31 healthy subjects. Cytokine released from sputum cells were measured by a home-made two-step sandwich immunoassay. Cytokines investigated were interleukin (IL)-4, IL-6, IL-10, tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Sputum cells from eosinophilic asthmatics produced more IL-4 than those from both healthy subjects (P < 0.05) and non-eosinophilic asthmatics (P < 0.05). Conversely, sputum cells from eosinophilic asthma were found to release lower amounts of TNF-alpha than those from healthy subjects (P < 0.05). The group of non-eosinophilic asthmatics did not distinguish from healthy subjects with respect to any cytokines measured. Sputum cells from asthmatics exhibiting eosinophilic airway inflammation release more IL-4 and less TNF-alpha than those of healthy subjects. By contrast, non-eosinophilic asthmatics did not distinguish from healthy subjects by abnormal cytokine release from their sputum cells.     

Role of eosinophils and their clinical significance in allergic inflammation

Eosinophils are believed to play roles in the pathophysiology of allergic inflammation, such as bronchial asthma. However, recent studies on anti-interleukin-5 monoclonal antibody treatment of asthmatic patients raised the possibility that eosinophils may play only a limited role. More recent studies established that eosinophils are essentially involved in the development of airway remodeling. Moreover, it is theoretically conceivable that eosinophils are a cellular source of lipid mediators, such as cysteinyl leukotrienes or platelet-activating factor in asthma. Even in the absence of interleukin-5, it is likely that the ‘T-helper Type 2 network’, including a cascade of vascular cell adhesion molecule-1, intercellular cell adhesion molecule-1, CC chemokines, granulocyte-macrophage colony-stimulating factor, for example, can maintain sufficient eosinophilic infiltration and effector functions, such as superoxide anion generation and degranulation. Long-term studies, wherein tissue eosinophils are eliminated effectively will be required to establish the exact roles of these cells in asthma. Finally, the authors will demonstrate that eosinophils have the potential for not only playing detrimental roles but also beneficial ones.     

Expression of the apoptosis inhibitor Bcl-2 in sputum eosinophils from children with acute asthma

BACKGROUND: Apoptosis of eosinophils is of increasingly important value in modulating allergic airway inflammation in asthma. Our purpose was to investigate the degree of expression of the antiapoptotic B-cell lymphoma/leukaemia-2 (Bcl-2) protein in sputum eosinophils during acute asthma exacerbation and its relationship with exacerbation severity. METHODS: Sputum was obtained from 33 asthmatic children and 15 healthy children as a control group. Patients were studied during an acute asthma exacerbation. They were classified according to the severity of exacerbation into mild, moderate and severe (n=11 for each). Patients with severe exacerbation were followed up until remission and another sputum sample was obtained. Number of sputum eosinophils was expressed as percentage of leucocytes. Bcl-2 expression in sputum eosinophils was assessed by immunohistochemical staining techniques; the results were expressed as percentage of positively stained cells over total eosinophils. RESULTS: Sputum eosinophils and Bcl-2(+) eosinophils' percentages were significantly higher in patients with acute exacerbation than controls (P<0.01). Patients with severe exacerbation had significantly higher sputum Bcl-2(+) eosinophils' percentage than those with mild-to-moderate exacerbation (mean+/-SD=42.4+/-31.96% vs. 5.7+/-14.5%, P<0.01). A significant negative correlation was found between Bcl-2(+) eosinophils' percentage and peak expiratory flow rate % predicted (P<0.05). After remission, patients with severe exacerbation showed a significant decrease of Bcl-2(+) eosinophils' percentage (P<0.05). CONCLUSION: Our findings suggest that Bcl-2 prolongs survival and decreases apoptosis of airway eosinophils in asthma especially during exacerbation. Eosinophil apoptosis and Bcl-2 represent a target for new and effective therapeutic strategies of asthma.     

Mediterranean diet is associated with reduced asthma and rhinitis in Mexican children

Background: Diet during pregnancy and childhood has been suggested to play an important role in children’s asthma risk. We assessed whether the adherence to a Mediterranean dietary pattern, for children in the last 12 months and their mothers during pregnancy, was associated with both childhood asthma and allergic rhinitis. Methods: A cross‐sectional study was conducted in 2004 using a random sample of 1476 children (6‐ to 7‐year old) from the Mexicali region, Mexico. Dietary data of children’s intake in the last 12 months and their mothers’ intake during pregnancy was collected, through a parental food frequency questionnaire. A Mediterranean diet score was computed [Trichopoulou et al., N Engl J Med 348 (2003), 2599]. Data on seven asthma and rhinitis‐related outcomes were obtained from the International Study of Asthma and Allergies in Childhood questionnaire. Results: Adherence to a Mediterranean dietary pattern was inversely associated with asthma ever (OR = 0.60, 95% CI = 0.40–0.91), wheezing ever (0.64, 0.47–0.87), rhinitis ever (0.41, 0.22–0.77), sneezing ever (0.79, 0.59–1.07), current sneezing (0.71, 0.52–0.96) and current itchy‐watery eyes (0.63, 0.42–0.95). No associations were found using the mothers’ pregnancy diet score, except for current sneezing (0.71, 0.53–0.97). Conclusions: Our findings suggest a protective effect of following a healthy dietary pattern on asthma and allergic rhinitis in Mexican children.