BACKGROUND: One mechanism of the eosinophil's contribution to airway inflammation in asthma is through release of cationic granule proteins to cause airway injury. Differences in either the intracellular concentration of granule proteins or the extent of activated degranulation between eosinophils from healthy patients and those with allergy and asthma could, therefore, relate to fundamental differences in this cell's function. OBJECTIVE: To identify phenotypic differences in eosinophil-derived neurotoxin (EDN) content and release in eosinophils from healthy patients, those with allergy, and those with allergy and asthma. METHODS: Peripheral blood eosinophils were isolated by negative anti-CD16 selection. Total intracellular and cytokine-activated release of EDN protein was measured by radioimmunoassay. EDN mRNA was assessed by real-time PCR. RESULTS: Eosinophils from patients with asthma contained significantly more EDN per cell than comparable cells from healthy patients, those with allergy but without asthma, or those with asthma treated with inhaled corticosteroids, but they had concentrations similar to airway eosinophils isolated from bronchoalveolar lavage fluid 48 hours after segmental bronchoprovocation with allergen. Furthermore, this increased granule protein was reflected in more EDN degranulation by IL-5- or GM-CSF-activated eosinophils when calculated as nanograms of protein secreted but not when calculated as a percentage of total EDN release. Levels of EDN mRNA were similar in all subject groups. CONCLUSIONS: These data suggest that peripheral blood eosinophils from subjects with untreated asthma have increased inflammatory capacity, as reflected by greater intracellular concentrations of EDN.
BACKGROUND: One mechanism of the eosinophil's contribution to airway inflammation in asthma is through release of cationic granule proteins to cause airway injury. Differences in either the intracellular concentration of granule proteins or the extent of activated degranulation between eosinophils from healthy patients and those with allergy and asthma could, therefore, relate to fundamental differences in this cell's function. OBJECTIVE: To identify phenotypic differences in eosinophil-derived neurotoxin (EDN) content and release in eosinophils from healthy patients, those with allergy, and those with allergy and asthma. METHODS: Peripheral blood eosinophils were isolated by negative anti-CD16 selection. Total intracellular and cytokine-activated release of EDN protein was measured by radioimmunoassay. EDN mRNA was assessed by real-time PCR. RESULTS: Eosinophils from patients with asthma contained significantly more EDN per cell than comparable cells from healthy patients, those with allergy but without asthma, or those with asthma treated with inhaled corticosteroids, but they had concentrations similar to airway eosinophils isolated from bronchoalveolar lavage fluid 48 hours after segmental bronchoprovocation with allergen. Furthermore, this increased granule protein was reflected in more EDN degranulation by IL-5- or GM-CSF-activated eosinophils when calculated as nanograms of protein secreted but not when calculated as a percentage of total EDN release. Levels of EDN mRNA were similar in all subject groups. CONCLUSIONS: These data suggest that peripheral blood eosinophils from subjects with untreated asthma have increased inflammatory capacity, as reflected by greater intracellular concentrations of EDN.
Authors: Michelle A Makiya; Jesica A Herrick; Paneez Khoury; Calman P Prussin; Thomas B Nutman; Amy D Klion Journal: J Immunol Methods Date: 2014-06-08 Impact factor: 2.303