The study protocol was approved by the University of Wisconsin-Madison Health Sciences Institutional Review Board. Informed written consent was obtained from subjects prior to participation. For studies of
in vitro eosinophil activation, peripheral blood eosinophils were obtained from allergic subjects with and without mild asthma. Subjects with prescriptions for low doses of inhaled corticosteroids did not use their corticosteroids the day of the blood draw. Eosinophils were purified by negative selection as previously described [20 (
link)]. Briefly, heparinized blood was diluted 1:1 in HBSS and was overlaid above Percoll (1.090 g/ml). After centrifugation at 700 ×
g for 20 min at room temperature, the mononuclear cells were removed from the plasma/percol interface and erythrocytes were eliminated from the cell pellet by hypotonic lysis. The remaining pellet was resuspended in 2% NCS in HBSS. Cells were then incubated with anti-CD16, anti-CD3, anti-CD14 and anti-Glycophorin-A beads from Miltenyi (San Diego, CA), and run through an AutoMACS (Miltenyi). Eosinophil preparations with purity > 99% and viability ∼98% were used the same day, ∼5 h after the blood draw.
For studies of
in vivo eosinophil activation, bronchoscopy and bronchoalveolar lavage (BAL) were performed 48 h after segmental bronchoprovocation with an allergen (SBP-Ag) in subjects with mild asthma who were allergic to ragweed, dust mite, or cat dander allergens [20 (
link)]. Eosinophils were purified, as previously described [12 (
link)], from the BAL cell preparation (BAL EOS) and from peripheral blood (BBL EOS) of the same allergen-challenged subject. On the same day, eosinophils were also purified from peripheral blood of a control unchallenged subject (control EOS (Ctrl)).
Esnault S., Johansson M.W., Kelly E.A., Koenderman L., Mosher D.F, & Jarjour N.N. (2017). IL-3 Up-regulates and Activates Human Eosinophil CD32 and αMß2 Integrin Causing Degranulation. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 47(4), 488-498.