Materials and Methods

Subjects Human eosinophils were isolated from volunteers with normal to mildly elevated eosinophil counts (211 to 510 eosinophils/ul of blood) according to a protocol approved by the University of Chicago Institutional Review Board. All subjects gave a history of allergic rhinitis and/or mild asthma. None were taking oral or parenteral corticosteroids at the time of the study. Neutrophils were isolated from patients with normal eosinophil and neutrophil counts. Informed consent was obtained from all human volunteers before participation in this study.

Granulocyte Isolation Normodense eosinophils were isolated from peripheral blood by a modification of the negative immunomagnetic selection method of Hansel and colleagues (14). A total of 60 to 120 ml of heparinized blood was diluted 1: 1 in Hanks' balanced salt solution (HBSS) without calcium. Thirtymilliliter aliquots of this suspension then were overlayered carefully onto 15 ml of Percoll (density= 1.084) and centrifuged at 1,000 X g for 30 min. The overlying serum, mononuclear cell layer, and excess Percoll then were aspirated, leaving a pellet containing red cells, neutrophils, and eosinophils. Red cell lysis was performed by adding 5 vol of ice-cold ammonium chloride solution (NaCI, 155 mM; KHCOh 10 mM; and EDTA, 0.1 mM) to 1 vol of this cell suspension and incubating for 20 min on ice. The resulting mixture of neutrophils and eosinophils then was washed twice in HBSS supplemented with 2% heat-inactivated fetal calf serum (HBSS/FCS). After centrifugation, the supernatant was aspirated completely from the cell pellet; 75 to 100 1/-1 of anti-CD16 immunomagnetic beads (Miltenyi Biotec, Bergisch-Gladbach, Germany) then was added to the cell pellet containing 1.5 to 2.0 X 108 granulocytes. This mixture was incubated for 45 min on ice with occasional gentle agitation. Four milliliters of cold HBSS/FCS then was added to the cell suspension, and the resulting mixture was transferred to the top of a magnetic separation column (type C column; Miltenyi Biotec) placed in the magnetic field of a MACS magnet (Miltenyi Biotec and Becton-Dickinson, San Jose, CA). Using a 23-gauge needle at the base of the column