NK cells and eosinophil incubations
For in vitro assays, peripheral blood was obtained from healthy subjects according to a protocol approved by the local Research Ethics Committee. Written informed consent was obtained from all subjects before their participation. Peripheral blood mononuclear cells (PBMCs) and granulocytes were isolated by density gradient centrifugation (Histopaque-1077 and Histopaque-1119 respectively; Sigma-Aldrich) as described earlier (12). NK cells, monocytes and T lymphocytes were isolated from PBMCs with a magnetically cell isolation kit (EasySep, Stemcell Technologies) according to the manufacturer’s instructions. Eosinophils and neutrophils were further sorted from a granulocyte suspension with an ARIA flow cytometer (Becton Dickinson) as described earlier (18). In some selected experiments, CD56dimand CD56bright NK cells were sorted with an ARIA flow cytometer (Becton Dickinson) from a total NK cell suspension obtained earlier with a magnetically NK cell isolation kit (EasySep, Stemcell Technologies). For NK cell/eosinophil co-cocultures, autologous human granulocytes or eosinophils were resuspended in RPMI 1640 medium (Gibco, BRL) supplemented with 10% heat-inactivated FCS (Gibco, BRL) and 2 mM L-glutamine (Gibco, BRL), in the absence or presence of total NK cells or CD56dim and/or CD56bright NK cells (1:5 ratio of granulocytes/NK cells or CD56dimor CD56bright subset) or autologous supernatants from earlier co-incubation assays (4 hours, 37°C, 5% CO2). Apoptosis was assayed by FACS with annexin V and 7-AAD staining (BD Pharmingen). For assays assessing superoxide generation, detection of superoxide radical anion was assessed by electron paramagnetic resonance (eScan, Bruker) in medium containing 200 μM of CMH (1-hydroxyl–3– methoxycarbonyl- 2,2,5,5–tetramethylpyrrolidine). In some experiments, cells were stimulated (10 min, 50 ng/ml) with Phorbol 12-myristate 13-acetate (PMA) (Sigma) and/or selectively exposed (15 min, 37°C) to LXA4 (1, 10 or 100 nM) or vehicle (0.1% ethanol) (Cayman Chemical).