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).