Lysasa

Blood eosinophils were activated for 30 min at 37°C with lysine aspirin (LysASA) (10 mM: Sanofi-Aventis; Athens, Greece), ketorolac (10 mM: Hospira; Lake Forest, IL), sodium salicylate (NaSal) (10 mM: Acros Organics; Geel, Belgium) or the combination of phorbol 12-myristate 13-acetate (PMA) (100 ng/ml) with ionomycin (2 μg/ml (Sigma)). For studies with PGE₂, butaprost, EP3 antagonist (L-798106) or the EP1 antagonist (SC-51089) (Cayman Chemical; Ann Arbor MI), these compounds were preincubated with eosinophils for 30 min prior to stimulation with LysASA for an additional 30 min at 37°C. Supernatants were collected and mediators quantified by enzyme immunoassay (EIA) as described below.

Purified blood or eosinophils differentiated with or without IFN-γ were either left in a resting state or stimulated with lysine aspirin (LysASA (Sanofi-Aventis, Athens, Greece)) (1 and 10 mM) for 30 min. For studies involving pretreatment with COX-1 inhibitors, eosinophils were incubated for 30 min with either 10 mM ibuprofen sodium salt (Santa Cruz, Dallas, TX) or 0.1 mM SC-560 (Santa Cruz) after which cells were washed in PBS and stimulated with varying doses of LysASA for an additional 30 min. Supernatants were collected and assayed by enzyme immunoassay (EIA) for PGD₂ (Cayman; Ann Arbor, MI) according to the manufacturer's directions: lower limit of detection 55 pg/ml.