Saecs

Three human airway epithelial cells (HAECs), including A549 cells, BEAS-2B cells, and primary small airway epithelial cells (SAECs), were purchased from American type Culture Collection (ATCC, Manassas, VA, USA) and were cultured following previously reported methods.17 (link) A549 and BEAS-2B cells were cultured in RPMI-1640 medium with 10% heat-inactivated FBS, penicillin (100 IU/mL), and streptomycin (50 µg/mL). SAECs were cultured in Airway Epithelial Cell Basal Medium (ATCC), supplemented with HSA 500 mg/mL, linoleic acid 0.6 mM, lecithin 0.6 mg/mL, L-glutamine 6 mM, extract P 0.4%, epinephrine 1.0 mM, transferrin 5 mg/mL, triiodothyronine 10 nM, hydrocortisone 5 mg/mL, epidermal growth factors 5 ng/mL, and insulin 5 mg/mL. Cells were maintained at 37℃ with 5% CO₂ in humidified air.
HAECs (1×10⁵ cells/mL) were treated with 10 µL of 1 µg/mL TDI-HSA conjugate and/or 10 µL of 1 mM NAC for 24 hours. Isolated neutrophils at 0.5×10⁵ cells/mL, an optimal count in previous experiments, were co-cultured with HAECs in transwells to interfere with direct contact between neutrophils and HAECs. Supernatants were collected and frozen at −80℃ until ready for use.

Two human AECs (type II alveolar epithelial cell line [A549] and primary small airway epithelial cells [SAECs]) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). A549 cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), and 1% penicillin-streptomycin (Gibco). SAECs were cultured in AEC basal medium (ATCC) plus bronchial epithelial cell growth kit (ATCC), 1% penicillin-streptomycin, and 25 ng/mL amphotericin B (Sigma-Aldrich, St. Louis, MO, USA).
SAECs (1 × 10⁵) or A549 cells (2 × 10⁵) were stimulated with poly I:C (1 or 10 µg/mL; Sigma-Aldrich), IL-13 (10 or 100 ng/mL; R&D Systems, Minneapolis, MN, USA), or phorbol myristate acetate (PMA)-induced EETs (100 ng/mL) for 24 hours or cocultured with 5 × 10⁵ peripheral blood eosinophils (PBEs) from asthmatics for 18 hours. AECs were pretreated with 1 or 10 µg/mL dexamethasone (Dex; Sigma-Aldrich) for 30 minutes to evaluate its suppressive effect. Supernatants were collected for ELISA.

Two kinds of AECs were used in this study, primary small airway epithelial cells (SAECs) and human AEC line (A549), both of which were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). SAECs were cultured in basal medium (ATCC) plus bronchial epithelial cell growth kit (ATCC), 10 UI/mL penicillin G sodium, 10 μg/mL streptomycin sulfate (Gibco, Grand Island, NY, USA), and 25 ng/mL amphotericin B (Sigma Aldrich, St. Louis, MO, USA) at 37°C in humidified air with 5% CO₂. A549 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, penicillin G sodium (100 UI/mL), and streptomycin sulfate (100 μg/mL). SAECs (1 × 10⁵ cells) or A549 (2 × 10⁵ cells) were seeded onto a 12-well plate and stimulated with 50 ng/mL IL-5/IL-13/IL-6/IL-1β (Sigma Aldrich), or 50 μg/mL Poly I-C (Sigma Aldrich) for 24 hours. In some experiments, AECs were stimulated with 50 μg/mL Poly I-C with/without 50 ng/mL human recombinant SAA1/SAA (LifeSpan Biosciences, Franklin Lakes, NJ, USA).