Tf 1 cells

TF‐1 cells (erythroleukemic cell line; R&D Systems, Abingdon, UK) maintained in 4 ng/ml human GM‐CSF (as supplier's instructions, in RPMI‐1640 with 5% FBS (heat inactivated) and penicillin/streptomycin) were washed 3 times to ensure complete removal of GM‐CSF. The cells were then treated with 0.25 ng/ml GM‐CSF (R&D Systems), in the presence or absence of a serial dilution of CAM3001 (blocking antibody specific to human GM‐CSFRα, MedImmune Ltd, Cambridge, UK) or isotype control (NIP228, MedImmune), with both ligand and antibody being added at the same time to the cultures. The cells were incubated for 72 hr. CellTiter‐Glo (Promega UK, Southampton, UK, G7570) was used to measure ATP as an indirect measure of the number of viable cells according to the manufacturer's instructions.

TF‐1 cells (R&D Systems) that had been maintained in human GM‐CSF (4 ng/ml, R&D Systems) were washed 3 times to remove GM‐CSF. GM‐CSFRα expression was assessed on the cells following culture for a further 18 hr in the presence or absence of GM‐CSF (4 ng/ml).
GM‐CSFRα expression was quantified on human neutrophils cultured in the presence of GM‐CSF (0.001‐10 ng/ml, R&D Systems), LPS (100 ng/ml, Sigma Aldrich UK, Poole, UK), TNFα (20 ng/ml, R&D Systems) or appropriate vehicle control. In certain experiments neutrophils were pretreated for 30 min with CAM‐3001 (blocking antibody specific to human GM‐CSFRα) or for 1 hr with the proteasomal inhibitor MG132 (20 μM, Sigma Aldrich), brefeldin A (10 μg/ml) to block lysosomal degradation, or the transcriptional inhibitor actinomycin D (2 μg/ml); in certain experiments IL‐8 was measured in the supernatants using an in‐house ELISA.20 GM‐CSFRα levels were also quantified on blood neutrophils and BALF neutrophils derived from patients with ARDS.