Inhibition of multiple Class I PI3K isoforms is required to abrogate the rhGM-CSF cytoprotective effect in human neutrophils.

<p><b>A.</b> Human neutrophils were incubated in IMDM +10% autologous serum in the presence (closed symbols) or absence (open circles) of rhGM-CSF 10 ng/ml for 6–24 h. Neutrophil apoptosis was quantitated after 20 hours of culture by flow cytometry following AnV and PI staining. Data represent %AnV positive cells (PI negative plus PI positive). Data represent n = 2 independent experiment performed in triplicate. <b>B.</b> Human neutrophils (5×10⁶/ml) were pre-incubated for 20 mins in IMDM +10% autologous serum containing the indicated inhibitors or vehicle control (DMSO), prior to treatment with rhGM-CSF 10 ng/ml. Neutrophil apoptosis was quantitated after 20 hours of culture by flow cytometry following AnV and PI staining. <b>C.</b> Human neutrophils (5×10⁶/ml) were pre-incubated for 20 mins in IMDM +10% autologous serum containing the indicated inhibitors, individually or in combination or vehicle control (DMSO concentration equivalent to that applied with the combination of all four isoform-selective inhibitors). Cells were then treated with rhGM-CSF 10 ng/ml and apoptosis was quantitated after 20 hours of culture by flow cytometry following AnV and PI staining. For B and C, data represent results of three independent experiments, performed in triplicate, expressed as mean % apoptotic neutrophils ± SEM values. *p<0.01 versus GM-CSF treatment alone; ns indicates no significant difference compared with GM-CSF alone.