Effect of LY294002 on ROS production and PKB/Akt phosphorylation in neutrophils from healthy volunteers.

(A) Isolated neutrophils were preincubated with 1, 10, or 100 μM LY294002 for 30 minutes and were stimulated for 20 minutes with 1 μM fMLP with or without priming with 5 ng/mL GM-CSF. ROS production was measured by FACS analysis. Results represent the mean increase in ROS produced after stimulation, compared with unstimulated cells, of 4 individual experiments. Significant reductions in respiratory burst by pretreatment of neutrophils with LY294002 are indicated by single asterisks (P < .05). Significant differences between the unprimed and the GM-CSF–primed groups in inhibition of ROS production by LY294002 are indicated with 2 asterisks (P < 0.05). (B) Neutrophils were stimulated with 1 μM fMLP, with or without priming with 5 ng/mL GM-CSF, or with GM-CSF alone. Serine-phosphorylated PKB/Akt was detected by Western blot analysis and immunodetection. ERK1/2 antibodies were used to determine equal loading (lower panel). (C) To better visualize GM-CSF–stimulated PKB/Akt phosphorylation, a longer exposure of another Western blot is shown. (D) Isolated human neutrophils were preincubated with increasing amounts of LY294002 for 30 minutes and were stimulated for 2 minutes with either 1 μM fMLP, with or without GM-CSF priming, or 5 ng/mL GM-CSF alone. Unstimulated cells were run as negative control (c). Cell lysates were analyzed by Western blotting, using an antibody against serine phosphorylated PKB/Akt (upper panel). Equal protein loading in the samples was confirmed by immunodetection of ERK1/2 (lower panel). A representative blot of 3 independent experiments is shown.