Figure 3

Human interleukin 23 activates the leukotriene B4 synthesis pathway in macrophages via phosphatidylinositol 3-kinase. (a) Graphical representation of the percentage of cells fluxing calcium and immunofluorescence imaging of phosphorylated phospholipase A₂ (p-PLA₂) in human peripheral blood mononuclear cells (PBMCs) cultured with macrophage colony-stimulating factor (M-CSF) for 8 days, followed by acute treatment of 10 nM leukotriene B4 (LTB4) with or without pretreatment of 100 nM BLT1 and BLT2 antagonists. (b) Graphical representation of the percentage of cells fluxing calcium and immunofluorescence imaging of p-PLA₂ in human PBMCs cultured with M-CSF for 8 days, acutely activated with 100 ng/ml of interleukin 23 (IL-23) with or without pretreatment of 1 μM Wortmannin for 30 minutes. For calcium experiments, cells were also prelabeled with fluo-4 AM calcium dye. For p-PLA₂ expression, cells were fixed, permeabilized and labeled for p-PLA₂, and mean fluorescence intensity (MFI) was measured for >30 cells. PI3-K, Phosphatidylinositol 3-kinase. 4′,6-diamidino-2-phenylindole (DAPI) is blue and p-PLA₂ expression is in red (scale bars represent 20 μm). Representative data and images from three experiments are shown. **P < 0.01 and ***P < 0.001.