Fig. 3.

ILT4 inhibits the CD32a-mediated production of ROS, and both ILT4 and CD32a colocalize within neutrophil lipid rafts. (A) Neutrophils were stimulated with coated anti-CD32 mAb in presence of either coated anti-ILT4 or isotype control mAb (n = 5). When indicated, neutrophils were previously incubated with 1 µM DPI or control DMSO (n = 4). The amount of ROS produced by neutrophils was detected using Amplex Red method. Fluorescence was measured every 30 s for 60 min. A representative experiment is shown. (B) Total areas under curves from the independent experiments were measured. Results obtained after stimulation with anti-CD32 + isotype control mAb were assigned a value of 100%. Mann−Whitney test was performed, and P values < 0.05 (*) and < 0.01 (**) were taken to be significant. (C) ILT4 colocalized with CD32a within neutrophil lipid rafts. CD32a (red) was cross-linked followed by ILT4 (blue), lipid rafts (CTβ, green), and nucleus (DAPI, dark blue) stainings. (Scale bars: 5 μm.) Images are representative of three independent experiments.