Fig. 4.
ILT4 is localized within neutrophil granules and is up-regulated on cell surface upon stimulation. (A) Confocal microscopy analysis of ILT4 localization. Permeabilized neutrophils were stained for ILT4 (green) and nucleus (DAPI, blue). (Scale bars: 5 μm.) (B) Surface ILT4 and CD11b expression on neutrophils after stimulation with 10−7 M fMLF, 1 µg/mL LPS, or 2 ng/mL TNF-α. (C) ILT4 and CD11b surface expression on resting and fMLF-stimulated neutrophils. Histograms represent ILT4 (n = 36) and CD11b (n = 23) MFI ratios. (D) Time course analysis of surface ILT4 and CD11b increase. Neutrophils were stimulated with 10−6, 10−7, or 10−8 M fMLF for 5, 15, 30, and 60 min before CD11b/ILT4 expression analysis (n = 3). (E) The fMLF-induced surface ILT4 increase is enhanced by actin polymerization inhibition. Neutrophils were stimulated or not with 10−7 M fMLF following pretreatment with 10 µM cytochalasin D (CytD), control DMSO, or PBS 1×. Surface ILT4 expression (MFI ratio) was analyzed by flow cytometry (n = 9). (F) Azurophil granules marker CD63 surface expression on neutrophils following previous treatments. Histograms are representative of three independent experiments. **P < 0.01; ***P < 0.001.