Effects of fMLP on CD157 expression and compartmentalization. (A) The expression of CD157, CD38, and CD11b was examined during stimulation of PMNs with fMLP. All the experiments were performed on whole blood to minimize the artifactual modulation attributable to cell purification procedures. Blood (100 μL) was treated with fMLP (10 nM, 37°C) for the indicated times, fixed, and incubated with anti-CD157, anti-CD38, anti-CD11b, and anti-HLA class I mAbs. After labeling with F(ab′)2-RaMIg-FITC, samples were treated with lysis buffer and analyzed by FACS. Forward- and right-angle scatters were used to selectively gate PMNs. Results showing the increasing rate of mean fluorescence intensity (MFI) are expressed as fMLP-induced expression of CD157, CD38, CD11b, or HLA class I/basal surface expression of each molecule at 37°C. The results are expressed as means of 3 separate experiments ± SD. (B) Confocal microscopy analysis of PMNs treated with fMLP. PMNs were incubated for 5 minutes at 37°C in the presence of 100 nM fMLP, then fixed and stained with anti-CD157 and cholera toxin-FITC. (C) PMNs treated with fMLP were fixed, permeabilized, and stained with anti-CD157-FITC and phalloidin-TRITC to visualize F-actin polarization. Samples were observed by differential interference contrast (DIC) and fluorescence confocal microscopy. Cells were mounted onto slides and imaged at 20°C using a confocal scanning laser microscope (FV300) mounted on an IX71 inverted microscope (both from Olympus) with a PlanApo 60 × oil, 1.4 numerical aperture (NA) objective lens. Data are representative of 5 experiments with cells from different donors. Bar, 10 μm.
