CD177 ligation impairs integrin internalization. (A) Peripheral blood neutrophils were incubated with Filipin III (inhibitor of calveolin-mediated endocytosis, 3 µg/mL), Dynasore (inhibitor of dynamin, required for clathrin or calveolin-mediated endocytosis, 80 µM), AEBSF (pan-serine protease inhibitor, 10 µM), or corresponding vehicles and allowed to migrate across a 3-µm pore transwell toward LTB4 100 nM × 2 h. Effect on migration is expressed as a proportion of corresponding vehicle control. Inhibition of endocytosis but not proteolytic cleavage impairs migration. (B) Neutrophils were preincubated with αCD177 or isotype 10 µg/mL RT for 10 min and then added to a transwell apparatus with a 0.4-µm pore membrane that was impermeable to neutrophil transit. After 2 h of attraction toward LTB4 100nM in the bottom chamber, attached neutrophils were liberated from the membrane using EDTA and assessed for CD11b expression. (C) Neutrophils exposed to isotype immunoglobulin G (IgG) or αCD177 10 µg/mL for 15 min at RT were allowed to adhere to 6-well plates and then incubated for with anti-CD11b-phycoerythrin (1 µg/mL, 30 min) at 4°C. After washing, cells were incubated at 37°C in prewarmed media containing 100 nM LTB4, as is indicated, acid-washed to strip off the surface signal, and then detached using EDTA for analysis of internalized fluorescence by flow cytometry. (D) Neutrophils exposed to isotype IgG, αCD177 10 µg/mL, or MEM148 for 10 min at RT were incubated with complement-opsonized 2-µm fluorescent latex beads for 1 h at 37°C, followed by washing in PBS 5-mM EDTA to remove adherent beads. Internalized fluorescence was then analyzed by flow cytometry. Each experiment is representative of at least 3 replicates. Means ± SEMs. *P < .05; **P < .01; ***P < .001; ****P < .0001; by unpaired t test (A-B), 2-way ANOVA (C) or 1-way ANOVA (D).
