Bivalent crosslinking of human monocyte/macrophage-expressed FcγRIIIA but not neutrophil-expressed FcγRIIIB inhibits bacterial phagocytosis and ROS production. (A) Human alveolar macrophages (5 × 105) were allowed to adhere on glass cover slips for 12 hours at 37°C followed by preincubation with 10 μg/mL anti–human FcγRIII F(ab′)2 (3G8) or irrelevant F(ab′)2 (320) for 30 minutes at 37°C. Phagocytosis assay was initiated by addition of alexa fluor 594-coupled S aureus for 30 minutes at 37°C and evaluated by confocal microscopy. Representative merged optical sections are presented showing internalized S aureus inside the cell. Scale bars: 5 μm. Internalized S aureus were quantified by determining red MFI inside each cell using LSM510 image analysis software (**P < .01; n = 3; nonparametric Mann-Whitney test). (B) Human blood monocytes (5 × 105 cells/0.5 mL Hanks buffer) were preincubated with the indicated concentration of 3G8 F(ab′)2 or 10 μg/mL of irrelevant 320 F(ab′)2 for 30 minutes at 37°C before cell activation with fLMF (10−6M), and luminol-amplified chemiluminescence was measured. (C) Human neutrophils (5 × 105 cells/0.5 mL Hanks buffer) were preincubated with the indicated concentrations of 3G8 F(ab′)2 or 10 μg/mL of irrelevant 320 F(ab′)2 for 30 minutes at 37°C before cell activation with fLMF (10−6M), and luminol-amplified chemiluminescence was measured. Blue and red curves represent 3G8 F(ab′)2 anti-FcγRIII at 10 and 5 μg/mL, respectively, whereas black curves indicate the control 320 F(ab′)2 at 10 μg/mL.
