Monovalent and bivalent targeting of FcγRIII induce SHP-1 dependent ITAMi signaling and inhibisome formation. (A) huFcγRIII+ RBL-2H3 transfectants were incubated with either 3G8 F(ab′)2 (10 μg/mL) or IVIg (10 mg/mL) for the indicated times at 37°C. Cells were solubilized in 1% digitonin lysis buffer and immunoprecipitated with sepharose-coupled protein G sepharose (left panel) or with 3G8 F(ab′)2 fragment coupled to CNBr-activated sepharose 4B (right panel), and then immunoblotted (IB) with anti-SHP1 or anti-Syk antibodies. Total lysates were analyzed for SHP-1 phosphorylation by immunoblotting with anti-phospho–SHP-1 (p-SHP1) antibody. The amounts of SHP-1 and Syk in lysates were analyzed in parallel using anti–SHP-1 or anti-Syk antibodies. (B) LPS-primed BMMs from WT and mev/mev mice were incubated with IVIg (18 mg/mL) or with BSA (18 mg/mL) for 30 minutes at 37°C. Alexa Fluor 488–coupled acLDL was added to allow endocytosis for 30 minutes followed by plasma membrane staining using anti-CD11b–biotin and streptavidin–Alexa 568 before confocal analyses. Left panels show representative merged optical sections of internalized acLDL inside the cell. Scale bars: 5 μm. Right panel indicates quantification of acLDL endocytosis using green MFI inside each cell as quantified by LSM510 analysis software that analyzes specific pixels in at least 5 confocal microscope fields (**P < .01; nonparametric Mann-Whitney test). Data are representative for at least 3 independent experiments. (C) IgE-sensitized huFcγRIII+ RBL-2H3 transfectants were incubated with either 3G8 F(ab′)2 (10 μg/mL) or IVIg (10 mg/mL) for 30 minutes at 37°C. Free intracellular Ca2+ was assessed after cell activation with DNP-HSA (0.1 μg/mL). After 7 minutes transfectants were stimulated with 1μM ionomycin to determine maximal calcium uptake (n = 30 cells in each group). (D) Alexa 488–IgE sensitized huFcγRIII+ RBL-2H3 transfectants were incubated for 30 minutes with etheir biotinylated 3G8 F(ab′)2 or biotinylated 320 F(ab′)2 at 10 μg/mL before addition of DNP-HSA antigen for 10 minutes. Cells were then fixed, permeabilized and further incubated with rabbit anti–SHP-1 antibodies followed by goat anti–rabbit Alexa Fluor 647. For detection of FcγRIII, Alexa Fluor 568–conjugated steptavidin was used. Receptors (FcϵRI in green, FcγRIII in red) and SHP-1 (in blue) were analyzed by confocal microscopy. DIC and representative single optical sections through individual cells, as well as the merged images (xy planes), are presented. Scale bars: 5 μm. Data are representative of at least 3 independent experiments.
