Figure 1
Figure 1. FcγRIIIB- and FcγRIIA-expressing neutrophils internalize immune complexes in vitro. (A) Mature neutrophils isolated from the bone marrow of γ−/− mice expressing FcγRIIA (IIA/γ−/−) or FcγRIIIB (IIIB/γ−/−) were incubated with FITC-BSA or FITC-BSA/anti-BSA IC (sIC) at 4°C. Fluorescence associated with samples was evaluated by flow cytometric analysis of Gr-1–positive gated neutrophils, a representative histogram of which is shown. (B) BMNs from indicated mice incubated with DQ-BSA alone or DQ-BSA/anti-BSA (DQ-sIC) at 4°C for 1 hour were further incubated for 1 hour at 4°C, or at 37°C to promote IC uptake. Fluorescence associated with samples was evaluated by flow cytometric analysis of Gr-1–positive gated neutrophils, representative histograms of which are shown (left panel). The average mean fluorescence intensities (M.F.I) obtained by flow cytometry are shown for each mouse strain (right panel). **P < .01, ***P < .001, compared with DQ-BSA alone within each genotype. (C) FITC-anti–Gr-1–labeled BMNs (green) from indicated mice were incubated with ICs labeled with Cy3 secondary antibody (red) at 37°C and subjected to analysis by laser scanning confocal microscopy. Serials of Z slices were acquired by confocal microscopy (left panels) followed by 3D reconstruction and surface analysis (right panels). IC-positive intracellular puncta (arrow) in FcγRIIIB-positive neutrophils were also positive for Gr-1, another GPI-anchored protein. (D) BMNs from FcγRIIIB/γ−/− mice or FcγRIIIB/γ−/− lacking Mac1−/− (IIIB/γ−/−/Mac-1) were incubated with DQ-BSA/anti-BSA and assessed as described in panel B. n.s. indicates not significant. (E) Internalization of DQ-BSA/anti-BSA sIC by FcγRIIIB (left panel) and FcγRIIA (right panel) expressing neutrophils pretreated with DMSO (−), cytochalasin D (cytD), MβCD, or dynasore (Dyn) was analyzed as in panel B. *P < .05, compared with DMSO control. (F) FcγRIIIB was transiently expressed in HEK293A cells. Transfected cells incubated with soluble ICs labeled with cy3 secondary antibody (red) combined with FITC-dextran (70 kDa, green, top panel) or Alexa-488–transferrin (green, bottom panel), were evaluated by laser scanning confocal microscopy. Intracellular ICs colocalized with Dextran-containing (arrowheads) but not transferrin-containing intracellular compartments. (G) FcγRIIA or FcγRIIIB stably expressing HEK293A cells were transfected with GFP or GFP-cdc42N (dominant negative) or GFP-cdc42L (constitutive active; top panels), flag-Control or myc-Rac1N (dominant negative), or myc-Rac1L (constitutively active; middle panels) constructs or pretreated with a Rho inhibitor CT04 (bottom panels). Cells were incubated with cy5-labeled sICs. Top panels: GFP-positive cells were analyzed for sIC uptake by flow cytometry. Middle panels: Cells were fixed, permeabilized, and stained with anti-myc or anti-flag antibody followed by Alexa488 anti–mouse antibody. Cells positive for myc or flag were analyzed for sIC uptake by flow cytometry. Bottom panels: Cells were directly analyzed by flow cytometry. Of all the treatments, only constitutively active cdc42 (arrows) decreased sIC uptake by FcγRIIIB and FcγRIIA. N = 3 or 4 independent experiments for panels A-E and G. N = 2 independent experiments for panel F.

FcγRIIIB- and FcγRIIA-expressing neutrophils internalize immune complexes in vitro. (A) Mature neutrophils isolated from the bone marrow of γ−/− mice expressing FcγRIIA (IIA/γ−/−) or FcγRIIIB (IIIB/γ−/−) were incubated with FITC-BSA or FITC-BSA/anti-BSA IC (sIC) at 4°C. Fluorescence associated with samples was evaluated by flow cytometric analysis of Gr-1–positive gated neutrophils, a representative histogram of which is shown. (B) BMNs from indicated mice incubated with DQ-BSA alone or DQ-BSA/anti-BSA (DQ-sIC) at 4°C for 1 hour were further incubated for 1 hour at 4°C, or at 37°C to promote IC uptake. Fluorescence associated with samples was evaluated by flow cytometric analysis of Gr-1–positive gated neutrophils, representative histograms of which are shown (left panel). The average mean fluorescence intensities (M.F.I) obtained by flow cytometry are shown for each mouse strain (right panel). **P < .01, ***P < .001, compared with DQ-BSA alone within each genotype. (C) FITC-anti–Gr-1–labeled BMNs (green) from indicated mice were incubated with ICs labeled with Cy3 secondary antibody (red) at 37°C and subjected to analysis by laser scanning confocal microscopy. Serials of Z slices were acquired by confocal microscopy (left panels) followed by 3D reconstruction and surface analysis (right panels). IC-positive intracellular puncta (arrow) in FcγRIIIB-positive neutrophils were also positive for Gr-1, another GPI-anchored protein. (D) BMNs from FcγRIIIB/γ−/− mice or FcγRIIIB/γ−/− lacking Mac1−/− (IIIB/γ−/−/Mac-1) were incubated with DQ-BSA/anti-BSA and assessed as described in panel B. n.s. indicates not significant. (E) Internalization of DQ-BSA/anti-BSA sIC by FcγRIIIB (left panel) and FcγRIIA (right panel) expressing neutrophils pretreated with DMSO (−), cytochalasin D (cytD), MβCD, or dynasore (Dyn) was analyzed as in panel B. *P < .05, compared with DMSO control. (F) FcγRIIIB was transiently expressed in HEK293A cells. Transfected cells incubated with soluble ICs labeled with cy3 secondary antibody (red) combined with FITC-dextran (70 kDa, green, top panel) or Alexa-488–transferrin (green, bottom panel), were evaluated by laser scanning confocal microscopy. Intracellular ICs colocalized with Dextran-containing (arrowheads) but not transferrin-containing intracellular compartments. (G) FcγRIIA or FcγRIIIB stably expressing HEK293A cells were transfected with GFP or GFP-cdc42N (dominant negative) or GFP-cdc42L (constitutive active; top panels), flag-Control or myc-Rac1N (dominant negative), or myc-Rac1L (constitutively active; middle panels) constructs or pretreated with a Rho inhibitor CT04 (bottom panels). Cells were incubated with cy5-labeled sICs. Top panels: GFP-positive cells were analyzed for sIC uptake by flow cytometry. Middle panels: Cells were fixed, permeabilized, and stained with anti-myc or anti-flag antibody followed by Alexa488 anti–mouse antibody. Cells positive for myc or flag were analyzed for sIC uptake by flow cytometry. Bottom panels: Cells were directly analyzed by flow cytometry. Of all the treatments, only constitutively active cdc42 (arrows) decreased sIC uptake by FcγRIIIB and FcγRIIA. N = 3 or 4 independent experiments for panels A-E and G. N = 2 independent experiments for panel F.

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