Figure 2.
Anti-CD44 inhibits macrophage FcγR-mediated phagocytosis of anti-GPIIb/IIIa–opsonized platelets. Macrophages (RAW 264.7) were treated with anti-CD44 or controls before incubation with platelets opsonized with anti-GPIIb/IIIa antibodies. (A) Confocal microscopy images of untreated macrophages (MΦ) (left panels) or macrophages treated with anti-CD44 (right panels) before incubation with anti-GPIIb/IIIa (clone MWReg30)–opsonized platelets. Scale bars, 10 μm. All platelets (internalized and external) are labeled with the green cytoplasmic dye 5-chloromethylfuorescein diacetate (upper panels). External nonphagocytosed platelets were distinguished after completion of phagocytosis and formaldehyde fixation with an anti–glycoprotein V (GPV) primary antibody plus Alexa Fluor 647 (AF-647) secondary antibody (lower panels). Arrowheads point to example macrophages with phagocytosed platelets. Images were taken using a Quorum multimodal imaging system with a 63× oil-immersion objective (NA 1.47). Fluorescent images are merged with differential interference contrast images. Images were Z-stacked every 330 nm and reconstructed in 3 dimensions for analysis using Imaris v8.0.2. At least 4 images were taken at the center of each well (>500 cells counted). (B) Magnitude of macrophage phagocytosis of anti-GPIIb/IIIa IgG1 (clone MWReg30)–opsonized platelets. Nonopsonized platelets (-) were incubated with PBS only during the opsonization step before addition to macrophages. The phagocytic index is the average number of phagocytosed platelets per 100 macrophages. Data are from 6 experiments (mean ± standard error of the mean [SEM]). **P = .0022, nonparametric Mann-Whitney t test. (C) Effect of anti-CD44 (α-CD44) antibodies of different IgG subclasses (rat IgG1 anti-CD44 clone KM114 and rat IgG2b anti-CD44 clone IM7) on macrophage phagocytosis of IgG1-opsonized platelets. FcγRIII block is antibody clone 2.4G2. Data (mean ± SEM) are from 4 or 5 experiments. Dotted line indicates the level of phagocytosis by untreated macrophages (100%). The percentage of phagocytosis was calculated as (PI treatment/PI untreated control) × 100%, where PI (phagocytic index) is the average number of phagocytosed platelets per 100 macrophages. ****P < .0001, 1-way analysis of variance with Tukey’s post hoc test. CMFDA, 5-chloromethylfuorescein diacetate.

Anti-CD44 inhibits macrophage FcγR-mediated phagocytosis of anti-GPIIb/IIIa–opsonized platelets. Macrophages (RAW 264.7) were treated with anti-CD44 or controls before incubation with platelets opsonized with anti-GPIIb/IIIa antibodies. (A) Confocal microscopy images of untreated macrophages (MΦ) (left panels) or macrophages treated with anti-CD44 (right panels) before incubation with anti-GPIIb/IIIa (clone MWReg30)–opsonized platelets. Scale bars, 10 μm. All platelets (internalized and external) are labeled with the green cytoplasmic dye 5-chloromethylfuorescein diacetate (upper panels). External nonphagocytosed platelets were distinguished after completion of phagocytosis and formaldehyde fixation with an anti–glycoprotein V (GPV) primary antibody plus Alexa Fluor 647 (AF-647) secondary antibody (lower panels). Arrowheads point to example macrophages with phagocytosed platelets. Images were taken using a Quorum multimodal imaging system with a 63× oil-immersion objective (NA 1.47). Fluorescent images are merged with differential interference contrast images. Images were Z-stacked every 330 nm and reconstructed in 3 dimensions for analysis using Imaris v8.0.2. At least 4 images were taken at the center of each well (>500 cells counted). (B) Magnitude of macrophage phagocytosis of anti-GPIIb/IIIa IgG1 (clone MWReg30)–opsonized platelets. Nonopsonized platelets (-) were incubated with PBS only during the opsonization step before addition to macrophages. The phagocytic index is the average number of phagocytosed platelets per 100 macrophages. Data are from 6 experiments (mean ± standard error of the mean [SEM]). **P = .0022, nonparametric Mann-Whitney t test. (C) Effect of anti-CD44 (α-CD44) antibodies of different IgG subclasses (rat IgG1 anti-CD44 clone KM114 and rat IgG2b anti-CD44 clone IM7) on macrophage phagocytosis of IgG1-opsonized platelets. FcγRIII block is antibody clone 2.4G2. Data (mean ± SEM) are from 4 or 5 experiments. Dotted line indicates the level of phagocytosis by untreated macrophages (100%). The percentage of phagocytosis was calculated as (PI treatment/PI untreated control) × 100%, where PI (phagocytic index) is the average number of phagocytosed platelets per 100 macrophages. ****P < .0001, 1-way analysis of variance with Tukey’s post hoc test. CMFDA, 5-chloromethylfuorescein diacetate.

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