Figure 3
Figure 3. Human IVIg inhibits acLDL endocytosis through FcγRIII. (A) LPS-primed BMM from FcγRIIB−/− or FcγRIII−/− 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 acDL 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). (B) LPS-primed BMMs from FcγRIIB−/− mice were preincubated with 10 mg/mL unfractionnated IVIg, with HPLC-purified monomeric (IVmIg) or HPLC-purified dimeric IVIg (IVdIg) or BSA as a control. Cells were also preincubated with 10 μg/mL of 2.4G2 F(ab′)2 or immune complexes (IC) made of 2.4G2 F(ab′)2 (10 μg/mL) plus goat anti–rat F(ab′)2 (GAR at 40μg/mL) for 30 minutes at 37°C. Endocytosis was induced by addition of Alexa Fluor 488–coupled acLDL. The plasma membrane was stained with CD11b-biotin and streptavidin–Alexa 568 on ice for 20 minutes and cells were analyzed using confocal microscopy. Representative merged optical sections are presented showing internalized acDL inside the cell. Scale bars: 5 μm. Green mean fluorescence intensity (MFI) inside each cell was quantified (right panel) using LSM510 image examiner software that analyzes specific pixels in at least 5 confocal microscope fields (**P < .01; n = 5; nonparametric Mann-Whitney test).

Human IVIg inhibits acLDL endocytosis through FcγRIII. (A) LPS-primed BMM from FcγRIIB−/− or FcγRIII−/− 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 acDL 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). (B) LPS-primed BMMs from FcγRIIB−/− mice were preincubated with 10 mg/mL unfractionnated IVIg, with HPLC-purified monomeric (IVmIg) or HPLC-purified dimeric IVIg (IVdIg) or BSA as a control. Cells were also preincubated with 10 μg/mL of 2.4G2 F(ab′)2 or immune complexes (IC) made of 2.4G2 F(ab′)2 (10 μg/mL) plus goat anti–rat F(ab′)2 (GAR at 40μg/mL) for 30 minutes at 37°C. Endocytosis was induced by addition of Alexa Fluor 488–coupled acLDL. The plasma membrane was stained with CD11b-biotin and streptavidin–Alexa 568 on ice for 20 minutes and cells were analyzed using confocal microscopy. Representative merged optical sections are presented showing internalized acDL inside the cell. Scale bars: 5 μm. Green mean fluorescence intensity (MFI) inside each cell was quantified (right panel) using LSM510 image examiner software that analyzes specific pixels in at least 5 confocal microscope fields (**P < .01; n = 5; nonparametric Mann-Whitney test).

Close Modal

or Create an Account

Close Modal
Close Modal