Figure 6

sPLA ₂ -IIA enhances phagocytosis on microglial cells. sPLA₂-IIA induces fluorescein-isothiocyanate (FITC)-dextan beads uptake in microglial cells. BV-2 microglial cells were treated with different doses of sPLA₂-IIA and IFNγ (A), or with 1 μg/ml of different sPLA₂ isoforms (B) for 24 h at 37°C. Primary microglial cells were treated with 1 μg/ml of sPLA₂-IIA and IFNγ (F). Then, cells were co-incubated with FITC-dextan beads and quantified using a fluorescent plate reader, as detailed in Methods. Results are the average of three separate experiments in quadruplicate and are expressed as the mean of the fluorescent intensity values ± SD (*P < 0.001 vs. control cells). (C) Images with a Leica TCS SP5X confocal microscope, under a ×60 oil objective, confirm that fluorescent dextran associated with BV-2 microglia is internalized by the cells and not only bound to the cell surface. (D) BV-2 cells were cultured in the absence or presence of 1 μg/ml of sPLA₂-IIA or 100 UI/ml of IFNγ for 24 h at 37°C. Then, they were incubated with FITC-dextran for 1 h and analyzed by flow cytometry. Histograms represent one experiment out of three. Solid grey curves represent unspecific endocytosis and open black curves endocytosis of cells cultured as indicated. (E, G) Primary and immortalized BV-2 microglial cells were pretreated with the indicated inhibitors for 30 minutes. Cells were then treated with 1 μg/ml of sPLA₂-IIA at 37°C for 24 h followed by co-incubation with FITC-dextran for 30 minutes at 37°C. Phagocytosis was quantified using a fluorescent plate reader as described under Methods. Values ± SD are the average of three separate experiments in quadruplicate (*P < 0.001 vs. control cells, **P < 0.001 compared to sPLA₂-IIA stimulated cells).