Figure 7
sPLA 2 -IIA induces efferocytosis in BV-2 cells. BV-2 cells were treated with 1 μg/ml of sPLA2-IIA or 100 UI/ml IFNγ for 24 h at 37°C, exposed to PrI-labelled apoptotic Jurkat cells for 2 h, and then stained with an anti-PE-CD68 antibody, as described under Methods. (A) Phagocytosis was analyzed by flow cytometry. FL2 fluorescence (left) shows BV-2 cells expressing CD68. The bar marks the population of BV-2 cells which was gated for detection of PrI-apoptotic cell uptake. FL3 fluorescence (right) shows the red fluorescence (PrI-apoptotic cells) in PE-CD68-labelled cells (on the gated BV-2 cells). As a measure of the phagocytosed cells, MIF values are shown. (B) Phagocytosis was also studied by confocal microscopy (final magnification ×60). Co-cultures of PrI-apoptotic cells with either resting or stimulated BV-2 cells were fixed and stained with Alexa-fluor-CD11b and DAPI. Laser confocal images confirm that fluorescent apoptotic cells associated with BV-2 microglia are internalized by the cells and not only bound to the cell surface. (C) Confocal images with reconstructed orthogonal projections presented as viewed in the x-z plane (bottom) and y-z plane (right): Fluorescent apoptotic cells (stained with PrI) are shown to be internalized by BV-2 microglia. (DAPI-labelled nuclei, blue). Scale bars: 10 μm in sPLA2-treated cells, and 7.5 μm in IFNγ-treated cells. (D) BV-2 cells were treated with the indicated inhibitor for 30 minutes before sPLA2-IIA stimulation and mixing with the apoptotic cells. Phagocytosis was analyzed as in (A). In all the histograms, the solid grey curve represent the resting/control cells. The results are representative of three independent experiments.