Apoptosis Is Essential for Neutrophil Functional Shutdown and Determines Tissue Damage in Experimental Pneumococcal Meningitis
Figure 3
Overexpression of Bcl-2 protects neutrophils from apoptosis and removal by macrophages.
(A) wt or Bcl-2-overexpressing neutrophils differentiated for 5 days were in addition cultured for 24 or 48 hours in the presence or absence of SCF. Cells were then stained with AnnexinV-FITC and propidium iodide (PI) and analysed by flow cytometry. Bars/error bars indicate mean/SEM of five independent experiments. (B) Neutrophils differentiated for 5 days were cultured for 8 or 48 hours in the presence or absence of SCF, followed by staining for active caspase-3. Error bars show the SEM of three independent experiments. (C) Neutrophils differentiated for 5 days were cultured in the absence of SCF for the indicated periods of time and stained with AnnexinV-PI or CFSE. Green-fluorescent (CFSE-stained) neutrophils were then added to cultures of RAW macrophages stained with the red dye PKH26 (ratio neutrophil∶macrophages, 5∶1). After 4 h of co-incubation, cultures were analysed by flow cytometric analysis. The proportion of phagocytic cells within the macrophage population is shown for wt or Bcl-2-expressing neutrophils at various time points. No double-positive cells were observed when reactions were carried out at 4°C (data not shown). Data are mean/SEM from 3 independent experiments.