Mechanisms of Induction of Autophagy in Macrophages

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Abstract

The development of autophagy was studied in the murine macrophage cell line RAW264.7 cells and in elutriated human peripheral blood monocytes. After transfection of cells with a plasmid expreessing PEGFP-LC3 (from Dr. N. Mizushima), the development of autophagic vesicles was determined by the identification of LC3 associated with the vesicle wall using confocal microscopy. The development of autophagy was confirmed by western blot of cellular lysate demonstrating conversion of LC3-1 to LC3-II. Treatment of cells with interferon-gamma induced autophagy as measured by confocal microscopy of LC3 associated vesicles or by western blot demonstrating the presence of LC3-II. Studies of the pathway leading from interferon-gamma stimulation to autophagic vesicle development are ongoing.