Novel fluorescence method to visualize antibody-dependent hydrogen peroxide-associated "killing" of liposomes by phagocytes.
We have developed a new methodology to examine effector-cell-mediated immune attack using liposomes as targets. Hydrogen-peroxide-associated killing of liposomes was observed with fluorescence intensification microscopy. Liposomes were composed of 98-99 mol % egg phosphatidylcholine and 1-2 mol % dinitrophenyl lipid hapten. Anti-dinitrophenyl IgG antibody was used to opsonize liposomes. Liposomes were loaded with dihydroxymandelic acid (DHMA) and peroxidase. Macrophage- or neutrophil-mediated recognition of liposomes triggers the release of H2O2 and other oxidative products. Upon interaction of H2O2 or OH radical with liposome contents, DHMA dimerizes forming a fluorescent derivative. Our studies indicate that individual living neutrophils and macrophages deposit oxidative products in a heterogeneous fashion among bound targets.
D000920:Antibody-Dependent Cell Cytotoxicity; D001770:Blood Bactericidal Activity; D006861:Hydrogen Peroxide / Q000502:physiology*; D008081:Liposomes; D008264:Macrophages / Q000502:physiology; D008333:Mandelic Acids; D008856:Microscopy, Fluorescence / Q000379:methods; D009504:Neutrophils / Q000502:physiology*; D010586:Phagocytes / Q000502:physiology*
D008081:Liposomes; D008333:Mandelic Acids; C000989:3,4-dihydroxymandelic acid; D006861:Hydrogen Peroxide