LOCALIZATION OF NRAMP1 IN MACROPHAGES - MODULATION WITH ACTIVATION AND INFECTION

The murine natural resistance-associated macrophage protein, Nramp1, h as multiple pleiotropic effects on macrophage activation and regulates survival of intracellular pathogens including Leishmania, Salmonella and Mycobacterium species. Nramp1 acts as an iron transporter, but pre cisely how this relates to macrophage activation and/or pathogen survi val remains unclear. To gain insight into function, anti-Nramp1 monocl onal and polyclonal antibodies are used here to localise Nramp1 follow ing activation and infection. Confocal microscope analysis in uninfect ed macrophages demonstrates that both the mutant (infection-susceptibl e) and wild-type (infection-resistant) forms of the protein localise t o the membranes of intracellular vesicular compartments. Gold labellin g and electron microscopy defines these compartments more precisely as electron-lucent late endosomal and electron-dense lysosomal compartme nts, with Nramp1 colocalizing with Lamp1 and cathepsins D and L in bot h compartments with macrosialin in late endosomes, and with BSA-5 nm g old in pre-loaded Lysosomes. Nramp1 is upregulated with interferon-gam ma and lipopolysaccaride treatment, coinciding with an increase in lab elling in lysosomes relative to late endosomes and apparent dispersion of Nramp1-positive vesicles from a perinuclear location towards the p eriphery of the cytoplasm along the microtubular network, In both cont rol and activated macrophages, expression of the protein is 3- to 4-fo ld higher in wild-type compared to mutant macrophages. In Leishmania m ajor-infected macrophages, Nramp1 is observed in the membrane of the p athogen-containing phagosomes, which retain a perinuclear localization in resting macrophages. In Mycobacterium avium-infected resting and a ctivated macrophages, Nramp1-positive vesicles migrated to converge, b ut not always fuse, with pathogen-containing phagosomes. The Nramp1 pr otein is thus located where it can have a direct influence on phagosom e fusion and the microenvironment of the pathogen, as well as in the m ore general regulation of endosomal/lysosomal function in macrophages.