Iron transport into Mycobacterium avium-containing phagosomes from an Nramp1(Gly169)-transfected RAW264.7 macrophage cell line

Nramp1 is an important determinant of innate resistance of macrophages to t he growth of intracellular microorganisms. We previously showed that Nramp1 functions to transport iron from the cytoplasm into phagosomes of Mycobact erium avium-infected macrophages. The purpose of this investigation was to further characterize the factors that regulate Nramp1-mediated iron transpo rt into phagosomes. Treatment of Nramp1(Gly169) macrophages with the lysomo trophic agents chloroquine or ammonium chloride reduced the import of iron significantly, We found that macrophage-activating cytokines, including TNF -alpha, IFN-gamma, IL-1 alpha, aid GM-CSF, when added prior to RI. avium, i ncreased the transport of iron into the phagosome. This increase in iron tr ansport was not a result of all increased amount Of Nramp1 protein in the p hagosome nor to new protein synthesis. Treatment of Nramp1(Gly169)-transfec ted macrophages with inhibitors of protein kinase C (PBC) diminished the im port of iron into the phagosomes. Iron import was inhibited by an anti-Nram p1 antibody against the putative fourth outer-loop region of Nramp1 I,nt no t by an anti-Nramp1 antibody against the carboxy terminus. The significance of these results on the orientation of Nramp1 ill the phagosome membrane a nd on the transport of iron is discussed.