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Iron transport into mycobacterium avium-containing phagosomes from an Nramp1(Gly169)-transfected RAW264.7 macrophage
D E Kuhn1, W P Lafuse, B S Zwilling
1Department of Microbiology and Molecular Virology, The Ohio State University, Columbus 43210, USA.
Abstract:
Nramp1 is an important determinant of innate resistance of macrophages to the growth of intracellular microorganisms. We previously showed that Nramp1 functions to transport iron from the cytoplasm into phagosomes of Mycobacterium avium-infected macrophages. The purpose of this investigation was to further characterize the factors that regulate Nramp1-mediated iron transport into phagosomes. Treatment of Nramp1(Gly169) macrophages with the lysomotrophic agents chloroquine or ammonium chloride reduced the import of iron significantly. We found that macrophage-activating cytokines, including TNF-alpha, IFN-gamma, IL-1alpha, and GM-CSF, when added prior to M. avium, increased the transport of iron into the phagosome. This increase in iron transport was not a result of an increased amount of Nramp1 protein in the phagosome nor to new protein synthesis. Treatment of Nramp1(Gly169)-transfected macrophages with inhibitors of protein kinase C (PKC) diminished the import of iron into the phagosomes. Iron import was inhibited by an anti-Nramp1 antibody against the putative fourth outer-loop region of Nramp1 but not by an anti-Nramp1 antibody against the carboxy terminus. The significance of these results on the orientation of Nramp1 in the phagosome membrane and on the transport of iron is discussed.
Insights
Host resistance to intracellular microbes relies on Nramp1 iron transport into phagosomes. Macrophage activation enhances this iron transport, regulated by factors like pH and protein kinase C, crucial for innate immunity.
Area of Science:
- Immunology
- Cell Biology
- Microbiology
Background:
- The Nramp1 protein is crucial for innate resistance against intracellular microorganisms.
- Nramp1 facilitates iron transport from macrophage cytoplasm into phagosomes during Mycobacterium avium infection.
- Understanding Nramp1 regulation is key to elucidating macrophage antimicrobial mechanisms.
Purpose of the Study:
- To investigate factors regulating Nramp1-mediated iron transport into phagosomes.
- To characterize the influence of lysosomotropic agents and cytokines on Nramp1 activity.
- To explore the role of protein kinase C and Nramp1 structure in iron transport.
Main Methods:
- Macrophages expressing Nramp1 (Nramp1(Gly169)) were treated with lysosomotropic agents (chloroquine, ammonium chloride).
- Macrophage-activating cytokines (TNF-alpha, IFN-gamma, IL-1alpha, GM-CSF) were added prior to M. avium infection.
- Nramp1-transfected macrophages were treated with protein kinase C inhibitors and specific anti-Nramp1 antibodies.
Main Results:
- Lysosomotropic agents significantly reduced iron import into phagosomes.
- Macrophage-activating cytokines increased Nramp1-mediated iron transport without altering Nramp1 protein levels or synthesis.
- Inhibitors of protein kinase C diminished iron import, and antibody binding suggested Nramp1's fourth outer-loop is critical for iron transport.
Conclusions:
- Phagosomal pH and protein kinase C activity are key regulators of Nramp1 iron transport.
- Cytokine-induced enhancement of Nramp1 function is independent of Nramp1 protein levels.
- Nramp1's orientation, particularly its fourth outer-loop, is vital for its role in iron transport and innate immunity.
