Nramp1 (natural resistance-associated macrophage protein one) regulates intracellular pathogen proliferation and macrophage inflammatory responses. Murine Nramp1 exhibits a natural polymorphism with alleles termed resistant and susceptible. Alleles restrict or allow the proliferation of intracellular pathogens, respectively. Structural predictions suggest that Nramp1 encodes the prototypic member of a transporter family. Nramp1 exhibits sequence identity to Nramp2, which regulates intestinal and reticulocyte iron uptake. Based on this sequence identity we have initiated experiments for Nramp1 to investigate its role in macrophage iron homoeostasis and using a transfection approach in the RAW264.7 murine macrophagelike cell line, which lacks a functional Nramp1 gene. Nramp1 expression supports increased acute cytoplasmic influx of iron, detected using the fluorescent iron sensor dye calcein. Analysis of the endogenous iron sensors, iron regulatory protein 1 and 2, reveals a greater flux of iron in Nramp1-expressing cells and in its exclusion from the cytoplasm. Other work supports the prediction that Nramp1 is a phosphoprotein and the extent of phosphorylation changes in response to inflammatory cytokines. Together these data support the hypothesis that control of intracellular iron homoeostasis is a vital element used by phagocytes to control the proliferation of intracellular pathogens.

Nramp1 controls responses to infection and encodes a biallelic (G169D) macrophage‐restricted divalent‐cation transporter. Nramp1D169 is phenotypically null.We demonstrate Nramp1 is implicated in iron regulation in vivo. In spleen, expression is exclusive to Nramp1G169 strains within the red pulp. By morphometric analysis, the distribution of splenic iron, following systemic overload, correlates with Nramp1 genotype. More iron is located within the red pulp in Nramp1D169 strains, whereas in Nramp1G169 strains iron deposits are localized within the marginal‐zone metallophilic cells. Nramp1 immunoreactive protein is not present in control brain, but inducible within a hemorrhagic lesion model in Nramp1G169 strains. Nramp1 protein expression demonstrates an inverse correlation to the presence of iron. Nramp1G169 strains show no Perl's stain‐reactive iron within the lesion. In contrast, Nramp1D169 strains display iron‐staining cells. The process of cellular iron regulation was investigated in vitro in Nramp1G169 transfectant Raw264.7 macrophages. Greater (30–50%) iron efflux from Nramp1G169 compared with Nramp1D169 cells was determined. The extent of Nramp1‐dependent iron‐release was influenced by bafilomycin A1, and endogenous nitric oxide synthesis, both inhibitors of vacuolar‐ATPase. This study demonstrates that Nramp1 regulates macrophage iron handling, and probably facilitates iron release from macrophages undergoing erythrophagocytosis in vivo.

Iron is essential for growth, and impaired iron homoeostasis through a non-conserved mutation within murine Nramp1, also termed Slc11a1, contributes to susceptibility to infection. Nramp1 depletes the macrophage cytosol of iron, with effects on iron-regulated gene expression and iron-dependent processes. Wu and colleagues (Wu, K.-J., Polack, A., and Dalla-Favera, R. A biallelic phenotype in mouse, termed Ity/Lsh/Bcg, was identified in response to infection by obligate intracellular macrophage pathogens (1-3). Nramp1 1 (also termed Slc11a1) was isolated as the positional gene candidate for Ity/Lsh/Bcg (4). In subsequent studies the full-length sequence of the encoded polypeptide was identified (5). Mouse strains resistant to infection encode Gly at codon 169 within Nramp1 whereas susceptible mice encode Asp (6). The G169D polymorphism is sufficient to explain the outcomes of model infections within inbred mouse strains at pre-T cell stages of infection, as confirmed by gene targeting and transgenesis experiments (7,8).Allele D169 is phenotypically null (7). When Nramp1 was cloned, the biochemical basis for its control over the proliferation of intracellular pathogens was not obvious, but the sequence suggested a transporter function (4). Subsequent studies showed the encoded polytopic integral membrane Nramp1 protein was expressed in a perinuclear location, on intracellular late endosomal/lysosomal membranes (9 -11). Nramp1 underwent rapid recruitment to the periphery of a pathogencontaining vesicle (10, 11) and displayed a more peripheral location in response to treatment with interferon-␥ (11). These observations led to the suggestion that growth control of microbial pathogens could be achieved by the transport of some toxin into the lumen of the phagosome or by the sequestration of some essential nutrient. The identity of a candidate transport substrate was revealed from studies on a highly sequencerelated gene, Nramp2 (DMT1/DCT1/SLC11A2). Nramp2 was initially isolated as an orphan gene (12) but was re-isolated by functional cloning through a divalent cation/iron uptake assay (13). In addition, identical mutations, G185R, within Nramp2 in the mk mouse and the Belgrade rat are associated with impaired intestinal and erythroid cell iron uptake (14). Based on the striking sequence similarity between the two polypeptides (15), Nramp1 was also predicted to transport divalent cations or iron within murine macrophages, and infection susceptibility occurs through impaired divalent cation transport. The many pleiotropic effects attributed to Nramp1 (16) should be explained by differential cation transport. Because Nramp1 protein is expressed within internal membranes, the differential partitioning of divalent cations between the cytosol and the lumen of the internal vesicle should contribute to the pleiotropic effects described (16). Inducible nitric-oxide synthase (iNos) is expressed at quantitatively higher levels in functional-Nramp1 macrophages (17)(18)(19)(20). Ferrous iron provides the link between iNos expressio...

Nramp1 (natural resistance-associated macrophage protein) controls resistance to infection by intracellular pathogens in mice. Nramp1 regulates the microenvironment of the invading pathogen by increasing the luminal iron that participates in the Haber-Weiss reaction, producing radicals that attack the pathogen. We have studied the effect of inflammatory stimuli, iron, and sodium nitroprusside on Nramp1 expression in bone marrow macrophages. Investigations show all three up-regulate Nramp1 expression with a parallel increase in immunoreactivity to an amino-terminal antibody and Nramp1 mRNA. Growth rates are reduced in macrophage cell lines expressing Nramp1. This is through a decrease in iron availability, shown by an increase in IRP2 activity and a reciprocal decrease in conventional protein kinase Cbeta-1 expression. We propose that Nramp1 activity may control its own expression via a negative autoregulatory loop that is important for iron homeostasis and maintenance of low cytoplasmic redox active iron levels in the macrophage.

The Nramp1 (natural resistance-associated macrophage protein 1) gene modulates the growth of intracellular pathogens and encodes a divalent cation transporter within lysosomes/late endosomes of macrophages. Nramp1 modulates the cytoplasmic iron pool. Wu, Polack and Dalla-Favera [(1999) Science 283, 676-679] showed reciprocal control of H-ferritin and IRP2 by c-Myc, and suggest that c-Myc regulates genes to increase cytoplasmic iron. A role for c-Myc in Nramp1 regulation was evaluated. Co-transfection studies show that c-Myc represses Nramp1 promoter function. Five non-canonical Myc-max binding sites (E-box) identified within the Nramp1 5'-flanking sequence are not responsible for the inhibitory effects of c-Myc on Nramp1 expression. An initiator(s) adjacent to the transcription-initiation site is a candidate for the inhibition observed. Results are consistent with a role for Nramp1 removing iron from the cytosol and antagonizing c-Myc function.