c-Myc represses the murine Nramp1 promoter.

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.

Nramp1 modulates iron homoeostasis in vivo and in vitro: evidence for a role in cellular iron release involving de-acidification of intracellular vesicles.

Nramp1 controls responses to infection and encodes a biallelic (G169D) macrophage-restricted divalent-cation transporter. Nramp1(D169) is phenotypically null. We demonstrate Nramp1 is implicated in iron regulation in vivo. In spleen, expression is exclusive to Nramp1(G169) 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 Nramp1(D169) strains, whereas in Nramp1(G169) 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 Nramp1(G169) strains. Nramp1 protein expression demonstrates an inverse correlation to the presence of iron. Nramp1(G169) strains show no Perl's stain-reactive iron within the lesion. In contrast, Nramp1(D169) strains display iron-staining cells. The process of cellular iron regulation was investigated in vitro in Nramp1(G169) transfectant Raw264.7 macrophages. Greater (30-50%) iron efflux from Nramp1(G169) compared with Nramp1(D169) 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.

A negative autoregulatory link between Nramp1 function and expression.

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.

Nramp1: a link between intracellular iron transport and innate resistance to intracellular pathogens.

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 macrophage-like 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.

A macrophage hippocampal slice co-culture system: application to the study of HIV-induced brain damage.

We have developed an in vitro system that allows the study of the effects of factors released from macrophages on neuronal and glial survival in cultured hippocampal slices. Organotypic hippocampal slice cultures are grown on semi-permeable membranes in stationary co-culture with a murine macrophage cell line (RAW 264.7). The two culture systems are separated by a semi-permeable membrane specifically allowing the study of diffusable factors between the two culture systems. The use of the fluorescent exclusion dye propidium iodide as an in vitro marker of cell viability allows the study of progressive toxicity as it evolves in the slice cultures. We demonstrate that the HIV-1 derived nuclear regulatory protein Tat induces toxicity in slice cultures via the production of soluble mediators. The advantages of organotypic cultures over other in vitro systems is discussed as well as the general applicability of this method to the study of other brain pathologies, where macrophage derived factors are thought to play a role in neuronal survival.

A soluble factor produced by macrophages mediates the neurotoxic effects of HIV-1 Tat in vitro.

OBJECTIVES: There is now a strong consensus that the neurotoxic properties of HIV-1 are likely to be mediated by an indirect mechanism in which neurones are damaged by infected mononuclear cells. The aim of this study was to determine the ability of HIV-1 Tat to induce neurotoxic properties in a murine macrophage cell line RAW264.7. DESIGN: Simple culture systems using dissociated neurones may not provide the appropriate microenvironment in which to observe the complex cell-cell interactions that occur in the brain. We have therefore developed a more physiological model in which rat organotypic hippocampal slices are co-cultured with the murine macrophage cell line RAW264.7. Effects of Tat were studied by using a stable Tat expressing RAW264.7 cell line or by addition of recombinant Tat protein to co-cultures. METHODS: Organotypic hippocampal slices prepared from 8-10 day rat pups were grown on membrane inserts that were placed into six-well plates on which RAW264.7 cells were growing as an adherent monolayer. Cell death in the slices was assessed using propidium iodide. Specific astrocytic (glial fibrillary acidophilic protein; GFAP) and neuronal (microtubule-associated protein; MAP2) markers were visualized by immunocytochemistry. RESULTS: RAW264.7 cells that either expressed or were exposed to HIV-1 Tat protein, produced a soluble factor that caused profound degeneration in brain slice cultures involving loss of both glial cells and neurones. By contrast treatment of slice cultures with Tat in the absence of RAW264.7 cells was not neurotoxic. CONCLUSIONS: The neurotoxic properties previously attributed to HIV-1 Tat are likely to be mediated via induction of macrophage derived soluble factor(s).

Induction of activator protein 1 (AP-1) in macrophages by human immunodeficiency virus type-1 NEF is a cell-type-specific response that requires both hck and MAPK signaling events.

Human immunodeficiency virus type 1 (HIV-1) Nef is important for viral infectivity and pathogenicity. HIV-1 infection is associated with inappropriate activation and defects in the function of monocytes/macrophages. We have studied the effects of HIV-1 Nef in the murine (RAW264.7) and human (THP-1) monocyte-macrophage cell lines. Investigation of the activator protein-1 (AP-1) transcription factor showed that Nef expression induced both its DNA binding and transcriptional activities. Increased AP-1 DNA binding activity in RAW264.7 cells was associated with raised levels of c-Fos expression and induction of mRNA for the AP-1 responsive tissue inhibitor of metalloproteinases-1 (TIMP-1) gene. Mutagenesis and kinase inhibition studies were employed to determine signaling pathways used by Nef to induce AP-1. Data from these studies indicated that induction of AP-1 by Nef is likely to be mediated through the MAPK (ERK1 and 2) signaling pathway and requires the proline-rich PxxP motif of Nef, suggesting the involvement of upstream protein kinases belonging to the Src family. Effects of Nef on AP-1 induction were cell lineage-specific, being stimulatory in macrophages, inhibitory in T cells and without effect in HeLa cells. These latter two observations led us to test the possibility that cell-specific interactions of Nef with Src family proteins may modulate AP-1 activity. To this end we demonstrated that a dominant-negative Hck mutant caused inhibition of Nef-mediated AP-1 DNA binding activity in RAW cells. In conclusion, induction of AP-1 by Nef is a specific feature of human and murine macrophage cell lines that requires signal transduction events involving Hck and MAPKs.

Regulated expression vectors demonstrate cell-type-specific sensitivity to human immunodeficiency virus type 1 Nef-induced cytostasis.

The nef gene product of both human and simian immunodeficiency viruses is critically important for virus replication and disease progression in vivo. However, the precise biological function of Nef remains poorly characterized in vitro, with previous reports suggesting that Nef might be either cytotoxic or cytostatic. As a result of difficulties encountered by several groups in establishing cell lines constitutively expressing Nef, we have developed two inducible systems resulting in stable Nef expression in various mammalian cell lines. Tetracycline-regulated Nef expression was achieved in HeLa cells but could not be established in human T cell lines. Jurkat E6-1 T cell and RAW264.7 murine macrophage cell lines expressing a regulated nef gene were generated using a system in which Nef expression was controlled by a mutated version of the heavy metal-inducible human metallothionein IIA promoter. Induction of high levels of Nef expression in HeLa-Nef and Jurkat-Nef cells resulted in a moderate (2-fold) and a dramatic (10-fold) retardation of cell growth respectively, supporting the contention that Nef may be a cytotoxic or cytostatic factor. This property was also observed at low basal levels of Nef expression in RAW264.7-Nef macrophage clones (5-fold reduction in growth) and was associated with an altered morphological phenotype suggesting that different cell types may be more susceptible to the cytostatic activity of Nef. The regulated Nef-expression systems provide tools for investigating the molecular basis of Nef function, including Nef-mediated cytopathogenicity, CD4 down-regulation and enhancement of virus infectivity.

The human immunodeficiency virus type 1 regulatory protein Tat inhibits interferon-induced iNos activity in a murine macrophage cell line.

Human immunodeficiency virus type 1 (HIV-1) infection is frequently associated with concurrent infection by opportunistic pathogens, against which production of nitric oxide by host macrophages provides a first line of defence. We have investigated whether regulatory HIV-1 proteins, such as Tat, can modulate the activity of the inducible nitric oxide synthase (iNos) gene when expressed in stable transfectant lines of RAW264.7 cells. A bioassay for Tat, based on transactivation of an HIV-1 LTR-CAT reporter gene, allowed selection of Tat-expressing cells. Parental and Tat-expressing macrophages accumulated identical levels of nitrite following lipopolysaccharide (LPS) stimulation. Interferon gamma (IFN-gamma) stimulation however, resulted in reduced levels of nitrite accumulation as a direct consequence of Tat expression. Conditioned media from Tat-expressing cells reduced the level of nitrite accumulation in parental cells following IFN-gamma stimulation but not stimulation with LPS. These results implicate HIV-1 Tat as a modulator of the IFN-gamma-specific signal transduction pathways leading to iNos expression.