Departments of Microbiology and Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, Ohio
Correspondence: Bruce S. Zwilling, Department of Microbiology, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210. E-mail: zwilling.1{at}osu.edu
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,
IFN-
, IL-1, 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 Nramp1Gly169-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.
Key Words: Fe-citrate • PKC • Nramp1Asp169 • cytokines
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several observations support the possibility that Nramp1 is a divalent cation transporter. First, Nramp1 has several structural similarities to families of ion channels and transporters, including a highly conserved ion transport motif [12 , 13 ]. Nramp1 homologues in species ranging from bacteria to mammals have been shown to transport divalent metal cations [14 ]. For example, the yeast homologs of Nramp1, SFM1, and SFM2, transport manganese, cadmium, copper, and cobalt into the cell [15 ]. A second member of the Nramp family in mammals, Nramp2 (DMT-1), is located almost exclusively in recycling endosomes and in the plasma membrane where it functions to transport iron and other divalent cations [16 17 18 ]. A missense mutation in Nramp2 from glycine to asparagine at amino acid 165 results in microcytic anemia in the mouse [17 ]. Nramp2 is expressed in a variety of different cell types and is thought to be responsible for intestinal iron absorption and transport of iron from transferring-containing endosomes into the cytoplasm [19 ]. Taken together, these results indicate that the physiological function of Nramp1 is to transport iron. Previous work by us has shown that Nramp1 transports iron into phagosomes where it can serve as a catalyst for the Haber-Weiss reaction, resulting in the production of highly reactive hydroxyl radicals [20 ].
The purpose of this investigation was to characterize the transport of iron mediated by Nramp1. We found that an acidic environment was required to transport iron into the phagosome. Treatment of an Nramp1Gly169-transfected macrophage cell line with macrophage-activating cytokines increased the amount of iron transported into the phagosomes. In contrast, the treatment of macrophages transfected with the susceptible Nramp1Asp169 allele was without effect. Western blot analysis indicated that the increase in iron was not the result of an increase of Nramp1 protein in the phagosomes. The transport of iron into phagosomes, as well as the increase following cytokine treatment, did not require new protein synthesis but was dependent on PKC.
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(TNF-), interferon- (IFN-), and transforming growth factor-ß
(TGF-ß) were obtained from Gibco BRL (Gaithersburg, MD). IL-1 and
granulocyte-macrophage colony-stimulating factor (GM-CSF) were
purchased from R & D Systems (Minneapolis, MN). The protein kinase
inhibitors bis-indolylmaleimide I and Gö6976 were from Calbiochem
(San Diego, CA).
Macrophage cultures
The RAW264.7 mouse macrophage cell lines [21
],
stably transfected with Nramp1Gly169 (7.5R) or
Nramp1Asp169 (10.S), were generously provided by
Jenefer Blackwell (University of Cambridge, UK). These cell lines were
created by cloning Nramp1 cDNAs into the pBabe vector, which contains a
puromycin-resistance marker under the control of a SV40 early promoter,
and introduced by electroporation into the RAW264.7 macrophage cell
line. These cell lines express Nramp1 under the control of a viral long
terminal repeat (LTR) and respond to a 24 h exposure to
IFN-/lipopolysaccharide (LPS) with an enhanced respiratory burst,
major histocompatibility complext (MHC) II expression, and nitrite
release [21
]. The cells were maintained in Iscove’s
modified Dulbecco’s minimum essential medium (IMDM, Gibco-BRL),
supplemented with 10% fetal bovine serum (FBS; <3 ng/mL LPS; Hyclone,
Logan, UT) and penicillin-streptomycin at 37°C in 5%
CO2. The cells were tested periodically for plasmid
retention by growth in puromycin and were not used for longer than 3
months in culture.
Isolation of M. avium-containing phagosomes
M. avium-containing phagosomes were prepared by
incubating macrophages (80–90% confluent) with a 10:1 ratio of
bacteria:cells for 1 h at 37°C in 5% CO2 in
IMDM. The cells were washed twice with IMDM and incubated an additional
1 h. Phagosomes were isolated as previously described by us using
a procedure based on that described by Sturgill-Koszycki et
al. [22
]. The infected macrophages were scraped
from the plates and collected by centrifugation at 800 g for
10 min. Combined cell pellets (1.2–1.4x108 cells) were
resuspended in 600 µl extraction buffer containing 20 mM Hepes (pH
7.2), 250 mM sucrose, 0.5 mM EGTA, 0.1% gelatin, and protease
inhibitors. The cells were lysed by repeatedly passing through a
21 g needle until >95% lysis was achieved. The resulting
homogenate was diluted 3:1 with phosphate-buffered saline (PBS),
aliquots were removed for radioactivity and protein determinations, and
the remainder was centrifuged for 5 min at 150 g to remove
unbroken cells and large debris. The supernatant was filtered through a
5 µm Nucleopore filter (Corning, Acton, MA). The filter was rinsed
with 1.5 mL PBS, and the rinse was added to the original filtrate. The
solution was layered on top of a discontinuos sucrose gradient,
consisting of 3 mL of a 12% sucrose (in Hepes buffer, pH 7.0) on top
of a 1 mL cushion of 50% sucrose (in Hepes buffer, pH 7.0), and
centrifuged at 800 g for 45 min at 4°C. The phagosomes
were recovered from the 12–50% sucrose interface, diluted threefold
with PBS, and placed on top of a 2 mL cushion of 12% Ficoll (in Hepes
buffer, pH 7.0). The solution was centrifuged at 1400 g for
45 min at 4°C, and the phagosomes were collected from the bottom of
the tube. Latex bead phagosomes were isolated as described previously
[20
]. The resulting phagosomes were dissolved in 2%
sodium dodecyl sulfate (SDS), and aliquots were taken for radioactivity
and protein measurements.
Iron import by M. avium-containing phagosomes
The import of Fe by mycobacterial-containing phagosomes from
macrophages was measured as previously described by us
[20
]. Macrophages were plated at 40–50% confluency in
150 mm tissue culture plates in IMDM (with penicillin and streptomycin)
containing 5 µM 55Fe-citrate and incubated for 18–22 h
at 37°C in 5% CO2. Iron was chelated to
citrate in 20 mM Hepes/Tris (pH 6.0), 100 mM HCl, 5 mM sodium citrate,
and 50µM [55Fe] ferric chloride (NEN, Boston, MA;
specific activity 
17 mCi/mg). This solution was neutralized by the
addition of IMDM and then diluted to the appropriate concentration for
use. Following this incubation, the cells were washed twice with IMDM
mycobacteria added, and the phagosomes were isolated as described
above.
In vitro Fe import by phagosomes from unlabeled-resistant macrophage cells was performed essentially as described earlier [20 ]. Phagosomes were isolated and resuspended in IMDM, aliquots containing 50–100 µg protein in 50 µl were added to 50 µl 55Fe-citrate (10 µM) substrate solution, and the reaction solution was incubated at 4°C or 37°C for 15 min. Reactions were terminated by the addition of 500 µl ice-cold Fe-citrate. Phagosomes were washed free of unincorporated 55Fe-citrate by filtration through Fe-citrate-saturated 0.2 µm Supor-200 filters (Gelman Sciences, Ann Arbor, MI). The filters were washed twice with 5 ml cold Fe-citrate, allowed to dry, and then counted by liquid scintillation. In some experiments, phagosomes were incubated at room temperature for 30 min with an anti-Nramp1 antibody prior to the addition of the substrate solution.
Western blot analysis M. avium phagosomes
Phagosomes from M. avium-infected, -resistant, and
-susceptible macrophages were analyzed by Western blotting using
antibodies against the phagosome proteins Nramp1, Lamp1, and Rab7 and
against the "early" phagosomal protein Rab5a. Aliquots containing
10 µg protein were mixed with sample buffer (Tris, SDS-PAGE
containing ß-mercaptoethanol; Novex, San Diego, CA), heated at 37°C
for 5 min, and then separated on 10–20% gradient Tris-Tricine
acrylamide gels using a Tris-Tricine/SDS running buffer (Novex). The
separated proteins were transferred to Hybond nitrocellulose membranes
using a semi-dry transfer-blotter apparatus (BioRad, Hercules, CA) at
15 V for 1 h. The blots were processed according to the enhanced
chemiluminescence (ECL) Western blotting protocol supplied by the
manufacturer (Amersham, Arlington Heights, IL). Bands were visualized
using ECL hyperfilm. The anti-mouse Nramp1 antibodies were raised in
rabbits against a glutathione-S-transferase-Nramp1 fusion protein
(containing Nramp1 amino acids 305–346 for the "loop" or amino
acids 514–548 for the C-terminus antibody), according to standard
protocols. The Nramp1 loop antibody was further purified using a
dihydropyridine (DHP)-Nramp1/loop4 fusion protein-affinity column.
LAMP1 protein was detected using a purified rat anti-mouse, CD107a
monoclonal antibody from Pharmingen (San Diego, CA). Rab5a and Rab7
proteins were detected using rabbit or goat anti-mouse polyclonal
antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish
peroxidase-conjugated secondary antibodies—sheep anti-rat and sheep
anti-rabbit—were obtained from Amersham and used at a 1:500 dilution
of stock. All commercial primary antibodies were used at a 1:250
dilution of stock. The anti-mouse Nramp1 antibodies were used at a
1:2000 dilution of stock. Protein molecular-weight standards were
obtained from Gibco-BRL.
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 View larger version (28K): [in a new window] |
Figure 1. The effects of chloroquine and ammonium chloride (NH4Cl) on
Fe import by phagosomes isolated from the
Nramp1Gly169 macrophage cell line. The cells
were incubated for 18–20 h in radiolabeled 55Fe-citrate to
label intracellular pools. The cells were washed, and then 30 µM
chloroquine or 15 mM ammonium chloride was added for 30 min prior to
M. avium exposure. Phagosomes were isolated from these
cells, and the amount of protein and radiolabeled 55Fe was
measured. The values represent the mean ± SE from
three independent measurements. The effects of chloroquine and
NH4Cl are significant (p<0.005), as determined
by Student’s t-test and are indicated by asterisks.
|
.
IFN- resulted in a 35% increase in phagosomal Fe import, and
IL-1 and GM-CSF increased Fe import by 20–25%. In contrast,
neither IL-10, PGE2, nor TGF-ß affected Fe import into
the phagosome.
 View larger version (32K): [in a new window] |
Figure 2. The effects of different cytokines on Fe import by phagosomes isolated
from the Nramp1Gly169 macrophage cell line. The
cells were grown for 18–20 h in radiolabeled 55Fe-citrate,
washed several times, and then incubated for 3 h at 37°C in IMDM
containing one of the listed cytokines. The cells were washed, M.
avium was added, and the phagosomes were isolated as described.
TNF-, IL-1, and TGF-ß were used at 10 ng/ml; GM-CSF, at 20
ng/ml; IL-10, at 1 ng/ml; PGE2, at 25 ng/ml; and IFN-,
at 100 units/106 cells (500 U/ml). The cytokine levels
represent optimal values obtained from dose and time experiments for
the individual cytokines. The results are normalized to phagosomal
protein and are expressed as the % increase in phagosomal Fe import
over untreated control cells. The results represent the mean ±
SE of five separate determinations for TNF- and IFN-
and three separate determinations for the other cytokines. The
significance levels were determined by Student’s t-test.
The effects of TNF-, IFN-, IL-1, and GM-CSF are significant
when compared with untreated cells, as indicated by asterisks
(p<0.001*, p<0.005**, and
p<0.01***).
|
(Fig. 3A)
or
IFN- (Fig. 3B)
increased the phagosomal import of Fe by macrophages
transfected with Nramp1Gly169 but not by
macrophages transfected with the susceptible
Nramp1Asp169 allele. The import of Fe into the
phagosomes following treatment with TNF- (Fig. 4A
) or IFN- (unpublished results) increased linearly and reached
a maximum at about 3 h. The dose of IFN- required for maximum
Fe uptake was 25 units/106 cells or 150 U/ml (Fig. 4B)
, and
that for TNF- was 10 ng/ml (unpublished results). Treatment of the
macrophages with cyclohexamide did not affect Fe import nor did it
affect the stimulation of Fe import by IFN- (Fig. 5
). The increase in phagosomal Fe import following treatment of the
macrophages with cytokines did not require the synthesis of new
protein.
 View larger version (19K): [in a new window] |
Figure 3. The effects of TNF- (A) and IFN- (B) on Fe import by phagosomes
isolated from M. avium-treated
Nramp1Gly169 and
Nramp1Asp169 macrophage cell lines. The cells
were incubated in 55Fe-citrate for 18–20 h and then washed
to remove the remaining radiolabeled Fe in the media. The cytokines
were added to cells in IMDM containing penicillin and streptomycin for
3 h before M. avium infection. Phagosomes were
isolated, dissolved in 2% SDS, and then analyzed for protein and
radiolabeled 55Fe content. R169 refers to
results obtained from the Nramp1Gly169 cell
line, and S169 refers to the results from the
Nramp1Asp169 cell line. The values shown
represent the mean ± SE of four separate experiments.
The significance levels were determined by Student’s
t-test. The effects of TNF- and IFN- on Fe import by
cells transfected with Nramp1Gly169 are
significant, as indicated by asterisks (p<0.005**). Iron
imported by Nramp1Asp169-transfected cells is
significantly less than that imported into phagosomes isolated from
Nramp1Gly169-transfected cells. There is no
difference in Fe import between
Nramp1Asp169-transfected cells treated with
either cytokine.
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 View larger version (26K): [in a new window] |
Figure 4. Cytokine stimulation of phagosomal Fe import by
Nramp1Gly169-transfected macrophages infected
with M. avium. (A) TNF- (10 ng/ml) was added to
55Fe-citrate-labeled cells for the indicated periods of
time prior to M. avium addition. (B) IFN-, at the
concentrations shown, was added to labeled cells 3 h prior to
adding M. avium. Phagosomes were isolated, and their content
of protein and radiolabeled 55Fe was determined. All values
represent the mean ± SE of three different
experiments. Significance levels were determined by analysis of
variance (ANOVA). The effect of time and concentration of cytokine was
significant at p < 0.01.
|
 View larger version (34K): [in a new window] |
Figure 5. The effect of cyclohexamide on IFN- stimulation of Fe import by
phagosomes from the Nramp1Gly169 macrophage cell
line. The cells were labeled with 55Fe, washed several
times, and then 4 µg/ml cyclohexamide [4
] was added
for 4 h. IFN- was then added to the cells for 3 h prior to
M. avium infection. Isolation and analysis of phagosomes
were performed as described previously. The treatments are listed at
the bottom left; (-) refers to no treatment, and (+) refers to the
addition of the particular treatment. The values represent the
mean ± SE of three independent measurements.
Significance levels were determined by Student’s t-test.
The effect of IFN- was significant, as indicated by asterisks
(p<0.01***). The effect of cyclohexamide was not
significant.
|
for 3 h prior to M.
avium infection did not increase the amount of Nramp1 protein,
although under the same condition, phagosomal Fe transport is
increased.
 View larger version (56K): [in a new window] |
Figure 6. Western blot analysis of phagosomes isolated from M.
avium-treated Nramp1Gly169 and
Nramp1Asp169 macrophage cell lines. Cells were
labeled overnight with 55Fe-citrate, washed, M.
avium was added, and phagosomes were isolated as described. This
figure shows the results using Lamp1, Rab5a, and Nramp1 antibodies.
Lanes 1 and 2 show the results obtained when blots were analyzed using
an anti-Lamp1 antibody; lanes 3 and 4, using an anti-Rab5a antibody;
lanes 5, 6, and 9–12, using an anti-Nramp1 loop antibody; and lanes 7
and 8, using an anti-Nramp1, C-terminus antibody. Lanes 9 and 10 are
the results of Nramp1Gly169 cells untreated or
treated with IFN- for 30 min prior to M. avium infection,
respectively, and lanes 11 and 12, of
Nramp1Asp169 cells with or without IFN-. The
molecular weight of each protein was determined by comparison with
protein standard markers. Lamp1 had an apparent molecular weight of 112
kD; Rab5a, 31 kD; and Nramp1, approximately 90 kD. The results shown
are representative of at least three independent experiments.
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 View larger version (23K): [in a new window] |
Figure 7. The effects of anti-Nramp1 antibodies on Fe import by phagosomes from
M. avium-infected Nramp1Gly169
macrophages. M. avium phagosomes were isolated from
resistant cells as described and resuspended in IMDM. Aliquots were
removed and incubated with 10 µg of the loop or C-terminus Nramp1
antibody at room temperature for 30 min before measuring Fe import.
Nonspecific background values were measured at 4°C and subtracted
from the import values obtained at 37°C. The % inhibition was
determined as the value from nontreated control cells over the value
from antibody-treated cells x 100. Phagosomes were isolated by
filtration, and the radioactivity was quantitated by liquid
scintillation. Results shown are representative of three separate
experiments. The difference in means between treated and untreated
cells was significant at p < 0.001*, as determined by
the Student’s t-test.
|
and IFN--activated macrophages. Fe import by
the TNF--treated cells was reduced by 64%, and import by the
IFN--treated cells was 54%.
 View larger version (45K): [in a new window] |
Figure 8. The effect of PKC inhibitors on phagosomal Fe import by M.
avium or latex bead-treated macrophages of the
Nramp1Gly169 cell line. Cells were labeled
overnight with 55Fe-citrate, washed, and then incubated
with PKC inhibitors bis-indolylmaleimide I (bis-indol) or Gö6976
for 30 min prior to M. avium or latex-bead addition (A) or
cytokines TNF- or IFN- for 3 h, followed by the PKC
inhibitor bis-indolylmaleimide I for 30 min before M. avium
(B). Phagosomes were isolated by the standard protocol, and aliquots
were taken for protein and radioactivity measurements. (A) The first
three bars in each box show the results obtained from
Nramp1Gly169 macrophages, and the second group
of bars shows the results from Nramp1Asp169
macrophages. The box on the left shows the results from M.
avium-treated cells and the box on the right, from latex-bead
treated cells. The (-) and (+) correspond to the absence or presence,
respectively, of either inhibitor. The results are the mean ±
SE of three separate determinations of each. Significance
levels were determined by Student’s t-tests. The effect of
bis-indolylmaleimide I and Gö6976 on Fe import by
Nramp1Gly169-transfected cells is significant
for M. avium-infected cultures and those fed latex beads, as
indicated by asterisks (p<0.005**, and
p<0.01***). (B) The effect of the PKC inhibitor
bis-indolylmaleimide I on the TNF- stimulation of phagosomal Fe
import is shown on the left, and the effects of IFN- are shown on
the right. The (-) and (+) refer to the absence or presence,
respectively, of either additive. The results shown are the mean ± SE from three independent determinations. The effect of
each cytokine is significant as is the effect of bis-indolylmaleimide,
as indicated by asterisks (p<0.001*, p<0.005**,
and p<0.01***).
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We also found that stimulation of macrophages by activating cytokines
resulted in an increase in the transport of iron into phagosomes of
macrophages expressing the resistant
Nramp1Gly169 allele. The increase in import
because of cytokine stimulation and from M. avium infection
could be the result of an increased synthesis of Nramp1, increased
fusion of Nramp1-containing vesicles, or an increase in
Nramp1-transport activity. We have ruled out the first two of these
possibilities by showing that inhibition of protein synthesis did not
affect the increase in Fe import resulting from the maturation of the
phagosome following the ingestion of M. avium or the
increase associated with cytokine treatment. The increase in Fe import
does not appear to be the result of an increased delivery of Nramp1 to
the phagosome because Western blot analysis did not detect increasing
levels of Nramp1 protein. The increase in Fe import into phagosomes
appears to be associated with a modulation of Nramp1 function. Nramp1
contains several sequence motifs that could be used to modulate
activity, including the putative SH3 binding domain and a number of
potential PKC and tyrosine phosphorylation sites. Thus, infection by
M. avium may result in cell activation, perhaps via
Toll-like receptors, the release of TNF-, activation of Nramp1, and
increased anti-mycobacterial activity.
Iron transport mediated by Nramp1 requires PKC activity. Nramp1
contains five potential PKC phosphorylation sites. The inhibition of
PKC activity resulted in a suppression of Fe transport by Nramp1 from
macrophages that had taken up M. avium or latex beads or
that had been treated with cytokines prior to infection. The PKC
inhibitors that we used, bis-indolylmaleimide I and Gö6976, have
an overlapping spectrum of activity, indicating that only
PKC or PKCß1 may be involved
in modulating Nramp1 activity. We do not yet know if the cytokine
treatment increased phagosomal Fe import because of increased
phosphorylation of Nramp1. However, we and others have demonstrated
that macrophages from Nramp1-resistant mice or from macrophages
transfected with Nramp1Gly169 have increased PKC
activity following treatment of the cells with IFN- or infection
with M. bovis [25
, 26
].
Also, it has been shown that Nramp1 becomes phosphorylated in response
to IFN- treatment [6
]. The possibility that Nramp1
may be directly phosphorylated by PKC is currently being explored.
We found that Nramp1 protein was detected in macrophages transfected with the Nramp1Gly169-resistant and Nramp1Asp169-susceptible alleles. This is consistent with the results of Atkinson and Barton [7 ] who also showed that Nramp1 could be detected in Western blots of macrophage proteins from resistant or susceptible mice. In contrast, Vidal et al. [6 ] showed that Nramp1 protein could not be detected in macrophages from susceptible mice. Our results that Nramp1 in macrophages has a molecular weight of 90–100 kD is consistent with others [6 , 7 ] but differs in that we did not observe minor bands of 45 and/or 65 kD. However, others have shown 45 kD and 60 kD Nramp1 proteins using whole-cell extracts. Thus, the presence of only the high molecular weight form of Nramp1 in our study is consistent with a mature form of the protein expected to be found in mature phagosomes.
An antibody directed against the loop peptide blocked phagosomal iron transport, and an antibody against the C-terminus did not. Taken together, these results indicate that Nramp1 is orientated in the phagosomal membrane with the glycosylated, "outer" loop side facing into the cytoplasm and the amino and carboxy termini facing into the phagosome. This is opposite of the orientation expected for expression of Nramp2 in the plasma membrane and recycling endosomes. Because of the opposite orientation, Nramp2 is able to transport iron out of the recycling endosome into the cytoplasm, and Nramp1 transports iron from the cytoplasm into the phagolysosome. Thus, the direction of iron import relative to the orientation of Nramp1 in the phagosomal membrane is the same as Nramp2 in outer cell and endosomal membranes.
The transport of iron by host cells is a tightly regulated process. Free iron, in its mobile Fe2+ form, is highly toxic because of its capacity to generate toxic reactive oxygen intermediates. During infection, the host and invading pathogen attempts to sequester iron; the pathogen needs iron for growth. The host has evolved an iron-withholding defense mechanism that limits the availability of iron to the pathogen. Thus, intestinal absorption of iron decreases by 80%. This suggests that the function of Nramp2, which transports iron from the intestinal lumen into the circulation, is somehow affected as a result of infection. Plasma iron therefore decreases by 70%, iron saturation of transferrin decreases to less than 50%, and the synthesis and expression of transferrin receptors by macrophages are reduced [27 ]. Thus, the host finds itself with an iron-deficient environment and at the same time requires iron to serve as an important catalyst for several anti-microbial pathways, including the synthesis of inducible nitric oxide synthase (iNOS) and the generation of toxic hydroxyl radicals via the Haber-Weiss reaction. Our data suggest that Nramp1, by transporting iron into phagosomes, serves to supply sufficient quantities of biologically active iron that results in the limitation of microbial growth.
Received August 26, 2000; revised September 22, 2000; accepted September 25, 2000.
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P. Courville, R. Chaloupka, F. Veyrier, and M. F. M. Cellier Determination of Transmembrane Topology of the Escherichia coli Natural Resistance-associated Macrophage Protein (Nramp) Ortholog J. Biol. Chem., January 30, 2004; 279(5): 3318 - 3326. [Abstract] [Full Text] [PDF]
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J. R. Forbes and P. Gros Iron, manganese, and cobalt transport by Nramp1 (Slc11a1) and Nramp2 (Slc11a2) expressed at the plasma membrane Blood, September 1, 2003; 102(5): 1884 - 1892. [Abstract] [Full Text] [PDF]
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G. Fritsche, M. Dlaska, H. Barton, I. Theurl, K. Garimorth, and G. Weiss Nramp1 Functionality Increases Inducible Nitric Oxide Synthase Transcription Via Stimulation of IFN Regulatory Factor 1 Expression J. Immunol., August 15, 2003; 171(4): 1994 - 1998. [Abstract] [Full Text] [PDF]
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O. Olakanmi, L. S. Schlesinger, A. Ahmed, and B. E. Britigan Intraphagosomal Mycobacterium tuberculosis Acquires Iron from Both Extracellular Transferrin and Intracellular Iron Pools. IMPACT OF INTERFERON-gamma AND HEMOCHROMATOSIS J. Biol. Chem., December 13, 2002; 277(51): 49727 - 49734. [Abstract] [Full Text] [PDF]
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S. Wyllie, P. Seu, F. Q. Gao, P. Gros, and J. A. Goss Disruption of the Nramp1 (also known as Slc11a1) gene in Kupffer cells attenuates early-phase, warm ischemia-reperfusion injury in the mouse liver J. Leukoc. Biol., November 1, 2002; 72(5): 885 - 897. [Abstract] [Full Text] [PDF]
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N. Boechat, B. Lagier-Roger, S. Petit, Y. Bordat, J. Rauzier, A. J. Hance, B. Gicquel, and J.-M. Reyrat Disruption of the Gene Homologous to Mammalian Nramp1 in Mycobacterium tuberculosis Does Not Affect Virulence in Mice Infect. Immun., August 1, 2002; 70(8): 4124 - 4131. [Abstract] [Full Text] [PDF]
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F. Canonne-Hergaux, J. Calafat, E. Richer, M. Cellier, S. Grinstein, N. Borregaard, and P. Gros Expression and subcellular localization of NRAMP1 in human neutrophil granules Blood, June 17, 2002; 100(1): 268 - 275. [Abstract] [Full Text] [PDF]
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S. L. Wardrop, C. Wells, T. Ravasi, D. A. Hume, and D. R. Richardson Induction of Nramp2 in activated mouse macrophages is dissociated from regulation of the Nramp1, classical inflammatory genes, and genes involved in iron metabolism J. Leukoc. Biol., January 1, 2002; 71(1): 99 - 106. [Abstract] [Full Text] [PDF]
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T. Ravasi, C. Wells, A. Forest, D. M. Underhill, B. J. Wainwright, A. Aderem, S. Grimmond, and D. A. Hume Generation of Diversity in the Innate Immune System: Macrophage Heterogeneity Arises from Gene-Autonomous Transcriptional Probability of Individual Inducible Genes J. Immunol., January 1, 2002; 168(1): 44 - 50. [Abstract] [Full Text] [PDF]
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W. Zhong, W. P. Lafuse, and B. S. Zwilling Infection with Mycobacterium avium Differentially Regulates the Expression of Iron Transport Protein mRNA in Murine Peritoneal Macrophages Infect. Immun., November 1, 2001; 69(11): 6618 - 6624. [Abstract] [Full Text] [PDF]
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