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Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 1125-1129
RED CELLS
Wild-type HFE protein normalizes transferrin iron accumulation in
macrophages from subjects with hereditary hemochromatosis
Giuliana Montosi,
Paola Paglia,
Cinzia Garuti,
Carlos A. Guzman,
Judy M. Bastin,
Mario P. Colombo, and
Antonello Pietrangelo
From the Unit for the Study of Disorders of Iron Metabolism,
Department of Internal Medicine, University of Modena and Reggio
Emilia, Modena; Immunotherapy and Gene Therapy Unit, Istituto Nazionale
Tumori, Milano, Italy; Division of Microbiology, GBF-National Research
Center for Biotechnology, Braunschweig, Germany; Institute of Molecular
Medicine, Oxford University, John Radcliffe Hospital, Oxford, United
Kingdom.
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Abstract |
Hereditary hemochromatosis (HC) is one of the most common
single-gene hereditary diseases. A phenotypic hallmark of HC is low
iron in reticuloendothelial cells in spite of body iron overload. Most
patients with HC have the same mutation, a change of cysteine at
position 282 to tyrosine (C282Y) in the HFE protein. The role of HFE in iron metabolism and the basis for the phenotypic
abnormalities of HC are not understood. To clarify the role of HFE in
the phenotypic expression of HC, we studied monocytes-macrophages from
subjects carrying the C282Y mutation in the HFE protein and
clinically expressing HC and transfected them with wild-type HFE by
using an attenuated Salmonella typhimurium strain as a gene
carrier. The Salmonella system allowed us to deliver genes of
interest specifically to monocytes-macrophages with high transduction
efficiency. The accumulation of 55Fe delivered by
55Fe-Tf was significantly lower in macrophages from
patients with HC than from controls expressing wild-type HFE.
Transfection of HC macrophages with the HFE gene resulted in a high
level of expression of HFE protein at the cell surface. The
accumulation of 55Fe delivered by 55Fe-Tf was
raised by 40% to 60%, and this was reflected by an increase in the
55Fe-ferritin pool within the HFE-transfected cells. These
results suggest that the iron-deficient phenotype of HC macrophages is a direct effect of the HFE mutation, and they demonstrate a role for
HFE in the accumulation of iron in these cells.
(Blood. 2000;96:1125-1129)
© 2000 by The American Society of Hematology.
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Introduction |
Hereditary hemochromatosis (HC) is one of the most
common single-gene hereditary diseases.1 A cornerstone of
HC genetics was laid in 1996, with the isolation of the hemochromatosis
gene, now called HFE.2 Most (83% to 100% in
different series) patients with HC carry the same mutation, resulting
in a change from cysteine at position 282 to tyrosine (C282Y)
in the HFE protein.2-4 The human HFE protein is closely
related to the family of major histocompatibility complex class I
molecules. The C282Y mutation disrupts a critical disulfide
bond in the  3 domain of the HFE protein and
abrogates binding of the mutant HFE protein to
 2-microglobulin (2M). This results in
reduced transport to and expression on the cell surface.5,6 Some light has been shed on the role of HFE in iron metabolism by the
observation that wild-type HFE makes a stable complex with transferrin
receptor (TfR)7 and that HFE, TfR, and transferrin (Tf)
form a ternary complex, with binding of HFE to TfR strictly dependent
on the pH.8 In spite of the growing data on the molecular interactions of TfR and HFE in transfected cell lines, little is known
about the biologic effect of this interaction and the phenotypic
consequences of a mutated HFE in HC.
In patients with HC9 and in murine models of HC (ie, HFE
and 2M knockout mice)10,11 iron
preferentially accumulates in parenchymal cells; little metal is stored
in reticuloendothelial (RE) cells. This indicates an abnormal
regulation of iron metabolism in RE cells in HC.9
Macrophages are the central portal of entry for Salmonella
infection.12 We took advantage of the fact that an
attenuated Salmonella typhimurium carrier strain harboring eukaryotic expression vectors can mediate gene transfer in
vivo.13,14 Differentiated, nondividing macrophages from
patients with HC carrying the C282Y mutation in HFE were transfected
with wild-type HFE. This allowed us to study the effect of a functional
HFE on specific parameters of iron metabolism in cells from patients with HC.
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Materials and methods |
Isolation and culture of monocytes
Patients with HC were characterized through full clinical evaluation
and HFE genotyping, as described.15 Monocytes from 8 patients (2 women, 6 men; mean age, 47 ± 10 years) with untreated HFE-associated HC (homozygous for the C282Y genotype) and
clinically expressing the disease were obtained by gradient
centrifugation of peripheral white blood cells as previously
described.9 For the iron incorporation experiments, cells
from HFE wild-type control subjects (3 women, 3 men; mean
age 40 ± 14 years) without abnormalities of iron metabolism, were
used. Yield, purity, viability, and recovery (usually between 95% and
98% as assessed by specific markers) of monocytes were as previously
reported.9 In some experiments contaminating lymphocytes
were depleted by complement-mediated lysis using a monoclonal antibody
to CD3 (clone HIT3a; PharMingen, San Diego, CA) and rabbit
complement (Cederlane, Hornby, Canada). To induce differentiation to
macrophages, monocytes (approximately 2.5 × 106
cells) were maintained in culture for 10 to 12 days in RPMI 1640 medium
containing 2 mmol/L glutamine, antibiotics, and 20% heat-inactivated human serum and kept in 5% CO2 at 37°C. The
medium and all reagents were endotoxin free.
Bacterial strains, plasmids, and media
The auxotrophic S typhimurium aroA strain
SL7207 (S typhimurium 2337-65 derivative hisG46,
DEL407-aroA::Tn10[Tc-s]) was kindly provided by B. A. D. Stocker (Stanford University School of Medicine, Stanford, CA).
Bacterial strains were routinely grown at 37°C in brain-heart
infusion broth or agar (BHI; Sigma, Milan, Italy), supplemented with 100 µg/mL ampicillin when required. The
eukaryotic pCMV-gal and the pCMV-GFP vectors that contains the
-galactosidase (gal) or the green fluorescent protein (GFP) gene,
respectively, under the control of the immediate early promoter of
cytomegalovirus (CMV) (Clontech, Palo Alto, CA),
were used to characterize gene transfer and transgene
expression. Full-length HFE cDNA was generated by
reverse transcription-polymerase chain reaction (RT-PCR) amplification of human lung RNA, and the PCR product was sequenced and
subcloned into the pCDNA3.1 expression vector (Invitrogen,
Groningen, The Netherlands) that contains a
c-myc epitope to produce an HFE/c-myc mRNA product.
In vitro assay for gene transfer and HFE expression
Human monocytes-macrophages were exposed in vitro to recombinant
Salmonella to test for transfection efficiency.
Salmonella infection was preferentially carried out after 10 to
12 days of in vitro culture. Medium was replaced with antibiotic free
medium containing recombinant Salmonella at a 10:1 to 50:1
ratio (bacteria/cell). Infection was carried out for 30 minutes at
37°C. Wells were then carefully washed and refilled with
gentamicin-containing fresh medium to kill any residual extracellular
Salmonella. Gene transfer was determined 24 to 48 hours after
infection by cytofluorometric analysis. HFE was detected by staining
with anti-HFE monoclonal antibody (HFE-JB1)16 followed by
biotinylated antimouse immunoglobulin and streptavidin-phycoerythrin
(PharMingen). Appropriate isotype-matched antibodies were used as
negative controls. Cells have also been stained with anti-CD14PE or
fluorescein isothiocyanate (PharMingen) and with anti-CD71PE for the
detection of TfR. Expression of gal, TfR, and HFE was determined on
CD14-positive gated cells using a FACScalibur Instrument (Becton
Dickinson, San Jose, CA) and cellQuest
software. Cells were processed for gal activity by X-gal staining,
as previously specified.13 HFE protein was also detected by
Western blot analysis. Briefly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed under denaturing conditions, followed by transfer to a polyvinylidene difluoride membrane (Amersham, Little Chalfont, UK). Immunostaining
was performed by incubation with a rabbit polyclonal antibody to the
3 domain of HFE. This antibody was made by immunizing a Sandy
half-lop rabbit with 0.3-mg peptide (KDKQPMDAKEFEPKD) glutaraldehyde
cross-linked to keyhole limpet hemocyanin. The serum was taken after 3 more immunizations of antigen, and the antiserum was purified using a
specific peptide-affinity column.17 Peroxidase-conjugated goat antirabbit antibody (Sigma) was then used and was followed by
chemiluminescence detection. Total RNA was extracted from in vitro
differentiated macrophages by cesium chloride gradient and subjected to
RT-PCR. First-strand DNA was generated from 1 µg RNA using Moloney
murine leukemia virus reverse transcriptase (Promega, Madison,
WI) for 1 hour at 42°C. The cDNA was amplified by PCR
with Taq DNA polymerase (Promega) using primers specific for
2m (direct, 5'-CTCGCGCTACTCTCTCTTTCTGG-3';
reverse, 5'-GCTTACATGTCTCGATCCCACTTAA-3'), HFE (direct,
5'-CTTGCTGCGTTCACACTCTCTG-3'; reverse,
5'-GGCTTGAAATTCTACTGGAAACCC-3'), gal (direct,
5'-CGTGACGTCTCGTTGCTGCAT-3'; reverse,
5'-CACCATCGTCTGCTCATCCATG-3'), and HFE/c-myc
(direct, 5'-GGGGAAGAGCAGAGATATACGTGC-3'; reverse, 5'-TGAGATGAGTTTTTGTTCGGGC-3'). The HFE/c-myc cDNA
encompasses the last HFE exon and the c-myc epitope. PCR was
carried out in a 30-µL volume (1 µmol/L primers, 1 U Taq DNA
polymerase, 2 mmol/L MgCl2, 25 µmol/L dNTPs) for 30 cycles (1 minute denaturation at 94 °C, 1 minute annealing at
60°C, 45-second extension at 72°C) using a thermal cycler
(PCT-200 Peltier Thermal Cycler; MJResearch). The PCR
products were then analyzed on an 8% to 10% polyacrylamide gel and
stained by ethidium bromide.
Transferrin-iron incorporation
Human (apo)Tf (Sigma) was passed over an S-200 (Pharmacia, Little
Chalfont, UK) column to remove any aggregated protein, and it showed a single peak on high-pressure liquid chromatography. Saturation of Tf with iron was performed as follows: a stock solution of 0.2 mmol/L 55FeCl3, > 5 mCi/mg Fe
(Amersham), and citric acid (molar ratio, 1:2) in Tris-HCl 100 mmol/L,
pH 8.8, was prepared and added to a solution of Tf dissolved in 10 µmol/L NaHCO3 (molar ratio Fe-Tf, 2:1) and incubated at
room temperature for 60 minutes. The 55Fe-Tf solution was
dialyzed against phosphate buffer, checked by nondenaturing PAGE, and
added to the culture medium at 0.5-µmol/L final Tf concentration.
Cells were kept for 24 hours after infection in fresh culture medium
before the addition of 55Fe-Tf. Cell-associated
radioactivity was measured at different incubation times by using
liquid scintillation counting (Beckman, Palo Alto, CA). In
addition to monitoring the incorporation of radioactive iron in the
macrophages, cell lysates were subjected to nondenaturing PAGE coupled
with autoradiography and densitometry. The main cellular radioactive
protein comigrated with control purified human ferritin (kindly
provided by P. Arosio, Milan, Italy). Relative quantities of ferritin
in cell lysates were normalized to cell protein content.
Statistical analysis
The values shown in Figure 4 are expressed as means ± SD. Significant differences between normal and HC macrophages
(Figure 4A) or HC macrophages transduced with the control vector or the HFE vector (Figure 4B) were evaluated by Student t test using the Stat View 4.0 program (Abacus Concept, Berkeley, CA).
| |
Results |
We first assessed the specificity of gene delivery in HC cells by
using an attenuated S typhimurium
aroA SL7207 strain harboring the
pCMV-gal vector that contains the gal gene under the control of
the immediate early promoter of CMV. After infection of peripheral
blood mononuclear cells, we found that gal expression was restricted
to macrophages and spared lymphocytes (Figure
1A). Figure 1B indicates that after
lymphocyte depletion, most of the remaining cells were infected and
expressed the transgene. Transduction efficiency after infection ranged from 85% to 95%, as assessed by cytofluorometric analysis.
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| Fig 1.
Gene delivery in HC monocytes-macrophages by
Salmonella carrier.
In vitro differentiated macrophages from patients with HC were infected
with Salmonella harboring the pCMV-gal plasmid. (A) gal
expression in peripheral blood mononuclear cells was restricted to
macrophages (large cells) and spared lymphocytes (small cells). (B) A
duplicate cell preparation of A after complement-lysis of lymphocytes
showing high gal expression in most cells.
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Once the DNA delivery system was optimized, we infected HC
macrophages with Salmonella carrying the pCMV-gal plasmid or
a human HFE cDNA under the control of the CMV promoter. At 24 hours after infection with the pCMV-HFE construct, we found a significant increase in HFE expression by RT-PCR that was maintained up to 48 hours, as with cells transduced with the pCMV-gal plasmid (Figure
2A-B). We also showed that the up-regulation of HFE expression correlated with the detection of RT-PCR product from the c-myc epitope linked to the HFE cDNA, proving transgene expression (Figure 2B). Up-regulation of HFE in transduced
cells was confirmed by Western blot analysis (Figure 2C) with a
polyclonal antibody to a peptide derived from the 3
domain.
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| Fig 2.
Salmonella mediates HFE gene transfer in HC
macrophages.
Expression of different genes in HC macrophages as detected by RT-PCR
at 24 hours (lanes 1 and 4), 30 hours (lanes 2 and 5), and 48 hours
(lanes 3 and 6) after Salmonella infection. Lanes 1 to 3, macrophages transfected with gal; lanes 4 to 6, macrophages
transfected with HFE; lane 7, noninfected HC macrophages. The same cDNA
preparations were divided and amplified in duplicate test tubes with
primers for either HFE and gal (A) or HFE and HFE/c-myc (B),
using 2M primers as an internal control for each
reaction. The presence of the transgene is demonstrated by the
detection of HFE/c-myc RNA. (C) Western blot of proteins from
infected cells. Proteins (30 µg) from cell homogenates were separated
by SDS-PAGE, blotted, and analyzed by Western blot using the anti-HFE
polyclonal antibody. A significant increase of HFE expression was
detected in cells infected with Salmonella carrying the
pCMV-HFE plasmid (lane 2) compared with those infected with the
pCMV-gal plasmid (lane 1).
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To demonstrate expression of the HFE protein on the cell surface after
HFE gene transduction in HC macrophages, cells were stained
with the HFE-specific monoclonal antibody HFE-JB116 and
were analyzed by FACS. As a control, Salmonella carrying a pCMV-gal plasmid was used to infect the same cell preparation. Results of a representative experiment are reported in Figure 3. HC macrophages were efficiently
transduced with pCMV-gal, and HFE gene transduction
increased HFE expression in HC macrophages. Interestingly,
Salmonella infection with HFE wild type led to a slight
reduction of the cell surface expression of TfR, as identified by
specific antibody to CD71.
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| Fig 3.
Cell surface expression of HFE in transfected
macrophages.
Human macrophages from a patient with HC were infected in vitro with
Salmonella strain SL7207 carrying either the pCMV-gal
(middle panels) or the pCMV-HFE (lower panels) plasmid. Results from a
representative experiment in which cells were analyzed for gene
expression by FACS 48 hours after infection using monoclonal antibodies
against major histocompatibility class I, HFE, CD71, and gal
followed by a PE-conjugated anti-murine IgG polyclonal
antibody. Appropriate isotype-matched monoclonal antibody controls have
been used for instrument settings (empty histograms). Detection of
gal expression was performed after saponin-mediated permeabilization
of monocytes. Percentage of positive cells and mean values of
fluorescence (in brackets) are reported for each single histogram.
Basal expression level for each marker in untreated HC-monocytes is
also shown (upper panel).
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In vitro differentiated HC macrophages showed a significantly
lower accumulation of iron than macrophages from normal
controls (Figure 4A). To evaluate the
effect of HFE reconstitution on iron metabolism in HC macrophages 24 hours after Salmonella infection, HC cells were incubated
for an additional 24 hours in the presence of 55Fe-Tf. The
HFE-transduced HC cells showed a 40% to 60% increase in the
accumulation of 55Fe delivered by
55Fe-Tf compared with HC cells transduced with
the control vector (Figure 4B). Reconstitution of HC macrophages with
wild-type HFE also led to expansion of the iron-ferritin pool,
which is inappropriately low in HC macrophages9
(Figure 4B, inset). Densitometric evaluation of radioactive ferritin
bands showed a 30% to 45% increase in 4 separate experiments after
normalization to protein content. No differences in cell
growth rate (and protein content) were detected in HFE-transduced cells
compared with cells transduced with the control plasmid.
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| Fig 4.
Transferrin-iron incorporation in HC macrophages
transfected with HFE.
(A) HC (solid bars) and normal (empty bars) macrophages were incubated
for 24 hours in the presence of 55Fe-Tf and cell-associated
radioactivity measured at the indicated time points. Data are mean ± SD from 6 separate experiments performed in triplicate.
*P < .001 versus HC macrophages. (B) HC
macrophages transduced with the pCMV-HFE (solid bars) or the
pCMV-gal (empty bars) plasmids were incubated for 24 hours in the
presence of 55Fe-Tf. Cell-associated radioactivity was
measured at the indicated incubation time points. Data are mean ± SD of experiments in macrophages from 3 subjects with HC (A, B, C)
performed in triplicate, representative of 8 separate experiments in HC
macrophages. *P < .001 versus HC macrophages
transduced with control vector. (Inset) Time-course of intracellular
55Fe-ferritin accumulation in HC macrophages transduced
with the pCMV-HFE or the pCMV-gal plasmid as evaluated by
nondenaturing PAGE of pooled cell lysates coupled with
55Fe-autoradiography. Ft, ferritin.
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Discussion |
Reticuloendothelial cells play a central role in iron metabolism.
They process more than 80% of the iron entering the plasma each day
and act as the main storage compartments for iron.18 Although erythrocyte phagocytosis accounts for most of the iron acquired by these cells in vivo, TfR-mediated uptake may also occur in
culture19 and in vivo.20 Iron metabolism in RE
cells is altered in HC. In patients and in murine models of HC, the metal accumulates preferentially in parenchymal cells, and little iron
is stored in RE cells despite the rise in total body
iron.10,11 Indeed, inappropriately high activity of the
iron regulatory protein, an intracellular sensor for iron, has been
documented in macrophages and in duodenal cells of patients with HC,
which suggests that these cells are actually iron
deficient.9,21 It is of interest that the macrophages of
the liver (Kupffer cells) and intestinal crypt cells are sites of
strong HFE expression in tissue-staining experiments with HFE-specific
antibodies (including JB-1, used in this study).16,22 The
mechanism that underlies this defect in iron storage by RE cells in HC
is unknown and led us to examine the effect of expressing wild-type HFE
in macrophages from patients with HC. We found that expression of HFE
enhanced iron and ferritin accumulation in these cells, detected after
exposure to 55Fe-Tf. In addition, we observed a small
reduction in surface expression of TfR consistent with iron
accumulation within the cells.
Recent work7,8,23,24 has shown that HFE can bind to TfR and
inhibit the binding of Fe-Tf. HFE might, therefore, be expected to
inhibit iron uptake by cells. Experiments involving the transfection of
HFE into HeLa cells in vitro23 certainly confirm that under
these conditions, the expression of HFE results in reduced uptake of
Fe-Tf and in a detectable reduction in cellular ferritin content. This
is the exact opposite of our results with HC macrophages, and it
suggests that HFE may serve a function in specialized RE cells
different from that detected in established cell lines in vitro.
The biochemical basis for the effect of HFE that we observed is as yet
unknown. We can speculate that it could be mediated by either enhanced
uptake of iron through the TfR cycle or by reduced egress of iron from
the cell. Our observation that an appreciable effect on iron
accumulation was seen only after a 24-hour exposure of macrophages to
55Fe-Tf would be more compatible with an inhibitory effect
of HFE on the egress of iron. This is also consistent with the enhanced release of iron observed in macrophages isolated from patients with
HC.25 It has been estimated that macrophages in vivo
deliver 10 million atoms of iron each minute to circulating
transferrin,18 but the mechanism for this movement of iron
out of the macrophage is unknown. Recently, a novel transporter of
Fe2+ ions, Ferroportin1, was shown to be expressed in
mature duodenal enterocytes, where it may mediate the transport of iron
across the basolateral membrane.26 Ferroportin1 was also
expressed in Kupffer cells (macrophages) in the liver,26 a
known site of iron storage and HFE expression,16 where it
may mediate iron egress. It is tempting to speculate that a function of
HFE or the HFE-TfR complex is to counterbalance (or to
directly inhibit?) the function of Ferroportin1 in RE macrophages and
thereby promote iron storage. The loss of HFE expression in HC would
result in an RE system incontinent of iron. In support of this
conclusion, spleens from HFE knock-out mice are relatively
resistant to dietary iron loading, possibly reflecting decreased
accumulation of Tf-bound iron by the HFE/
splenic macrophages.27 Because the iron
content of RE cells may have a role in controlling intestinal iron
absorption through a negative feedback mechanism exerted on
enterocytes,18,20 the chronically iron-deficient RE cells
in HC may determine or contribute to enhanced intestinal iron absorption.
| |
Acknowledgment |
We thank Prof Alain Townsend for reading the manuscript and offering
helpful comments.
| |
Footnotes |
Submitted January 5, 2000; accepted March 21, 2000.
Supported by grants E609 and A102 from Comitato Telethon
Fondazione Onlus and by a grant from Cofinanziamento MURST.
Reprints: Antonello Pietrangelo, Unit for the Study of
Disorders of Iron Metabolism, Department of Internal Medicine, Policlinico, Via del Pozzo 71, 41100 Modena, Italy; e-mail:
pietra{at}unimo.it.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Barton JC, Bertoli LF.
Haemochromatosis: the genetic disorder of the twenty-first century.
Nat Med.
1996;2:394-395[Medline]
[Order article via Infotrieve].
2.
Feder JN, Gnirke A, Thomas W, et al.
A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.
Nat Genet.
1996;13:399-408[Medline]
[Order article via Infotrieve].
3.
Jazwinska EC, Cullen L, Busfield F, et al.
Haemochromatosis and HLA-H.
Nat Genet.
1996;14:249-251[Medline]
[Order article via Infotrieve].
4.
Jouanolle AM, Gandon G, Jesequel P, et al.
Haemochromatosis and HLA-H.
Nat Genet.
1996;14:251-255[Medline]
[Order article via Infotrieve].
5.
Feder JN, Tsuchihashi Z, Irrinki A, et al.
The haemochromatosis founder mutation in HLA H disrupts beta(2) microglobulin interaction and cell surface expression.
J Biol Chem.
1997;272:14025-14028[Abstract/Free Full Text].
6.
Waheed A, Parkkila S, Zhou XY, et al.
Hereditary haemochromatosis: effects of C282Y and H63D mutations on association with beta(2)-microglobulin, intracellular processing, and cell surface expression of the HFE protein in COS-7 cells.
Proc Natl Acad Sci U S A.
1997;94:12384-12389[Abstract/Free Full Text].
7.
Feder JN, Penny DM, Irrinki A, et al.
The haemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding.
Proc Natl Acad Sci U S A.
1998;95:1472-1477[Abstract/Free Full Text].
8.
Lebron JA, Bennett MJ, Vaughn DE, et al.
Crystal structure of the haemochromatosis protein HFE and characterization of its interaction with transferrin receptor.
Cell.
1998;93:111-123[Medline]
[Order article via Infotrieve].
9.
Cairo G, Recalcati S, Montosi G, Castrusini E, Conte D, Pietrangelo A.
Inappropriately high iron regulatory protein activity in monocytes of patients with genetic hemochromatosis.
Blood.
1997;89:2546-2553[Abstract/Free Full Text].
10.
Zhou XY, Tomatsu S, Fleming RE, et al.
HFE gene knockout produces mouse model of hereditary hemochromatosis.
Proc Natl Acad Sci U S A.
1998;95:2492-2497[Abstract/Free Full Text].
11.
Rothenberg BE, Voland JR.
Beta2 knockout mice develop parenchymal iron overload: a putative role for class I genes of the major histocompatibility complex in iron metabolism.
Proc Natl Acad Sci U S A.
1996;93:1529-1534[Abstract/Free Full Text].
12.
Jones BD, Falkow S.
Salmonellosis: host immune responses and bacterial virulence determinants.
Annu Rev Immunol.
1996;14:533-561[Medline]
[Order article via Infotrieve].
13.
Darji A, Guzman CA, Gerstel B, et al.
Oral somatic transgene vaccination using attenuated S typhimurium.
Cell.
1997;91:765-775[Medline]
[Order article via Infotrieve].
14.
Paglia P, Medina E, Arioli I, Guzman CA, Colombo MP.
Gene transfer in dendritic cells, induced by oral DNA vaccination with Salmonella typhimurium, results in protective immunity against a murine fibrosarcoma.
Blood.
1998;92:3172-3176[Abstract/Free Full Text].
15.
Pietrangelo A, Montosi G, Totaro A, et al.
Hereditary hemochromatosis in adults without pathogenic mutations in the hemochromatosis gene.
N Engl J Med.
1999;341:725-732[Abstract/Free Full Text].
16.
Bastin JM, Jones M, O'Callaghan CA, Schimanski L, Mason DY, Townsend AR.
Kupffer cell staining by an HFE-specific monoclonal antibody: implications for hereditary haemochromatosis.
Br J Haematol.
1998;103:931-941[Medline]
[Order article via Infotrieve].
17.
Kleijmeer MJ, Kelly A, Geuze HJ, Slot J, Townsend A, Trowsdale J.
Location of MHCencoded transporters in the endoplasmic reticulum and cis-Golgi.
Nature.
1992;357:342-344[Medline]
[Order article via Infotrieve].
18.
Finch CA, Huebers HA.
Iron metabolism.
Clin Physiol Biochem.
1986;4:5-10[Medline]
[Order article via Infotrieve].
19.
Testa U, Pelosi E, Peschle C.
The transferrin receptor.
Crit Rev Oncog.
1993;4:241-276[Medline]
[Order article via Infotrieve].
20.
Bjorn-Rasmussen E, Hageman J, Van den Dungen P, Prowit-Ksiazek A, Biberfeld P.
Transferrin receptors on circulating monocytes in hereditary haemochromatosis.
Scand J Haematol.
1985;34:308-311[Medline]
[Order article via Infotrieve].
21.
Pietrangelo A, Casalgrandi G, Quaglino D, et al.
Duodenal ferritin synthesis in genetic hemochromatosis.
Gastroenterology.
1995;108:208-217[Medline]
[Order article via Infotrieve].
22.
Waheed A, Parkkila S, Saarnio J, et al.
Association of HFE protein with transferrin receptor in crypt enterocytes of human duodenum.
Proc Natl Acad Sci U S A.
1999;96:1579-1584[Abstract/Free Full Text].
23.
Gross CN, Irrinki A, Feder JN, Enns CA.
Co-trafficking of HFE, a nonclassical major histocompatibility complex class I protein, with the transferrin receptor implies a role in intracellular iron regulation.
J Biol Chem.
1998;273:22068-22074[Abstract/Free Full Text].
24.
Salter-Cid L, Brunmark A, Li Y, et al.
Transferrin receptor is negatively modulated by the hemochromatosis protein HFE: implications for cellular iron homeostasis.
Proc Natl Acad Sci U S A.
1999;96:5434-5439[Abstract/Free Full Text].
25.
Moura E, Noordermeer MA, Verhoeven N, Verheul AF, Marx JJ.
Iron release from human monocytes after erythrophagocytosis in vitro: an investigation in normal subjects and hereditary hemochromatosis patients.
Blood.
1998;92:2511-2519[Abstract/Free Full Text].
26.
Donovan A, Brownlie A, Zhou Y, et al.
Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter.
Nature.
2000;403:776-781[Medline]
[Order article via Infotrieve].
27.
Levy JE, Montross LK, Cohen DE, Fleming MD, Andrews NC.
The C282Y mutation causing hereditary hemochromatosis does not produce a null allele.
Blood.
1999;94:9-11[Abstract/Free Full Text].
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S. Xiong, H. She, A.-S. Zhang, J. Wang, H. Mkrtchyan, A. Dynnyk, V. R. Gordeuk, S. W. French, C. A. Enns, and H. Tsukamoto
Hepatic macrophage iron aggravates experimental alcoholic steatohepatitis
Am J Physiol Gastrointest Liver Physiol,
September 1, 2008;
295(3):
G512 - G521.
[Abstract]
[Full Text]
[PDF]
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P. A. Swanson II, C. D. Pack, A. Hadley, C.-R. Wang, I. Stroynowski, P. E. Jensen, and A. E. Lukacher
An MHC class Ib-restricted CD8 T cell response confers antiviral immunity
J. Exp. Med.,
July 7, 2008;
205(7):
1647 - 1657.
[Abstract]
[Full Text]
[PDF]
|
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O. Olakanmi, L. S. Schlesinger, and B. E. Britigan
Hereditary hemochromatosis results in decreased iron acquisition and growth by Mycobacterium tuberculosis within human macrophages
J. Leukoc. Biol.,
January 1, 2007;
81(1):
195 - 204.
[Abstract]
[Full Text]
[PDF]
|
|
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K. Dassler, M. Zydek, K. Wandzik, M. Kaup, and H. Fuchs
Release of the Soluble Transferrin Receptor Is Directly Regulated by Binding of Its Ligand Ferritransferrin
J. Biol. Chem.,
February 10, 2006;
281(6):
3297 - 3304.
[Abstract]
[Full Text]
[PDF]
|
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H. Makui, R. J. Soares, W. Jiang, M. Constante, and M. M. Santos
Contribution of Hfe expression in macrophages to the regulation of hepatic hepcidin levels and iron loading
Blood,
September 15, 2005;
106(6):
2189 - 2195.
[Abstract]
[Full Text]
[PDF]
|
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H. Carlson, A.-S. Zhang, W. H. Fleming, and C. A. Enns
The hereditary hemochromatosis protein, HFE, lowers intracellular iron levels independently of transferrin receptor 1 in TRVb cells
Blood,
March 15, 2005;
105(6):
2564 - 2570.
[Abstract]
[Full Text]
[PDF]
|
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P. S. Davies and C. A. Enns
Expression of the Hereditary Hemochromatosis Protein HFE Increases Ferritin Levels by Inhibiting Iron Export in HT29 Cells
J. Biol. Chem.,
June 11, 2004;
279(24):
25085 - 25092.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
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A. M. Giannetti and P. J. Bjorkman
HFE and Transferrin Directly Compete for Transferrin Receptor in Solution and at the Cell Surface
J. Biol. Chem.,
June 11, 2004;
279(24):
25866 - 25875.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
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A. Pietrangelo
Hereditary Hemochromatosis -- A New Look at an Old Disease
N. Engl. J. Med.,
June 3, 2004;
350(23):
2383 - 2397.
[Full Text]
[PDF]
|
|
|
|
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A.-S. Zhang, S. Xiong, H. Tsukamoto, and C. A. Enns
Localization of iron metabolism-related mRNAs in rat liver indicate that HFE is expressed predominantly in hepatocytes
Blood,
February 15, 2004;
103(4):
1509 - 1514.
[Abstract]
[Full Text]
[PDF]
|
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|
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A.-S. Zhang, P. S. Davies, H. L. Carlson, and C. A. Enns
Mechanisms of HFE-induced regulation of iron homeostasis: Insights from the W81A HFE mutation
PNAS,
August 5, 2003;
100(16):
9500 - 9505.
[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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H. Drakesmith, E. Sweetland, L. Schimanski, J. Edwards, D. Cowley, M. Ashraf, J. Bastin, and A. R. M. Townsend
The hemochromatosis protein HFE inhibits iron export from macrophages
PNAS,
November 26, 2002;
99(24):
15602 - 15607.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Burke, S. Sumner, N. Maitland, and C. E. Lewis
Macrophages in gene therapy: cellular delivery vehicles and in vivo targets
J. Leukoc. Biol.,
September 1, 2002;
72(3):
417 - 428.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D Trinder, C Fox, G Vautier, and J K Olynyk
Molecular pathogenesis of iron overload
Gut,
August 1, 2002;
51(2):
290 - 295.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
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D. Trinder, J. K. Olynyk, W. S. Sly, and E. H. Morgan
Iron uptake from plasma transferrin by the duodenum is impaired in the Hfe knockout mouse
PNAS,
April 8, 2002;
(2002)
82112299.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
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|
A. Rolfs, H. L. Bonkovsky, J. G. Kohlroser, K. McNeal, A. Sharma, U. V. Berger, and M. A. Hediger
Intestinal expression of genes involved in iron absorption in humans
Am J Physiol Gastrointest Liver Physiol,
April 1, 2002;
282(4):
G598 - G607.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
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A. Pietrangelo
Physiology of iron transport and the hemochromatosis gene
Am J Physiol Gastrointest Liver Physiol,
March 1, 2002;
282(3):
G403 - G414.
[Abstract]
[Full Text]
[PDF]
|
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|
|
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A. Waheed, J. H. Grubb, X. Y. Zhou, S. Tomatsu, R. E. Fleming, M. E. Costaldi, R. S. Britton, B. R. Bacon, and W. S. Sly
Regulation of transferrin-mediated iron uptake by HFE, the protein defective in hereditary hemochromatosis
PNAS,
February 20, 2002;
(2002)
42701499.
[Abstract]
[Full Text]
[PDF]
|
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|
|
|
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|
S. Fargion, L. Valenti, P. Dongiovanni, A. Scaccabarozzi, A. L. Fracanzani, E. Taioli, M. Mattioli, M. Sampietro, and G. Fiorelli
Tumor necrosis factor {alpha} promoter polymorphisms influence the phenotypic expression of hereditary hemochromatosis
Blood,
June 15, 2001;
97(12):
3707 - 3712.
[Abstract]
[Full Text]
[PDF]
|
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|
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A. Waheed, J. H. Grubb, X. Y. Zhou, S. Tomatsu, R. E. Fleming, M. E. Costaldi, R. S. Britton, B. R. Bacon, and W. S. Sly
Regulation of transferrin-mediated iron uptake by HFE, the protein defective in hereditary hemochromatosis
PNAS,
March 5, 2002;
99(5):
3117 - 3122.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
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D. Trinder, J. K. Olynyk, W. S. Sly, and E. H. Morgan
Iron uptake from plasma transferrin by the duodenum is impaired in the Hfe knockout mouse
PNAS,
April 16, 2002;
99(8):
5622 - 5626.
[Abstract]
[Full Text]
[PDF]
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Top
Abstract
Introduction