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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2383-2389
Regulation of Iron Metabolism in Murine J774 Macrophages: Role of
Nitric Oxide-Dependent and -Independent Pathways Following Activation
With Gamma Interferon and Lipopolysaccharide
By
Victoriano Mulero and
Jeremy H. Brock
From the Department of Immunology and Bacteriology, Western
Infirmary, University of Glasgow, Glasgow, UK; and the Department of
Cell Biology, Faculty of Biology, University of Murcia, Murcia, Spain.
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ABSTRACT |
To elucidate the pathways by which nitric oxide (NO) influences
macrophage iron metabolism, the uptake, release, and intracellular distribution of iron in the murine macrophage cell line J774 has been
investigated, together with transferrin receptor (TfR) expression and
iron-regulatory protein (IRP1 and IRP2) activity. Stimulation of
macrophages with interferon- (IFN-) and/or lipopolysaccharide (LPS) decreased Fe uptake from transferrin (Tf), and there was a
concomitant downregulation of TfR expression. These effects were
mediated by NO-dependent and NO-independent mechanisms. Addition of the
NO synthase (NOS) inhibitor N-monomethyl arginine (NMMA) partially restored Fe uptake but either had no effect on or
downregulated TfR expression, which suggests that NO by itself is able
to affect iron availability. Analysis of the intracellular distribution of incorporated iron revealed that in IFN-/LPS-activated macrophages there was a decreased amount and proportion of ferritin-bound iron and
a compensatory increase in insoluble iron, which probably consists
mainly of iron bound to intracellular organelles. Finally, although NO
released by IFN-/LPS-activated macrophages increased the
iron-responsive element (IRE)-binding activity of both IRP1 and IRP2,
IFN- treatment decreased IRP2 activity in an NO-independent manner.
This study demonstrates that the effect of IFN- and/or LPS on
macrophage iron metabolism is complex, and is not entirely due to
either NO-or to IRP-mediated mechanisms. The overall effect is to
decrease iron uptake, but not its utilization.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
IRON METABOLISM and macrophage physiology
are closely connected. Macrophages, through processing of
hemoglobin-iron from senescent erythrocytes, are responsible for iron
supply to peripheral tissues, including the bone marrow.1
Moreover, changes in macrophage iron content can affect the function of
these cells in the inflammatory response. For example, iron plays a
critical role in macrophage-mediated cytotoxicity by contributing to
the production of highly toxic hydroxyl radicals via the Fenton
reaction2 and by controlling the production of nitric oxide
(NO) after activation by immunologic stimuli.3
Intracellular iron homeostasis is controlled by cytoplasmic iron
regulatory proteins (IRP1 and IRP2), which regulate several mRNAs
containing iron-responsive elements (IREs) in their untranslated regions.4 IRP binding to the IREs in the 5'
untranslated regions of ferritin (Ft) and erythroid-specific
5-aminolaevulinate synthase mRNAs represses their
translation,5 whereas binding of IRP to multiple IREs in
the 3' untranslated region of transferrin receptor (TfR) mRNA
confers stability against targeted endonucleolytic degradation.6
IRP1 is a bifunctional protein that can act either as a cytoplasmic
aconitase or as an IRE-binding protein.7 In iron-replete cells, IRP1 bears a 4Fe-S cluster and shows aconitase activity, but in
iron-depleted cells reversible disassembly of the cluster converts IRP1
to its RNA-binding form. IRP2, despite conservation of the
cluster-ligating cysteines at the active site, is unable to assemble an
Fe-S cluster in vitro and therefore is unable to exhibit aconitase
activity.8 Unlike the regulation of IRP1 by iron, loss of
IRE binding of IRP2 in iron-replete cells is due to iron-dependent
oxidation, ubiquitinylation, and degradation by the
proteasome.9
In addition to iron availability, other signals such as NO and
oxidative stress (ie, H2O2 and peroxynitrite)
can modulate the activity of both IRPs and thus influence cellular iron
metabolism.10-12 Stimulation of macrophages with IFN-
and lipopolysaccharide (LPS) induces NO synthesis and was originally
reported to activate IRE-binding by IRP1 and IRP2.10,13
However, other investigators have recently reported that stimulation of
macrophages with IFN- and LPS results in an increase of IRP1
activity, but a strong reduction of IRP2 activity.12,14 A
further point of controversy is that while Bouton et al14
reported that the decrease of IRP2 activity is not due to NO, Recalcati
et al12 showed that inhibition of IRP2 activity was
NO-dependent.
Although the effect of NO on IRP activity has been well studied, no
data regarding the effect of NO on iron uptake, release, and
distribution after macrophage stimulation with cytokines are available.
Such information is important if the role of inflammatory stimuli and
NO in macrophage iron metabolism is to be understood. In the present
study, we examined cellular iron uptake, release, and distribution, as
well as TfR expression and IRP1/2 activities in stimulated murine J774
macrophages to better clarify the regulation of iron metabolism in
activated macrophages. We found that treatment of these cells with
IFN- and LPS strongly inhibits iron uptake from transferrin despite
the fact that IRP1 is upregulated, and that both NO-dependent and
-independent mechanisms are involved.
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MATERIALS AND METHODS |
Reagents.
RPMI 1640 culture medium and fetal calf serum (FCS) were purchased from
GIBCO (Paisley, UK). Human apotransferrin (apoTf) was obtained from
Sigma (Poole, UK) and when required saturated with
59Fe using Fe-nitrilotriacetate (FeNTA; Fe:NTA molar ratio,
1:4)15 prepared from Na-NTA and
59FeCl3 (Amersham, Amersham, UK).
Human serum albumin (HSA; Tf-free), NMMA, LPS, leupeptin,
phenylmethylsulfonyl fluoride (PMSF), benzamidine, and pepstatin A were
all obtained from Sigma; desferrioxamine (DFO) was from Novartis
(Horsham, UK); ( -32P) uridine triphosphate (UTP) was
from Amersham; and RNAsin ribonuclease inhibitor, XbaI
restriction enzyme, and T7 RNA polymerase were from Promega
(Southampton, UK). R-phycoerythrin (R-PE)-conjugated rat
IgG1, anti-mouse CD71 (TfR) monoclonal antibody and
R-PE-conjugated rat IgG1, monoclonal immunoglobulin
isotype standard were obtained from Pharmingen (Oxford, UK). Rabbit
anti-mouse ferritin was produced in our laboratory.15 Mouse
recombinant IFN- was kindly provided by Dr E. Esfandiari, Department
of Immunology, University of Glasgow.
Cell cultures and treatments.
The mouse macrophage cell line J774 was kindly supplied by Dr G.-J.
Feng (Department of Immunology, University of Glasgow) and grown in
RPMI-1640 supplemented with 10% heat-inactivated FCS, 100 U/mL
penicillin, and 100 µg/mL streptomycin at 37°C in 5%
CO2. To avoid problems of variability in levels of iron and transferrin caused by the presence of serum, all of the treatments were
performed in serum-free RPMI 1640 containing 1 mg/mL HSA and
50 µg/mL human apoTf unless otherwise indicated. Cells were incubated
overnight with additives as required, then washed twice with ice-cold
phosphate-buffered saline (PBS) and subjected to further procedures as
indicated below. To ensure that equal numbers of cells were used,
cultures were always seeded with 106 viable cells per
milliliter, monitored daily by microscopic observation to check for
uniform cell number and viability, and experiments performed when the
cultures had reached near-confluence.
Iron uptake assay.
After overnight incubation with appropriate additives, cells were
pulsed for 8 hours with 50 µg/mL 59Fe-Tf to allow
incorporation of 59Fe, then washed and lysed with 2%
sodium dodecyl sulfate (SDS). The radioactivity in cell lysates and
culture supernatants was determined and 59Fe uptake calculated.
Iron release assay.
After overnight stimulation, cells were pulsed for 4 hours with 50 µg/mL 59Fe-Tf, washed twice with prewarmed PBS, and then
incubated for 2 hours in the presence of "cold" Fe-Tf.
Thereafter, the cells were washed, lysed in 2% SDS and the
radioactivity present in cell lysates and supernatants determined.
Alternatively, cells were exposed to 59Fe-Tf for 24 hours,
washed, and then incubated overnight with cold Fe-Tf in the presence of
the different treatments. After stimulation the radioactivity was
determined as above.
Intracellular distribution of iron.
Cells were treated as described for iron uptake assays, then lysed in
100 µL cytoplasmic lysis buffer (1% Triton X-100, 40 mmol/L KCl, 25 mmol/L Tris-HCl pH 7.4, 50 µg/mL leupeptin, 200 µg/mL PMSF, 1 mmol/L benzamidine, and 50 µg/mL pepstatin A) and the intracellular
iron distribution determined as described by Alvarez-Hernández et
al16 with slight modifications. Briefly, this consisted of
centrifugation at 10,000g for 5 minutes and immunoprecipitation
of ferritin with a rabbit anti-ferritin antibody. This allows
separation of intracellular iron into 3 compartments containing
predominantly insoluble material (consisting mainly of hemosiderin and
iron bound to intracellular organelles), ferritin-bound iron, and
nonferritin cytoplasmic iron, respectively. Separation of the soluble
nonferritin cytoplasmic iron into high- and low-molecular-weight fractions was omitted.
TfR expression.
Aliquots of approximately 5 × 105 cells were washed
in PBS containing 2% FCS and 0.05% sodium azide, and stained for 20 minutes at 4°C with saturating amounts of R-PE-conjugated rat
anti-mouse TfR monoclonal antibody. Cells were washed twice and then
fixed in PBS containing 1% formaldehyde. The percentage of fluorescent cells and the mean fluorescence intensity were measured by flow cytometry using a fluorescence-activated cell sorter (Becton Dickinson, Franklin Lakes, NJ). Specificity of staining was checked
by using a monoclonal immunoglobulin isotype standard.
IRP activity.
IRP activity was determined by mobility shift assay, using a
32P-labeled probe containing the ferritin IRE sequence
obtained by in vitro transcription of an IRE-CAT plasmid (kindly
provided by Dr N. Gray, European Molecular Biology
Laboratory [EMBL], Heidelberg) linearized with XbaI. Cell
extracts in cytoplasmic lysis buffer were freshly prepared as described
above and aliquots (20 µg of protein) loaded with a molar excess of
transcript (15,000 cpm) and subjected to nondenaturing gel
electrophoresis.17 Total IRP1 was determined by incubating
samples before the addition of the probe with 2% 2-mercaptoethanol
(2ME), which converts inactive IRP1 to the RNA binding
form.18 Autoradiographs were densitometrically scanned and
the proportion of spontaneously active IRP calculated.
Measurement of nitrite.
Nitrite was determined in the culture medium by the Griess
reagent.19
Protein determination.
The protein concentration of cell lysates was estimated by the BCA
protein assay reagent (Pierce, Chester, UK) using bovine serum albumin
as a standard.
Statistical analysis.
Data were analyzed by 1-way analysis of variance (ANOVA) and unpaired
Student's t-test to determine difference between each group.
Differences were considered statistically significant when P < .05.
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RESULTS |
Iron uptake and release.
Stimulation of J774 macrophages with IFN- or LPS caused a strong
dose-dependent inhibition of Fe-Tf uptake (Fig
1), there being a correlation between
nitric oxide release by macrophages (assayed as
NO2 accumulation in the medium) and
inhibition of Fe uptake. To confirm whether the observed inhibition of
Fe uptake was mediated by the nitric oxide released after macrophage
activation by IFN-/LPS, we stimulated macrophages in the presence of
NMMA, an inhibitor of NOS. The results show that NMMA partially
restored Fe incorporation (Fig 1).
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| Fig 1.
Inhibition of 59Fe uptake in J774 macrophages
by IFN-/LPS stimulation. Macrophages were incubated overnight with
the indicated concentrations of IFN- (U/mL), LPS (ng/mL), and/or
NMMA (500 µmol/L) and then exposed to 50 µg/mL 59Fe-Tf
for 8 hours. Nitrite production was assayed in the culture medium by
the Griess reaction. Results are expressed as percent inhibition of
iron uptake compared with the control, and are representative of 5 separate experiments. Values are given as the mean ± SE of triplicate
cultures. Versus control: *P < .01, **P < .001, ***P < .0001; versus IFN/LPS: #P < .001.
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Since earlier studies indicated that NO causes iron release from
cells,20,21 it seemed possible that the apparent inhibition of iron uptake might be due to increased efflux of iron from
IFN-/LPS-activated J774 cells during the incubation. To test this
possibility, we performed two different experiments. First, cells were
stimulated with IFN-/LPS immediately before the addition of
59Fe-Tf. This had no significant impact on 59Fe
released by the cells during a subsequent chase with unlabeled Fe-Tf
(Fig 2), indicating that these mediators
did not cause increased release of Fe during the acquisition process,
for example, by preventing intracellular release of Fe from transferrin
and allowing it to be recycled out of the cell. Addition of
59Fe-Tf followed by a chase in the presence of IFN-/LPS
actually caused a modest but significant decrease in iron release,
which was not affected by NMMA (Fig 3).
Thus, the mediators did not provoke release of iron already
incorporated into the cell. Therefore, these results confirm that the
main effect of IFN-/LPS treatment was to decrease macrophage Fe
uptake rather than to increase its release.
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| Fig 2.
Iron released by IFN-/LPS-activated J774 macrophages.
Cells were stimulated overnight with IFN- (50 U/mL), LPS (10 ng/mL),
and/or NMMA (500 µmol/L), pulsed for 4 hours with 50 µg/mL
59Fe-Tf, and then incubated 2 more hours in the presence of
unlabeled Fe-Tf. Nitrite production was assayed in the culture medium
by the Griess reaction. Results shown are representative of 3 separate
experiments. Values are given as the mean ± SE of triplicate
cultures.
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| Fig 3.
Iron released by IFN-/LPS-activated J774 macrophages.
Macrophages were incubated for 24 hours with 50 µg/mL
59Fe-Tf and then stimulated overnight with the indicated
concentrations of IFN- (U/mL), LPS (ng/mL), and/or NMMA (500 µmol/L) in the presence of unlabeled Fe-Tf. Nitrite production was
assayed in the culture medium by the Griess reaction. Results shown are
representative of 3 separate experiments. Values are given as the mean ± SE of triplicate cultures. Versus control: *P < .05, **P < .001, ***P < .0001.
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Intracellular distribution of iron.
We next investigated the distribution of incorporated iron in
nonactivated and IFN-/LPS-activated macrophages. J774 macrophages were pulsed for 8 hours with 50 µg/mL 59Fe-Tf to allow
incorporation of 59Fe, lysed, and the lysates analyzed by a
fractionation method, which provides a useful means of making
comparative analysis of intracellular iron distribution.15
After the 8-hour pulse with 59Fe-Tf, nonactivated
macrophages contained approximately 40% soluble iron, 10% insoluble
iron, and 50% ferritin-bound iron (Fig 4). In contrast, DFO-treated macrophages had approximately 80% of total
incorporated iron as soluble nonferritin iron and very little insoluble
iron. Activation with IFN-/LPS downregulated the total iron
incorporated into all 3 fractions because of the previously observed
inhibition of iron uptake. However, there was also a noticeable
decrease in the proportion of ferritin-bound iron and relatively more
insoluble iron, the effects being mediated at least in part by nitric
oxide as they were to some extent reversed by NMMA. The soluble iron
pool was proportionally much greater in DFO-treated cells, due probably
to iron complexed with DFO itself, but unchanged upon activation with
IFN/LPS. Thus, treatment of J774 cells with IFN-/LPS not only
decreased total iron uptake, but also altered intracellular
distribution, with ferritin-iron being particularly decreased and
insoluble iron least so. NMMA increased the proportion of
ferritin-iron, but had less effect on insoluble iron, which suggests
that the former but not the latter compartment was downregulated by NO.
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| Fig 4.
Intracellular iron distribution in J774 macrophages.
Macrophages were treated as described in the legend to Fig 1 and then
lysed in cytoplasmic lysis buffer containing protease inhibitors and
the intracellular iron distribution determined as described in the
Methods. Nitrite production was assayed in the culture medium by the
Griess reaction. Results shown are representative of 5 separate
experiments. Values are given as the mean ± SE of triplicate
cultures. Versus control: aP < .05, bP < .01, cP < .001; versus
IFN/LPS: #P < .01, ##P < .001.
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TfR expression.
The above results prompted us to examine the effect of IFN- and LPS
on TfR expression. Incubation with either IFN- or LPS for 16 hours
downregulated macrophage TfR expression, even though NO production was
extremely low (Fig 5A). In cultures
containing both IFN- and LPS, TfR expression was further inhibited,
but NMMA produced only a small and statistically insignificant reversal of inhibition. Thus, the inhibition of TfR expression 16 hours after
stimulation of the cells appeared to be largely NO-independent. However, when the macrophages were stimulated for 24 hours instead of
16 hours with IFN- alone or with IFN- plus LPS, a less strong TfR
downregulation was found and this correlated with a greater NO
production. Furthermore, in the case of cells activated with IFN-
plus LPS, downregulation of TfR was enhanced by addition of NMMA (Fig
5B). This suggests that stimulation of macrophages with IFN-/LPS has
2 opposing effects on TfR expression: an NO-independent inhibitory
effect, and an opposing upregulation by NO when the latter is produced
in sufficient quantity. The changes in TfR expression between 16 hours
and 24 hours, though modest, were consistently observed, and even
modest changes in TfR expression are likely to have significant effects
on iron uptake given the efficiency and rapidity with which the TfR
delivers iron to the cell.
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| Fig 5.
Modulation of TfR expression by IFN-/LPS in J774
macrophages. Cells were stimulated for 16 hours (A) or 24 hours (B)
with the indicated concentrations of IFN- (U/mL), LPS (ng/mL),
and/or NMMA (500 µmol/L) and the expression of TfR determined by flow
cytometry as described in the Methods. Nitrite production was assayed
in the culture medium by the Griess reaction. Results shown are
representative of 5 separate experiments. Values are given as the mean ± SE of triplicate cultures. Versus control: *P < .05, **P < .01; versus IFN: #P < .05; versus
IFN/LPS: &P < .001.
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IRP1 and IRP2 activity.
Since uptake and utilization of cellular iron is regulated by the iron
regulatory proteins IRP1 and IRP2,4 it was of interest to
determine whether the activity of these proteins in stimulated J774
cells could explain the effects of IFN-/LPS on iron uptake and
distribution in J774 cells. Figure 6 shows
a representative gel retardation assay using extracts prepared from
nonactivated control (C), DFO-treated (D), and IFN- (I)- and/or LPS
(L)-activated cells, with or without NMMA (N). DFO, included as a
positive control, activated both IRP1 and IRP2 as expected. Stimulation
with either IFN- or LPS, or both together, caused an increase in
IRE-binding activity of IRP1 that appeared to depend on NO, as it was
largely reversed by NMMA. The effect on activity of IRP2 was more
complex; a low dose of IFN- (5 U/mL), which caused minimal NO
production, decreased activity of IRP2, but at higher IFN- doses, at
which significant amounts of NO were produced, IRP2 activity was
progressively regained. LPS alone had no effect on IRP2, even at doses
sufficient to cause significant NO production, while IFN- plus LPS
caused a decrease in IRP2 activity that was not reversed by NMMA.
Downregulation of IRE-binding activity of IRP2 was still observed when
the cells were treated with a combined low dose of IFN- plus LPS
that was insufficient to cause significant NO production (data not
shown). These observations provide evidence that while IRP1 is
upregulated by NO, IRP2 is downregulated by IFN- alone, in an
NO-independent manner.
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| Fig 6.
Regulation of IRE-binding activity of IRPs by IFN-/LPS
in J774 macrophages. (A) Cells were treated overnight in RPMI culture
medium containing 10% FCS with 100 µmol/L DFO (D), 5 to 500 U/mL
IFN- (I), 10 to 1,000 ng/mL LPS (L), and/or 500 µmol/L NMMA (N),
or remained untreatred (control, C). Twenty micrograms of detergent
cell extract was assayed for IRE-binding activity of IRP1 and IRP2 by
gel-shift assay in the absence or presence of 2% 2-ME. Nitrite
production was assayed in the culture medium by the Griess reaction.
Results shown are representative of 4 separate experiments. (B)
Autoradiographs were densitometrically scanned and the proportion of
spontaneous IRP1 activity was expressed as a percentage of the value
obtained after exposure to 2% 2-ME, which allows calculation of total
IRE binding activity of IRP1. IRP2 activity was plotted in arbitrary
units.
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DISCUSSION |
Iron homeostasis is of central importance for the regulation of
macrophage functions.22 Increased intracellular free iron decreases the effectiveness of IFN- action on monocytic
cells23,24 and reduces the transcription of
inducible NO synthase (iNOS) mRNA and subsequent NO
formation in J774 macrophages.3 Nevertheless, adequate iron
levels are required for macrophage-mediated cytotoxicity to catalyze
the production of highly toxic hydroxyl radicals via the Fenton
reaction.2
Intracellular iron levels are maintained by regulating the uptake,
utilization, and storage of iron. The most important molecules involved
in this process are TfR, which mediates cellular uptake of Tf-bound
iron,25 and Ft, which stores iron acquired in
excess.26 Two cytoplasmic protein, IRP1 and IRP2, interact
with both TfR and Ft mRNAs during iron deficiency or in cells exposed
to oxidative stress or NO, thereby blocking Ft translation and
increasing TfR mRNA stability.10-12 In addition to this,
several cytokines influence iron metabolism by IRP-independent
transcriptional and posttrancriptional regulation of TfR and Ft
expression.22
Although the effect of NO on IRP activity has been well studied, the
combined effects of NO and cytokine stimulation on the actual handling
of iron by macrophages has not been investigated in any detail. In this
study, we have shown that in J774 macrophages, cell activation by
IFN- and/or LPS inhibits Fe uptake with a concomitant downregulation
of TfR expression. This inhibition of iron uptake was partially but not
completely reversed by NMMA, which indicates that NO, despite its
previously reported ability to activate IRP1,10 can
actually inhibit iron uptake by macrophages. The failure of NMMA to
completely reverse inhibition of iron uptake may indicate that the
effect is not entirely NO-mediated, or may simply reflect that fact
that, as found by others,3,12,14 NMMA does not completely
inhibit NO synthesis. Incubation of J774 cells with the NO generator
S-nitroso-N-acetyl-penicillamine (SNAP) also inhibited iron
uptake, but this result is difficult to interpret because penicillamine
itself, which does not generate NO, also caused some inhibition (data
not shown), perhaps because penicillamines can chelate iron. Iron
release by IFN-/LPS-activated cells was not greater than from
control cells, indicating that the effect is actually caused by
inhibition of Fe uptake from Tf, rather than accelerated release of
iron during the incubation period. This contrasts with earlier work
with K562 erythroleukemia cells, in which NO (produced exogenously from
SNAP) did cause an increase in iron release.21,27 Although
inhibition of NO production in IFN-/LPS-treated cells by NMMA
increased Fe incorporation, it did not cause a corresponding increase
in TfR expression, and if incubation was extended to 24 hours, TfR
expression was actually further reduced by NMMA. These apparently
contradictory findings may indicate that NO directly affects iron
availability to the cell. One possible explanation is that NO removes
iron from transferrin at the cell membrane immediately before
endocytosis of the transferrin-TfR complex, thus reducing the
efficiency of iron uptake but not the expression of TfR. However, a
previous study found that NO could not remove iron from Tf at neutral
pH.27 A further possibility is that NO reduces the rate of
recycling of TfR without reducing surface expression, as has been
reported in spleen cells exposed to oxidative stress in
vivo.28 Our results clearly indicate that NO by itself
affects iron uptake independently of TfR expression, and this needs
further investigation.
The effect of IFN- and LPS on TfR expression by J774 macrophages
appears to be complex and regulated by both NO-dependent and
-independent mechanisms. IFN- can by itself downregulate TfR
expression, this being NO-independent, but correlating with a
concomitant decrease in IRE-binding activity of IRP2. However, if
incubation is continued long enough to allow significant production of
NO, TfR expression can be at least partially restored, accompanied by
activation of IRP1 and, to a lesser extent, of IRP2. In contrast, LPS
by itself decreases TfR expression, even though NO is produced and IRP1
is activated. Costimulation of macrophages with both IFN- and LPS
also leads to a downregulation of TfR expression that is initially
NO-independent, but may be modulated by NO after a longer (24 hours)
incubation period. Previous reports have also suggested that the
regulation of TfR expression in activated macrophages is complex and
may depend on experimental differences among groups.22 Thus, cells exposed to NO-releasing drugs or overexpressing NOS exhibit
higher TfR mRNA expression,10,21,29,30 whereas
IFN-/LPS-stimulated macrophages show decreased TfR mRNA levels,
despite production of NO and subsequent activation of IRP1 and
IRP2.8,12,13,31 These apparently contradictory data can be
reconciled by the fact that IFN/LPS can decrease TfR expression via an
IRE/IRP-independent mechanism,13,32 as well as by the
NO-independent IFN--mediated downregulation of IRP2 described in
this study and by others.14 Overall, it is clear that the
major effect of IFN-/LPS on both TfR expression and iron uptake by
J774 macrophages is downregulatory. Although NO activates IRP1, this is
only able to compensate for the other inhibitory effects on TfR
expression when high concentrations of NO are present.
The regulation of ferritin mRNA expression and protein content in
IFN-/LPS-activated macrophages is also controversial. Weiss et
al33,34 reported that IFN-/LPS treatment increases
ferritin mRNA expression in J774 macrophages but decreases ferritin
translation, indicating that IRP activation mediated by NO overcomes
the increased mRNA expression. In contrast Recalcati et
al12 more recently demonstrated increased ferritin
synthesis and accumulation in IFN-/LPS-stimulated macrophages,
accompanied by an NO-dependent increase in IRP1 and decrease in IRP2
activity. However, the effect on iron incorporation into ferritin has
not been previously reported. In the present study, we found that
IFN-/LPS treatment results in a strong decrease in both the amount
and the proportion of iron incorporated into ferritin, together with a
corresponding compensatory increase in insoluble iron (consisting
mainly of hemosiderin and/or iron bound to intracellular organelles).
These findings probably indicate an increase in iron bound to
mitochondria in the more highly metabolic activated macrophages, rather
than increased formation of hemosiderin, and the overall picture is one
of iron being used for metabolic activity rather than being diverted to
an enlarged ferritin compartment. Nevertheless, an NO-dependent
downregulation of ferritin-bound iron in activated macrophages, perhaps
as a consequence of NO-mediated iron release from
ferritin,35 should not be discounted, as NMMA partially increased this intracellular iron fraction.
The cytoplasmic proteins IRP1 and IRP2 have a key role in the
regulation of iron metabolism by binding specifically to stem-loop structures (IRE) of several mRNAs. Recent evidence indicates that these
two proteins respond preferentially to different immunologic stimuli.12,14 In this work, we found that
IFN-/LPS-stimulated macrophages showed an increase in IRP1 activity
and a strong reduction of IRP2 activity, as also reported by
others.12,14 Although both of these previous studies
demonstrated that upregulation of IRP1 was NO-mediated, Bouton et
al14 showed that the decrease of IRP2 activity is not
mediated by NO, whereas Recalcati et al12 reported it to be
NO-dependent. Our results accord with those of Bouton et
al,14 as we found that NO released by IFN-/LPS-activated macrophages increased IRE-binding activity of IRP1, whereas low levels
of IFN-, insufficient to cause significant NO production, were able
to decrease IRP2 activity. These results strongly suggest that (1) NO
is not involved in the IFN-/LPS-induced downregulation of IRP2
activity despite significant NO production, as LPS treatment is unable
to downregulate IRP2 activity even when NO is produced; and (2) NO can
mediate the activation of both IRP1 and IRP2, probably by affecting
iron availability.
In conclusion, this study demonstrates that activation of IRPs,
expression of TfR, and iron uptake and utilization in
IFN-/LPS-activated macrophages is complex, and that a model based
solely on NO-mediated activation of IRP1 is not tenable as it appears
to affect TfR expression positively only at high NO concentrations. The
overall picture is one of reduced iron uptake but increased cellular
utilization for metabolic activity, mediated partly by an
NO-independent downregulation of IRP2, and partly by IRP-independent
mechanisms, some of which may also involve NO. Such a scenario would
allow activated macrophages to fine-tune their iron uptake to a level
which permits optimal activation of antimicrobial and cytotoxic
activities but without accumulating excess iron that might facilitate
intracellular microbial growth.
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ACKNOWLEDGMENT |
We thank Dr C. Guillén for her helpful advice with the band shift assay.
| |
FOOTNOTES |
Submitted January 6, 1999; accepted May 27, 1999.
V.M. was supported by a grant from CajaMurcia (Spain).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Jeremy H. Brock, PhD,
Department of Immunology and Bacteriology, Western
Infirmary, Glasgow G11 6NT, UK; e-mail: jhb1h{at}clinmed.gla.ac.uk.
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Perspectives in iron metabolism.
N Engl J Med
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ABSTRACT
INTRODUCTION