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1040-9238/03/$.50 2003 by CRC Press LLC
Critical Reviews in Biochemistry and Molecular Biology,
38(1):6188 (2003)
Iron Metabolism in the ReticuloendothelialSystem
Mitchell Knutson* and Marianne Wessling-ResnickDepartment of
Nutrition, Harvard School of Public Health, 665 Huntington
Avenue,Boston, MA 02115
* To whom correspondence should be addressed at: Harvard School
of Public Health, Department of Nutrition,665 Huntington Avenue,
Boston, MA 02115. E-mail: [email protected]
ABSTRACT: Comprised mainly of monocytes and tissue macrophages,
the reticuloendot-helial system (RES) plays two major roles in iron
metabolism: it recycles iron fromsenescent red blood cells and it
serves as a large storage depot for excess iron. Although
ironrecycling by the RES represents the largest pathway of iron
efflux in the body, the precisemechanisms involved have remained
elusive. However, studies characterizing the functionand regulation
of Nramp1, DMT1, HFE, FPN1, CD163, and hepcidin are rapidly
expandingour knowledge of the molecular aspects of RE iron
handling. This review summarizesfundamental physiological and
biochemical aspects of iron metabolism in the RES andfocuses on how
recent studies have advanced our understanding of these areas.
Alsodiscussed are novel insights into the molecular mechanisms
contributing to the abnormalRE iron metabolism characteristic of
hereditary hemochromatosis and the anemia of chronicdisease.
KEY WORDS: CD163, DMT1, ferroportin1, hepcidin, HFE, Nramp1.
TABLE OF CONTENTS
I. Introduction
.........................................................................
62
II. The Reticuloendothelial System (RES)
.............................. 63A. Definition and Functions
.............................................. 63B. The Study of
Iron Metabolism in the RES .................. 63
III. Iron Acquisition by the RES
............................................... 65A.
Erythrophagocytosis
..................................................... 65
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B. Receptor-Mediated Uptake of Hemoglobin ................. 65C.
Receptor-Mediated Uptake of Transferrin ................... 66
IV. Intracellular Iron Metabolism in the RES
.......................... 66A. Iron Homeostasis and IRE-IRP
.................................... 66B. Cellular Iron Transport:
Roles of Nramp1 and
Nramp2
(DMT1)...........................................................
67C. Iron Storage
..................................................................
68
V. Iron Release by the RES
..................................................... 69A. Iron
Release and Plasma Iron ...................................... 69B.
Kinetics and Chemical Forms of Released Iron .......... 70C. Effect
of Transferrin and Ceruloplasmin ..................... 70D.
Potential Roles for Ferroportin1 and Nramp1 ............. 73E.
Regulation of Iron Release
........................................... 74
VI. Perturbations of RE Iron
Metabolism................................. 74A. Hereditary
Hemochromatosis (HH) ............................. 74B. Anemia of
Chronic Disease (ACD) ............................. 76C. Possible
Role of Hepcidin ............................................
77
VII. Unanswered Questions
........................................................ 78
I. INTRODUCTION
One of the most distinguishing featuresof iron metabolism is the
degree to whichbody iron is conserved. Of the typical 3 to4 g of
iron contained in the normal adulthuman, only about 0.03% (or ~1
mg) is lostper day, mainly the result of obligatory lossesof
exfoliated mucosal cells, bile, and ex-travasated red cells. To
replace these basallosses and remain in iron balance, the bodymust
absorb a roughly equivalent amount ofiron from the diet. This
relatively small dailyexchange of iron between body and
envi-ronment contrasts sharply with the
comparatively large exchange of this metalbetween internal
organs. For example, eachday the bone marrow utilizes
approximately24 mg of iron to produce over 200 billionnew
erythrocytes. To meet the demand forheme production necessary for
erythropoie-sis, iron must be recycled from senescentred cells;
this process is carried out by mac-rophages of the
reticuloendothelial system(RES). Despite this critical role of the
RESin body iron conservation, iron recycling bythe RE cell has
remained one of the leastwell-understood areas of iron
metabolism(Aisen, 1990). However, in the last decadefive new genes
involved in iron metabolismhave been discovered: Nramp1 (Vidal
et
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al., 1993), HFE (Feder et al., 1996), DMT1(Fleming et al., 1997;
Gunshin et al., 1997),FPN1 (Abboud and Haile, 2000; Donovanet al.,
2000; McKie et al., 2000), and CD163(Kristiansen et al., 2001).
These genes areabundantly (or exclusively) expressed in REcells,
and characterization of their functionsis starting to reveal how
the RES handlesiron at the molecular level. Another note-worthy
advance has been the identificationof hepcidin, a serum peptide
that appears toaffect iron storage in the RES (Nicolas,2001). A
list of these new factors, alongwith other proteins known to
participate inRE iron metabolism, is presented in Table 1.Here we
review iron metabolism in theRESfrom iron acquisition,
intracellulariron processing, and iron releasehighlight-ing how
recent studies have contributed toour understanding of these highly
dynamicprocesses.
II. THE RETICULOENDOTHELIALSYSTEM (RES)
A. Definition and Functions
Also known as the mononuclear ph-agocyte system (Weinberg and
Athens,1993), the RES is composed of monocytes,macrophages, and
their precursor cells.Monocytes arise from progenitor cells inthe
bone marrow and are released into theblood. After migration to
different tissues,they differentiate into macrophages
withcharacteristic morphologic and functionalqualities. Studies
using an antibody againstthe macrophage-specific antigen F4/80
showthat mouse organs with the most macroph-ages are, in descending
order, the liver, largeintestine, small intestine, bone
marrow,spleen, and kidney (Lee et al., 1985). Al-though RE cells
residing in various tissues
likely have different or highly specializedfunctions (e.g.,
immunoregulation, antimi-crobial activity, antitumorical activity),
onecommon task involves the clearance of par-ticulate matter and
damaged or effete cells.The removal of damaged or senescent
eryth-rocytes, with the subsequent recycling ofiron, directly links
the RES and iron me-tabolism. This process is mainly carried outby
RE cells of the spleen, liver, and bonemarrow. The splenic red pulp
appears to beone of the most active sites of red cell de-struction.
However, after splenectomy, redcell survival time does not increase
(Ath-ens, 1993), indicating that macrophages ofthe liver and bone
marrow (or elsewhere)can rapidly compensate for this function ofthe
spleen.
B. The Study of Iron Metabolismin the RES
A variety of systems are available forthe study of iron
metabolism in the RES,each with advantages and disadvantages.In
vivo studies are clearly the most physi-ologic, but interpretation
of the results canbe complicated by the diffuse distributionand
specialized functions of different REcells. Pure primary cultures
of liver mac-rophages (Kupffer cells) can be used, buttheir
extraction from tissues is laboriousand involves tissue disruption,
producingcell populations with different degrees ofactivation and
differentiation (Olynyk andClarke, 1998). More readily
availablesources of macrophages include the lungand the peritoneum.
Peripheral blood mono-cytes can also be relatively easily
obtainedand studied in culture, either before or af-ter
differentiation to macrophages. How-ever, the extent to which
monocytes oralveolar and peritoneal macrophages areinvolved in
normal RE iron metabolism is
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unknown. In recent years, cell lines thatdisplay many of the key
characteristics ofbona fide macrophages are being used
morefrequently. It is worthwhile to note that thecommonly used J774
and RAW264.7 mac-rophage cell lines do not express functionalNramp1
protein (Vidal et al., 1996). Thus,iron metabolism studies using
these cellsmust be interpreted carefully (see below).It is also
difficult to compare results fromdifferent studies of RE iron
metabolism
because of the disparate forms of iron used(e.g., erythrocytes,
hemoglobin, heme, iron-transferrin, iron-transferrin-immune
com-plex, iron dextran, ferric ammonium cit-rate, ferric
nitrilotriacetic acid). Moreover,the metabolism of some of these
iron com-pounds can differ depending on whetherthe iron is acquired
via phagocytosis orendocytosis. Thus, gaining a comprehen-sive
understanding of RES function hasproven difficult.
TABLE 1Proteins Involved in RE Iron Metabolism*
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III. IRON ACQUISITION BY THERES
A. Erythrophagocytosis
Macrophages of the RES acquire mostof their iron by
phagocytosing senescentred blood cells. With each red cell
ingested,the macrophage accrues approximately onebillion iron
atoms. It has been estimatedthat fixed macrophages of rat liver,
spleen,and bone marrow phagocytose an averageof one red cell per
macrophage per day(Kondo et al., 1988). Interestingly, the
cel-lular and molecular mechanisms of the seem-ingly simple
clearance of effete erythrocytesfrom the circulation remains the
subject ofa great deal of controversy (reviewed byBratosin et al.,
1998). After erythrophago-cytosis, hydrolytic enzymes present in
thephagolysosome degrade the red blood cell.Proteolytic digestion
of hemoglobin liber-ates heme, which is assumed to cross
thephagolysosomal membrane either by diffu-sion or by a specific
transporter in order toreach heme oxygenase (HMOX). Threeisoforms
of HMOX have been described inmammals: an inducible HMOX1; a
consti-tutively active but uninducible HMOX2; andHMOX3, a form
nearly devoid of catalyticcapability (Elbirt and Bonkovsky,
1999).HMOX2 appears predominant in all organsmeasured, except for
the rat spleen, whichnormally expresses five times more HMOX1than
HMOX2 (Braggins et al., 1986).The strong splenic HMOX1
expressionlikely reflects the high concentration
oferythrophagocytosing RE cells in this or-gan. Although HMOX1
appears to be largelyresponsible for heme catabolism in RE
cells,studies of mice lacking HMOX1 reveal theexistence of other
significant, but less-effi-cient pathways of heme degradation
(Possand Tonegawa, 1997).
HMOX proteins are localized to the en-doplasmic reticulum (ER),
where theycatabolize heme to produce biliverdin, car-bon monoxide,
and Fe2+ (Maines, 1997).The iron thus liberated is then either
re-leased from the macrophage or stored (seebelow). Baranano et al.
(2000) propose thatthe iron freed from heme transiently be-comes
part of the cytoplasmic labile ironpool before being transported to
the lume-nal side of the ER by a novel ATPase. Thisiron-inducible
ATP-dependent transporterlocalizes with HMOX1 to microsomal
mem-branes and is greatly enriched in the spleen(Baranano et al.,
2000), but rigorous identi-fication of a gene product is still
needed. Analternative site of heme catabolism is sug-gested by
recent analyses of J774 macroph-ages in the process of
erythrophagocytosis.Using electron microscopy and two-dimen-sional
gel electrophoresis, Gagnon et al.(2002) provide compelling
evidence that partof the phagosomal membrane is derived fromER.
This observation raises the intriguingpossibility that
ER-associated HMOX pro-teins may catalyze heme degradation andiron
liberation within the phagolysosome. Ifso, a nonheme iron
transporter would berequired to translocate iron into the
cytosol.Future studies need to explore whetherHMOX proteins are
recruited to thephagolysosomal membrane after erythroph-agocytosis
to determine the exact site ofheme catabolism.
B. Receptor-Mediated Uptake ofHemoglobin
From kinetic studies of hemoglobin turn-over in humans, it has
been calculated that10 to 20% of normal erythrocyte destruc-tion
occurs intravascularly, resulting in therelease of hemoglobin
(Garby and Noyes,1959a). Under normal circumstances, all of
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this hemoglobin is rapidly bound by hapto-globin, which is then
cleared from the cir-culation by parenchymal cells of the
liver(Deiss, 1999). However, recent studies haveidentified a
hemoglobin scavenger receptor,CD163, expressed exclusively on
monocytesand macrophages (Kristiansen et al., 2001).Found in the
highest concentrations in thespleen and the liver, CD163 scavenges
he-moglobin by mediating endocytosis and sub-sequent degradation of
the hemoglobin-hap-toglobin complex (Kristiansen et al.,
2001).Thus, uptake of hemoglobin-haptoglobin viaCD163 may represent
a significant pathwayof normal iron acquisition by the RES.
Underconditions associated with increased intra-vascular hemolysis
(e.g., hemolytic anemia,thalassemia, and certain bacterial
infections),the hemoglobin-binding capacity of hapto-globin can be
exceeded such that free he-moglobin appears in the plasma. Some
ofthe circulating free hemoglobin degradesand releases heme, which
then becomesbound to the plasma glycoprotein hemo-pexin. Specific
hemopexin receptors onhepatocytes clear the heme-hemopexin com-plex
from the circulation (Alam and Smith,1989). The detection of
hemopexin recep-tors on human monocytic cell lines (Alamand Smith,
1989; Taketani et al., 1990) alsosuggests that the RES is able to
acquireheme from this pathway, but the amounttaken up is probably
not significant undernormal circumstances.
C. Receptor-Mediated Uptake ofTransferrin
Iron is delivered to most tissues via en-docytosis of the plasma
iron-binding pro-tein transferrin bound to its cell surface
re-ceptor. The transferrin receptor is a dimerof 90-kDa subunits
that associates with aregulatory molecule called HFE (Parkkila
et al., 1997; Feder et al., 1998). Isolatedhuman monocytes
express transferrin re-ceptors (Bjorn-Rasmussen et al., 1985)
andare able to take up iron from transferrin(Sizemore and Bassett,
1984). When cul-tured monocytes differentiate into macroph-ages,
the expression of transferrin receptorincreases greatly (Andreesen
et al., 1984).Transferrin-binding activity has also
beendemonstrated in various macrophages frommice (Hamilton et al.,
1984), rats (Nishisatoand Aisen, 1982; Kumazawa et al., 1986),and
humans (Andreesen et al., 1984; Testaet al., 1987; Testa et al.,
1989; Montosi etal., 2000). Although macrophages in cul-ture can
acquire iron from transferrin, theextent to which this occurs in
vivo remainsunknown. Human studies have failed to findevidence of
significant iron uptake by REcells after injection of radiolabeled
transfer-rin-bound iron (Finch et al., 1970).
IV. INTRACELLULAR IRONMETABOLISM IN THE RES
A. Iron Homeostasis and IRE-IRP
Cellular iron homeostasis is regulatedposttranscriptionally by
two cytoplasmiciron regulatory proteins, IRP-1 and IRP-2.IRPs
control cellular iron uptake and stor-age by binding to
iron-responsive elements(IRE) present in mRNAs of factors
involvedin iron metabolism, and in particular, tran-scripts for the
transferrin receptor and fer-ritin. When cytoplasmic iron
concentrationsare low, IRPs bind to IRE and coordinatelyincrease
the stability of transferrin receptormRNA and decrease the
translation of fer-ritin. Conversely, when iron is plentiful IRPsdo
not bind to IRE, and transferrin receptormRNA is degraded while
iron storage inferritin predominates. The many factors that
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influence IRP function have been reviewedelsewhere in detail
(Eisenstein, 2000). Inhuman peripheral blood monocytes, IRE-IRP
binding activities increase in responseto iron depletion and
decrease with ironloading (Cairo et al., 1997). Similar regula-tion
of IRE-IRP binding activities has beendemonstrated in THP-1 cells
(Weiss et al.,1996), mouse peritoneal
macrophages(Kuriyama-Matsumura et al., 1998), J774cells (Recalcati
et al., 1998; Pinero et al.,2001), and RAW264.7 cells (Kim
andPonka, 1999; Kim and Ponka, 2000;Wardrop and Richardson, 2000).
Accord-ingly, increased transferrin receptor mRNAlevels are
associated with increased IRE-IRP binding (Kim and Ponka, 1999;
Kimand Ponka, 2000; Wardrop and Richardson,2000). Thus, it appears
that the IRE-IRPregulatory system functions in RE cells as itdoes
in other cell types.
B. Cellular Iron Transport: Rolesof Nramp1 and Nramp2 (DMT1)
Two proteins of the NRAMP (naturalresistance associated
macrophage protein)family have been identified: Nramp1 (Vidalet
al., 1993) and Nramp2 (Gruenheid et al.,1995). Nramp1 is a highly
hydrophobic 56-kDa protein with 12 predicted transmem-brane regions
that is expressed exclusivelyin monocytes and macrophages. The
pro-tein sequence of Nramp1 shares 64% aminoacid sequence identity
with Nramp2, whichis ubiquitously expressed. The associationbetween
Nramp2 and iron transport wasestablished by Fleming et al. (1997)
andGunshin et al. (1997). Also known as DCT1(divalent cation
transporter1), Nramp2 isnow more commonly referred to as
DMT1(divalent metal transporter1).
Nramp1 localizes to lysosomes and lateendosomes and is rapidly
recruited to mem-
branes of maturing phagosomes (Gruenheidet al., 1997; Searle et
al., 1998; Govoni etal., 1999). As its name implies, Nramp1
isinvolved in determining the ability of in-bred mouse strains to
resist infection withcertain intracellular pathogens.
Susceptibil-ity is associated with a single G169D sub-stitution in
the protein (Vidal et al., 1993):mice expressing the wild-type
Nramp1G169allele are resistant, whereas those express-ing the
Nramp1D169 allele are susceptible.The Nramp1D169 allele encodes a
nonfunc-tional protein that is rapidly degraded inmacrophages
(Vidal et al., 1995). A role forNramp1 in intracellular iron
transport wasestablished in studies using the Nramp1-deficient
RAW264.7 macrophage cell line.The transport of iron into phagosomes
con-taining latex beads (Kuhn et al., 1999) ormycobacteria
(Zwilling et al., 1999; Kuhnet al., 2001) was shown to be higherin
RAW264.7 cells transfected withNramp1G169 than in cells
expressingNramp1D169 . These observations are consis-tent with the
hypothesis that Nramp1 func-tions to transport iron into the
bacterium-containing phagosome and thereby limitmycobacterial
growth by catalyzing the for-mation of reactive oxygen species. In
con-trast, data from other studies in intact cellshave been
interpreted to suggest that Nramp1transports metals out of the
phagosome(Atkinson and Barton, 1999; Barton et al.,1999; Jabado et
al., 2000), a function thatmay restrict the growth of
phagocytosedpathogens by decreasing the availability ofiron as an
essential nutrient. These two seem-ingly contradictory functions of
Nramp1may be reconciled by a recent study charac-terizing Nramp1
transport activity. Whenexpressed in Xenopus oocytes, Nramp1
iscapable of transporting iron bidirectionally,depending on pH
(Goswami et al., 2001).Clearly, more work is needed to defineNramp1
function in intracellular iron trans-port. For a more detailed
review of Nramp1
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and macrophage iron metabolism, the readeris referred to Wyllie
et al. (2002).
The recruitment of Nramp1 to thephagolysosome has fostered
speculation thatNramp1 may function to transport
erythro-cyte-derived iron into the cytosol (Fleming etal., 1998;
Atkinson and Barton, 1999). Thisidea, however, presupposes that
iron is re-leased from heme inside the phagolysosome,and this seems
unlikely if HMOX1 functionsin the ER (Tenhunen et al., 1968).
Moreover,if Nramp1 were the sole mediator of erythro-cyte iron
transport out of the phagolysosome,then inbred mouse strains
homozygous for themutant Nramp1D169 allele (e.g., BALB/c
andC57BL/6) would be expected to have iron-deficiency anemia due to
inefficient iron recy-cling. The demonstration that these mice
havenormal hematological profiles (Leboeuf et al.,1995) suggests
that lack of Nramp1 does notdisrupt this process.
The effect of iron status on Nramp1mRNA and protein levels has
been inves-tigated recently using in vitro and in vivosystems.
Nramp1 mRNA levels increasedin bone marrow-derived cells exposed
tohemin (Biggs et al., 2001) or ferric am-monium sulfate (Baker et
al., 2000),whereas no change was observed inRAW267.4 or J774 cells
treated with fer-ric ammonium citrate or the iron
chelatordesferrioxamine (Wardrop and Richardson,2000). At the
protein level, Nramp1 in-creased in bone marrow-derived
macroph-ages in response to ferric ammonium sul-fate (Baker et al.,
2000) and after treatingsplenic cells with red blood cells (Biggset
al., 2001). Using immunohistochemis-try, Biggs et al. (2001) did
not detectchanges in Nramp1 protein levels insplenic macrophages in
mice given anintraperitoneal injection of iron dextran.Discordant
findings between these stud-ies may reflect inherent variability
amongdifferent types of macrophages or the useof different chemical
forms of iron.
Although discovered after Nramp1,DMT1 has been more fully
characterized interms of its biochemical function (Gunshinet al.,
1997). Studies in HEp-2, HeLa, andCOS-7 cells reveal that DMT1
localizes torecycling endosomes where it transports ironfrom
transferrin into the cytosol (Fleming etal., 1998; Tabuchi et al.,
2000). Becausethis transporter co-localizes with transferrinin RAW
264.7 macrophages (Gruenheid etal., 1999), it is likely that DMT1
performsa similar function in RE cells. The observa-tion that DMT1
also becomes associatedwith the phagolysosome in J774 macroph-ages
(Gruenheid et al., 1999) suggests thatit may transport
erythrocyte-derived ironinto the cytosol. As with Nramp1, this
modelwould require iron liberation from hemeinside the
phagolysosome.
Of the two splice variants of DMT1 thathave been identified, one
has an atypical IRE inits 3'UTR, suggesting that it may be
regulatedlike transferrin receptorthat is, DMT1 mRNAwould be
stabilized under low iron conditionsand degraded under iron
loading. Studies ofintestinal and liver cell lines support this
idea(Gunshin et al., 2001). However, studies ofmacrophage cell
lines (RAW264.7 and J774)indicate that DMT1 transcript levels do
notchange in parallel with changes in transferrinreceptor mRNA
(Wardrop and Richardson,2000). The lack of iron responsiveness of
mac-rophage DMT1 mRNA levels is consistent withresults from studies
of cultured fibroblastsand erythroleukemic cells (Wardrop
andRichardson, 1999). To better characterizeDMT1s function in RE
iron metabolism, fu-ture studies need to determine the effect of
ironstatus on DMT1 protein levels.
C. Iron Storage
The main sites of body iron stores arethe hepatic parenchyma and
the RES, par-
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ticularly the RE cells of the bone marrow,spleen, and liver. The
liver and the totalbone marrow each contain approximately100 to 300
mg of storage iron in healthyWestern individuals (Gale et al.,
1963;Bothwell et al., 1979). The concentrationsof iron in liver and
bone marrow have beenshown to correlate well over a wide range(up
to 9000 g/g tissue) (Gale et al., 1963).
Iron in the RES most likely accumu-lates secondary to the
catabolism of red cellheme. RE iron acquired via
erythrophago-cytosis that is not utilized or released is
firstdestined for storage in ferritin, a cytosolicprotein comprised
of 24 subunits of twotypes, H and L. In RE cells, ferritin is
com-prised mainly of the L-subunit (Invernizziet al., 1990), the
form most associated withiron storage (Levi et al., 1994). Cell
culturestudies using monocytes and macrophagesdocument the
formation of ferritin proteinwithin hours after red cell ingestion
(Custeret al., 1982; Bornman et al., 1999). Threehours after
erythrophagocytosis, both H- andL-subunits of ferritin are
upregulated in equalamounts (Bornman et al., 1999), whereasafter 18
, the L-form predominates (Raha-Chowdhury et al., 1993). Although
ferritinsynthesis after red cell ingestion can be regu-lated via
IRP-IRE interactions effected bychanges in iron levels, some
evidence indi-cates that reactive oxygen species formedduring
phagocytosis may also play a role(Bornman et al., 1999), perhaps
throughupregulation of ferritin transcription (Tsujiet al., 2000).
Recent serial analyses of geneexpression in human
monocyte-derivedmacrophages highlight the importance offerritin in
the RE cell (Hashimoto et al., 1999).Out of 35,000 genes identified
by this method,ferritin L- and H-chains were the first andthird
most abundant mRNA species, repre-senting nearly 5% of all
transcripts. Under-standably, targeted deletion of the
murineH-ferritin gene in Fth/ mice leads to earlyembryonic death
(Ferreira et al., 2000), but it
is of interest that heterozygous Fth+/ mice,which have markedly
increased ratios ofL-to-H subunits, show no abnormalities iniron
metabolism, including no changes insplenic iron stores (Ferreira et
al., 2001).
The storage of iron from the uptake ofhemoglobin appears to be
influenced by ge-netic polymorphisms in haptoglobin. Of thethree
haptoglobin polymorphisms in humans(Langlois and Delanghe, 1996),
the multimericHp2-2 phenotype has the highest functionalaffinity
for the hemoglobin scavenger receptor,CD163 (Kristiansen et al.,
2001). In a study of717 healthy Caucasian subjects, males with
theHp2-2 phenotype had significantly increasedserum iron levels and
twofold higher monocyteL-ferritin concentrations than other Hp
pheno-types (Langlois et al., 2000). These associa-tions, along
with observations from early stud-ies of hemoglobin iron metabolism
(Garby andNoyes, 1959b), have led to the hypothesis thathemoglobin
iron acquired via CD163 on REcells is shunted into slowly
exchanging storagecompartments normally bypassed by iron recy-cling
pathways (Delanghe and Langlois, 2002).More work will be needed to
better define thequantitative contribution of hemoglobin to
ironstores within the RES.
As the amount of iron in the cell increases,a larger percentage
deposits in hemosiderin,an insoluble, aggregated form of partially
di-gested ferritin. Diversion of excess iron intohemosiderin
permits storage of more iron perunit volume in the cell, and, in
fact, the highestconcentrations of hemosiderin in the body arefound
in the RES (Bothwell et al., 1979).
V. IRON RELEASE BY THE RES
A. Iron Release and Plasma Iron
Normal adult human plasma containsabout 3 to 4 mg of iron,
essentially all bound
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to transferrin. About 80% of the circulatingiron is en route
between the RES and thebone marrow. Small amounts of plasma ironare
contributed by hepatic iron stores andby the absorption of dietary
iron from theduodenum, but most circulating iron is con-tributed by
the RES through the release ofiron from catabolized senescent red
cells(Figure 1). Cyclic fluctuations in RE ironrelease appear to
cause the pronounced cir-cadian variation in plasma iron
concentra-tions (Fillet et al., 1974). Neither the mecha-nism nor
the significance of this diurnalvariation in iron output from the
RES isknown.
B. Kinetics and Chemical Formsof Released Iron
In vivo ferrokinetic studies have charac-terized RE iron release
using trace amountsof 59Fe heat-damaged red blood
cells(59FeHDRBCs). After injection into the cir-culation,
59FeHDRBCs are rapidly scav-enged and processed by the RES. Studies
indogs (Fillet et al., 1974) and humans (Filletet al., 1989) show
that iron given in thismanner is released in two distinct phases:an
early phase, in which two-thirds of theiron freed from hemoglobin
is returned tothe plasma within the first few hours, and alate
phase, in which the remainder is re-leased from RE stores over days
and weeks.A similar biphasic pattern of iron releaseafter
erythrophagocytosis has been observedin isolated human monocytes
(Moura et al.,1998b) and macrophages (Custer et al.,1982), cultured
rat peritoneal macrophages(Saito et al., 1986), and Kupffer cells
(Kondoet al., 1988). The efficient release of eryth-rocyte-derived
iron appears to require hemecatabolism by HMOX1, as mice
lackingthis enzyme develop iron-deficiency ane-mia (Poss and
Tonegawa, 1997).
Most of the iron released into the plasmais bound by
transferrin. Studies of culturedmacrophages confirm that iron is
released asa low-molecular-weight species that readilybinds to
plasma transferrin (Haurani andOBrien, 1972; Kondo et al., 1988;
Rama etal., 1988; Moura et al., 1998b). A number ofstudies also
indicate that RE cells releasesignificant amounts of
erythrophagocytosediron in the form of hemoglobin (Custer et
al.,1982; Saito et al., 1986; Kondo et al., 1988;Costa et al.,
1998; Moura et al., 1998b),heme (Kleber et al., 1981; Costa et al.,
1998),or ferritin (Kleber et al., 1981; Custer et al.,1982; Kondo
et al., 1988; Rama et al., 1988;Moura et al., 1998b). It has been
speculatedthat hemoglobin release results from mac-rophage cell
death after the ingestion of toomany erythrocytes (Kondo et al.,
1988),whereas others argue that hemoglobin re-lease represents a
normal physiological pro-cess (Custer et al., 1982; Moura et al.,
1998b).Interestingly, Moura et al. (1998b) note thatmost early
release consists of hemoglobin,whereas ferritin and
low-molecular-weightiron are the main forms released
subsequently.
C. Effect of Transferrin andCeruloplasmin
Because most of the iron recycled bythe RES after
erythrophagocytosis binds tocirculating transferrin, studies have
ad-dressed whether the iron-binding capacityof transferrin affects
iron mobilization. In-creasing plasma iron-binding capacity
byinjecting apotransferrin into rats does notaffect the release of
radioiron after infusionof 59FeHDRBCs (Lipschitz et al.,
1971).Similarly, apotransferrin had no effect oniron release after
erythrophagocytosis byisolated rat peritoneal macrophages (Saitoet
al., 1986). In other studies, however, thepresence of
apotransferrin slightly increased
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FIG
URE
1.
Rec
yclin
g of
ery
thro
cyte
iron
by
the
RES
in re
latio
n to
oth
er p
athw
ays
of in
tern
al ir
onex
chan
ge.
Phag
ocyt
osis
of s
enes
cent
ery
thro
cyte
s is
perfo
rmed
prim
arily
by
RE m
acro
phag
es lo
cate
d in
the
sple
en, l
iver,
and
bone
mar
row.
Nu
mbe
rs in
dica
te th
e ap
prox
imat
e da
ily fl
ow o
f iro
n th
roug
h ea
chpa
thwa
y (B
othwe
ll et a
l., 19
74).
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iron efflux from rat Kupffer cells (Kondo etal., 1988) or
dramatically enhanced ironrelease from rat bone marrow
macrophages(Rama et al., 1988). Decreasing plasma iron-binding
capacity by intravenous iron infu-sion before administering
59FeHDRBCs hasbeen shown to reduce RE iron release inmost
(Lipschitz et al., 1971; Bergamaschiet al., 1986; Siegenberg et
al., 1990) but notall studies (Fillet et al., 1974). A slight
sup-pression in iron release from isolated ratbone marrow
macrophages incubated withsaturated transferrin has also been
reported(Rama et al., 1988). Thus, although thesestudies do not
allow definitive conclusionsto be drawn regarding the effect of
iron-binding capacity of transferrin on iron re-lease, it should be
noted that neither theprotein nor its iron-binding capacity
appearsto be essential for RE iron release. Culturedmacrophages can
release iron in the ab-sence of apotransferrin in the culture
media(Saito et al., 1986; Kondo et al., 1988; Mouraet al., 1998b),
and patients with hemochro-matosis or bone marrow aplasia can
releaseiron despite transferrin saturation (Fillet etal., 1989).
Moreover, the conspicuous lackof iron accumulation in the spleen
ofhypotransferrinemic mice, which can sur-vive at least 9 months
without exogenoustransferrin injections (Trenor et al., 2000),also
suggests that iron is recycled throughthis organ in the virtual
absence of transfer-rin.
Normal iron release does seem to requireceruloplasmin, a
multicopper ferroxidase.Early studies showed that
copper-deficientpigs developed iron-deficiency anemia de-spite
having normal or elevated iron stores(Lee et al., 1968). The iron
deficiency ap-peared to result from inefficient release ofiron from
the RES because serum iron con-centrations did not increase
significantlyafter intravenous administration of
damagederythrocytes. The subsequent observationthat the defective
iron mobilization in cop-
per deficiency could be promptly correctedby the intravenous
administration of cerulo-plasmin (Ragan et al., 1969) indicated
thatthis protein plays a role. Ceruloplasminappears to mobilize
iron from storage sitesby catalyzing the oxidation of ferrous
ironto the ferric form, which can be incorpo-rated into
apotransferrin (Osaki et al., 1971).More direct evidence for
ceruloplasminsrole in RE iron release is provided by stud-ies of
aceruloplaminemic (Cp/) mice (Har-ris et al., 1999), which have
normal coppermetabolism (Meyer et al., 2001). As in
cop-per-deficient animals, serum iron concen-trations of Cp/ mice
do not change signifi-cantly after the administration of damagedred
cells, but do increase after the adminis-tration of ceruloplasmin
and not apocerulo-plasmin (Harris et al., 1999). The observa-tion
that Kupffer cells of Cp/ mice displaymarkedly increased iron
levels (Harris etal., 1999) is also consistent with a role
forceruloplasmin in RE iron release. Curiously,Cp/ mice do not
develop iron-deficiencyanemia, indicating that other sources
offerroxidase activity capable of mobilizingiron from storage sites
exist in this animalmodel. Patients with
aceruloplasminemiaconsistently present with
mild-to-moderateiron-deficiency anemia (Miyajima et al.,1987;
Yoshida et al., 1995), sometimes as-sociated with iron loading in
Kupffer cells(Logan et al., 1994; Bosio et al., 2002;Hellman et
al., 2002). Future work is neededto determine whether ceruloplasmin
pro-motes RE iron release extracellularly or in-tracellularly.
Recent gene mapping studies have iden-tified a ceruloplasmin
homologue, hephaestin,that is expressed predominantly in the
smallintestine (Vulpe, 1999). Mutations inhephaestin result in
impaired iron exportfrom the duodenum into the portal circula-tion,
producing the phenotype of the sex-linked anemia (sla) mouse.
Althoughhephaestin mRNA has been detected in the
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spleen (Frazer et al., 2001), its potentialinvolvement in iron
export from the REShas not been studied. The observation thatthe
anemia of the sla mouse is rapidly cor-rected by a single
intraperitoneal injectionof iron dextran (Bannerman and
Cooper,1966), which is taken up and recycled bythe RES, suggests
that hephaestin is notnecessary for RE iron release.
D. Potential Roles forFerroportin1 and Nramp1
Ferroportin1 (FPN1) is a 62-kDa iron-export protein with 9 or 10
predicted trans-membrane regions (Donovan et al., 2000).The protein
is also known as iron-regulatedtransporter 1, IREG1 (McKie et al.,
2000)and metal transporter protein 1, MTP1(Abboud and Haile, 2000).
FPN1 mRNAcontains an IRE sequence in the 5UTR,suggesting that iron
regulates its expressionin a manner similar to ferritin. Northern
blotanalyses of the tissue distribution of humanand mouse FPN1 mRNA
reveals most abun-dant expression in liver, spleen, kidney,
pla-centa, and duodenum (Abboud and Haile,2000; Donovan et al.,
2000; McKie et al.,2000). Immunostaining indicates
particularlystrong FPN1 expression in hepatic Kupffercells and
splenic macrophages (Abboud andHaile, 2000; Donovan et al., 2000).
Recentdouble immunofluorescence staining usingantibodies to FPN1
and F4/80, a macroph-age-specific cell surface antigen, has
con-firmed the localization of FPN1 to RE cellsin liver, spleen,
and bone marrow (Yang etal., 2002).
Several lines of evidence indicate thatFPN1 functions as an iron
exporter in vari-ous cell types. First, in duodenal mucosalcells
and syncytiotrophoblasts of the pla-centa, FPN1 localizes to the
site of ironexport, the basolateral membrane. Second,
FPN1 mutations in zebrafish are associatedwith severe
iron-deficiency anemia, whichcan be partially rescued by exogenous
ex-pression of wild-type FPN1 (Donovan etal., 2000). Third,
iron-loaded Xenopus oo-cytes injected with FPN1 cRNA
displayincreased iron release (Donovan et al., 2000;McKie et al.,
2000), and HEK293T cellstransfected with FPN1 cDNA have de-creased
levels of cytosolic iron (Abboudand Haile, 2000). Incidentally, it
should benoted that increased iron efflux after FPN1expression in
Xenopus required the pres-ence of either ceruloplasmin (McKie et
al.,2000) or high concentrations of transferrin(Donovan et al.,
2000) in the culture media.
The expression profile of FPN1 in mac-rophages of the liver,
spleen, and bonemarrow implicates the protein in iron recy-cling by
the RES. Consistent with this pos-sibility is the observation that
loading theRES with iron dextran enhances mouseKupffer cell FPN1
expression (Abboud andHaile, 2000). In this case, the increased
FPN1expression may serve to export the acquirediron. However, it
remains to be determinedhow FPN1 expression changes in responseto
erythrophagocytosis. Causal relationshipsbetween RE iron release
and FPN1 expres-sion also need to be demonstrated. None-theless,
recent clinical reports continue tostrengthen the link between FPN1
and REiron metabolism. Patients with FPN1 muta-tions exhibit an
autosomal dominant formof hemochromatosis (Montosi et al.,
2001;Njajou et al., 2001; Devalia et al., 2002;Roetto et al., 2002)
in which hepatic ironloads primarily in Kupffer cells (Devalia
etal., 2002; Pietrangelo, 2002; Roetto et al.,2002). One caveat is
that an iron exportprotein would be expected to reside exclu-sively
at the plasma membrane, whereas theavailable immunofluorescence
data inKupffer cells and RAW267.4 cells indicatean intracellular
distribution of FPN1. There-fore, it is possible that FPN1 mediates
intra-
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cellular transit of iron released by hemecatabolism after
erythrophagocytosis.
As discussed above, Nramp1 has alsobeen implicated in iron
release. This pos-sible function has been studied in
RAW264.7macrophage lines stably transfected witheither wild-type
Nramp1G169 or nonfunc-tional mutant Nramp1D169. No differencesin
iron release were found between the twocell lines after loading
with iron using 55Fe-citrate (Kuhn et al., 1999),
59Fe-transferrin(Mulero et al., 2002a), or
59Fe-transferrin-anti-transferrin immune complex (Biggs etal.,
2001; Mulero et al., 2002a), which isphagocytosed by the
macrophage. How-ever, after loading with
59Fe-transferrin-anti-transferrin immune complex andtreatment with
interferon- and li-popolysaccharide, macrophages express-ing
functional Nramp1 released signifi-cantly more iron than those
expressingnonfunctional Nramp1 (Biggs et al., 2001;Mulero et al.,
2002a). Interestingly, therelease of iron from the phagocytosed
im-mune complex is reduced if the activity ofinducible nitric oxide
synthase (iNOS) isinhibited (Biggs et al., 2001; Mulero etal.,
2002a; Mulero et al., 2002b). Bonemarrow macrophages from mice
lackingiNOS also have reduced iron release(Mulero et al., 2002b),
further indicatingthat NO influences iron efflux. Althoughthese
studies suggest a role for Nramp1 iniron release, the localization
of this pro-tein to the phagolysosome makes it anunlikely candidate
for performing the ul-timate step in iron export from the REcell.
Moreover, it remains to be deter-mined if the RE cell handles the
transfer-rin-anti-transferrin immune complexiron in the same
fashion as erythro-phagocytosed iron, which must first beliberated
by ER-bound HMOX. As thesestudies indicate, the precise roles of
FPN1and Nramp1 in iron release from the RESremain to be better
defined.
E. Regulation of Iron Release
Marrow iron requirements appear to bean important factor in the
physiological regu-lation of iron release from the RES. Whenbody
(marrow) requirements increase, as iniron deficiency or
venesection, iron releaseincreases (Noyes et al., 1960; Beamish
etal., 1971; Lipschitz et al., 1971). Withinhours after being given
59FeHDRBCs, iron-deficient individuals released 100% of theiron,
whereas normal subjects had a meanrelease of 64% (Fillet et al.,
1989). Con-versely, decreased marrow requirementsresulting from
either hypertransfusion (Finchet al., 1982) or bone marrow aplasia
(Filletet al., 1989) are associated with decreasediron release.
Interestingly, patients withaplasia and suppressed erythropoiesis
stillrelease 22% of iron in the early phase. Asnoted by Fillet et
al. (1989), this releasemay represent the limit of the RES to
retainiron from recycled erythrocytes. How a dis-tant stimulus from
the bone marrow regu-lates RE iron release is not
understood.Recently, Pietrangelo (2002) has proposedthat the extent
of transferrin saturation re-lays information about the iron status
of thebone marrow to the RES. Another modelwith a signaling role of
transferrin satura-tion, in combination with levels of
solubletransferrin receptor in plasma, has been sug-gested by
Townsend and Drakesmith (2002).
VI. PERTURBATIONS OF REIRON METABOLISM
A. Hereditary Hemochromatosis(HH)
In normal individuals, any dietary ironabsorbed in excess
deposits in roughly equal
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amounts in parenchymal cells of the liverand RE cells. In
contrast, the abnormallyelevated iron absorption of HH patients
leadsto preferential iron accumulation in the pa-renchymal cells of
the liver; it is only late inthe disease that iron starts to accrue
in theKupffer cells of the liver and RE cells of thebone marrow
(Bothwell et al., 1965; Valberget al., 1975; Brink et al., 1976).
This uniquepattern of iron deposition in HH suggeststhat there is a
defect in the RE cells abilityto accumulate iron. Indeed, because
intesti-nal iron absorption is inversely related toRE iron stores
(Rosenmund et al., 1980),abnormal iron handling by RE cells mightbe
responsible for both excess deposition inparenchymal cells and the
lack of feedbackregulation of duodenal iron uptake (Valberg,1978).
Such a defect could result from analtered ability of the RE cell to
acquire,store, or release iron. Decreased erythroph-agocytosis,
which has been observed incultured monocyte-derived macrophagesfrom
HH patients (Moura et al., 1998b;Moura et al., 1998a), may indicate
a defectin iron acquisition from senescent erythro-cytes. No
abnormalities in iron acquisitionfrom transferrin have been
identified in stud-ies of HH monocytes (Jacobs and Summers,1981;
Sizemore and Bassett, 1984).
Given that one of the normal functionsof the RES is to store
iron, it has beenspeculated that the low RE iron levels inHH result
from defective synthesis of theiron storage protein ferritin.
Studies of HHmonocytes incubated with transferrin-boundiron,
however, have failed to detect anyabnormalities in their ability to
synthesizeferritin or to incorporate iron into the stor-age protein
(Jacobs and Summers, 1981;Bassett et al., 1982). Recently, Cairo et
al.(1997) have studied the activity of IRP, theintracellular
regulator of ferritin synthesis,in monocytes from HH patients.
Unexpect-edly, they found that HH monocyte IRPactivity was 50%
higher than normal. The
increased activity does not appear to be dueto an inherent
defect in IRP control, be-cause changes in cellular iron status
modu-lated IRP activities similarly in HH mono-cytes as in
controls. As noted by Cairo et al.(1997), the increased IRP
activity likelyreflects a reduction in the labile iron pool,which
could be due to either decreased ironuptake or increased release.
The increase inIRP activity would be expected to decreaseferritin
mRNA translation and thus maycontribute to the inability of the RE
cell tostore iron in ferritin.
Fillet et al. (1989) used 59FeHDRBCs tostudy in vivo iron
release from the RES ofHH patients. In these patients, the early
iron-release phase was similar to that of healthyindividuals, but
did not negatively correlatewith iron stores as it did in normal
subjects.This result suggests that the RES in HH isunable to
efficiently downregulate iron re-lease in the face of high iron
stores. Abnor-mally elevated iron release from the RESthus may
contribute to the high serum ironlevels characteristic of HH. Using
isolatedmonocytes from HH patients, Moura et al.(1998b)
investigated iron efflux after eryth-rophagocytosis. Similar to the
in vivo stud-ies of Fillet et al. (1989), iron release wasidentical
in control and HH monocytes;however, HH monocytes released twice
asmuch iron in a low-molecular-weight formas did control cells.
Moura et al. (1998b)speculate that the released
low-molecular-weight iron, which readily binds to transfer-rin, may
contribute to the high plasma trans-ferrin saturation and
nontransferrin boundiron observed in HH patients.
Significantlyincreased ferritin release by HH monocyteshas also
been reported (Flanagan et al.,1989).
The recent discovery of the genetic ba-sis of HH is providing
insight into the ab-normal RE iron metabolism in the disease.The
majority of HH cases are caused by amutation of amino acid 282
(C282Y) in the
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HFE gene (Feder et al., 1996). HFE en-codes a protein similar in
structure to MHCclass I molecules in that it associates
with2-microglobulin at the cell surface. TheC282Y mutant protein
demonstrates dimin-ished binding with 2-microglobulin anddecreased
cell-surface expression (Waheedet al., 1997). Functional HFE
protein ap-pears to be required for normal iron deposi-tion in the
RES, as mice without HFE (Zhouet al., 1998; Levy et al., 1999) or
with C282YHFE (Levy et al., 1999) do not accumulateappreciable
amounts of iron in Kupffer cellsand in the spleen, despite hepatic
iron over-load. HFE protein is abundantly ex-pressed in monocytes
(Parkkila et al.,1997), tissue macrophages (Parkkila etal., 1997),
and Kupffer cells (Bastin etal., 1998; Griffiths et al., 2000). In
mono-cytes from HH patients, the C282Y pro-tein is detectable by
immunohistochem-istry, but at reduced levels (Parkkila etal.,
2000). Although the exact functionof HFE remains unknown
(Philpott,2002), its association with the transfer-rin receptor
(Parkkila et al., 2000) impli-cates its involvement in the
metabolismof transferrin-bound iron. Support for thisrole is
provided by a study showing thatmonocyte-derived macrophages from
HHpatients accumulate less iron from trans-ferrin than macrophages
from normal in-dividuals (Montosi et al., 2000). Theadditional
demonstration that the HHmacrophages accumulated 50%
moretransferrin-iron after transfection withwild-type HFE directly
implicates a rolefor HFE in RE iron accumulation. Thesefindings
suggest that, in these cells, HFEeither enhances the uptake of iron
ordecreases its release. Townsend andDrakesmith (2002) have
proposed amodel in which HFE not associated withthe transferrin
receptor inhibits RE ironrelease by inhibiting FPN1.
B. Anemia of Chronic Disease(ACD)
Patients with infection, inflammation,or other chronic diseases
often develop amild-to-moderate anemia after severalmonths. This
type of anemia is most com-monly known as ACD; other
designationsinclude anemia of chronic disorders (Lee,1993), anemia
of inflammation (Schilling,1991), primary defective
iron-reutilizationsyndrome (Besa et al., 2000), and hypo-ferremic
anemia with reticuloendothelialsiderosis (Cartwright and Lee,
1971). Thelow serum iron concentrations and anemiaof ACD appear to
result primarily from thedecreased flow of iron from cells to
plasma.Although diminished iron flux occurs inenterocytes (Cortell
et al., 1967) and hepa-tocytes (Hershko et al., 1972), the
decreasediron flow from RE cells is most importantquantitatively.
Impaired RE iron releasefrom 59FeHDRBCs has been observed in
ratmodels of acute infection (Kampschmidt etal., 1964) and
inflammation (Konijn andHershko, 1977). Similarly, ferrokinetic
stud-ies using 59FeHDRBCs in patients with in-flammation
demonstrate that the early ironrelease phase is decreased about 20%
(Filletet al., 1989). This modest decrease in REiron release may
account for the mild andnonprogressive nature of the anemia in
ACD.The inhibition of iron release in vitro has alsobeen observed
in inflammatory mouse peri-toneal macrophages (Esparza and
Brock,1981) and J774 macrophages treated withlipopolysaccharide
(Mulero and Brock, 1999).
The molecular mechanisms responsiblefor the decreased iron
release from the RESremain unidentified. Early studies
suggestedthat ferritin levels, which increase markedlyin
inflammatory and malignant conditions,impair release by diverting
iron into storage(Konijn and Hershko, 1977). However,
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studies in mouse peritoneal macrophagesfound that the reduced
iron release afterinjection of an inflammatory agent was
as-sociated with decreased ferritin synthesis(Alvarez-Hernandez et
al., 1986). The di-version of iron into more inert storage
formssuch as hemosiderin, which did increaseafter inflammation, was
thus proposed as amechanism for impaired release (Alvarez-Hernandez
et al., 1986). Various cytokines,especially tumor necrosis factor
alpha andinterleukin-1, have also been implicated inthe impairment
of RE iron release, but re-sults from different groups are
inconsistent(Kondo et al., 1988; Alvarez-Hernandez etal., 1989;
Uchida et al., 1991; Mabika andLaburn, 1999). Recent studies
suggest thatdownregulation of FPN1 plays a role. Usinga model of
acute inflammation in mice, Yanget al. (2002) found that treatment
withlipopolysaccharide resulted in a downregu-lation of FPN1
expression in RE cells of thespleen, liver, and bone marrow. Time
courseexperiments revealed that the LPS-inducedhypoferremia
preceded the downregulationof splenic FPN1 protein levels,
indicatingthat the initial hypoferremia results frommechanisms
other than FPN1 in the spleen.Yang et al. (2002) speculate that
downregu-lation of splenic FPN1 may serve to main-tain the
hypoferremia rather than induce it.
C. Possible Role of Hepcidin
It is interesting to note that the perturba-tions in RE iron
metabolism in HH andACD are, for the most part, exactly oppo-site.
Recent studies have led to the proposalthat this reciprocal
regulation may be medi-ated a novel plasma peptide called
hepcidin(Fleming and Sly, 2001). Also known asLEAP-1
(liver-expressed antimicrobial pep-tide) (Krause et al., 2000),
hepcidin is syn-thesized by the liver in the form of an 84
amino acid propeptide and is detected in theplasma as a peptide
of 25 amino acids(Krause et al., 2000). A link betweenhepcidin and
iron metabolism was first madeby Pigeon et al. (2001), who
demonstratedthat hepatic hepcidin mRNA levels increasedwith various
forms of iron loading and de-creased with iron deprivation.
Subsequently,Nicolas et al. (2001) observed that micelacking
hepcidin develop severe tissue ironoverload. Based on these two
studies,Nicolas et al. (2001) proposed that hepcidinmay serve as an
iron-status signaling mol-ecule between tissues involved in iron
mo-bilization. According to this model, an iron-loaded liver would
secrete increased amountsof hepcidin into the plasma, which in
turnwould signal the intestine to downregulateiron absorption and
the RES to downregulateiron release. The demonstration that
li-popolysaccharide, a classic inducer of theinflammatory response,
also enhanced he-patic hepcidin mRNA expression raised
thepossibility that the diminished iron absorp-tion and impaired RE
iron release of ACDare mediated through changes in plasmahepcidin
levels. This connection has beenstrengthened by recent studies
showing in-creased hepatic hepcidin mRNA levels inanimal models of
infection (Shike et al.,2002) and in anemic patients with
hepaticadenomas (Weinstein et al., 2002). On theother hand, in the
absence of hepcidin, in-testinal iron absorption and RE iron
releasewould be expected to continue unabated asliver iron
accumulates. Over time, this wouldrecapitulate the cardinal
features of HH:abnormally increased iron absorption, el-evated
plasma iron levels, and increasediron deposition in the hepatic
parenchymalcells, but not in the RES. Indeed, all of thesefeatures
are displayed by mice lackinghepcidin (Nicolas et al., 2001;
Nicolas etal., 2002). Although these studies are con-sistent with
the hypothesis that plasmahepcidin can mediate the perturbations
of
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iron metabolism characteristic of both HHand ACD, future studies
will need to deter-mine plasma hepcidin levels in these dis-ease
states affecting the RES.
VI. UNANSWERED QUESTIONS
Figure 2 summarizes our understandingof the major pathways of
iron handling bythe RE cell. As indicated by the figure
andthroughout this review, several key ques-tions remain:
Where is iron liberated from heme? If lib-erated at the
endoplasmic reticulum, dointracellular heme transporters exist? If
freedwithin the phagolysosome, what transporteris responsible for
the efflux of iron into thecytosol (Nramp1, DMT1, FPN1, or
someother factor)?
How is iron released? Does FPN1 exportiron from the RE cell as
it appears to do forother cell types? Does FPN1 act at theplasma
membrane or does it function withinthe cell? Does the release of
hemoglobin,heme, and ferritin represent a normal physi-
ologic process? If so, how significant istheir release in
quantitative terms?
How is iron release coordinated with bodyiron status? Do the
plasma proteins trans-ferrin or hepcidin serve as signaling
mol-ecules between the bone marrow and theRE cell? If hepcidin
plays such a role, whatchanges does it elicit in the RE cell?
Doesit interact with or regulate FPN1, HFE, and/or transferrin
receptor?
What molecular mechanisms mediate theperturbations in RE iron
metabolism thatcharacterize HH and ACD? What iron re-lease pathways
are upregulated in HH anddownregulated in ACD? Is hepcidin amarker
or a mediator of these changes?
While the recent discoveries of Nramp1,DMT1, HFE, FPN1, CD163,
and hepcidinhave significantly advanced our knowledgeof iron
metabolism in the RES, it is clearthat these outstanding questions
(and oth-ers) need to be addressed. Given the rapidadvances in
characterizing the proteins re-sponsible for iron transport, the
molecularpathways mediating the movement of ironinto and out of the
RES should soon berevealed.
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FIG
URE
2.
Iron
hand
ling
by th
e RE
cel
l.
Ques
tion
mar
ks in
dicat
e th
at e
ither
the
path
way o
r the
tran
spor
t mec
hanis
m h
as n
ot b
een
elucid
ated
.Ar
row
s w
ith d
otte
d lin
es in
dica
te fo
rms
of ir
on re
leas
ed b
y th
e ce
ll.
CD16
3, he
mog
lobi
n sc
aven
ger r
ecep
tor;
CP, ce
rulo
plas
min
; DM
T1, di
vale
ntm
eta
l tra
nspo
rter
1; F
PN1,
fe
rropo
rtin;
Hb,
he
mog
lobi
n;
Hp,
ha
ptog
lobi
n; H
MO
X, he
me
oxyg
enas
e; N
ram
p1, natu
ral
resi
stan
ce-a
ssoc
iate
dm
acr
oph
age
prot
ein
1; T
F, tra
nsfe
rrin;
TFR
, tra
nsfe
rrin
rece
ptor
.
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