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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: mknutson@hsph.harvard.edu

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

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