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Dendritic CellsMonocytes, Intestinal Macrophages, andfor IgG Is Functionally Expressed in MHC Class I-Related Neonatal Fc Receptor

Richard S. BlumbergRobert, Benyan Wu, Phillip D. Smith, Wayne I. Lencer and

CarolineLi, Emiko Mizoguchi, Lili Miao, Yuansheng Wang, Xiaoping Zhu, Gang Meng, Bonny L. Dickinson, Xiaotong

http://www.jimmunol.org/content/166/5/3266doi: 10.4049/jimmunol.166.5.3266

2001; 166:3266-3276; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2001 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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MHC Class I-Related Neonatal Fc Receptor for IgG IsFunctionally Expressed in Monocytes, Intestinal Macrophages,and Dendritic Cells1

Xiaoping Zhu,* Gang Meng,§ Bonny L. Dickinson,‡ Xiaotong Li,† Emiko Mizoguchi,†

Lili Miao,* Yuansheng Wang,† Caroline Robert,2† Benyan Wu,* Phillip D. Smith,§

Wayne I. Lencer,‡ and Richard S. Blumberg3*

The neonatal Fc receptor (FcRn) for IgG, an MHC class I-related molecule, functions to transport IgG across polarized epithelialcells and protect IgG from degradation. However, little is known about whether FcRn is functionally expressed in immune cells.We show here that FcRn mRNA was identifiable in human monocytes, macrophages, and dendritic cells. FcRn heavy chain wasdetectable as a 45-kDa protein in monocytic U937 and THP-1 cells and in purified human intestinal macrophages, peripheral bloodmonocytes, and dendritic cells by Western blot analysis. FcRn colocalized in vivo with macrosialin (CD68) and Ncl-Macro, twomacrophage markers, in the lamina propria of human small intestine. The heavy chain of FcRn was associated with theb2-microglobulin (b2m) light chain in U937 and THP-1 cells. FcRn bound human IgG at pH 6.0, but not at pH 7.5. This binding couldbe inhibited by human IgG Fc, but not Fab. FcRn could be detected on the cell surface of activated, but not resting, THP-1 cells.Furthermore, FcRn was uniformly present intracellularly in all blood monocytes and intestinal macrophages. FcRn was detectableon the cell surface of a significant fraction of monocytes at lower levels and on a small subset of tissue macrophages that expressedhigh levels of FcRn on the cell surface. These data show that FcRn is functionally expressed and its cellular distribution isregulated in monocytes, macrophages, and dendritic cells, suggesting that it may confer novel IgG binding functions upon thesecell types relative to typical FcgRs: FcgRI, FcgRII, and FcgRIII. The Journal of Immunology,2001, 166: 3266–3276.

T he neonatal Fc receptor (FcRn)4 is structurally related tothe MHC class I family (1–3) and consists of a mem-brane-bound heavy chain (45 kDa for human, 51 kDa for

rodents) in nonconvalent association withb2-microglobulin (b2m;12 kDa). FcRn was originally characterized as a transport receptorinvolved in the uptake of maternal IgG by an intestinal route inrodents (4–8) and probably via syncytiotrophoblastic cells withinhuman placenta, respectively (9–13). Additionally, FcRn has beenconsidered to function in the protection of IgG from degradation.This idea was first proposed by Brambell (14) and is supported by

recent observations that mice deficient inb2m exhibit significantreduction in the serum half-life of IgG (15–17). Recent evidencefor FcRn expression by endothelial cells suggested that this may bethe cell type most prominently involved in IgG protection (18).

A hallmark of FcRn interaction with its ligand is its strictlypH-dependent IgG binding in both epithelial and endothelial cells.FcRn preferentially binds IgG at acidic pH (6–6.5), but is unableto bind IgG at neutral pH (7–7.4) (19–21). FcRn is expressed in avariety of cell types and tissues, including intestinal epithelial cells(IECs) of neonatal rodents, syncytiotrophoblasts of humans, en-dothelial cells of adult rodents and humans, adult rat hepatocytes,and adult epithelial cells of bovine mammary gland, human intes-tine, and human kidney (22–27).

Immune cells, such as B lymphocytes, macrophages, dendriticcells, NK cells, mast cells, and granulocytes, typically express sin-gle or multiple receptors for the Fc portion of IgG, includingFcgRI (CD64), FcgRII (CD32), FcgRIII (CD16), and their splicevariants. These FcgRs play a pivotal role in linking the cellular andhumoral arms of the immune response. Specifically, these recep-tors are involved in internalization of immune complexes, Ag pre-sentation, Ab-dependent cellular cytotoxicity, negative regulationof effector functions of FcgR-bearing cells, regulation of the in-flammatory cascade, and autoimmunity (28–31). However, FcRnexpression has not been characterized in immune cells, especiallyin FcgR1 cells. Therefore, we tested the hypothesis in this studythat FcRn is functionally expressed in human immune cells. Wefound by several criteria that FcRn was expressed in human mono-cytes, macrophages, and dendritic cells and in human monocyticcell lines and exhibits pH-dependent binding of IgG in these cells.Moreover, the cellular distribution of FcRn expression betweenintracellular and cell surface locations appears to be differentiallyregulated. These studies indicate that FcRn is the fourth FcR for

*Gastroenterology Division, Brigham and Women’s Hospital, and†Departments ofMedicine and Pathology, and‡Combined Program in Pediatric Gastroenterology andNutrition, Children’s Hospital, Harvard Medical School, Boston, MA 02115; and§Department of Medicine, University of Alabama and Veteran’s Affairs Medical Cen-ter, Birmingham, AL 35294

Received for publication August 22, 2000. Accepted for publication December20, 2000.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by research grants from the National Institutes of Health(DK/AI-53056 (to R.S.B. and W.I.L.), DK44319 and DK51362 (to R.S.B.), DK48107(to W.I.L.), and DK-47322, AI-41530, DK-54495, and DE-72621 (to P.D.S.)), aDepartment of Veterans Affairs Merit Review Award (to P.D.S.), and the HarvardDigestive Disease Center. X.Z. was supported by a Career Development Award fromthe Crohn’s and Colitis Foundation of American.2 Current address: Department of Dermatology, Institute Gustave, Roussy, 39 rueCamille, Desmoulin, 94805 Villejuif, France.3 Address correspondence and reprint requests to Dr. Richard S. Blumberg, Gastro-enterology Division, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA02115. E-mail address: rblumberg@rics.bwh.harvard.edu4 Abbreviations used in this paper: FcRn, neonatal Fc receptor;b2m, b2-microglobu-lin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; MFI,mean fluorescence intensity; IEC, intestinal epithelial cell; MIIC, MHC class IIcompartment.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

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IgG to be defined on macrophages and dendritic cells and signif-icantly extend the potential function of FcRn and the cell typesinvolved in the known functions of this novel MHC class I-likemolecule.

Materials and MethodsHuman cell lines and tissues

HeLa (cervical epithelial cell line), Jurkat (thymoma cell line), U937(monocyte cell line), Raji (B cell line), and 721.721 (HLA-A-, -B-, and-C-negative B cell line) were purchased from American Type Culture Col-lection (Manassas, VA). THP-1 (monocytic cell line), NK3.3 (NK cellline), and NKL (NK cell line) were gifts from Dr. Mark Birkenbach (Uni-versity of Chicago, Chicago, IL), Dr. Paul Anderson (Harvard MedicalSchool, Boston, MA), and Dr. Marco Colonna (Basel Institute for Immu-nology, Basel, Switzerland), respectively. The U937 (promonocytic cellline), Raji, and 721.721 cell lines were cultivated in suspension in RPMI1640 medium (Life Technologies, Gaithersburg, MD) supplemented with10% FCS, 1%L-glutamine, and 1% penicillin/streptomycin. A CD1d-trans-fected cell line, 721.721CD1d, generated by transfecting the 721.221 cellline with the full-length CD1day cDNA in the PSRaneo expression vector,was cultivated in the same medium supplemented with 500mg/ml G418(Life Technologies). The THP-1 cell line was cultivated in the same me-dium with 5 3 1025 M 2-ME (Sigma, St. Louis, MO). NK3.3 and NKLwere cultivated in 10% RPMI 1640 medium with 10% human serum. HeLacells were cultivated with 10% FCS in DMEM (Life Technologies). Cellviability was assessed by trypan blue dye exclusion.

Production of human FcRn domain-specific serum Abs

The human FcRn codons (11) corresponding to thea1 (1–87), thea2(88–177), and thea3 (178–274) domains were amplified by PCR andsubcloned into theEcoRI sites of the pGEX4T-1 (Amersham PharmaciaBiotech, Piscataway, NJ) expression vector. The primer pairs fora1 (59-CCGGAATTCGCAGAAAGCCACCTCTCCCT-39, 59-GGCGAATTCTCAACCTTTTCCCCCCAA-39),a2 (59-GGCGAATTCTACACTCTGCAGGGCCTGCT-39, 59-CGCGAATTCTCACTTCCACTCCAGGTTT-39),and a3 (59-CCGGAATTCGAGCCCCCCTCCAT, 59-GGCGAATTCGGAGGACTTGGCTGGAGATT-39) were used for amplification byPfupolymerase (Stratagene, La Jolla, CA). TheEcoRI site in the primers areunderlined, and human FcRn sequences are italicized. The plasmid encod-ing the full-length human FcRn, provided by Dr. Neil Simister (BrandeisUniversity, Waltham, MA), was used as a template. All subclones wereverified by sequencing. The production of recombinant proteins was per-formed by a method modified from that previously described (32) andanalyzed by SDS-PAGE electrophoresis. Five micrograms of the purifiedGST-a1, GST-a2, and GST-a3 proteins were respectively emulsified inCFA and injected s.c. into each BALB/c mouse. Mice were boosted twiceat 3-wk intervals with fusion protein emulsified with IFA. Sera were sam-pled 2 wk following the final dose. Furthermore, the immunization of rab-bits with purified fusion protein was performed by Charles River BreedingLaboratories (Wilmington, MA).

Isolation of lamina propria macrophages, blood monocytes, anddendritic cells

Lamina propria macrophages were isolated from surgical human normaltissue by neutral protease digestion of intestinal tissue sections with coun-terflow centrifugal elutriation as previously described (33, 34). Briefly,sections of normal human jejunum were incubated in 0.2 M EDTA (FisherScientific, Norcross, GA) plus 10 mM 2-ME (Sigma) to remove the epi-thelium, minced, and then treated twice (45 min, 200 rpm, 37°C) in RPMI(Mediatech, Washington, DC) containing 100mg/ml DNase and 75mg/mlof the neutral protease dispase (grade I; Roche, Indianapolis, IN) to releasethe lamina propria mononuclear cells (33). After straining to remove debrisand gradient sedimentation to remove residual nonmononuclear cells, thecells were separated into highly purified populations of lamina propriamacrophages and lymphocytes by counterflow centrifugal elutriation usinga J-6 M elutriation centrifuge (Beckman, Palo Alto, CA) (33, 34). The cellsisolated by this procedure contained,1% CD31 lymphocytes and dis-played the size distribution, morphological features, ultrastructure, andphagocytic activity of macrophages (33).

Peripheral blood monocytes were isolated from leukopaks from healthydonors by elutriation. Both cell populations were rested for 2 days inDMEM (Quality Biologicals, Gaithersburg, MD) plus 50 mg/ml gentami-cin and 10% human AB serum (Atlanta Biologicals, Atlanta, GA) beforestudy. Cell purity was assessed by flow cytometry as previously described(3–5) using mAbs against the following cell markers: HLA-DR, CD3,

CD13, CD14, CD20, and CD80 (Becton Dickinson, San Jose, CA) andCD103 and CD83 (Immunotech, Westbrook, ME). Isotype-matched irrel-evant mAbs were used as controls.

The monocyte-derived dendritic cells were obtained by a previouslydescribed method (35). Briefly, monocytes were isolated from PBMCs byadherence to plastic for 2 h and were cultured for 8 days in RPMI 1640(Life Technologies) supplemented with 10% FCS, 10 mM HEPES, 2 mML-glutamine, 53 1025 2-mecaptoethanol, penicillin (100 U/ml), strepto-mycin (100 mg/ml), recombinant human GM-CSF (100 U/ml), and recom-binant human IL-4 (1000 U/ml). The medium was replaced every 3–4days. After 8 days, cells displaying dendritic morphology and predomi-nantly expressing CD1a and HLA-DR, but that had lost most of the ex-pression of the monocyte marker CD14, were obtained. Immature dendriticcells were obtained by culturing the adherent fraction of normal humanPBMCs in the presence of GM-CSF and IL-4 for 3 days.

RT-PCR

Cells were pelleted and resuspended at 106 cells/ml in Tri-Reagent (Mo-lecular Research Center, Cincinnati, OH). Total RNA was extracted ac-cording to the method recommended by the manufacturer. First-strandcDNA was synthesized from 1mg of total RNA using Moloney murineleukemia virus reverse transcriptase (Promega, Madison, WI) and an oli-go(dT) primer (Promega) as recommended by manufacturer. The humanFcRn gene was amplified from cDNA by a primer pair (59-CCGGAATTCGCAGAAAGCCACCTCTCCCT, 59-CGGAATTCTTAGCAGTCGGAATGGCGGA-39) that containedEcoRI sites in the 59extension to facilitatecloning. Amplification was performed by hot start PCR using 35 cycleseach consisting of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min. Atthe end of the 35 cycles, samples were run for an additional 10 min at 72°Cand then maintained at 4°C until analyzed by agarose gel electrophoresis.The mRNA was also amplified by GAPDH-specific primers as an internalcontrol to monitor the quality of the RNA purification and cDNA synthesis.

Transfection of HeLa cells with plasmid encoding human FcRnand b2m

The FcRn codon (1–343) was amplified from an FcRn-containing plasmid(11) with the primer pair 59-ATAAGAATGCGGCCGCGGCAGAAAGCCACCTCTCCCT-39and 59-TGCTCTAGATTAGGCGGTGGCTGGAATCA-39. The upstream primer introduced aNotI site, and the downstreamprimer introduced anXbaI site to facilitate cloning (underline). Amplifi-cation was performed using Pfu DNA polymerase with initial heating to95°C for 5 min, followed by 35 cycles each consisting of 95°C for 1 min,58°C for 1 min, and 74°C for 1.5 min, and was terminated by a finalextension step at 72°C for 10 min. The PCR product was purified by aga-rose gel using a GeneClean II kit (Bio 101, Vista, CA). The DNA fragmentwas digested withNotI andXbaI and ligated into the plasmid pFlagCMV-1(Sigma) to generate the plasmid, pFlagCMVhFcRn. In this plasmid a Flagepitope (DYKDDDDK, single-letter amino acid code) was fused into the Nterminus of the FcRn gene. The plasmid pCDNAhb2m was constructed aspreviously described (36). The open reading frames of plasmidspCDNAhb2m and pFlagCMVhFcRn were verified by sequencing bothstrands to confirm the fidelity of amplification and cloning.

Transfection of HeLa was performed by electroporation (ElectroporatorII; Invitrogen, San Diego, CA) using 20mg of pFlagCMVhFcRn and 2mgof pCDNAhb2m to ensure thatb2m concentrations were not substrate lim-iting for FcRn expression. Transfected cells were grown under selectionwith 1 mg/ml of G418 (Life Technologies). Single colonies of transfectedHeLa were expanded under 500mg/ml of G418. Positive colonies wereconfirmed by Western blotting using the FcRn anti-a2-specific serum asdescribed. The chosen positive transfectant was designated HeLaFcRn1b2m.

Western blotting, immunoprecipitation, and immunofluorescence

Cell lysates were prepared in PBS with 0.5% Nonidet P-40, 0.5% sodiumdeoxycholate, and 0.1% SDS by adding a protease inhibitor cocktail (Sig-ma). A postnuclear supernatant was analyzed for total protein concentra-tions by the Bradford method with BSA as a standard (Bio-Rad, Hercules,CA). The proteins were separated on 12% SDS-PAGE gels under reducingconditions and transferred onto nitrocellulose (Schleicher & Schuell,Keene, NH). The membranes were blocked with 5% nonfat milk andprobed with mouse anti-human FcRna2 Ab (1/500) for 1 h, then withHRP-conjugated goat anti-mouse IgG Fc Ab (1/10,000). All blocking, in-cubation, and washing steps were performed in PBS containing 0.05%Tween 20 and 5% milk. The final product was visualized by ECL (Pierce,Rockford, IL).

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Immunoprecipitations were performed as previously described (22).Briefly, 5 3 105 log-phase-grown THP-1 and U937 cells were metaboli-cally labeled with 0.5 mCi oftrans-35S-labeled methionine and cysteine(ICN Biomedicals, Costa Mesa, CA) in methionine- and cysteine-freeRPMI 1640 medium (ICN Biomedicals) supplemented with 10% dialyzedFCS and incubated at 37°C for 5 h. After washing with PBS, cells werelysed in buffer (0.15 M NaCl, 1 mM EDTA, 50 mM Tris (pH 8), and 10mM iodoacetamide) with protease inhibitors and detergent as describedabove. Cells were lysed and subsequently centrifuged at 14,0003 g for 30min. Radioimmunoprecipitations were performed using mouse anti-a2-specific serum coupled to protein G-Sepharose beads (Pierce). Ab2mmAb (Sigma) was used to depleteb2m from cell lysates.

For immunofluorescence assays, HeLaFcRn1b2m was grown on glasscoverslips overnight. A total of 13 105 U937 and THP-1 were mountedonto adhesive microscope slides, air-dried, and fixed in 3.7% paraformal-dehyde. After washes, cells were permeabilized with 0.1% digitonin inPBS for 10 min at room temperature, washed, and blocked for 30 min atroom temperature with 10% heat-inactivated goat serum (Sigma) in PBS.Cells were then incubated with a mouse anti-a2-specific serum in PBS(1/250) containing 10% goat serum (Sigma) for 1 h at room temperature.Primary Ab was detected with an FITC-conjugated F(ab)2 goat anti-mouseAb (1/100) for 1 h at room temperature. As a negative control, cells wereincubated with normal mouse serum. Nuclei were stained with 0.1mg/ml4969-diamidino-2-phenylindole (Molecular Probes, Eugene, OR) in PBSfor 5 min. After final washes, cells were mounted. Images were capturedusing a fluorescence microscope (Microphot FXA; Nikon, Tokyo, Japan)and processed with Adobe Photoshop 5.0. Positive samples and negativecontrols were viewed using the same contrast and brightness settings.

IgG binding and Fc blocking assay

IgG Fc binding assays were performed as previously described (1, 18, 22)with the following modifications. Cells were lysed by shaking in sodiumphosphate buffer (pH 6.0 or 7.5) with 0.5% 3-[(3-cholamidopropyl)dim-ethylammonio]-1-propanesulfonate (CHAPS; Sigma) and protease inhibi-tor cocktail on ice for 1 h. Postnuclear supernatants containing 0.5–1 mg ofsoluble proteins was diluted with an equal volume of sodium phosphatebuffer containing 0.1% CHAPS and incubated with human IgG-Sepharose(Amersham Pharmacia Biotech) at 4°C for 4 h or overnight. The unboundproteins were removed with sodium phosphate buffer (pH 6.0 or 7.5) con-taining 0.1% CHAPS. The adsorbed proteins were eluted with sodiumphosphate buffer (pH 8) or boiled with electrophoresis sample buffer at100°C for 5 min. The eluted fractions were subjected to 12% reducingSDS-PAGE analysis. Proteins were visualized by Western blot using anti-a2-specific serum. For the blocking experiments, 250–500mg/ml of hu-man Fc or F(ab)2 (ICN Pharmaceuticals, Aurora, OH) were added to IgG-Sepharose beads before adding FcRn cell lysates. For the removal ofCD64, CD32, and CD16 molecules, cell lysates (pH 7.5) were incubatedwith protein G that was previously incubated with mAbs specific for CD64,CD32, and CD16 (Caltag, Burlingame, CA) at 4°C overnight with shaking.

Cell surface biotinylation

Cell surface biotinylation was performed as previously described (18).THP-1 and U937 cells (53 107) were suspended in 5 ml of PBS, pH 7.5,to which 2.5 ml of sulfo-NHS-biotin in PBS (1 mg/ml; Pierce) was added.The mixture was incubated at room temperature with rotation for 30 min.After washing with sodium phosphate buffer (pH 6.0) containing 0.1%CHAPS, the pellet was resuspended in 5 ml of sodium phosphate buffer(pH 6.0) with 0.5% CHAPS. A postnuclear supernatant was diluted 2-foldby sodium phosphate buffer (pH 6.0) with 0.1% CHAPS, then incubatedwith IgG-Sepharose. Following washings at pH 6.0, the bound protein waseluted in loading buffer at 100°C or with sodium phosphate buffer, pH 7.5.The eluted proteins were resolved by SDS-PAGE followed by blotting withstreptavidin-HRP (Pierce). To confirm the specificity, the proteins elutedwith sodium phosphate buffer were immunoprecipitated by mouse anti-a2-specific serum bound to protein G-Sepharose beads. Following incubationat 4°C on ice, the beads were washed, resuspended in loading buffer, re-solved by SDS-PAGE, transferred onto nitrocellulose, and blotted withstreptavidin-HRP. The final product was visualized using ECL (Pierce).

Flow cytometry

Surface and intracellular expressions of FcRn were examined in eitherfixed or permeabilized monocytes, macrophages, or THP-1 cells by flowcytometry. For staining, 13 106 cells were washed with PharMingen stainbuffer (FBS; PharMingen, San Diego, CA), followed by blocking with PBScontaining 10% normal goat sera (Jackson ImmunoResearch, West Grove,PA) on ice for 20 min. For surface Ag staining, 10ml of diluted anti-a2-specific serum was added to each tube and incubated for 20 min at room

temperature. Surface staining was also conducted at 4°C to minimize in-ternalization, and the results were identical with those observed at roomtemperature (data not shown). For intracellular staining, the cells were firstpermeabilized with Cytofix/Cytoperm (PharMingen) on ice for 20 min andthen washed with 13perm/wash buffer. Anti-a2-specific serum was addedas described. After washing, 20ml of 1/50 diluted goat anti-mouse IgG-FITC Ab (Jackson ImmunoResearch) was added to each tube and incu-bated at room temperature for 15 min. After washing, cells were fixed withCytofix and analyzed using a FACScan flow cytometer and CellQuest soft-ware (Becton Dickinson). The mouse IgG (0.5mg/million cells) was usedas a negative control.

Immunohistochemistry

Normal adult human small intestine was obtained from patients undergoinggastric bypass surgery under a protocol that was approved by the humanstudies committee of the Brigham and Women’s Hospital. Tissue was em-bedded in Tissue-Tek OCT compound (Sakura-Finite, Torrance, CA).Samples were sectioned on a Leach CM3050 cryomicrotome (Leica, Nus-sloch, Germany). A frozen section (5mm) was air-dried at room temper-ature, fixed in 4% paraformaldehyde in PBS, washed in PBS, and blockedin 10% nonimmune goat serum (Zymed, South San Francisco, CA). Sec-tions were stained with an affinity-purified FcRn-specific anti-peptide Ab(aa 174–188; provided by Dr. Neil Simister, Brandeis University) oragainst Ncl-Macro (Novocastra, Newcastle upon Tyne, U.K.) diluted inPBS containing 10% nonimmune goat serum and 0.02% Tween 20. Pri-mary Abs were detected with appropriate fluorophore-conjugated second-ary Abs for epifluorescence microscopy. All staining reactions were ac-companied by a negative control that consisted of an affinity-purified,isotype-matched irrelevant Ab. Sections were mounted in ProLong antifadereagent (Molecular Probes, Eugene, OR) and viewed with a Zeiss Axiophotmicroscope (Zeiss, New York, NY) equipped with a Spot digital camera(Diagnostic Instruments, Sterling Height, CA). Electronic images werecaptured and edited using Adobe Photoshop. The sections were stainedwith either mouse anti-a2-specific serum or an FcRn-specific anti-peptideAb and anti-CD68 (Santa Cruz Biotechnology, Santa Cruz, CA) using anavidin-biotin complex method (37).

ResultsGeneration of FcRn domain-specific Abs

To generate FcRn-specific serum Abs, we fused the codons cor-responding to thea1, a2, or a3 domains of FcRn in-frame to theGST gene. The anti-GST Abs in the mouse sera were removed byincubation with GST bound to glutathione-Sepharose beads. Seracontained only Abs specific for the FcRn as shown by Westernblot. We selected the GST-a2 for further immunization of rabbits.To show specificity of the mouse and rabbit anti-humana2-spe-cific serum Abs, we probed cell lysates from 721.221,721.221CD1d, Jurkat, and HeLa cells by Western blotting. Thea2domain-specific serum did not react with classical MHC or non-classical MHC class I-like CD1d molecules (data not shown). De-spite the 22–29% similarity between thea2 domains of MHC classI and FcRn (2), the anti-FcRn Abs recognized only a 45-kDa pro-tein from HeLa cells transfected with plasmids encoding bothFcRn heavy chain and humanb2m as defined by Western blotting(Fig. 1) and immunoprecipitation of metabolically labeled protein(data not shown). Mock-transfected HeLa cells were negative.

Expression of FcRn in macrophage and dendritic cells

Expression of FcRn heavy chains in immune cells was examinedby RT-PCR from a variety of cell lines and from isolated mono-cytes, macrophages, and dendritic cells with FcRn-specific prim-ers. The purity of the isolated cells is shown in Fig. 2. The resultsof RT-PCR screening are shown in Fig. 3. The amplified PCRproducts had a size similar to that of a product amplified from anFcRn-encoding plasmid or T84 cells, a polarized IEC line express-ing functional FcRn (25). Moreover, the FcRn heavy chain mRNAdetected in the human macrophage and dendritic cells had a DNAsequence identical with that previously described from human pla-centa as defined by sequencing five independent bacterial colonies(11) (data not shown). These results showed that human FcRn

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transcripts were expressed in monocytes, macrophages, and den-dritic cells, but not in NK, T, and B cell lines (Fig. 3).

Western blotting studies confirmed these results. Fig. 4A showsthat a band of 45 kDa was detected by the FcRn anti-a2-specificserum Abs in the promonocytic U937 and monocytic THP-1 celllines as well as in freshly isolated monocytes, macrophages, anddendritic cells. A band was also observed in transfected, but not inuntransfected, HeLa cells. It should be noted that the size of theband in the transfected HeLa cells was slightly larger than that ofthe band detected in the monocytic cell lines, monocytes, macro-phages, and dendritic cells due to a Flag epitope that was inserted

into the N terminus of the FcRn gene. Untransfected and trans-fected HeLa cells also exhibited two minor nonspecific bands withWestern blotting that migrated above the 45-kDa specific band(Fig. 4A). However, these were not observed when immunopre-cipitation of radiolabeled HeLahFcRn1b2m cells was performed(data not shown). Furthermore, Jurkat and untransfected HeLacells lacked this 45-kDa band. In Jurkat, a band smaller than 45kDa was detected, similar to a minor weak band present in themonocytic cell lines as well as macrophages and dendritic cells.This band probably represents a nonspecific immunoreactive pro-tein given the absence of FcRn-specific mRNA in Jurkat cells (Fig.3). In addition, immunoprecipitation of radiolabeled proteins fromthe Jurkat and Raji cell lines failed to show expression of FcRn(data not shown).

To further document the expression of FcRn in mononuclearcells, we performed immunofluorescence studies on the monocyticcell lines. Using the anti-a2-specific serum, we observed thatTHP-1 (Fig. 4B,panel C) and U937 (Fig. 4B,panel E) cell linesstained positively. For comparison, staining of the HeLaFcRn1b2m

transfectants is shown inpanel A. The negative control, normalmouse serum, failed to stain HeLa (data not shown), THP-1 (datanot shown), or U937 cells (Fig. 4B, panel G), and the anti-a2-specific serum failed to stain the untransfected HeLa cell line (datanot shown). Nuclear staining of all four cell lines is provided forreference (panels B,D, F, andH).

Association of human FcRn heavy chain withb2m in THP-1 andU937 cells

FcRn is expressed as a 45-kDa membrane-bound heavy chain innonconvalent association with 12-kDab2m. Immunoprecipitationof [35S]methionine- and [35S]cysteine-labeled THP-1 and U937

FIGURE 2. Purity of isolated dendritic cells andintestinal lamina propria macrophages.A, Primarylamina propria macrophages were isolated and pu-rified from normal human jejunum as described inMaterials and Methodsand then analyzed by flowcytometry for the indicated surface Ags. Gates wereset to include the total cell population. Insets displayFACS profiles using the CD-specific Abs with thefollowing purified control cells: CD103, intestinallymphocytes; CD14, blood monocytes; CD3, bloodlymphocytes; CD20, blood lymphocytes; and CD83,blood monocyte-derived dendritic cells. The FACSprofiles are representative of lamina propria macro-phages from intestinal tissue from a single donor(n 5 6). The MFI is shown on thex-axis, and therelative cell number on they-axis. B, Monocyteswere isolated from PBMCs and stimulated with re-combinant human GM-CSF and recombinant humanIL-4 for 8 days. After 8 days, cells displaying den-dritic morphology, as shown by forward and sidescatter, and predominantly expressed CD1a andHLA-DR but which had lost most of the expressionof the monocyte marker CD14 were isolated. Stain-ing is shown with specific mAbs directly conjugatedto PE.

FIGURE 1. Detection of human FcRn heavy chain by immunoblottingwith the mouse anti-a2-specific serum. Total cellular proteins (60mg) fromeither HeLamock or HeLaFcRn1b2m transfectant were resolved on a 12%SDS-PAGE gel under reducing conditions. Immunoblotting was performedwith the mouse anti-a2-specific serum and HRP-conjugated goat anti-mouse IgG, with detection accomplished by ECL. The arrow indicates theheavy chain of human FcRn. TheMr markers in kilodaltons are indicatedon theright.

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cell lysates with an anti-a2-specific serum produced two bands, 45-and 12-kDa proteins, that were coimmunoprecipitated by the pres-ence of anti-a2-specific serum, but not by preimmune serum (Fig.4C). This is consistent with an association between the FcRn heavychain andb2m in other cells. Further confirmation that the 12-kDaband wasb2m was obtained when lysates from metabolically la-beled THP-1 cells were subjected to three rounds of depletion withan anti-b2m mAb that removed the 12-kDa band from the autora-diogram. The molecular size of FcRn immunoprecipitated fromU937 lysates was slightly larger than that of FcRn from THP-1.The explanation for this result is not clear. However, it may reflectdifferent post-translational modifications of FcRn in the two celllines, which are immortalized at different stages of monocyte mat-uration. We also observed that the associatedb2m band did notappear to be stoichiometric with FcRn. Becauseb2m containsthree methionine and cysteine residues compared with the ninesuch residues contained in human FcRn, we believe that this is anartifact of the metabolic labeling technique. Another possibility isthat since mutations of theb2m molecule have been described intransformed cell lines (36), it may be that ab2m mutation hasoccurred in the monocytic cell lines, resulting in a low affinity ofassociation with FcRn. Therefore, we sequenced the cDNA ofb2mderived from the U937 cell line. The sequence aligned perfectlywith the sequence ofb2m deposited in GenBank (accession no.GI4757825), thus ruling out this possibility.

Colocalization of FcRn and macrophage markers in vivo

There is a large population of macrophages in the normal humanintestinal mucosa (33). To determine whether FcRn is expressed inmacrophages in vivo, we stained intestinal macrophages for FcRnand Ncl-Macro, a marker for human macrophages. Crypt and vil-lus enterocytes exhibited a punctate apical membranous stainingpattern for FcRn visible at the apical plasma membrane and in theapical cytoplasm (Fig. 5,a, e, andf, arrowheads) as previouslydescribed (25). Resident lamina propria macrophages also ex-pressed FcRn (Fig. 5a, arrow). FcRn staining was absent from bothenterocytes and macrophages in the presence of an irrelevant an-tiserum (Fig. 5b). Abs against Ncl-Macro specifically stained lam-ina propria macrophages (Fig. 5c, arrows), and this staining wasnot observed in the presence of an irrelevant isotype-matched mAb(Fig. 5d). Double labeling with both anti-FcRn and anti-NCL-Macro Abs revealed colocalization of FcRn and Ncl-Macroin lamina propria macrophages of the villi (Fig. 5e, arrows) andcrypts (Fig. 5f, arrows). We also colocalized FcRn and macrosialin(CD68), an activated macrophage marker, in intestinal macro-phages by an avidin-biotin complex method (37, 38). FcRn- andCD68 positively stained cells were clearly detectable in normallamina propria of intestine with either the mouse anti-a2-specificserum or with an FcRn-specific anti-peptide Ab and an anti-CD68

mAb. Additionally, double-color staining revealed that someFcRn- and CD68-positive cells colocalized (data not shown).Therefore, the same result was obtained when two different Absspecific for FcRn in macrophages were used, thus confirming theexpression of FcRn in tissue macrophages.

pH-dependent IgG binding by FcRn in macrophages anddendritic cells

IgG binding assays were performed at both pH 6.0 and 7.5. Be-cause macrophages and dendritic cells express conventional Fcreceptors for IgG, which could confound the interpretation of func-tional IgG binding assays, we assessed pH-dependent binding bybiochemical methods. FcRn was specifically immunoprecipitatedfrom U937 and THP-1 cell lysates using human IgG bound toSepharose 4B as the ligand at pH 6.0, but not at pH 7.5 (Fig. 6A).An ;32-kDa band was also detected in binding assays using theU937 cells at both pH 7.5 and 6.0. Because this band was detect-able in FcRn-negative cells (data not shown), it is presumed thatthis represents a nonspecific precipitated protein. Isolated intestinalmacrophages and monocyte-derived, peripheral blood dendriticcells also displayed the same pattern of pH-dependent binding(Fig. 6B). Because it is possible that FcRn failed to bind IgG at pH7.5 due to competition from other FcgRs, especially high affinityFcgRI (CD64), we removed CD64, CD32, and CD16 molecules byincubating THP-1 cell lysates with excess amounts of anti-CD64,CD32, and CD16 mAbs immunoadsorbed to protein G at pH 7.5.Despite preclearing the THP-1 cell lysates of these IgG bindingproteins, the 45-kDa protein binding of IgG could still not be de-tected at pH 7.5 (data not shown).

pH-dependent binding by FcRn in macrophage is Fc mediated

To further demonstrate that the Fc portion of IgG is responsible forthe FcRn interaction, we performed an IgG binding assay at pH 6.0in the presence of soluble human IgG Fc and F(ab)2. The resultsare shown in Fig. 7 and reveal that the binding of FcRn to IgG-Sepharose was inhibited by the presence of excess human IgG Fcfragments, but not by the presence of excess human IgG F(ab)2

(Fig. 7). Furthermore, this inhibition of IgG binding to FcRn by Fcfragments was concentration dependent, indicating that the bindingof IgG Fc to FcRn in macrophage was specific and saturable.

Cellular distribution of FcRn in monocytes and macrophages

Because FcRn binds IgG in a pH-dependent manner, it is importantto know whether FcRn is expressed on the cell surface and/orintracellularly. First, cell surface biotinylation experiments wereperformed. Following cell surface biotinylation, FcRn could not bedetected on the cell surface of monocytic THP-1 cells (Fig. 8A).The failure to detect FcRn on the cell surface may be associated

FIGURE 3. RT-PCR amplification of FcRn cDNAfrom immune cells. First-strand cDNA was prepared asdescribed inMaterials and Methods. Amplified PCRproducts (800 bp) were electrophoresed in 1.2% agarosegels and stained with ethidium bromide. Similar PCRproducts amplified with a GAPDH-specific primer pairwas also fractionated in 1.2% agarose gels as internalcontrols. The arrow indicates the location of the ampli-fication products for human FcRn heavy chain (hFcRn)and GAPDH. The m.w. markers in base pairs are indi-cated on theleft. SI, small intestine; Mf, macrophages;DC, dendritic cells.

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A

B

C

FIGURE 4. FcRn expression in freshly isolated cellsand cell lines.A, Detection of human FcRn protein infreshly isolated monocytes, small intestinal macrophages,dendritic cells, and cell lines by Western blot. SDS-PAGE gels were loaded with 60mg of total protein perlane from the indicated sources, and the proteins wereresolved under reducing conditions. The proteins weretransferred onto nitrocellulose and probed with a rabbitanti-a2-specific serum and HRP-conjugated donkey anti-rabbit IgG used for development. The protein bands werevisualized by ECL. The arrow indicates the location ofthe human FcRn heavy chain (hFcRn).B, Immunofluo-rescence staining of monocyte-like cell lines THP-1 andU937. The THP-1 and U937 cell lines were grown onglass coverslips, fixed with 3.7% paraformaldehyde, andpermeabilized in 0.1% digitonin. Subsequently, the cellswere incubated with mouse anti-a2-specific serum,followed by staining with a FITC-conjugated F(ab)2 goatanti-mouse Ab (panels Cand E). HeLaFcRn1b2m cellswere stained as positive controls (A). The U937 cellline was stained with normal mouse serum as nega-tive control (panel G). The nucleus was stained with4969-diamidino-2-phenylindole (panels B,D, F, andH) and photographed through a fluorescence micro-scope. Positive samples and negative controls wereviewed using the same contrast and brightness set-tings.C, Detection of FcRn association withb2m inmonocyte-like cell lines. Metabolically labeledTHP-1 and U937 cells were immunoprecipitatedwith either rabbit anti-a2-specific serum or nonim-mune serum and analyzed by SDS-PAGE and auto-radiography. The 45- and 12-kDa bands were copre-cipitated in the presence of hFcRn-specific immuneserum, but not in the presence of nonimmune serum.The Mr markers in kilodaltons are indicted on theleft. The locations of human FcRn heavy chain(hFcRn) andb2m are indicated by arrows.

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with either the activation state or the degree of cellular differen-tiation, because THP-1 is a monocyte-like cell without completematuration. PMA treatment can activate THP-1 cells with mor-phological changes consistent with differentiation. When THP-1cells were labeled with biotin after PMA treatment, FcRn wasreadily detectable on the cell surface (Fig. 8A). Similar resultswere obtained by flow cytometry (Fig. 8B). Whereas restingTHP-1 cells expressed FcRn solely intracellularly, THP-1 cellsactivated by PMA expressed FcRn both on the cell surface andintracellularly. This appearance of FcRn on the cell surface wasdetectable within 6 h of PMA activation and was sustained for upto 48 h. During this time period, intracellular levels of FcRn ex-pression were maintained or even increased, suggesting that redis-tribution of FcRn to the cell surface was associated with increasesin total cellular FcRn levels. Because PMA is reported to induceapoptosis in the HL-60 cell line (39), it is possible that apoptosiscould result in leakage of cell membranes in THP-1 cells. How-ever, we found that PMA-activated THP-1 cells did not stain withtrypan blue (data not shown). These data suggest that the cellular

distribution of FcRn may be regulated by either cellular maturationand/or activation in cells of the monocyte lineage.

To assess the in vivo relevance of these observations with theTHP-1 cell line, we performed flow cytometry for FcRn expressionon monocytes and macrophages whose purities were described inFig. 2. This type of analysis provided the following results (Fig.8C). Virtually all peripheral blood monocytes expressed FcRn in-tracellularly, and the majority (72.8%) exhibited detectable FcRnon the cell surface, albeit at lower levels (mean fluorescence in-tensity (MFI): surface, 9.02; intracellular, 22.56). These data in-dicate that monocytes uniformly express FcRn with the majority ofthe FcRn contained within intracellular compartments, and alesser, but still substantive, proportion displayed on the cell surfaceon a majority of cells. Although the macrophages purified from thesmall intestine were also uniformly positive for FcRn expression(95.7% positive), only a small subset (23.2%) of these cells dis-played FcRn on the cell surface. Interestingly, the MFI of theseFcRn surface-positive macrophages was equivalent to that ob-served intracellularly (MFI: surface, 40.59; intracellular, 42.51).

FIGURE 5. Immunolocalization of FcRn in macrophages ofthe lamina propria in adult human small intestine. Frozen sec-tions of tissue samples obtained from normal human jejunumwere stained with either rabbit anti-FcRn Ab or anti-Ncl-MacromAb. a, e, andf, arrowheads, Crypt and villus enterocytes showa punctuate staining pattern of FcRn expression visible at theapical plasma membrane and in the apical cytoplasm.a, arrow,A nearby resident lamina propria macrophage expresses FcRn.b, FcRn staining was not observed in the presence of an irrel-evant antiserum.c, arrows, Abs against Ncl-Macro-stained lam-ina propria macrophages.d, Macrophage staining was absent inthe presence of an irrelevant isotype-matched mAb. Double la-beling with both anti-FcRn and anti-Ncl-Macro Abs revealedcolocalization (yellow, arrow) of FcRn and Ncl-Macro in lam-ina propria macrophages of the villus (e) and crypt (f).

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Thus, with differentiation to a macrophage, FcRn expression per-sists, but is redistributed intracellularly, except for a minor subsetof cells that exhibits extremely high levels of surface FcRn ex-pression. Taken together with the observations generated with theTHP-1 cell line, these results suggest that the cellular distribution

of FcRn is regulated in monocytes and macrophages. Moreover,they suggest that FcRn may function intracellularly and extracel-lularly in monocytes and predominantly intracellularly in the ma-jority of tissue macrophages.

DiscussionFcRn is highly expressed in mouse and rat during the first 3 wkafter birth. In IECs it plays a major function in the passive acqui-sition of neonatal immunity. Following weaning, the expression ofFcRn in the IECs is rapidly and profoundly diminished (1, 4).However, it is also known that FcRn expression persists into adultlife in human IECs and in a limited range of other cell types inmammalian, including hepatocytes and endothelial cells (22–24).This expression beyond neonatal life is potentially relevant toother postnatal functions, including, importantly, the protection ofIgG from catabolism.

This study examined the hypothesis that FcRn, an MHC classI-related Fc receptor for IgG, is functionally expressed in mono-cytes, tissue macrophages, and dendritic cells that are already wellknown to abundantly express other conventional FcRs for IgG.Our study for the first time has demonstrated that FcRn is ex-pressed by monocytes, macrophages, and dendritic cells. The pres-ence of FcRn heavy chain in macrophages from small intestine anddendritic cells was specifically demonstrated by RT-PCR amplifi-cation with FcRn-specific primer pairs (Fig. 3), Western blotting(Fig. 4A), and immunofluorescence staining with FcRn specificserum Abs (Fig. 4B) in vitro, and immunohistochemical colocal-ization of FcRn heavy chain with the macrophage-specific markerNcl-Macro (Fig. 5) and CD68 (data not shown) in the lamina pro-pria of human small intestine. We reason that macrophages inother tissues would also express FcRn, because monocytes expressFcRn, although this should be further confirmed. Additional evi-dence to support this conclusion was that we were able to detectmurine FcRn, a homologue of human FcRn, in a macrophage cellline, RAW264.7 (data not shown). The association between FcRnandb2m was also demonstrated in monocyte-like cell lines (Fig.4C), proving that FcRn is structurally intact in this cell type.Therefore, our results support the previous finding that FcRn isexpressed beyond neonatal life. In our examination of FcRn ex-pression, we found that established cell lines derived from B lym-phocyte, T lymphocyte, and NK cell lineages failed to expressFcRn heavy chain (Fig. 3). However, we cannot exclude the pos-sibility that FcRn is expressed in freshly isolated or activated Tlymphocytes, B lymphocytes, and NK cells. We also do not knowwhether FcRn is expressed in other myeloid-derived lineages, suchgranulocytes and platelets. These issues will need furtherinvestigation.

FcRn binds IgG at acidic pH in macrophages and dendritic cells.As described in the intestine of neonatal rodent, FcRn binds IgG inthe slightly acidic pH of gut lumen and releases IgG into the blood-stream of newborn animals at the neutral pH of the interstitium, pH7.4 (1, 40). The amino acid residues isoleucine 254 and histidine310 within the CH2 domain and the sequence -H-N-H-Y (aa 433–436) of the CH3 domain in mouse and human IgG1 appear to beof particular functional significance in this pH-dependent binding(41–43). Our results show that FcRn displays complete pH-depen-dent binding of IgG binding in monocyte-like cell lines and in vivoisolated macrophage and dendritic cells (Fig. 6). This pH-depen-dent IgG binding can be inhibited by Fc fragments that contain theIgG binding motifs, but not by Fab that do not contain these motifs(Fig. 7), supporting specificity for the Fc portion of IgG.

Studies on transcytosis of IgG through yolk sac (44) and humanplacenta (12, 13) have suggested that FcRn resides primarilywithin acidified vesicles where ligand binding is likely to occur

FIGURE 6. Detection of pH-dependent FcRn binding of IgG in macro-phages and dendritic cells. IgG binding assays were performed at both pH6.0 and 7.5 as described inMaterials and Methods. The U937, THP-1,monocyte-derived dendritic cells, and intestinal macrophages were lysed insodium phosphate buffer (pH 6.0 or 7.5) with 0.5% CHAPS. Approxi-mately 0.5–1 mg of soluble proteins were incubated with human IgG-Sepharose at 4°C. The eluted proteins were subjected to 12% SDS-PAGEanalysis under reducing conditions. Proteins were probed with a rabbitanti-a2-specific serum and developed with HRP-conjugated donkey anti-rabbit Abs with visualization by ECL. Lysates of HeLaFcRn1b2m wereprobed similarly as a positive control. TheMr markers in kilodaltons areindicated on theright. The location of the human FcRn heavy chain isindicated by an arrow.

FIGURE 7. Blockade of FcRn-mediated IgG binding by IgG Fc frag-ment. IgG binding assays were performed as described in Fig. 6. For block-ing, 250–500mg of human Fc or F(ab)2 were added to IgG-Sepharosebeads before adding lysates from the THP-1 cell line. The eluted proteinswere analyzed by 12% SDS-PAGE under reducing conditions, probed witha rabbit anti-a2-specific serum, and developed with HRP-conjugated sec-ondary Abs with visualization by ECL. Lysates of HeLaFcRn1b2m wereprobed similarly as a positive control. TheMr markers in kilodaltons areindicated on theright.

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after fluid phase uptake. Also, several in vitro studies that havemodeled transcytosis of IgG in polarized epithelial cells supportthis idea (25, 45, 46). For example, pH gradient disruption in in-tracellular vesicles with bafilomycin A1 and monensin completelyinhibited IgG transcytosis in a model human intestinal or rat kid-ney epithelial cell line (25, 46). We also reason that FcRn is likelyto reside primarily within acidic vesicular compartments of cells ofmonocyte lineage. Our data support this conclusion, because FcRnwas barely detectable on the cell surface of a resting monocyte-likecell line (Fig. 8A), and the majority of FcRn expressed by mono-cytes and tissue macrophages was intracellular (Fig. 8B).

Interestingly, FcRn could also be expressed on the cell surface.When the THP-1 cell line was treated with the phorbol ester, PMA,which also drives THP-1 differentiation toward a macrophage-likephenotype (47), FcRn was readily detectable on the cell surface(Fig. 8, A and B). Similarly, a significant fraction of peripheralblood monocytes and a subset of tissue macrophages were ob-served to express FcRn on the cell surface, albeit at lower levelsthan intracellularly, except in the case of the tissue macrophagesubset that expressed extremely high levels. This suggests that thecellular distribution of FcRn may be related to the activationand/or differentiation state of the cell, which has not been previ-ously appreciated in other cell types. Because FcRn binds IgGstrongly in a pH-dependent manner, the appearance of FcRn on thecell surface would suggest that FcRn may be nonfunctional on thecell surface in terms of IgG binding under physiological condi-tions. However, it is possible that FcRn tethered on the cell surfaceof monocytes, macrophages, and dendritic cells might be func-tional in pathological conditions such as tissue inflammation (48,49) and tumor infiltration (50, 51), where acidic conditions arecreated by alterations in tissue metabolism. The interstitial pHwithin solid tumors has been observed to be below physiologicallevels, ranging from 5.6–7.7, which includes the pH optimum ofFcRn binding (50). Macrophages are recruited in the earliestphases of inflammation such as inflammatory bowel disease (52),and they are widely infiltrated in solid tumor tissues (53).

Alternatively, the expression of FcRn on the cell surface mayreflect other significant functions of FcRn on these cell types underphysiological conditions: a role in shuttling IgG from the intracel-lular to extracellular milieu in protecting IgG from catabolism.With regard to IgG protection, there is a significant body of evi-dence that suggests that FcRn is directly involved in the control ofserum IgG levels (14–17, 42). The proposed model is that pino-cytotic vacuole formation by cells expressing FcRn results in up-take of IgG from surrounding fluids, and following a lowering ofpH in early endosomes, some IgG molecules bind to FcRn. En-zymes present in organelles downstream of endosomes, such aslysosomes, digest the unbound IgG, but the IgG bound to FcRn isprotected and recycled into the surrounding tissue fluid. Data tosupport this model are the decrease in serum half-life of IgG inb2m

2/2 mice (16), because loss ofb2m presumably disables thefunction of FcRn, and the fact that mutated Fc fragments that ex-hibit a higher affinity for FcRn have a longer serum half-life thanwild-type Fc fragments (42). Currently, the cell type responsiblefor this protection of IgG has not been clearly defined, althoughendothelial cells have been proposed. The monocytic U937 cellline was shown to be capable of recycling monomeric IgG by anunknown mechanism (54). Therefore, we reason that the expres-sion of FcRn by virtually all monocytes in peripheral blood and thesignificant levels of FcRn expression detectable on the cell surfaceof this cell type may reflect a role of monocytic FcRn in the pro-tection of IgG from catabolism and the maintenance of IgG levelsin peripheral blood. Therefore, the prominent expression of FcRn

on the cell surface of monocytes may reflect highly active sortingof IgG by FcRn from the endocytic pathway to the cell surface.

However, the relative distribution of FcRn on tissue macro-phages was distinct from monocytes, with most FcRn in the former

FIGURE 8. Cellular distribution of FcRn expression patterns of FcRn onTHP-1, monocytes, and macrophages.A, Surface biotinylation of FcRn onresting THP-1 and PMA-activated THP-1 cell lines. THP-1 cells were treatedwith 100 nm/ml PMA for 48 h. Cell surface proteins were biotinylated andsolubilized at pH 6.0 as described in the text. Lysates were incubated withIgG-Sepharose beads. The eluted proteins were analyzed by SDS-PAGE elec-trophoresis under reducing conditions, blotted with streptavidin-HRP, and de-veloped with ECL. This experiment was conducted in a duplicate sample withidentical results. The specificity of the band identified in the avidin blot wasprovided by immunoprecipitation with an FcRn-specific Ab followed by avi-din blotting (data not shown).B, Analysis of the surface and intracellularexpression of FcRn on resting and PMA-activated THP-1 cells. Indirect im-munofluorescence staining was performed on untreated cells or cells treatedwith PMA for the indicated time periods (hours). Cell surface and intracellularexpression of FcRn on resting or activated THP-1 cells was described inMa-terials and Methods. Results are expressed as histograms of MFI (log scale) onthex-axis. The open peak represents staining of cells with the anti-hFcRn Ab,and the filled peak represents cells stained with irrelevant IgG.C, Expressionof FcRn in monocytes and macrophages analyzed by flow cytometry. Cellsurface and intracellular expression patterns of FcRn in either fixed or perme-abilized blood monocytes and small intestinal macrophages were measured byflow cytometry. Cells were stained as described inMaterials and Methods.Results are expressed as histograms of fluorescence intensity (log scale). Thefilled histograms represent staining of cells with anti-a2-specific serum, andthe open histograms represent cells stained with irrelevant IgG. Values in thetop right of each rectangle correspond to the proportion of cells stained withthe anti-hFcRn Ab relative to the control Ab. The staining for macrophagesand monocytes was conducted three times with similar results.

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cell type intracellularly except for a small subset of cells that re-sembled the distribution of FcRn in monocytes, i.e. intracellularand cell surface (Fig. 8C). This suggests that the function of FcRnin most macrophages may be distinct and skewed toward protect-ing IgG from degradation intracellularly and thus prolonging theintracellular half-life of IgG. For a macrophage involved in Agpresentation, such a property may be advantageous, and this sug-gests that FcRn may influence Ag presentation. In macrophagesand dendritic cells, FcgRs can promote the internalization of im-mune complexes into the endosomes, lysosomes, and MHC classII compartment (MIIC) to increase the efficiency of MHC class IIpresentation to CD41 T lymphocytes (28, 55, 56). FcRn, in con-trast, may influence Ag presentation pathways by protecting theseimmune complexes once inside cells in acidic compartments suchas early endosomes (pH 6.0–6.5), late endosomes (pH 5.0–6.0),lysosomes (pH 4.5–5.0), and MIIC (57). Generally, antigenic pep-tides, which are ultimately associated with MHC class II mole-cules, are generated from internalized exogenous Ags by themovement of MHC class II sequentially through early endosomes,late endosomes, lysosomes, and MIICs (58). Several lines of ev-idence support this probability. First, because FcRn is able to bindimmune complexes (59), it may be able to maintain high levels ofthese immune complexes at the sites of Ag processing. Second,FcRn binds IgG in the pH range of endosomes and lysosomes (pH4.5–6.5; data not shown). Third, the appearance of a dileucine-based motif in the cytoplasmic tails of FcRn and the MHC classII-associated invariant chain suggests that FcRn and MHC class IImolecules might be colocalized primarily in acidic compartments.The invariant chain has been shown to target MHC class II toacidic compartments (60). Therefore, the role of FcRn in protect-ing IgG may have an influence on Ag presentation in APCs suchas macrophages and dendritic cells.

In summary, FcRn, the only known Fc receptor for IgG withMHC class I-like structure, is functionally expressed by mono-cytes, macrophages, and dendritic cells. Furthermore, the cellulardistribution of FcRn expression on these cell types is regulatedbetween intracellular and extracellular sites. These features ofFcRn expression may confer upon monocytes, macrophages, anddendritic cells novel functions involving protection of IgG fromcatabolism that may relate to prolonging the IgG half-life in theextracellular (monocytes) and intracellular (macrophages and den-dritic cells) milieu, which may impact the Ag presentation func-tions of these cells. Future studies must be aimed at testing thesehypotheses.

AcknowledgmentsWe thank Dr. Sheldon Randall (Department of Surgery, Brigham andWomen’s Hospital) for human intestinal tissue. We gratefully acknowledgethe FcRn-containing plasmid and peptide Ab from Dr. Neil Simister. Wethank Drs. Victor M. Morales and Neil Simister for critically reviewing themanuscript. We thank Dr. Kamran Badizadegan and Atul Bhan for adviceabout immunohistochemistry. We thank Dr. Tom Kupper for advice inpurification of dendritic cells. We thank Drs. Mark Birkenbach, PaulAnderson, and Marco Colonna for the cell lines THP-1, NK3.3, andNKL. We also thank Dr. Jianhua Xu for human cDNA from PBMC, andSteven M. Claypool for technical help.

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