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*
Gastroenterology Division, Brigham and Women’s Hospital, and
Departments of Medicine and Pathology, and
Combined Program in Pediatric Gastroenterology and Nutrition, Children’s Hospital, Harvard Medical School, Boston, MA 02115; and
Department of Medicine, University of Alabama and Veteran’s Affairs Medical Center, Birmingham, AL 35294
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2-microglobulin (2m) light chain in U937
and THP-1 cells. FcRn bound human IgG at pH 6.0, but not at pH 7.5.
This binding could be 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 detectable on
the cell surface of a significant fraction of monocytes at lower levels
and on a small subset of tissue macrophages that expressed high levels
of FcRn on the cell surface. These data show that FcRn is functionally
expressed and its cellular distribution is regulated in monocytes,
macrophages, and dendritic cells, suggesting that it may confer novel
IgG binding functions upon these cell types relative to typical
Fc
Rs: FcRI, FcRII, and FcRIII. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-microglobulin (2m;
12 kDa). FcRn was originally characterized as a transport receptor
involved in the uptake of maternal IgG by an intestinal route in
rodents (4, 5, 6, 7, 8) and probably via syncytiotrophoblastic
cells within human placenta, respectively (9, 10, 11, 12, 13).
Additionally, FcRn has been considered 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 in 2m exhibit significant reduction
in the serum half-life of IgG (15, 16, 17). Recent evidence
for FcRn expression by endothelial cells suggested that this may be the
cell type most prominently involved in IgG protection
(18). A hallmark of FcRn interaction with its ligand is its strictly pH-dependent IgG binding in both epithelial and endothelial cells. FcRn preferentially binds IgG at acidic pH (6–6.5), but is unable to bind IgG at neutral pH (7–7.4) (19, 20, 21). FcRn is expressed in a variety of cell types and tissues, including intestinal epithelial cells (IECs) of neonatal rodents, syncytiotrophoblasts of humans, endothelial cells of adult rodents and humans, adult rat hepatocytes, and adult epithelial cells of bovine mammary gland, human intestine, and human kidney (22, 23, 24, 25, 26, 27).
Immune cells, such as B lymphocytes, macrophages, dendritic cells, NK
cells, mast cells, and granulocytes, typically express single or
multiple receptors for the Fc portion of IgG, including FcRI (CD64),
FcRII (CD32), FcRIII (CD16), and their splice variants. These
FcRs play a pivotal role in linking the cellular and humoral arms of
the immune response. Specifically, these receptors are involved in
internalization of immune complexes, Ag presentation, Ab-dependent
cellular cytotoxicity, negative regulation of effector functions of
FcR-bearing cells, regulation of the inflammatory cascade, and
autoimmunity (28, 29, 30, 31). However, FcRn expression has not
been characterized in immune cells, especially in
FcR+ cells. Therefore, we tested the
hypothesis in this study that FcRn is functionally expressed in human
immune cells. We found by several criteria that FcRn was expressed in
human monocytes, macrophages, and dendritic cells and in human
monocytic cell lines and exhibits pH-dependent binding of IgG in these
cells. Moreover, the cellular distribution of FcRn expression between
intracellular and cell surface locations appears to be differentially
regulated. These studies indicate that FcRn is the fourth FcR for IgG
to be defined on macrophages and dendritic cells and significantly
extend the potential function of FcRn and the cell types involved in
the known functions of this novel MHC class I-like molecule.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 Collection (Manassas, VA). THP-1 (monocytic cell line),
NK3.3 (NK cell line), and NKL (NK cell line) were gifts from Dr. Mark
Birkenbach (University of Chicago, Chicago, IL), Dr. Paul Anderson
(Harvard Medical School, Boston, MA), and Dr. Marco Colonna (Basel
Institute for Immunology, Basel, Switzerland), respectively. The U937
(promonocytic cell line), Raji, and 721.721 cell lines were cultivated
in suspension in RPMI 1640 medium (Life Technologies, Gaithersburg, MD)
supplemented with 10% FCS, 1% L-glutamine, and 1%
penicillin/streptomycin. A CD1d-transfected cell line,
721.721CD1d, generated by transfecting the
721.221 cell line with the full-length CD1day cDNA in the PSR
neo
expression vector, was cultivated in the same medium supplemented with
500 µg/ml G418 (Life Technologies). The THP-1 cell line was
cultivated in the same medium with 5 x
10-5 M 2-ME (Sigma, St. Louis, MO). NK3.3 and
NKL were cultivated in 10% RPMI 1640 medium with 10% human serum.
HeLa cells were cultivated with 10% FCS in DMEM (Life Technologies).
Cell viability was assessed by trypan blue dye exclusion.
Production of human FcRn domain-specific serum Abs
The human FcRn codons (11) corresponding to the
1 1–87(1–87), the 2 88–177(88–177), and the 3 178–274(178–274) domains were
amplified by PCR and subcloned into the EcoRI sites of the
pGEX4T-1 (Amersham Pharmacia Biotech, Piscataway, NJ) expression
vector. The primer pairs for 1
(5'-CCGGAATTCGCAGAAAGCCACCTCTCCCT-3',
5'-GGCGAATTCTCAACCTTTTCCCCCCAA-3'),
2 (5'-GGCGAATTCTACACTCTGCAGGGCCTGCT-3',
5'-CGCGAATTCTCACTTCCACTCCAGGTTT-3'), and 3
(5'-CCGGAATTCGAGCCCCCCTCCAT,
5'-GGCGAATTCGGAGGACTTGGCTGGAGATT-3') were
used for amplification by Pfu polymerase (Stratagene, La
Jolla, CA). The EcoRI site in the primers are underlined,
and human FcRn sequences are italicized. The plasmid encoding the
full-length human FcRn, provided by Dr. Neil Simister (Brandeis
University, Waltham, MA), was used as a template. All subclones were
verified by sequencing. The production of recombinant proteins was
performed by a method modified from that previously described
(32) and analyzed by SDS-PAGE electrophoresis. Five
micrograms of the purified GST-1, GST-2, and GST-3 proteins
were respectively emulsified in CFA and injected s.c. into each BALB/c
mouse. Mice were boosted twice at 3-wk intervals with fusion protein
emulsified with IFA. Sera were sampled 2 wk following the final dose.
Furthermore, the immunization of rabbits with purified fusion protein
was performed by Charles River Breeding Laboratories (Wilmington,
MA).
Isolation of lamina propria macrophages, blood monocytes, and dendritic cells
Lamina propria macrophages were isolated from surgical human normal tissue by neutral protease digestion of intestinal tissue sections with counterflow centrifugal elutriation as previously described (33, 34). Briefly, sections of normal human jejunum were incubated in 0.2 M EDTA (Fisher Scientific, Norcross, GA) plus 10 mM 2-ME (Sigma) to remove the epithelium, minced, and then treated twice (45 min, 200 rpm, 37°C) in RPMI (Mediatech, Washington, DC) containing 100 µg/ml DNase and 75 µg/ml of the neutral protease dispase (grade I; Roche, Indianapolis, IN) to release the lamina propria mononuclear cells (33). After straining to remove debris and gradient sedimentation to remove residual nonmononuclear cells, the cells were separated into highly purified populations of lamina propria macrophages and lymphocytes by counterflow centrifugal elutriation using a J-6 M elutriation centrifuge (Beckman, Palo Alto, CA) (33, 34). The cells isolated by this procedure contained <1% CD3+ lymphocytes and displayed the size distribution, morphological features, ultrastructure, and phagocytic activity of macrophages (33).
Peripheral blood monocytes were isolated from leukopaks from healthy donors by elutriation. Both cell populations were rested for 2 days in DMEM (Quality Biologicals, Gaithersburg, MD) plus 50 mg/ml gentamicin and 10% human AB serum (Atlanta Biologicals, Atlanta, GA) before study. Cell purity was assessed by flow cytometry as previously described (3, 4, 5) using mAbs against the following cell markers: HLA-DR, CD3, CD13, CD14, CD20, and CD80 (Becton Dickinson, San Jose, CA) and CD103 and CD83 (Immunotech, Westbrook, ME). Isotype-matched irrelevant mAbs were used as controls.
The monocyte-derived dendritic cells were obtained by a previously described method (35). Briefly, monocytes were isolated from PBMCs by adherence to plastic for 2 h and were cultured for 8 days in RPMI 1640 (Life Technologies) supplemented with 10% FCS, 10 mM HEPES, 2 mM L-glutamine, 5 x 10-5 2-mecaptoethanol, penicillin (100 U/ml), streptomycin (100 mg/ml), recombinant human GM-CSF (100 U/ml), and recombinant human IL-4 (1000 U/ml). The medium was replaced every 3–4 days. After 8 days, cells displaying dendritic morphology and predominantly expressing CD1a and HLA-DR, but that had lost most of the expression of the monocyte marker CD14, were obtained. Immature dendritic cells were obtained by culturing the adherent fraction of normal human PBMCs 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 (Molecular Research Center, Cincinnati, OH). Total RNA was extracted according to the method recommended by the manufacturer. First-strand cDNA was synthesized from 1 µg of total RNA using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) and an oligo(dT) primer (Promega) as recommended by manufacturer. The human FcRn gene was amplified from cDNA by a primer pair (5'-CCGGAATTCGCAGAAAGCCACCTCTCCCT, 5'-CGGAATTCTTAGCAGTCGGAATGGCGGA-3') that contained EcoRI sites in the 5' extension to facilitate cloning. Amplification was performed by hot start PCR using 35 cycles each consisting of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min. At the end of the 35 cycles, samples were run for an additional 10 min at 72°C and then maintained at 4°C until analyzed by agarose gel electrophoresis. The mRNA was also amplified by GAPDH-specific primers as an internal control to monitor the quality of the RNA purification and cDNA synthesis.
Transfection of HeLa cells with plasmid encoding human FcRn and
2m
The FcRn codon 1–343(1–343) was amplified from an FcRn-containing
plasmid (11) with the primer pair
5'-ATAAGAATGCGGCCGCGGCAGAAAGCCACCTCTCCCT-3'
and 5'-TGCTCTAGATTAGGCGGTGGCTGGAATCA-3'. The
upstream primer introduced a NotI site, and the downstream
primer introduced an XbaI site to facilitate cloning
(underline). Amplification was performed using Pfu DNA polymerase with
initial heating to 95°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 final extension step at 72°C for 10 min.
The PCR product was purified by agarose gel using a GeneClean II kit
(Bio 101, Vista, CA). The DNA fragment was digested with
NotI and XbaI and ligated into the plasmid
pFlagCMV-1 (Sigma) to generate the plasmid, pFlagCMVhFcRn. In this
plasmid a Flag epitope (DYKDDDDK, single-letter amino acid code) was
fused into the N terminus of the FcRn gene. The plasmid
pCDNAh2m was constructed as previously
described (36). The open reading frames of plasmids
pCDNAh2m and pFlagCMVhFcRn were verified by
sequencing both strands to confirm the fidelity of amplification and
cloning.
Transfection of HeLa was performed by electroporation (Electroporator
II; Invitrogen, San Diego, CA) using 20 µg of pFlagCMVhFcRn and 2
µg of pCDNAh2m to ensure that
2m concentrations were not substrate limiting
for FcRn expression. Transfected cells were grown under selection with
1 mg/ml of G418 (Life Technologies). Single colonies of transfected
HeLa were expanded under 500 µg/ml of G418. Positive colonies were
confirmed by Western blotting using the FcRn anti-2-specific
serum as described. The chosen positive transfectant was designated
HeLaFcRn+2m.
Western blotting, immunoprecipitation, and immunofluorescence
Cell lysates were prepared in PBS with 0.5% Nonidet P-40, 0.5%
sodium deoxycholate, and 0.1% SDS by adding a protease inhibitor
cocktail (Sigma). A postnuclear supernatant was analyzed for total
protein concentrations by the Bradford method with BSA as a standard
(Bio-Rad, Hercules, CA). The proteins were separated on 12% SDS-PAGE
gels under reducing conditions and transferred onto nitrocellulose
(Schleicher & Schuell, Keene, NH). The membranes were blocked with 5%
nonfat milk and probed with mouse anti-human FcRn 2 Ab (1/500)
for 1 h, then with HRP-conjugated goat anti-mouse IgG Fc Ab
(1/10,000). All blocking, incubation, 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).
Immunoprecipitations were performed as previously described
(22). Briefly, 5 x 105
log-phase-grown THP-1 and U937 cells were metabolically labeled with
0.5 mCi of trans-35S-labeled
methionine and cysteine (ICN Biomedicals, Costa Mesa, CA) in
methionine- and cysteine-free RPMI 1640 medium (ICN Biomedicals)
supplemented with 10% dialyzed FCS and incubated at 37°C for 5
h. After washing with PBS, cells were lysed in buffer (0.15 M NaCl, 1
mM EDTA, 50 mM Tris (pH 8), and 10 mM iodoacetamide) with protease
inhibitors and detergent as described above. Cells were lysed and
subsequently centrifuged at 14,000 x g for 30 min.
Radioimmunoprecipitations were performed using mouse
anti-2-specific serum coupled to protein G-Sepharose beads
(Pierce). A 2m mAb (Sigma) was used to deplete
2m from cell lysates.
For immunofluorescence assays,
HeLaFcRn+2m was grown on
glass coverslips overnight. A total of 1 x
105 U937 and THP-1 were mounted onto adhesive
microscope slides, air-dried, and fixed in 3.7% paraformaldehyde.
After washes, cells were permeabilized with 0.1% digitonin in PBS for
10 min at room temperature, washed, and blocked for 30 min at room
temperature with 10% heat-inactivated goat serum (Sigma) in PBS. Cells
were then incubated with a mouse anti-2-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-mouse Ab (1/100) for 1
h at room temperature. As a negative control, cells were incubated with
normal mouse serum. Nuclei were stained with 0.1 µg/ml
4'6'-diamidino-2-phenylindole (Molecular Probes, Eugene, OR) in PBS for
5 min. After final washes, cells were mounted. Images were captured
using a fluorescence microscope (Microphot FXA; Nikon, Tokyo, Japan)
and processed with Adobe Photoshop 5.0. Positive samples and negative
controls 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 sodium phosphate buffer (pH 6.0 or 7.5) with 0.5%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS;
Sigma) and protease inhibitor cocktail on ice for 1 h. Postnuclear
supernatants containing 0.5–1 mg of soluble proteins was diluted with
an equal volume of sodium phosphate buffer containing 0.1% CHAPS and
incubated with human IgG-Sepharose (Amersham Pharmacia Biotech) at
4°C for 4 h or overnight. The unbound proteins were removed with
sodium phosphate buffer (pH 6.0 or 7.5) containing 0.1% CHAPS. The
adsorbed proteins were eluted with sodium phosphate buffer (pH 8) or
boiled with electrophoresis sample buffer at 100°C for 5 min. The
eluted fractions were subjected to 12% reducing SDS-PAGE analysis.
Proteins were visualized by Western blot using anti-2-specific
serum. For the blocking experiments, 250–500 µg/ml of human Fc or
F(ab)2 (ICN Pharmaceuticals, Aurora, OH) were added to
IgG-Sepharose beads before adding FcRn cell lysates. For the removal of
CD64, CD32, and CD16 molecules, cell lysates (pH 7.5) were incubated
with 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 (5 x
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-fold
by sodium phosphate buffer (pH 6.0) with 0.1% CHAPS, then incubated
with IgG-Sepharose. Following washings at pH 6.0, the bound protein was
eluted 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
with streptavidin-HRP (Pierce). To confirm the specificity, the
proteins eluted with sodium phosphate buffer were immunoprecipitated by
mouse anti-2-specific serum bound to protein G-Sepharose beads.
Following incubation at 4°C on ice, the beads were washed,
resuspended in loading buffer, resolved by SDS-PAGE, transferred onto
nitrocellulose, and blotted with streptavidin-HRP. The final product
was visualized using ECL (Pierce).
Flow cytometry
Surface and intracellular expressions of FcRn were examined in
either fixed or permeabilized monocytes, macrophages, or THP-1 cells by
flow cytometry. For staining, 1 x 106 cells
were washed with PharMingen stain buffer (FBS; PharMingen, San Diego,
CA), followed by blocking with PBS containing 10% normal goat sera
(Jackson ImmunoResearch, West Grove, PA) on ice for 20 min. For surface
Ag staining, 10 µl of diluted anti-2-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 internalization, and
the results were identical with those observed at room temperature
(data not shown). For intracellular staining, the cells were first
permeabilized with Cytofix/Cytoperm (PharMingen) on ice for 20 min and
then washed with 1x perm/wash buffer. Anti-2-specific serum was
added as described. After washing, 20 µl of 1/50 diluted goat
anti-mouse IgG-FITC Ab (Jackson ImmunoResearch) was added to each
tube and incubated at room temperature for 15 min. After washing, cells
were fixed with Cytofix and analyzed using a FACScan flow cytometer and
CellQuest software (Becton Dickinson). The mouse IgG (0.5 µg/million
cells) was used as a negative control.
Immunohistochemistry
Normal adult human small intestine was obtained from patients
undergoing gastric bypass surgery under a protocol that was approved by
the human studies committee of the Brigham and Women’s Hospital.
Tissue was embedded in Tissue-Tek OCT compound (Sakura-Finite,
Torrance, CA). Samples were sectioned on a Leach CM3050 cryomicrotome
(Leica, Nussloch, Germany). A frozen section (5 µm) was air-dried at
room temperature, fixed in 4% paraformaldehyde in PBS, washed in PBS,
and blocked in 10% nonimmune goat serum (Zymed, South San Francisco,
CA). Sections were stained with an affinity-purified FcRn-specific
anti-peptide Ab (aa 174–188; provided by Dr. Neil Simister,
Brandeis University) or against Ncl-Macro (Novocastra, Newcastle upon
Tyne, U.K.) diluted in PBS containing 10% nonimmune goat serum and
0.02% Tween 20. Primary Abs were detected with appropriate
fluorophore-conjugated secondary Abs for epifluorescence microscopy.
All staining reactions were accompanied by a negative control that
consisted of an affinity-purified, isotype-matched irrelevant Ab.
Sections were mounted in ProLong antifade reagent (Molecular Probes,
Eugene, OR) and viewed with a Zeiss Axiophot microscope (Zeiss, New
York, NY) equipped with a Spot digital camera (Diagnostic Instruments,
Sterling Height, CA). Electronic images were captured and edited using
Adobe Photoshop. The sections were stained with either mouse
anti-2-specific serum or an FcRn-specific anti-peptide Ab
and anti-CD68 (Santa Cruz Biotechnology, Santa Cruz, CA) using an
avidin-biotin complex method (37).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To generate FcRn-specific serum Abs, we fused the codons
corresponding to the 1, 2, or 3 domains of FcRn in-frame to
the GST gene. The anti-GST Abs in the mouse sera were removed by
incubation with GST bound to glutathione-Sepharose beads. Sera
contained only Abs specific for the FcRn as shown by Western blot. We
selected the GST-2 for further immunization of rabbits. To show
specificity of the mouse and rabbit anti-human 2-specific serum
Abs, we probed cell lysates from 721.221,
721.221CD1d, Jurkat, and HeLa cells by Western
blotting. The 2 domain-specific serum did not react with classical
MHC or nonclassical MHC class I-like CD1d molecules (data not shown).
Despite the 22–29% similarity between the 2 domains of MHC class I
and FcRn (2), the anti-FcRn Abs recognized only a
45-kDa protein from HeLa cells transfected with plasmids encoding both
FcRn heavy chain and human 2m as defined by
Western blotting (Fig. 1
) and
immunoprecipitation of metabolically labeled protein (data not shown).
Mock-transfected HeLa cells were negative.
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Expression of FcRn heavy chains in immune cells was examined by
RT-PCR from a variety of cell lines and from isolated monocytes,
macrophages, and dendritic cells with FcRn-specific primers. The purity
of the isolated cells is shown in Fig. 2.
The results of RT-PCR screening are shown in Fig. 3. The amplified PCR products had a size
similar to that of a product amplified from an FcRn-encoding plasmid or
T84 cells, a polarized IEC line expressing functional FcRn
(25). Moreover, the FcRn heavy chain mRNA detected in the
human macrophage and dendritic cells had a DNA sequence identical with
that previously described from human placenta as defined by sequencing
five independent bacterial colonies (11) (data not shown).
These results showed that human FcRn transcripts were expressed in
monocytes, macrophages, and dendritic cells, but not in NK, T, and B
cell lines (Fig. 3).
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A shows that a band of 45 kDa
was detected by the FcRn anti-2-specific serum Abs in the
promonocytic U937 and monocytic THP-1 cell lines as well as in freshly
isolated monocytes, macrophages, and dendritic cells. A band was also
observed in transfected, but not in untransfected, HeLa cells. It
should be noted that the size of the band in the transfected HeLa cells
was slightly larger than that of the band detected in the monocytic
cell lines, monocytes, macrophages, and dendritic cells due to a Flag
epitope that was inserted into the N terminus of the FcRn gene.
Untransfected and transfected HeLa cells also exhibited two minor
nonspecific bands with Western blotting that migrated above the 45-kDa
specific band (Fig. 4A). However, these were not observed
when immunoprecipitation of radiolabeled
HeLahFcRn+2m cells was
performed (data not shown). Furthermore, Jurkat and untransfected HeLa
cells lacked this 45-kDa band. In Jurkat, a band smaller than 45 kDa
was detected, similar to a minor weak band present in the monocytic
cell lines as well as macrophages and dendritic cells. This band
probably represents a nonspecific immunoreactive protein given the
absence of FcRn-specific mRNA in Jurkat cells (Fig. 3
). In addition,
immunoprecipitation of radiolabeled proteins from the Jurkat and Raji
cell lines failed to show expression of FcRn (data not shown).
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2-specific serum, we observed that THP-1 (Fig. 4B, panel C) and U937 (Fig. 4B,
panel E) cell lines stained positively. For comparison,
staining of the
HeLaFcRn+2m
transfectants is shown in panel A. The negative control,
normal mouse serum, failed to stain HeLa (data not shown), THP-1 (data
not shown), or U937 cells (Fig. 4B, panel G), and
the anti-2-specific serum failed to stain the untransfected HeLa
cell line (data not shown). Nuclear staining of all four cell lines is
provided for reference (panels B, D,
F, and H).
Association of human FcRn heavy chain with 2m in
THP-1 and U937 cells
FcRn is expressed as a 45-kDa membrane-bound heavy chain in
nonconvalent association with 12-kDa 2m.
Immunoprecipitation of [35S]methionine- and
[35S]cysteine-labeled THP-1 and U937 cell
lysates with an anti-2-specific serum
produced two bands, 45- and 12-kDa proteins, that were
coimmunoprecipitated by the presence of anti-2-specific serum,
but not by preimmune serum (Fig. 4C). This is consistent
with an association between the FcRn heavy chain and
2m in other cells. Further confirmation that
the 12-kDa band was 2m was obtained when
lysates from metabolically labeled THP-1 cells were subjected to three
rounds of depletion with an anti-2m mAb
that removed the 12-kDa band from the autoradiogram. The molecular size
of FcRn immunoprecipitated from U937 lysates was slightly larger than
that of FcRn from THP-1. The explanation for this result is not clear.
However, it may reflect different post-translational modifications of
FcRn in the two cell lines, which are immortalized at different stages
of monocyte maturation. We also observed that the associated
2m band did not appear to be stoichiometric
with FcRn. Because 2m contains three
methionine and cysteine residues compared with the nine such residues
contained in human FcRn, we believe that this is an artifact of the
metabolic labeling technique. Another possibility is that since
mutations of the 2m molecule have been
described in transformed cell lines (36), it may be that a
2m mutation has occurred in the monocytic cell
lines, resulting in a low affinity of association with FcRn. Therefore,
we sequenced the cDNA of 2m derived from the
U937 cell line. The sequence aligned perfectly with the sequence of
2m 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 human
intestinal mucosa (33). To determine whether FcRn is
expressed in macrophages in vivo, we stained intestinal macrophages for
FcRn and Ncl-Macro, a marker for human macrophages. Crypt and villus
enterocytes exhibited a punctate apical membranous staining pattern for
FcRn visible at the apical plasma membrane and in the apical cytoplasm
(Fig. 5, a, e, and
f, arrowheads) as previously described (25).
Resident lamina propria macrophages also expressed FcRn (Fig. 5a, arrow). FcRn staining was absent from both enterocytes
and macrophages in the presence of an irrelevant antiserum (Fig. 5b). Abs against Ncl-Macro specifically stained lamina
propria macrophages (Fig. 5c, arrows), and this staining was
not 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-Macro in
lamina propria macrophages of the villi (Fig. 5e, arrows)
and crypts (Fig. 5f, arrows). We also colocalized FcRn and
macrosialin (CD68), an activated macrophage marker, in intestinal
macrophages by an avidin-biotin complex method (37, 38).
FcRn- and CD68 positively stained cells were clearly detectable in
normal lamina propria of intestine with either the mouse
anti-2-specific serum or with an FcRn-specific anti-peptide
Ab and an anti-CD68 mAb. Additionally, double-color staining
revealed that some FcRn- and CD68-positive cells colocalized (data not
shown). Therefore, the same result was obtained when two different Abs
specific for FcRn in macrophages were used, thus confirming the
expression of FcRn in tissue macrophages.
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IgG binding assays were performed at both pH 6.0 and 7.5. Because
macrophages and dendritic cells express conventional Fc receptors for
IgG, which could confound the interpretation of functional IgG binding
assays, we assessed pH-dependent binding by biochemical methods. FcRn
was specifically immunoprecipitated from U937 and THP-1 cell lysates
using human IgG bound to Sepharose 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 the U937 cells
at both pH 7.5 and 6.0. Because this band was detectable in
FcRn-negative cells (data not shown), it is presumed that this
represents a nonspecific precipitated protein. Isolated intestinal
macrophages and monocyte-derived, peripheral blood dendritic cells also
displayed the same pattern of pH-dependent binding (Fig. 6B). Because it is possible that FcRn failed to bind IgG at
pH 7.5 due to competition from other FcRs, especially high affinity
FcRI (CD64), we removed CD64, CD32, and CD16 molecules by incubating
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 binding proteins, the 45-kDa protein
binding of IgG could still not be detected at pH 7.5 (data not
shown).
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To further demonstrate that the Fc portion of IgG is responsible
for the FcRn interaction, we performed an IgG binding assay at pH 6.0
in the presence of soluble human IgG Fc and
F(ab)2. The results are shown in Fig. 7 and reveal that the binding of FcRn to
IgG-Sepharose was inhibited by the presence of excess human IgG Fc
fragments, but not by the presence of excess human IgG
F(ab)2 (Fig. 7). Furthermore, this inhibition of
IgG binding to FcRn by Fc fragments was concentration dependent,
indicating that the binding of IgG Fc to FcRn in macrophage was
specific and saturable.
|
Because FcRn binds IgG in a pH-dependent manner, it is important
to know whether FcRn is expressed on the cell surface and/or
intracellularly. First, cell surface biotinylation experiments were
performed. Following cell surface biotinylation, FcRn could not be
detected on the cell surface of monocytic THP-1 cells (Fig. 8A). The failure to detect
FcRn on the cell surface may be associated with either the activation
state or the degree of cellular differentiation, because THP-1 is a
monocyte-like cell without complete maturation. PMA treatment can
activate THP-1 cells with morphological changes consistent with
differentiation. When THP-1 cells were labeled with biotin after PMA
treatment, FcRn was readily detectable on the cell surface (Fig. 8A). Similar results were obtained by flow cytometry (Fig. 8B). Whereas resting THP-1 cells expressed FcRn solely
intracellularly, THP-1 cells activated by PMA expressed FcRn both on
the cell surface and intracellularly. This appearance of FcRn on the
cell surface was detectable within 6 h of PMA activation and was
sustained for up to 48 h. During this time period, intracellular
levels of FcRn expression were maintained or even increased, suggesting
that redistribution of FcRn to the cell surface was associated with
increases in total cellular FcRn levels. Because PMA is reported to
induce apoptosis in the HL-60 cell line (39), it is
possible that apoptosis could result in leakage of cell membranes in
THP-1 cells. However, we found that PMA-activated THP-1 cells did not
stain with trypan blue (data not shown). These data suggest that the
cellular distribution of FcRn may be regulated by either cellular
maturation and/or activation in cells of the monocyte lineage.
|
. This type of
analysis provided the following results (Fig. 8C). Virtually
all peripheral blood monocytes expressed FcRn intracellularly, and the
majority (72.8%) exhibited detectable FcRn on the cell surface, albeit
at lower levels (mean fluorescence intensity (MFI): surface, 9.02;
intracellular, 22.56). These data indicate that monocytes uniformly
express FcRn with the majority of the FcRn contained within
intracellular compartments, and a lesser, but still substantive,
proportion displayed on the cell surface on a majority of cells.
Although the macrophages purified from the small intestine were also
uniformly positive for FcRn expression (95.7% positive), only a small
subset (23.2%) of these cells displayed FcRn on the cell surface.
Interestingly, the MFI of these FcRn surface-positive macrophages was
equivalent to that observed intracellularly (MFI: surface, 40.59;
intracellular, 42.51). Thus, with differentiation to a macrophage, FcRn
expression persists, but is redistributed intracellularly, except for a
minor subset of cells that exhibits extremely high levels of surface
FcRn expression. Taken together with the observations generated with
the THP-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
extracellularly in monocytes and predominantly intracellularly in the
majority of tissue macrophages. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study examined the hypothesis that FcRn, an MHC class I-related Fc
receptor for IgG, is functionally expressed in monocytes, tissue
macrophages, and dendritic cells that are already well known to
abundantly express other conventional FcRs for IgG. Our study for the
first time has demonstrated that FcRn is expressed by monocytes,
macrophages, and dendritic cells. The presence of FcRn heavy chain in
macrophages from small intestine and dendritic cells was specifically
demonstrated by RT-PCR amplification with FcRn-specific primer pairs
(Fig. 3), Western blotting (Fig. 4A), and immunofluorescence
staining with FcRn specific serum Abs (Fig. 4B) in vitro,
and immunohistochemical colocalization of FcRn heavy chain with the
macrophage-specific marker Ncl-Macro (Fig. 5) and CD68 (data not shown)
in the lamina propria of human small intestine. We reason that
macrophages in other tissues would also express FcRn, because monocytes
express FcRn, although this should be further confirmed. Additional
evidence to support this conclusion was that we were able to detect
murine FcRn, a homologue of human FcRn, in a macrophage cell line,
RAW264.7 (data not shown). The association between FcRn and
2m 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 is expressed beyond neonatal life. In our examination of FcRn
expression, we found that established cell lines derived from B
lymphocyte, T lymphocyte, and NK cell lineages failed to express FcRn
heavy chain (Fig. 3). However, we cannot exclude the possibility that
FcRn is expressed in freshly isolated or activated T lymphocytes, B
lymphocytes, and NK cells. We also do not know whether FcRn is
expressed in other myeloid-derived lineages, such granulocytes and
platelets. These issues will need further investigation.
FcRn binds IgG at acidic pH in macrophages and dendritic cells. As
described in the intestine of neonatal rodent, FcRn binds IgG in the
slightly acidic pH of gut lumen and releases IgG into the bloodstream
of newborn animals at the neutral pH of the interstitium, pH 7.4
(1, 40). The amino acid residues isoleucine 254 and
histidine 310 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 be of
particular functional significance in this pH-dependent binding
(41, 42, 43). Our results show that FcRn displays complete
pH-dependent binding of IgG binding in monocyte-like cell lines and in
vivo isolated macrophage and dendritic cells (Fig. 6). This
pH-dependent IgG binding can be inhibited by Fc fragments that contain
the IgG 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
human placenta (12, 13) have suggested that FcRn resides
primarily within acidified vesicles where ligand binding is likely to
occur after fluid phase uptake. Also, several in vitro studies that
have modeled transcytosis of IgG in polarized epithelial cells support
this idea (25, 45, 46). For example, pH gradient
disruption in intracellular vesicles with bafilomycin A1 and monensin
completely inhibited IgG transcytosis in a model human intestinal or
rat kidney epithelial cell line (25, 46). We also reason
that FcRn is likely to reside primarily within acidic vesicular
compartments of cells of monocyte lineage. Our data support this
conclusion, because FcRn was barely detectable on the cell surface of a
resting monocyte-like cell line (Fig. 8A), and the majority
of FcRn expressed by monocytes 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-like phenotype
(47), FcRn was readily detectable on the cell surface
(Fig. 8, A and B). Similarly, a significant
fraction of peripheral blood monocytes and a subset of tissue
macrophages were observed to express FcRn on the cell surface, albeit
at lower levels than intracellularly, except in the case of the tissue
macrophage subset that expressed extremely high levels. This suggests
that the cellular distribution of FcRn may be related to the activation
and/or differentiation state of the cell, which has not been previously
appreciated in other cell types. Because FcRn binds IgG strongly in a
pH-dependent manner, the appearance of FcRn on the cell surface would
suggest that FcRn may be nonfunctional on the cell surface in terms of
IgG binding under physiological conditions. However, it is possible
that FcRn tethered on the cell surface of monocytes, macrophages, and
dendritic cells might be functional in pathological conditions such as
tissue inflammation (48, 49) and tumor infiltration
(50, 51), where acidic conditions are created by
alterations in tissue metabolism. The interstitial pH within solid
tumors has been observed to be below physiological levels, ranging from
5.6–7.7, which includes the pH optimum of FcRn binding
(50). Macrophages are recruited in the earliest phases 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 may reflect
other significant functions of FcRn on these cell types under
physiological conditions: a role in shuttling IgG from the
intracellular to extracellular milieu in protecting IgG from
catabolism. With regard to IgG protection, there is a significant body
of evidence that suggests that FcRn is directly involved in the control
of serum IgG levels (14, 15, 16, 17, 42). The proposed model is
that pinocytotic vacuole formation by cells expressing FcRn results in
uptake of IgG from surrounding fluids, and following a lowering of pH
in early endosomes, some IgG molecules bind to FcRn. Enzymes present in
organelles downstream of endosomes, such as lysosomes, digest the
unbound IgG, but the IgG bound to FcRn is protected and recycled into
the surrounding tissue fluid. Data to support this model are the
decrease in serum half-life of IgG in
2m-/- mice
(16), because loss of 2m
presumably disables the function of FcRn, and the fact that mutated Fc
fragments that exhibit a higher affinity for FcRn have a longer serum
half-life than wild-type Fc fragments (42). Currently, the
cell type responsible for this protection of IgG has not been clearly
defined, although endothelial cells have been proposed. The monocytic
U937 cell line was shown to be capable of recycling monomeric IgG by an
unknown mechanism (54). Therefore, we reason that the
expression of FcRn by virtually all monocytes in peripheral blood and
the significant levels of FcRn expression detectable on the cell
surface of this cell type may reflect a role of monocytic FcRn in the
protection of IgG from catabolism and the maintenance of IgG levels in
peripheral blood. Therefore, the prominent expression of FcRn on the
cell surface of monocytes may reflect highly active sorting of IgG by
FcRn from the endocytic pathway to the cell surface.
However, the relative distribution of FcRn on tissue macrophages was
distinct from monocytes, with most FcRn in the former cell type
intracellularly except for a small subset of cells that resembled the
distribution of FcRn in monocytes, i.e. intracellular and cell surface
(Fig. 8C). This suggests that the function of FcRn in most
macrophages may be distinct and skewed toward protecting IgG from
degradation intracellularly and thus prolonging the intracellular
half-life of IgG. For a macrophage involved in Ag presentation, such a
property may be advantageous, and this suggests that FcRn may influence
Ag presentation. In macrophages and dendritic cells, FcRs can
promote the internalization of immune complexes into the endosomes,
lysosomes, and MHC class II compartment (MIIC) to increase the
efficiency of MHC class II presentation to CD4+ T
lymphocytes (28, 55, 56). FcRn, in contrast, may influence
Ag presentation pathways by protecting these immune complexes once
inside cells in acidic compartments such as 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 peptides, which are
ultimately associated with MHC class II molecules, are generated from
internalized exogenous Ags by the movement of MHC class II sequentially
through early endosomes, late endosomes, lysosomes, and MIICs
(58). Several lines of evidence support this probability.
First, because FcRn is able to bind immune complexes (59),
it may be able to maintain high levels of these immune complexes at the
sites of Ag processing. Second, FcRn binds IgG in the pH range of
endosomes and lysosomes (pH 4.5–6.5; data not shown). Third, the
appearance of a dileucine-based motif in the cytoplasmic tails of FcRn
and the MHC class II-associated invariant chain suggests that FcRn and
MHC class II molecules might be colocalized primarily in acidic
compartments. The invariant chain has been shown to target MHC class II
to acidic compartments (60). Therefore, the role of FcRn
in protecting IgG may have an influence on Ag presentation in APCs such
as macrophages and dendritic cells.
In summary, FcRn, the only known Fc receptor for IgG with MHC class I-like structure, is functionally expressed by monocytes, macrophages, and dendritic cells. Furthermore, the cellular distribution of FcRn expression on these cell types is regulated between intracellular and extracellular sites. These features of FcRn expression may confer upon monocytes, macrophages, and dendritic cells novel functions involving protection of IgG from catabolism that may relate to prolonging the IgG half-life in the extracellular (monocytes) and intracellular (macrophages and dendritic cells) milieu, which may impact the Ag presentation functions of these cells. Future studies must be aimed at testing these hypotheses.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Department of Dermatology, Institute Gustave, Roussy, 39 rue Camille, Desmoulin, 94805 Villejuif, France.
3 Address correspondence and reprint requests to Dr. Richard S. Blumberg, Gastroenterology Division, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115.
4 Abbreviations used in this paper: FcRn, neonatal Fc receptor; 2m, 2-microglobulin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; MFI, mean fluorescence intensity; IEC, intestinal epithelial cell; MIIC, MHC class II compartment.
Received for publication August 22, 2000. Accepted for publication December 20, 2000.
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, macrophages; DC,
dendritic cells.
