INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 35: 1273-1289, 2015

Fc gamma receptor IIb participates in maternal IgG traffickingof human placental endothelial cells
TOMOKO ISHIKAWA1,5*, TAKAMI TAKIZAWA1*, JUN IWAKI1*, TAKUYA MISHIMA1, KUMIKO UI-TEI2,TOSHIYUKI TAKESHITA3, SHIGEKI MATSUBARA4 and TOSHIHIRO TAKIZAWA1
1

Department of Molecular Medicine and Anatomy, Nippon Medical School, Tokyo 113-8602;
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033;
3
Department of Obstetrics and Gynecology, Nippon Medical School, Tokyo 113-8602;
4Department of Obstetrics and Gynecology, Jichi Medical University, Tochigi 329-0498, Japan

2

Received January 21, 2015; Accepted March 17, 2015
DOI: 10.3892/ijmm.2015.2141
Abstract. The human placental transfer of maternal IgG is crucial
for fetal and newborn immunity. Low-affinity immunoglobulin
gamma Fc region receptor IIb2 (FCGR2B2 or FcγRIIb2) is exclusively expressed in an IgG-containing, vesicle-like organelle (theFCGR2B2 compartment) in human placental endothelial cells;
thus, we hypothesized that the FCGR2B2 compartment functions
as an IgG transporter. In this study, to examine this hypothesis,
we performed in vitro bio-imaging analysis of IgG trafficking by
FCGR2B2 compartments using human umbilical vein endothelial cells transfected with a plasmid vector containing enhanced
GFP-tagged FCGR2B2 (pFCGR2B2-EGFP). FCGR2B2-EGFP
signals were detected as intracellular vesicular structures similar
to FCGR2B2 compartments in vivo. The internalization andtranscytosis of IgG was significantly higher in the pFCGR2B2EGFP-transfected cells than in the mock-transfected cells, andthe majority of the internalized IgG was co-localized with the
FCGR2B2-EGFP signals. Furthermore, we isolated FCGR2B2
compartments from the human placenta and found that the Rab
family of proteins [RAS-related protein Rab family (RABs)]
were associated with FCGR2B2 compartments. Among the
RABs, RAB3D was expressed predominantly in placental
endothelial cells. The downregulation of RAB3D by smallinterfering RNA (siRNA) resulted in a marked reduction in the
FCGR2B2-EGFP signals at the cell periphery. Taken together,
these findings suggest that FCGR2B2 compartments participate
in the transcytosis of maternal IgG across the human placental

Correspondence to: Professor Toshihiro Takizawa, Departmentof Molecular Medicine and Anatomy, Nippon Medical School,
1-1-5 Sendagi, Tokyo 113-8602, Japan
E-mail: t-takizawa@nms.ac.jp

Present address: 5Institute of Environmental Science for Human
Life, Ochanomizu University, Tokyo 112-8610, Japan*

Contributed equally

Key words: Fcγ region receptor IIb, maternal IgG, human placenta,
placental endothelial cells, RAS-related protein Rab-3D

endothelium and that RAB3D plays a role in regulating the intracellular dynamics of FCGR2B2 compartments.
Introduction
Immunoglobulin G transport from the maternal circulation to
the fetus in the human placenta is crucial for the immunity of the
fetus and the newborn, whose own immune system is maturing.
This mechanism, which is not yet well understood, requires IgG
movement across two cell layers of the human placenta: thesyncytiotrophoblast and the fetal placental endothelium (1,2).
Transport across the syncytiotrophoblast is thought to be mediated by endosomes containing the MHC-related Fc receptor,
FCGRT (also known as FcRn) (3,4). An efficient transcytotic
mechanism to move the IgG from the apical to the basolateralsurface of the syncytiotrophoblast via the FCGRT-containing
endosomes can readily be envisioned, but has not yet been
substantiated. The mechanisms through which IgG crosses the
fetal endothelium are much less clear.
Low-affinity gamma immunoglobulin Fc region receptor IIb(FCGR2B; also designated as FcγRIIb) is a negative regulatorof immune cell function that is encoded by a single FCGR2B
gene; the two most common isoforms, B1 (FCGR2B1) andB2 (FCGR2B2), are generated by the alternative splicing of
the corresponding mRNA sequences (5). FCGR2B is generally
expressed on the surface of immune cells, e.g., neutrophils,
B-lymphocytes and monocytes (6,7), but is not expressedin any other endothelial cells of the adult human body, apartfrom placental endothelial cells (8) and hepatic sinusoidal
endothelial cells (9). Human placental endothelial cells abundantly and predominantly express FCGR2B2 (10-12). We
previously identified an FCGR2B2-defined, IgG-containing
organelle (tentatively designated as the FCGR2B2 compartment)in placental endothelial cells by immunoelectron microscopy;
the FCGR2B2 compartments did not overlap with various
marker proteins of well-recognized intracellular organelles[e.g., caveolae, secretory granules and (early, late and recycling)endosomes] (11). These previous in vivo findings suggested that
FCGR2B2 compartments mediate the transfer of IgG acrossthe placental endothelium, independent of caveolae. However,
the molecular mechanisms underlying the formation and intra-

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cellular dynamics of FCGR2B2 compartments and their IgG
trafficking in placental endothelial cells remain to be elucidated.
In this study, we performed bio-imaging analysis of
IgG trafficking of FCGR2B2 compartments using humanumbilical vein endothelial cells (HUVECs) transfected with aplasmid vector containing enhanced GFP-tagged FCGR2B2(pFCGR2B2-EGFP) as an in vitro system for the analysis of
FCGR2B2 expression in placental endothelial cells. We also
isolated FCGR2B2 compartments from the human placenta
and performed proteomic analysis of the vesicles to identify themolecules involved in the regulation of the FCGR2B2 compartment trafficking; we found that the Rab family of proteins[RAS-related protein Rab family (RABs)] were associated with
FCGR2B2 compartments in placental endothelial cells. Amongthe RABs, RAB3D was expressed predominantly in placental
endothelial cells. Furthermore, we generated small interfering
RNAs (siRNAs) targeting RAB3D to investigate the role of the
RAB3D in the FCGR2B2 compartment in our in vitro system.
Materials and methods
Sample collection. Human first-trimester placentas and full‑term
placentas with umbilical cords from patients who provided
informed consent were obtained according to the protocols
approved by the Nippon Medical School Hospital Ethics
Committee (Tokyo, Japan) and the Jichi Medical University
Ethics Committee (Tochigi, Japan). Tissue samples were
processed as soon as possible following delivery (within 20 min).
Isolation of endothelial cells from the human placenta. Humanumbilical cords were processed to obtain the HUVECs throughcollagenase digestion and subsequent magnetic bead isolation(Dynabeads CD31; catalog no. DB11128; Invitrogen, Carlsbad,
CA, USA). The HUVECs were maintained with the endothelial
cell growth medium MV2 kit (catalog no. C-22121; PromoCell,
Heidelberg, Germany) at 37˚C in a humidified incubator with
5% CO2. The placental endothelial cells were isolated fromhuman placental tissues as described in a previous study (3).
Dynabeads CD31 was used instead of Dynabeads that were coatedwith QB-End/40 monoclonal antibody to thrombomodulin.
Plasmid construction and transient transfection by electroporation. The open reading frame of human FCGR2B2 cDNA
was amplified as previously described (12). The PCR productwas inserted at the HindIII and AgeI sites of a pEGFP-Hyg-N1
vector, as previously described (13), which was generated bymodifying the commercially available pDsRed-Monomer-HygN1 vector (BD Biosciences Clontech, Mountain View, CA, USA).
These plasmids were designed to express EGFP fused to the
C-terminus of the FCGR2B2 protein avoiding the signal peptide.
The final construct was designated as pFCGR2B2-EGFP. A
pFCGR2B2 vector lacking EGFP was also constructed by the
PCR method and used to provide experimental foundations
for the bio-imaging analysis of IgG trafficking using HUVECs
transfected with pFCGR2B2-EGFP. As a mock control, cells
were transfected in parallel with vector pEGFP-N1.
For transfection, 100 µl HUVECs were trypsinized, resuspended with serum-free Opti-MEM (Invitrogen) at a densityof 1x106 cells/ml, and placed in 2 mm cuvettes with 2 µg
plasmid. Electroporation was performed using an exponential

wave pulse of 100 V and 500 µF using the Gene Pulser Xcell
electroporation system (Bio-Rad, Hercules, CA, USA). The thecells were then replated with 200 µl medium and cultured for
up to 72 h. Validation of our plasmid construction and transfection procedure was performed by transfection of the HUVECs
with pFCGR2B2-EGFP followed by immunocytochemistry.
Antibodies. Rabbit anti-human FCGR2B antibody directedagainst the C-terminal cytoplasmic tail of human FCGR2B(MHPDALEEPDDQNRI) and rabbit anti-human FCGRT antibody (DADLKDVNVIPATA) directed against the C-terminal
cytoplasmic tail of human FCGRT were generated (Peptide
Institute, Osaka, Japan). Rabbit anti-human low-affinity immunoglobulin FCGR2A antibody directed against the C-terminal
cytoplasmic tail of human FCGR2A (YLTLPPNDHVNSNN)
was also generated and used to test the specificity of the antiFCGR2B antibody. Cos-7 cells (Cell Resource Center for
Biomedical Research Institute of Development, Aging andCancer Tohoku University, Miyagi, Japan) were transfectedwith a plasmid designed to overexpress EGFP or EGFP-tagged
FCGRT, FCGR1A, FCGR2A, FCGR2B1 or FCGR2B2; thespecificity of these affinity-purified antibodies was validated
by western blot analysis (Fig. 1A). Rabbit anti-von Willebrandfactor antibody (catalog no. F3520; Sigma-Aldrich), mouse anticaveolin-1 antibody (catalog no. C-37120-150; BD Transduction
Laboratories, Franklin Lakes, NJ, USA), mouse anti-CD31
antibody (catalog no. BBA7; R&D Systems, Minneapolis, MN,
USA), mouse anti-CD144 (clone 16B1; eBioscience, San Diego,
CA, USA; CD144 also known as VE-cadherin and CDH5),
rabbit anti-human RAB1 antibody (catalog no. FL-205), rabbit
anti-human RAB3 antibody (catalog no. FL-220) (both fromSanta Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit
anti-human RAB3D (catalog no. 12320-1-AP; ProteinTech
Group, Chicago, IL, USA), mouse anti-human RAB11 antibody(catalog no. 610656; BD Transduction Laboratories), mouse antihuman RAB33B (catalog ID H00083452-M01; Abnova, Taipei,
Taiwan), mouse anti-human Ras-related protein Ral-A antibody(catalog no. 610221; BD Transduction Laboratories), Alexa Fluorlabeled secondary antibodies (Invitrogen), and HRP-conjugatedsecondary antibodies (Jackson ImmunoResearch Laboratories,
West Grove, PA, USA) were obtained from commercial sources.
Immunocytochemistry. The cultured HUVECs were fixed with
4% paraformaldehyde in PBS at room temperature for 2 h andpermeabilization was performed with 0.2% Triton X-100. For
the detection of caveolin-1, additional permeabilization was
performed with 0.5% sodium dodecyl sulfate (SDS). Dilutions
for the primary antibodies were 1:500 (FCGR2B), 1:200(von Willebrand factor), 1:100 (caveolin-1), 1:500 (CD31), and1:20 (CD144). Alexa Fluor-labeled secondary antibodies wereused at 1:200 dilutions. Following incubation with the antibodies,
the cells were subjected to nuclear staining with 4',6-diamidino2-phenylindole (DAPI) for 10 min and were mounted in
ProLong Gold (Invitrogen). Fluorescence images were collectedusing a BX60 microscope (Olympus, Tokyo, Japan) equippedwith a Spot RT SE6 CCD camera (Diagnostic Instruments,
Sterling Heights, MI, USA) and captured with the MetaMorphimage analysis system (Universal Imaging, Dowingtown, PA,
USA). The figures were compiled using Photoshop CS software(Adobe Systems, Mountain View, CA, USA).

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 35: 1273-1289, 2015

Binding affinity of FCGR2B2-EGFP for human IgG. The
binding property of FCGR2B2-EGFP for human IgG wasanalyzed by enzyme-linked immunosorbent assay (ELISA),
as previously described (14,15). Rabbit anti-human FCGR2B
antibody was biotinylated using a Biotin Labeling kit-NH 2(Dojindo Molecular Technologies, Kumamoto, Japan).
MaxiSorp 96 well microtiter plates (Nunc, Roskilde, Denmark)were coated with 100 µg/ml human IgG (catalog no. 31154;
Thermo Fisher Scientific Pierce, Waltham, MA, USA) at 4˚Covernight. The plates were blocked with 5% non-fat dry milk
in PBS containing 0.05% Tween-20 (PBST) for 1 h at room
temperature and then incubated with serially diluted cell lysate
samples from the transfected HUVECs for 2 h at 37˚C. Afterwashing with Tween-20-containing PBS, the plates were incubated with the biotinylated anti-FCGR2B antibody at 1:1,000
dilutions for 2 h at 37˚C. High Sensitivity Streptavidin HRP
conjugates at 1:10,000 dilutions (catalog no. 21130; Thermo
Fisher Scientific Pierce) was added to each well followed byincubation for 1 h at 37˚C. ELISA was developed using 1-Step
Ultra TMB-ELISA (Thermo Fisher Scientific Pierce,). The
absorbance of each well was measured at 450 nm using an
iMark Microplate Absorbance Reader (Bio-Rad).
Incorporation of human IgG and fluorescence imaging. We
evaluated the ability of the pFCGR2B2-EGFP-transfected
HUVECs to incorporate IgG using a modification of a previously described method (16,17). Alexa 555-human IgG and
Alexa 633-human IgG were prepared using human IgG(Thermo Fisher Scientific Pierce) and an Alexa Fluor proteinlabeling kit (Invitrogen). Alexa 555-F(ab')2 was generated bydigesting Alexa-human IgG using a Pierce F(ab')2 Preparation
kit (catalog no. 44988; Thermo Fisher Scientific Pierce) andpurified by chromatography and dialysis using the methods
recommended by the manufacturer.
Preliminary experiments of the cells exposed to different
concentrations of Alexa 555-IgG or Alexa 555-F(ab')2 (0, 1,
10 and 100 µg/ml) indicate that the minimum concentrationof detectable fluorescence signals in imaging analysis was
10 µg/ml. After 24 h transfection with pFCGR2B2-EGFP
or pEGFP-N1, HUVECs were washed with HBSS (catalog
no. H4891; Sigma-Aldrich) containing 10 mM HEPES (pH 7.4),
serum starved for 20 min, and then exposed to 10 µg/ml
Alexa 555-IgG or equimolar Alexa 555-F(ab')2 for 10 min.
The cells were immediately washed with PBS and incubatedwith fresh medium for another 10 min. For quantitative image
analysis of Alexa 555-IgG and Alexa 555-F(ab')2, the cells were
fixed with 4% paraformaldehyde, counterstained with DAPI,
and mounted in ProLong Gold. The signals were detected using
a fluorescence microscope (IX71; Olympus) or a confocal laserscanning microscope (LSM 710; Carl Zeiss, Jena, Germany).
The quantitative image analysis of the pFCGR2B2-EGFPtransfected or mock-transfected HUVECs was performed
using the MetaMorph image analysis system as follows: using
the auto-fluorescence threshold on merged images, the cellular
profiles were demarcated as a region of interest. The area within
the region of interest occupied by the fluorescence signals of theinternalized Alexa 555-IgG or Alexa 555-F(ab')2 were detectedwith an appropriate fluorescence threshold. Approximately
120 cells were used for this analysis. The figures were compiledusing Photoshop CS software.

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The ability of IgG to incorporate from the basolateralsurface was evaluated as follows: the HUVECs transfected withpFCGR2B2-EGFP were plated on Transwell membrane inserts
for 24-well plates (0.4 µm pores, catalog no. 353495, BD Falcon,
Franklin Lakes, NJ, USA) at a density of 2x105 cells/insert
and cultured for 24 h. Confluent HUVECs were washed withHBSS containing 10 mM HEPES (pH 7.4) and serum starved
for 20 min. The Alexa 633-IgG (100 µg/ml) was added to thewells of the lower chambers of the 24-well plates. Following
incubation for 60 min, the cells were immediately washed andfixed. Super-resolution microscopy detection of FCGR2B2EGFP and Alexa 633-IgG incorporation was performed
using a stimulated emission-depletion (STED) microscope(TSC STED CW; Leica, Wetzlar, Germany). Images werecompiled using Photoshop CS software.
Transcytosis of human IgG. We evaluated the ability of thepFCGR2B2-EGFP-transfected HUVECs to transcytose IgG
using a modification of a previously described method (18,19).
Human IgG (Thermo Fisher Scientific Pierce) was biotinylated using a Biotin Labeling kit-NH 2. The HUVECs
transfected with pFCGR2B2-EGFP or pEGFP-N1 were platedon Transwell membrane inserts for 24-well plates at a densityof 1x105 cells/insert and cultured for 24-36 h.
To confirm the confluence of the polarized monolayers,
we validated the passage of Lucifer yellow and the formationof cell-cell junctions (Fig. 1B and C). Before conducting IgG
transcytosis experiments, 100 µM Lucifer yellow (catalog
no. L-1177; Invitrogen-Molecular Probes, Eugene, OR, USA)was added to the basolateral chamber of HUVECs growing
on the culture inserts. After 4 h, the concentration of Lucifer
yellow of apical media in the culture inserts was measured
using a GloMax 96 microplate luminometer (Promega,
Madison, WI, USA). The integrity of the monolayer was
confirmed with <2% leakage of Lucifer yellow flux observedin all the experiments. Immunofluorescence analysis revealed
that adherens junction (CD144) and adhesive molecule (CD31)
were expressed on cell to-cell contacts. These results indicatedthat the HUVECs developed a confluent polarized monolayer.
After testing the cell monolayer integrity, 15 µg/ml biotinlabeled human IgG were added to in the basolateral donor
chamber. After 24 h, transcytosed, biotin-labeled human IgG in
the apical receiver chamber was detected by western blot analysis
using high sensitivity streptavidin HRP conjugates at 1:10,000
dilutions (catalog no. 21130; Thermo Fisher Scientific Pierce).
Signal detection was carried out using Immobilon Western HRP
Substrate (Millipore, Billerica, MA, USA) and an LAS-4000
lumino-image analyzer (Fujifilm, Tokyo, Japan). Densitometricanalysis of the band intensities of the heavy chain of the transcytosed, biotin-labeled IgG was quantitatively analyzed using
Multi Gauge software (GE Healthcare, Little Chalfont, UK).
Gene silencing. The siRNA-mediated knockdown of FCGRTand RAB3D in the HUVECs was performed as follows: two
distinct types of siRNA duplexes for each target gene were
designed using siDirect (http://sidirect2.rnai.jp/) as previously
described (20), which is based on an algorithm to increase theknockdown efficiency and minimize off-target silencing. The
designed siRNAs were synthesized by Nippon EGT (Toyama,
Japan). The transfection of the siRNAs was performed using

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Lipofectamine 2000 (Invitrogen) according to the manu­
facturer's instructions. The efficacy of gene silencing at themRNA level was assessed after 6-72 h by reverse transcriptionquantitative polymerase chain reaction (RT-qPCR). The
sequences of the designed siRNAs were as follows: siRNAs
for FCGRT, siFCGRT-1 [21 nt guide (5'→3'): acuuuugacuguua
gugacGA; 21 nt passenger (5'→3'): gucacuaacagucaaaaguGG)]and siFCGRT-2 [21 nt guide: uuuacauccuucaaa ucagCA; 21 nt
passenger: cugauuugaaggauguaaaUG)]; siRNAs for RAB3D,
siRAB3D-1 [21 nt guide: uaucaguagcaguuugaacAU; 21 nt
passenger: guucaaacugcuacugauaGG)] and siRAB3D-2 [21 nt
guide: acauugugaaggaaugagcCA; 21 nt passenger: gcucauuccuu
cacaauguCG)]; control siRNA [21 nt guide: uucuccgaacguguc
acguTT; 21 nt passenger: acgugacacguucggagaaTT)]. Nucleotides
shown by uppercases indicate those of 3'-overhangs.
Western blot analysis. Proteins in the HUVECs were extractedusing M-PER Mammalian Protein Extraction Reagent (Thermo
Fisher Scientific Pierce) containing protease inhibitors. Proteinsof isolated FCGR2B2 compartments were directly extractedwith SDS-containing loading buffer. Dilutions for the primary
antibodies were 1:5,000 (FCGR2B, Ral-A and ACTB), 1:250(FCGRT), 1:200 (RAB1 and RAB3), 1:400 (RAB3D) and
1:1,000 (RAB11 and RAB33B). HRP-conjugated secondary
antibodies were diluted to 1:10,000. Signals were detectedusing Immobilon reagent and visualized using a LAS-4000
Lumino image analyzer. The intensity of the visualized signals
was quantitatively analyzed using Multi Gauge software.
RT-qPCR. Total RNA was extracted from the samples using
Isogen reagent (Nippon Gene, Tokyo, Japan) according to the
manufacturer's instructions. cDNA synthesis was performed
using a PrimeScript RT reagent kit (Takara Bio, Shiga,
Japan). For conventional RT-PCR, ExTaq Hot Start Version(Takara Bio) was used for amplification using gene-specificprimers, some of which were designed using DNASIS software(Hitachi, Tokyo, Japan).
The primers used were as follows: FCGR2B forward,
CCACTAATCCTGATGAGGCTGACA and reverse, CTC
AAATCCCAATGCAAGACAATG; FCGRT forward, CAC
GCCTCGTCGTCACTAACA and reverse, AGTAGCAAG
ACACCGATGACGATTC; RAB1A forward, TATGGGACACAGCAGGCCAGG and reverse, ACGGAATTCCAAGGG
AATCAGC; RAB1B forward, TGAACCCCGAATATGAC
TAC and reverse, GTGTACGTGTCATCAGCAAA; RAB3A
forward, CAGACCTGTTCTGACCTCAT and reverse,
CTTTATTGGGTGCGTGTAGT; RAB3B forward, AGATGG
TCCCAGTAATAGATACTC and reverse, GACTGTTCTCTAAGTCCCTGTAGT; RAB3C forward, GCGACAAAATGTCAGAGAGT and reverse, GAGGAGGAGTTTCCTTGAGT;RAB3D forward, TCTGGAACTATGGACCACAT andreverse, CTCCTGGCTCTGAGGTTAAT; RAB2A forward,
CAGGTGTTGGTAAATCATGC and reverse, CACCGAACTCTACACCAATAG; RAB15 forward, ATCAAGACC
TATGCCACATC and reverse, CCACTGCCCAATATAA
CTTC; RAB18 forward, TACCCC TCAGTAAGATTCCA andreverse, TATTGCAAAGGTGGTCACTC; RAB35 forward,
CCAGGATGTGTTTCCTTAGA and reverse, AACTGCA
GTGTGATCTGTGA; RAB39A forward, ACGTCAAGTTACAAGGGAAG and reverse, TGTCTCTCGTCAAG

ATTGTG; ACTB forward, ATTGCCGACAGGATGCAGA
and reverse, GAGTACTTGCGCTCAGGAGGA; and GAPDH
forward, GCACCGTCAAGGCTGAGAAC and reverse, ATG
GTGGTGAAGACGCCAGT. All primers were synthesized by
Nippon EGT. The PCR products were separated by electro­
phoresis on a 2% agarose gel. Quantitative PCR (qPCR) was
performed using SYBR Premix ExTaq (Takara Bio) on an
ABI 7300 apparatus (Applied Biosystems, Foster City, CA, USA).
GAPDH was used for the ormalization of gene expression.
Isolation of FCGR2B2 compartments from the human placenta.
FCGR2B2-containing vesicles were purified from full-term
placentas as follows: human placental terminal villus-richfractions were collected from full-term placentas as previously
described (12). The terminal villus-rich fractions were resuspended in homogenization buffer (20 mM Tris-HCl pH 7.4,
5 mM MgCl2, 0.25 M sucrose, 1 mM PMSF, 1 µM aprotinin
and 1 µM pepstatin A) and disrupted using a Bioruptor (CellDisruption Bomb; Parr Instrument, Moline, IL, USA). After
15 min of shaking under high pressure at 1,300 psi on ice, cells
in terminal villi were disrupted by passage through a narrowvalve. The collected homogenate was centrifuged twice at 4˚C,
first at 600 x g for 10 min and then at 10,000 x g for 20 min. The
supernatant was transferred to ultracentrifuge tubes and centrifuged at 105,000 x g for 1 h at 4˚C. The pellets, representing the
microsomal fraction, were resuspended in phosphate-bufferedsaline (PBS) containing 5 mM MgCl2 and 1% BSA. Magnetic
Dynabeads M280 sheep anti-rabbit IgG (catalog no. 112.04;
Invitrogen) was used for immunoprecipitation. The microsomal
fraction was pre-incubated with Dynabeads bound to the nonimmune rabbit IgG whole molecule (catalog no. 011-000-003;
Jackson ImmunoResearch Laboratories) at 4˚C overnight toreduce non-specific binding. To capture FCGR2B2-containing
vesicles, the pre-incubated microsomal fraction was incubatedwith Dynabeads and either anti-FCGR2B antibody or the
non-immune rabbit IgG as a control at 4˚C for 2 h. The beads
were then washed 5 times with PBS at room temperature andsubjected to the following procedures.
Transmission electron microscopy of FCGR2B2 compartments.
The FCGR2B2 compartments were fixed overnight at room
temperature with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 containing
0.05% CaCl 2. The pellet was post-fixed with 2% osmium
tetroxide and 1.6% potassium ferrocyanide in 0.1 M sodium
cacodylate at room temperature for 30 min. The pellet wasdehydrated in ethanol and then embedded in epoxy resin. The
sections were electron stained with uranyl acetate and leadcitrate and examined under a transmission electron microscope(H-7600; Hitachi) at 80 kV.
Two-dimensional differential in-gel electrophoresis (2D-DIGE)
and mass spectrometry. 2D-DIGE was performed as previously described (21). The isolated FCGR2B2 compartments
were lysed with thiourea lysis buffer. As a control, an equalvolume of the control beads bound to the non-immune rabbit
IgG whole molecule was used. The proteins extracted from thepurified FCGR2B2 compartments and the control beads werelabeled with CyDye DIGE Fluor Cy3 and Cy5 minimal Dyes
(GE Healthcare), respectively. The CyDye-labeled proteins andINTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 35: 1273-1289, 2015

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Figure 1. Validation of antibodies and characterization of pFCGR2B2-EGFP-transfected human umbilical vein endothelial cells (HUVECs). (A) Validationof antibodies. The anti-FCGR2A, anti-FCGR2B and anti-FCGRT antibodies used in this study were validated by western blot analysis. Lysates of Cos-7
cells transfected with a plasmid designed to overexpress EGFP or EGFP-tagged FCGRT, FCGR1A, FCGR2A, FCGR2B1 or FCGR2B2 were used for thisvalidation. The upper band in each lane corresponds to an EGFP-conjugated Fc receptor and the lower band corresponds to an Fc receptor without EGFP.
(B and C) Representative immunofluorescence images of CD31 and CD144 in HUVECs plated on cell culture inserts. (B) CD31 and (C) CD144 are localizedalong the lateral membrane of adjacent cells. (D and E) Comparison of the subcellular localization and IgG binding property of FCGR2B2-EGFP with thoseof FCGR2B2. (D) Representative immunofluorescence image of a pFCGR2B2-transfected HUVEC. FCGR2B2 signals are detected as intracellular vesicularand tubular structures; some signals are also present on the cell surface. Note that a pFCGR2B2-EGFP-transfected cell (Fig. 2A) shows similar localization of
FCGR2B2 in the pFCGR2B2-transfected cell. (E) The affinities of human IgG for FCGR2B2-EGFP and FCGR2B2 detected by enzyme-linked immunosorbent
assay (ELISA). FCGR2B2-EGFP (white circles) shows an almost identical affinity for FCGR2B2 (black circles). By contrast, EGFP from mock-transfected
pEGFP-N1 cells do not exhibit selectivity for IgG (black triangles). Three independent experiments were performed; error bars represent the means ± SD.
FCGR2B2, low-affinity immunoglobulin gamma Fc region receptor IIb2.

non-labeled microsomal proteins (as a spot reference) werethen mixed and subjected to 2D-DIGE. The CyDye-labeledprotein spots in the gel were visualized using a Typhoon 9400
imager and analyzed using differential in-gel analysis with
DeCyder 2D Differential Analysis Software (both fromGE Healthcare). Protein spots that exhibited 2-fold higher
intensity in the FCGR2B2 compartments compared to thecontrol were selected as candidate FCGR2B2 compartmentassociated proteins. All proteins, including the non-labeled
microsomal proteins, on the same gel were then stained with
SYPRO Ruby (Takara Bio) to determine the positions of the

FCGR2B2 compartment-associated proteins. The eluted
proteins were analyzed by liquid chromatography-tandem mass
spectrometry (LC-MS/MS) (LCQ DECA, XP Plus; Thermo
Finnigan, San Jose, CA, USA). Data were analyzed using
Mascot Search on the Matrix Science website (http://www.
matrixscience.com/). The specificity of trypsin digestion wasused as the cutting enzyme, allowing 3 missed cleavages.
Peptide mass tolerance and fragment mass tolerance were
set to ±1.2 and ±0.6 Da, respectively. Carbamidomethyl ofcysteine was selected as the fixed modification and oxidation ofmethionine was searched as the variable modification. Identity

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Figure 2. Localization of FCGR2B2 in pFCGR2B2-EGFP-transfected human umbilical vein endothelial cells (HUVECs). (A and B) Representative confocal
fluorescence images of HUVECs transfected with either (A) pFCGR2B2-EGFP or (B) pEGFP-N1. (A) In a pFCGR2B2-EGFP-transfected cell, numerous
FCGR2B2-EGFP signals (green) are distributed throughout the cytoplasm; green signals are detected as intracellular vesicular (arrowheads) and tubular structures(arrows). The signals are also evident on the Golgi apparatus. (B) By contrast, in a mock-transfected cell, the EGFP signals are diffusely distributed within the cytoplasm and the nuclei. (C-E) Representative fluorescence microscopy images of pFCGR2B2-EGFP-transfected HUVECs immunostained with (C) anti-FCGR2B
antibody, (D) anti-von Willebrand factor antibody, or (E) anti-caveolin-1antibody. Alexa Fluor 594-labeled secondary antibodies were used to detect the primary
antibodies. Each inset shows a higher magnification view of the region in the white frame. (C) FCGR2B2-EGFP signals (green) and Alexa Fluor 594 signals
detected with anti-FCGR2B2 antibody (red) are co-localized (yellow arrowheads). By contrast, FCGR2B2-EGFP signals (green arrowheads) are not co-localizedwith either (D) von Willebrand factor or (E) caveolin-1 (red arrowheads). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue). Scalebars represent 10 µm. Data are representative of 3 or more independent experiments. FCGR2B2, low-affinity immunoglobulin gamma Fc region receptor IIb2.

or extensive homology is indicated for peptides with individual
ion scores >42 (p<0.05).
Statistical analysis. We conducted all the analyses using the
SPSS statistical software package (Windows version 20;
IBM-SPSS). The significance of between-group differences
was assessed using the Student's t-test or ANOVA with
Dunnett's post test, and p-values <0.05 were considered toindicate statistically significant differences.
Results
Expression and localization of FCGR2B2 in pFCGR2B2EGFP-transfected HUVECs. In the pFCGR2B2-EGFP-transfected HUVECs, fluorescence signals indicating the expressionof FCGR2B2 (FCGR2B2-EGFP signals) were transiently
detected within 18-48 h after electroporation. In the transfected
cells, FCGR2B2-EGFP signals were detected as intracellular
vesicular and tubular structures and were widely distributedin the cytoplasm (Fig. 2A). Some signals were also present on
the cell surface. The pFCGR2B2-EGFP-transfected HUVECs
showed a similar expression of FCGR2B2 in placental endothelial cells in vivo as previously reported (11). By contrast, inthe mock-transfected pEGFP-N1 cells, the fluorescence signals
showed a diffuse distribution throughout the cytoplasm andthe nucleus and did not exhibit intracellular structure-specificlocalization (Fig. 2B).
Subsequently, we wished to investigate whether the
localization of the FCGR2B2-EGFP reflected that of theendogenous protein. In the pFCGR2B2-EGFP-transfected
HUVECs, the majority of the EGFP signals were co-localizedwith the Alexa Fluor 594 fluorescence signals detected withan anti-FCGR2B antibody (Fig. 2C), thus suggesting that the
EGFP signals can be used to reliably monitor FCGR2B2 in thetransfected cells. Although an endogenous endothelial marker,
von Willebrand factor, and a caveola marker, caveolin-1,
were present in the FCGR2B2‑EGFP-expressing HUVECs,
they were not co-localized with the FCGR2B2-EGFP signals(Fig. 2D and E). These results are consistent with those ofprevious in vivo findings that the intracellular FCGR2B2
compartments do not overlap with caveolae and secretory
granules in the placental endothelium (22). In addition, inorder to provide an experimental foundation for bio-imaging
analysis, we compared the subcellular localization and IgGbinding property of FCGR2B2-EGFP with those of FCGR2B2.
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1279

transfected HUVECs showed a localization of FCGR2B2
similar to the pFCGR2B2-transfected cells (Fig. 1D). We also
examined the affinities of human IgG for FCGR2B2-EGFP
and FCGR2B2 by ELISA (Fig. 1E). FCGR2B2-EGFP showed
an almost identical affinity of human IgG for FCGR2B2.
Taken together, these results indicate that the pFCGR2B2EGFP-transfected HUVECs are a useful model for placental
endothelial cells in vitro.

IgG in the apical receiver chamber was detected by western
blot analysis. The band intensities of the heavy chain of thetranscytosed, biotin-labeled IgG were significantly higher inthe pFCGR2B2-EGFP-transfected cells than in the mocktransfected cells (P<0.05) (Fig. 4I). Taken together, these results
suggest that the IgG internalization and transcytosis observed inthis study was caused by the enforced expression of FCGR2B2
in the HUVECs and that it was an Fc fragment-specific event.

Human IgG incorporation from the basolateral surface of
FCGR2B2-EGFP-expressing cells. As maternal IgG is internalized from the basolateral surface of the placental endotheliumin vivo, we then examined the subcellular IgG incorporationfrom the basolateral surface of FCGR2B2‑EGFP‑expressing
HUVECs by both STED and confocal imaging. STED
microscopy is a technique that uses the non-linear de-excitation of fluorescent dyes to overcome the resolution limit ofstandard confocal microscopes that is imposed by diffraction (23). Confluent pFCGR2B2-EGFP-transfected HUVECs
that were cultured on Transwell filters were exposed toAlexa Fluor 633-labeled IgG (Alexa 633-IgG) from the basolateral surface. Super-resolution imaging with STED and confocal
imaging revealed that oval and tubular FCGR2B2‑EGFP
signals were often found and frequently co-localized with
Alexa Fluor 633-IgG signals that were incorporated from thebasolateral surface (Fig. 3). By three-dimensional analysis, itappeared that some FCGR2B2-EFGP compartments extended
or fused along the axis of apical-basal cell polarity (Fig. 3B, YZ
and XZ).

IgG internalization and transcytosis in FCGR2B2-EGFPexpressing cells that are FCGRT-deficient. HUVECs also
express FCGRT; thus, we examined the effects of endogenous
FCGRT on IgG incorporation by siRNA-mediated knockdown. We constructed 2 types of siRNA duplexes against
FCGRT (designated as siFCGRT-1 and siFCGRT-2). First, weevaluated the efficiency of the siRNA-mediated knockdown
using RT-qPCR and western blot analysis. After 48 h ofsiRNA transfection, the mRNA expression level of FCGRT
was 9.2 and 6.6% of the control cell levels with siFCGRT-1
and siFCGRT-2, respectively. After 72 h, the expression
level was 10.6% (siFCGRT-1) and 3.4% (siFCGRT-2) of
the control (Fig. 5A). Furthermore, the protein expressionof FCGRT was markedly decreased by siFCGRT-1 andsiFCGRT-2 at both 48 and 72 h, as determined by western blot
analysis (Fig. 5B). As these siRNAs efficiently suppressed theexpression of both the FCGRT mRNA and FCGRT protein,
they can be used for subsequent experiments to investigate
the effect of endogenous FCGRT on IgG incorporation in
FCGR2B2-EGFP-expressing HUVECs.
After 18-24 h of transfection with pFCGR2B2-EGFP, the
HUVECs were transfected with siRNAs and incubated for afurther 18 h. The transfection of siFCGRT-1 and siFCGRT-2
did not affect the expression and distribution of FCGR2B2EGFP (Fig. 5C-E). The internalization of Alexa 555-IgG was
not altered in the cells in which FCGRT had been knocked
down (Fig. 5C and D). Furthermore, we investigated the abilityof the cells in which FCGRT had been knocked down to transcytose IgG using the above-mentioned biotin-labeled human
IgG. No significant differences in the band intensities of theheavy chain of the transcytosed, biotin-labeled IgG were identified between the cells in which FCGRT had been knocked down
and the mock cells (Fig. 5F). Thus, our results suggested that the
IgG internalization and transcytosis were mediated primarily
by the enforced expression of FCGR2B2 rather than that ofendogenous FCGRT in the HUVECs.

Internalization and transcytosis of human IgG in the
FCGR2B2-EGFP-expressing cells. The ability of FCGR2B2EGFP-expressing HUVECs to incorporate human IgG
was evaluated by monitoring the fluorescence signals in
the cells exposed to Alexa Fluor 555-labeled human IgG(Alexa 555-IgG). In the cells, the majority of the internalizedAlexa 555-IgG signals were co-localized with the FCGR2B2EGFP signals (Fig. 4A-D). Internalized Alexa 555-IgG
was more easily detected as small dots of fluorescence inthe pFCGR2B2-EGFP-transfected cells than in the EGFP
(mock)-transfected cells (Fig. 4E). The ability of the FCGR2B2EGFP-expressing cells to incorporate IgG was evaluated bycomparing the ratios of the Alexa 555-positive area to the total
cellular area in the pFCGR2B2-EGFP-transfected cells (n=47)
and mock-transfected cells (n=48); this ratio was significantly
higher in the pFCGR2B2-EGFP-transfected cells than in the
mock-transfected cells (P<0.01) (Fig. 4G). Furthermore, tovalidate the Fc fragment-specific internalization, FCGR2B2EGFP-expressing cells were incubated with equimolar
concentrations of IgG or F(ab')2 fragments (Fig. 4F). The ratiosof the Alexa 555-IgG and Alexa 555-labeled-F(ab')2 fragment[Alexa 555-F(ab')2] positive areas were measured in 43 and48 cells, respectively. While the Alexa 555-F(ab')2 was detectedin the FCGR2B2-EGFP-expressing cells (Fig. 4F), the ratioof Alexa 555-F(ab')2 was significantly lower than the ratio of
Alexa 555-IgG (P<0.01) (Fig. 4H).
Subsequently, we aimed to investigate the ability of thepFCGR2B2-EGFP-transfected HUVECs to transcytose IgG.
Biotin-labeled human IgG was added to in the basolateral
donor chamber. After 24 h, transcytosed, biotin-labeled human

Identification of FCGR2B2 compartment-associated proteinsfrom the human placenta. We performed a proteomic analysis
to identify FCGR2B2 compartment-associated proteins, someof which may be involved in the regulation of the trafficking
of FCGR2B2 compartments. FCGR2B2 compartments wereisolated from human placental terminal villus-rich fractions
by immunoprecipitation using magnetic beads bound to a
FCGR2B-specific antibody as described in the Materials
and methods. Imaging by electron microscopy revealed
that FCGR2B2 compartments were round, oval and tubular
membranous components (Fig. 6A and B). These morphological characteristics were similar to those observed by the
immunoelectron microscopy of FCGR2B2 compartments inplacental endothelial cells (11). Western blot analysis revealed

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ISHIKAWA et al: FCGR2B IN HUMAN PLACENTAL ENDOTHELIAL CELLS

Figure 3. Super-resolution nanoscopic observation of human IgG internalization from the basolateral surface of FCGR2B2-EGFP-expressing human umbilicalvein endothelial cells (HUVECs) using stimulated emission-depletion (STED) microscopy. (A) Combined image of FCGR2B2-EGFP (STED in green) andAlexa Fluor 633-IgG (confocal in red) shows that most of the internalized Alexa 633-IgG signals are co-localized with FCGR2B2-EGFP signals. (B) Sets ofhigher magnification images of 3 regions indicated with numbered arrowheads in (A). Each set is arranged with the following images, from left to right: STED
microscopy image showing FCGR2B2-EGFP (FCGR2B2-EGFP), confocal image with Alexa 633-IgG (IgG), the merged image (merge), a virtual slice cut
at a vertical line indicated with YY in merged image (YZ), and a virtual slice cut at a horizontal line indicated with XX in merged image (XZ). Apical (AP)and basolateral (BL) sides are evident. Oval and tubular FCGR2B2-EGFP signals are co-localized with Alexa Fluor 633-IgG signals that are incorporated
from basolateral surface (arrows). Scale bars represent (A) 2 µm and (B) 200 nm. Three independent experiments were performed and yielded similar results.
FCGR2B2, low-affinity immunoglobulin gamma Fc region receptor IIb2.

that the FCGR2B2 compartments were specifically captured
on the beads coated with the anti-FCGR2B antibody (Fig. 6C).
Proteomic analysis of the FCGR2B fraction was performedby 2D-DIGE. Twenty protein spots showed a >2-fold higher
volume ratio in the FCGR2B2 compartment fraction comparedwith the control fraction. However, 4 of these spots were partof a horizontal streaking tail; thus, they were not subjected tofurther analyses. Of the 16 spots, 2 minor spots could not be
separated or detected on gels for protein identification. The
remaining 14 spots were isolated and analyzed by LC-MS/MS.

The FCGR2B2 compartment-associated proteins identified by
LC-MS/MS are summarized in Table I. Of note, LC-MS/MS
revealed that these proteins contained a number of RAB family
proteins.
Subsequently, we examined the expression of some of the
RAB family proteins identified by LC-MS/MS in the isolated
FCGR2B2 compartments (Table I). Western blot analysis
revealed that both RAB3 and RAB1 were detectable in the
FCGR2B2 compartments (Fig. 6D). However, specific antibodies against the other RABs identified by LC-MS/MS wereINTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 35: 1273-1289, 2015

1281

Figure 4. Internalization and transcytosis of human IgG in FCGR2B2-EGFP-expressing cells. (A-D) Representative confocal fluorescence images of internalized Alexa Fluor 555-IgG (red) in FCGR2B2-EGFP (green)-expressing human umbilical vein endothelial cells (HUVECs). (A) A confocal optical sliceof 0.6 µm thickness at the position closest to the basal surface. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue). (B) Higher
magnification images of the regions indicated with the numbered arrowheads in (A). (C and D) Virtual slices cut at the horizontal gray line denoted by (C and D)
in (A). In each virtual slice, the apical surface of the HUVECs is indicated as the upper side. Internalized Alexa Fluor 555-IgG signals appear as small dots inthe cells. Co-localization of internalized Alexa Fluor 555‑IgG and FCGR2B2-EGFP signals is frequently observed (arrows in B-D). (E) Higher magnification
confocal images of Alexa Fluor 555-IgG (red) in FCGR2B2-EGFP- and EGFP (green)-expressing cells. Internalized Alexa Fluor 555-IgG signals appear as
small vesicular structures colocalized with FCGR2B2-compartments in a pFCGR2B2-EGFP-transfected cell. By contrast, in an EGFP (mock)-transfected cell,the level of internalized Alexa Fluor 555-IgG is very low. (F) Higher magnification confocal images of Alexa Fluor 555-IgG or Alexa Fluor 555-F(ab')2 fragments(red) in FCGR2B2-EGFP-expressing cells. The internalization of the Alexa Fluor 555-F(ab')2 signal is obviously lower than that of Alexa Fluor 555-IgG. Scalebars are 10 µm. (G and H) Quantification of internalized Alexa Fluor 555-IgG or Alexa Fluor 555-F(ab')2 signals. The area of intracellular Alexa Fluor 555-IgG or
Alexa Fluor 555-F(ab')2 signals was quantified. Values are the means ± SE [FCGR2B2-EGFP-expressing cells, n=47; EGFP (mock)-expressing cells, n=48 (G)]and the mean ± SE [Alexa 555-IgG-internalized cells, n=43; Alexa 555-F(ab')2-internalized cells, n=48) (H)]. **p<0.01, Student's t-test. Three or more experiments were performed and yielded similar results. (I) Quantification of transcytosed, biotin-labeled human IgG by western blot analysis. The band intensitiesof the heavy chain of the transcytosed, biotin-labeled IgG are significantly higher in pFCGR2B2-EGFP-transfected cells than in EGFP (mock)-transfected cells(*p<0.05, Student's t-test). The data are the means from 3 measurements; error bars represent the means ± SD. FCGR2B2, low-affinity immunoglobulin gamma
Fc region receptor IIb2.

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ISHIKAWA et al: FCGR2B IN HUMAN PLACENTAL ENDOTHELIAL CELLS

Figure 5. IgG internalization and transcytosis in FCGR2B2-EGFP-expressing cells following the downregulation of FCGRT. (A and B) The efficiency of siRNAmediated knockdown of FCGRT in human umbilical vein endothelial cells (HUVECs). (A) RT-qPCR reveals that both siFCGRT-1 and siFCGRT-2 significantly
reduce the FCGRT mRNA level in the HUVECs after 48 and 72 h. The data are the means from 3 measurements. Error bars represent the means ± SD. **p<0.01,
one-way ANOVA with Dunnett's post test. (B) Western blot analysis reveals that the FCGRT protein levels in the HUVECs are markedly downregulated atboth 48 and 72 h after transfection of siRNAs. (C-E) Representative confocal fluorescence images of internalized Alexa Fluor 555‑IgG in FCGR2B2-EGFPexpressing HUVECs after FCGRT knockdown. After 18-24 h to transfect with pFCGR2B2-EGFP, HUVECs were transfected with siRNAs and incubated foranother 18 h. Transfection of (C) siFCGRT-1 and (D) siFCGRT-2 do not affect the expression and distribution of FCGR2B2-EGFP or the internalization of
Alexa 555-IgG signals compared to the control siRNA (E). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue). Scale bar represents
10 µm. Three independent experiments were performed with similar results. (F) Quantification of transcytosed, biotin-labeled human IgG by western blot
analysis. No significant differences in the band intensities of the heavy chain of the transcytosed, biotin-labeled IgG are identified between FCGRT-knockdown(siFCGRT-1 and siFCGRT-2) and mock (control) cells (one-way ANOVA). The data are the mean from 3 measurements; error bars represent the means ± SD.
FCGR2B2, low-affinity immunoglobulin gamma Fc region receptor IIb2.

not commercially available for use in western blot analysis. In
order to examine which isoforms may be associated with the
FCGR2B2 compartments and which isoforms may be highly
expressed in placental endothelial cells, we investigated themRNA expression levels of each isoform (i.e., RAB1A, RAB1B,
RAB2A, RAB3A, RAB3B, RAB3D, RAB15, RAB18, RAB35
and RAB39A) in placental tissues (first-trimester and full-term
placentas) and isolated endothelial cells (placental endothelial
cells and HUVECs). At first, we examined 3 RAB3 isoforms,
RAB3A, RAB3B and RAB3D, and another highly homologous
RAB3 isoform, RAB3C. RT-qPCR revealed that RAB3A andRAB3C were absent in the full-term placentas (Fig. 7A). The

mRNA expression levels of RAB3B, RAB3D, and other RABs
were subsequently compared among first-trimester placentas,
full-term placentas, placental endothelial cells and HUVECs by
RT-qPCR. RAB1A, RAB1B, RAB2A, RAB3D, RAB15, RAB18,
RAB35 and RAB39A were expressed in all the samples examined; RAB3B was detected in the placental tissues, but not in theendothelial cells (Figs. 7B and 8). Among these RAB mRNAs,
only RAB3D was expressed predominantly in the placental
endothelial cells (Fig. 7B). Therefore, we focused on RAB3D
as a candidate FCGR2B2 compartment-associated protein to
investigate its involvement in the formation and/or intracellular
dynamics of FCGR2B2 compartments.

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1283

Table I. FCGR2B2 compartment-associated proteins identified by LC-MS/MS.
GI no.

Gene			Western blot analysis
symbolProtein name
Fold change of RAB proteins

4504517HSPB1
20147713
RALA
4758984
RAB11A
181178CTSB
182518FTL
4504747
ITGA3
119610452 MYH1032490572
EPB41L3558436DLG1
115298659 SPTA1
5729877
HSPA871773329
ANXA614585873
DYNC1I2
2352945		
|2851393
POR
7706706SNX94506467
RDX
340217VIL213569962
RAB1B
229451		
4758988
RAB1A
5803135RAB3538371739
RAB15
13786129
RAB33B
1491714
RAB39A
1710248ERP5550062RAB210880989
RAB18
27734452
RAB15
4758988
RAB1A
4506365
RAB2A
4759000
RAB3D
1060888
PSMD2
179468HSD3B
4505257MSN
4758304
PDIA46470150HSPA519923750
RAB3B
4506367
RAB3A
20178293
KRT7
4759140
SLC9A3R1
178027ACTA3Heat shock 27 kDa protein 1
Ras-related protein Ral-A (precursor)
Ras-related protein Rab-11A
Lysosomal proteinase cathepsin B
Ferritin light subunit
Integrin alpha 3 isoform a precursor
Myosin, heavy polypeptide 10, non-muscle, isoform CRA_a
Erythrocyte membrane protein band 4.1-like 3
Homolog of Drosophila discs large protein, isoform 2
Spectrin, alpha, erythrocytic 1
Heat shock 70 kDa protein 8 isoform 1
Annexin VI isoform 1
Cytoplasmic dynein intermediate chain
Smooth muscle myosin heavy chain SM2
NADPH-cytochrome P450 reductase
Sorting nexin 9
Radixin
Cytovillin 2
RAB1B, member RAS oncogene family
Placental lactogen
RAB1A, member RAS oncogene family
RAB35, member RAS oncogene family
RAB15, member RAS onocogene family
RAB33B, member RAS oncogene family
Rab-related GTP-binding protein, Rab39A
Protein disulfide isomerase-related protein 5
GTP-binding protein, RAB2A
RAB18, member RAS oncogene family
Ras-related protein Rab-15
RAB1A, member RAS oncogene family
RAB2A, member RAS oncogene family
RAB3D, member RAS oncogene family
Human 26S proteasome subunit p97
3-beta-Hydroxysteroid dehydrogenase
MoesinProtein disulfide isomerase-associated 4
BiP protein, heat shock 70 kDa protein 5
RAB3B, member RAS oncogene family
RAB3A, member RAS oncogene family
Cytokeratin-7
Solute carrier family 9, isoform 3 regulator 1
Alpha-actin

3.03
3.03
3.03
2.48, 2.02
2.48
2.22
2.22
2.22
2.22
2.22
2.22, 2.05
2.22
2.22
2.22
2.22
2.22, 2.05
2.22
2.22
2.19, 2.14
2.19, 2.14
2.19, 2.14
2.19, 2.14, 2.12
2.19, 2.12
2.19, 2.12
2.19, 2.12
2.17
2.14
2.14
2.14
2.12
2.12
2.12, 2.02
2.06
2.05
2.05
2.05
2.03
2.02
2.02
2.02
2.02
2.02

(-)
(+/-)a

(+)b
(+)b
ND
ND
(+/-)
ND
ND
ND
ND
(+)b
ND
(+)c

(+)c
(+)c

RAB11A was detected using mouse anti-human RAB11 antibody. bRAB1 isoforms were detected using rabbit anti-human RAB1 antiserum.
RAB3 isoforms were detected using rabbit anti-human RAB3 antiserum. FCGR2B2, low-affinity immunoglobulin gamma Fc region receptor IIb2;
LC-MS/MS, liquid chromatography-tandem mass spectrometry; +, positive; +/-, faint; -, no band; ND, not determined.
ac

Association of the downregulation of RAB3D with FCGR2B2
compartments using gene specific siRNAs. We observed

FCGR2B2-EGFP expression in the cells in which RAB3D
expression was downregulated by siRNA-mediated knock-

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Figure 6. Validation of isolated FCGR2B2 compartments. FCGR2B2 compartments were isolated from human full-term placenta. (A and B) Electron microscopy
analysis reveals that on the surface of the magnetic beads bound to the anti-FCGR2B antibody, round (small arrows) and tubular (large arrows) membranouscompartments are visible. These morphological characteristics are similar to FCGR2B2 compartments previously observed in human placental endothelial cells
in vivo (11). Scale bars represent (A) 2 µm and (B) 200 nm. (C and D) Western blot analysis confirms that FCGR2B2 compartments are specifically capturedby the beads coated with the anti-FCGR2B antibody (C). RAB3 and RAB1 proteins are detected on the FCGR2B2 compartments (D). The broad smear of thecontrol fraction in the RAB3 panel is considered to be a non-specific signal because its molecular weight is >24 kDa. FCGR2B2, low-affinity immunoglobulin
gamma Fc region receptor IIb2.

Figure 7. RT-qPCR of RAB3 isoforms in human placental tissues and isolated endothelial cells. (A) RT-qPCR of full-term placentas (FT-P) and human brain(Brain) as a positive control. RAB3A and RAB3C are absent in FT-P. Other isoforms of RAB3, RAB3B and RAB3D, are expressed in FT-P. (B) RT-qPCR of humanplacental endothelial cells (HPECs), human umbilical vein endothelial cells (HUVECs), first-trimester placentas (1st-P) and FT-P. HPECs and HUVECs wereisolated simultaneously from FT-P and umbilical cords, respectively. FCGR2B is highly expressed in HPECs, but is not expressed in HUVECs. RAB3D is highly
expressed in HPECs compared to 1st-P, FT-P, and HUVEC. Expression levels were normalized to GAPDH. Relative expression levels in FT-P were assigned avalue of 1. Data are indicated as the mean from the results using 3 pregnant women. Error bars represent the means ± SD. *p<0.05 and **p<0.01, one-way ANOVA
with Dunnett's post test. FCGR2B2, low-affinity immunoglobulin gamma Fc region receptor IIb2.

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Figure 8. RT-qPCR of mRNAs encoding other RAB family proteins in human placental tissues and isolated endothelial cells. RT-qPCR of RAB mRNAs inhuman placentas and isolated endothelial cells. The expression levels of RAB mRNAs (RAB1A, RAB1B, RAB2A, RAB12, RAB18, RAB35 and RAB39) identified
from isolated FCGR2B2 compartments by proteomic analysis were examined in human placental endothelial cells (HPECs), human umbilical vein endothelial
cells (HUVECs), first-trimester placentas (1st-P) and full-term placentas (FT-P). The expression levels were normalized to GAPDH. The relative expression
levels in FT-P were assigned a value of 1. Data are the mean from the results using 3 pregnant women. Error bars represent the means ± SD. *p<0.05, one-way
ANOVA with Dunnett's post test. FCGR2B2, low-affinity immunoglobulin gamma Fc region receptor IIb2.

down. We constructed 2 types of siRNA duplexes against
RAB3D (designated as siRAB3D-1 and siRAB3D-2). First, weevaluated the efficiency of the siRNA-mediated knockdownby RT-qPCR. In the HUVECs, siRAB3D-1 and siRAB3D-2

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ISHIKAWA et al: FCGR2B IN HUMAN PLACENTAL ENDOTHELIAL CELLS

Figure 9. Intracellular dynamics of FCGR2B2 compartments in cells in which RAB3D was knocked down. (A and B) Validation of the siRNA-mediatedknockdown efficiency of RAB3D in human umbilical vein endothelial cells (HUVECs). (A) RT-qPCR reveals that both siRAB3D-1 and siRAB3D-2 efficiently
reduce the RAB3D mRNA level in the HUVECs after 24 and 48 h. Data are the mean from 3 measurements. Error bars represent the means ± SD. **p<0.01, oneway ANOVA with Dunnett's post test. (B) Western blot analysis reveals that RAB3D protein levels in the HUVECs are downregulated at both 24 and 48 h after
transfection of siRNAs. (C-E) Representative fluorescence images of FCGR2B2-EGFP (green) with 4',6-diamidino-2-phenylindole (DAPI) (blue) in HUVECs
transfected with 2 distinct siRNAs directed against RAB3D mRNA [(C) siRAB3D-1 and (D) siRAB3D-2] and (E) control non-targeting siRNA. After 18-24 h oftransfection with pFCGR2B2-EGFP, the HUVECs were transfected with siRNAs and incubated for a further 18 h. (C and D) RAB3D knockdown results in themarked reduction of FCGR2B2-EGFP signals at the cell periphery and accumulation of FCGR2B2-EGFP as large vesicular compartments in the juxtanuclear
area. Scale bar represents 10 µm. FCGR2B2, low-affinity immunoglobulin gamma Fc region receptor IIb2.

inhibited the expression of each mRNA by 9.8 and 14.2% after
24 h and by 9.5 and 10.7% after 48 h, respectively (Fig. 9A).
The protein level of RAB3D was also reduced by siRNAmediated knockdown (Fig. 9B).
We cultured the pFCGR2B2-EGFP-transfected HUVECs
until an EGFP signal could be observed and then treatedthem with the siRAB3Ds. Following RAB3D knockdown, we
observed a redistribution of the FCGR2B2-EGFP signals in thetransfected cells; the FCGR2B2-EGFP signals were markedly
reduced at the cell periphery and accumulated as large vesicularcompartments in the juxtanuclear area (Fig. 9C and D). These
results suggest that RAB3D participates in the formation of
FCGR2B2 compartments in the transfected cells.
Discussion
In this study, we performed bio-imaging analysis of IgG trafficking in FCGR2B2-transfected HUVECs and demonstrated
that FCGR2B2 compartments were involved in IgG transport
following basolateral IgG internalization in the transfected
cells. Maternal IgG is transferred across two cell layers of
the human placenta, the syncytiotrophoblast and the fetal
placental endothelium. In the syncytiotrophoblast, the maternal
IgG incorporated by non-specific pinocytosis is protected by

FCGRT in endosomes and is subsequently transcytosed tothe basolateral surface of the cell, i.e., the placental villous
stroma. Therefore, the villous stroma contains large amountsof maternal IgG. It is important to understand the mechanisms
through which the maternal IgG accumulated in the villous
stroma is transported across the fetal endothelium, the second
placental barrier. FCGR2B2 is abundantly and exclusively
expressed in the placental endothelium (10,11). We have previously suggested that a distinct FCGR2B2-containing organelle
in the placental endothelium mediates the transcytosis ofmaternal IgG (11). However, it is unclear as to how IgG negotiates the placental endothelial cells. We can envision at least
three possible mechanisms: passive movement along a constitutive endocytic pathway, movement down an IgG concentration
gradient, or transfer of IgG mediated by FCGR2B2. Thus,
we developed an in vitro model system for the analysis of
IgG transport in the placental endothelium. We transfectedpFCGR2B2-EGFP vectors into HUVECs and generated
FCGR2B2-EGFP-expressing fetal endothelial cells as mimics
of placental endothelial cells. Indeed, EGFP signals indicating
FCGR2B2 were distributed in vesicular and tubular compartments that were distinct from caveolae and secretory granules.
The majority of the internalized IgG was associated with
FCGR2B2-EGFP-positive compartments. Our in vitro findings

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 35: 1273-1289, 2015

are consistent with our previous in vivo results showing that
the FCGR2B2 compartments are intracellular organelles of theplacental endothelium, not associated with caveolae or secretory granules (11).
FCGR2B is thought to bind preferentially to IgG in theform of immune complexes (24,25). However, crystallographic
studies of FCGR2A and FCGR2B have suggested that thereceptor dimers can also bind to monomeric IgG (26,27).
Moreover, functional mapping using synthetic peptides corresponding to stretches of the IgG Fc peptides has also indicatedthat the IgG1 CH2 domain binds to both soluble and cellsurface-expressed forms of FCGR2B (14). FCGR2B2 mediates
endocytosis and transcytosis, while its companion isoform,
FCGR2B1, fails to enter endocytic compartments (28,29). Of
note, FCGR2B is typically undetectable in endothelial cells
in adult human tissues apart from the placental endothelium
and the hepatic sinusoid, and both organs express FCGR2B2
and not FCGR2B1. Mousavi et al demonstrated that FCGR2B2
expression in rat hepatic sinusoidal endothelial cells serves asa recycling receptor (30); however, their study focused on thereceptor-mediating endocytosis of immune complexes. Taken
together with previous observations, the present study lends
support to the idea that FCGR2B2 plays an important rolein the intracellular trafficking of IgG in the human placental
endothelium.
Based on our data, it can also be concluded that the internalization and transcytosis of IgG is accelerated by FCGR2B2
rather than FCGRT in our in vitro model of the humanplacental endothelium. Although FCGRT transports both IgG
and albumin (31), albumin cannot easily be transported likematernal IgG across the human placenta (32,33). One possible
explanation of the selective transfer of IgG across the placental
barrier for our findings may be that FCGRT of the syncytiotrophoblast transports both ligands but FCGR2B2 of the placental
endothelium allows only IgG to pass. However, we have notillustrated the whole picture of Fc receptors and their associated regulatory factors involved in IgG transcytosis in thehuman placental endothelium. FCGRT is expressed not onlyin the syncytiotrophoblast, but also in adult endothelial cells; it
regulates the serum levels of IgG (34,35). There is still limited
knowledge concerning intracellular trafficking in endothelial
cells. It would be of interest to investigate whether FCGRT
cooperates with FCGR2B2 to transcytose IgG in the humanplacental endothelium by imaging analysis. However, the antiFCGRT antibody generated in this study could not work wellin immunocytochemistry. The involvement of FCGRT in IgGtranscytosis in the placental endothelium remains an issue.
The mouse placenta does not transport IgG; rather, the yolk
sac is the organ responsible for maternal-fetal IgG transport in
the mouse. Maternal IgG is transported across the mouse yolksac endoderm by FCGRT (36). The yolk sac also expresses
FCGR2B2. However, FCGR2B2 is not present in the yolk sac
in endothelial cells; non-endothelial FCGR2B2 is not required
for the transport of IgG to the fetus (37,38). The vasculararchitecture of the human placenta is quite distinct from thatof the mouse yolk sac, and FCGR2B2 is expressed in different
cell types along the IgG transport pathway between the two
species. In addition, the carboxy-terminal sequence involved inphagocytosis by murine FCGR2B2 is not conserved in human
FCGR2B2 (39). This may also be a difference in the behavior

1287

of human and mouse FCGR2B2 which could impinge on theintracellular trafficking. Therefore, it is unlikely that the yolk
sac is suitable for study as a model for human IgG transport
across placental endothelial cells. In vivo studies using Fc
receptor gene-knockout nonhuman primate models are important future directions.
To identify molecules that regulate FCGR2B2 compartment trafficking in the human placenta, proteomic analysis
of FCGR2B2 compartments isolated from human placental
tissues was performed. A large number of FCGR2B2 compartment-associated proteins was identified, including RAB family
proteins, which have also been identified in several previous
proteomic studies on synaptic vesicles (40), insulin secretory
granules (41), endocytic vesicles (42), clathrin-coated vesicles (43) and clathrin-independent carriers (44). RAB proteins
are small GTPases and members of the Ras superfamily ofmonomeric G proteins. RAB proteins regulate each step in themembrane trafficking process: vesicle or tubule formation by
budding from donor membranes, intracellular vesicle delivery,
vesicle tethering and fusion to specific acceptor membranes withseveral specific RAB effectors (45). The localization and novel
functional aspects of RAB proteins have been revealed (46).
In this study, we focused on RAB3D as only RAB3D wasexpressed predominantly in placental endothelial cells. Using
siRNA analysis, we found that RAB3D was involved in theintracellular dynamics of FCGR2B2 compartment formation.
RAB3 proteins, known as secretory RABs, are primarily localized in secretory vesicles and intracellular granules together
with several other RABs and are involved in the regulationof each step of exocytosis with their effectors (47). RAB3D
plays an important role in the maturation and/or maintenanceof secretory granules (48-51). RAB3D also participates in theregulation of the apically directed transcytosis in rat hepatocytes (52). In endothelial cells, RAB3D regulates the secretionof von Willebrand factor that is stored in Weibel-Palade bodies,endothelial-specific secretory granules (53). While FCGR2B2
compartments were distinct from Weibel-Palade bodies in
FCGR2B2‑EGFP-expressing HUVECs (Fig. 2D), RAB3D
may serve as an important regulator for intracellular traffickingof IgG, as well as secretory proteins in placental endothelial
cells. Although we focused on RAB3D in this study, other
RAB proteins were also expressed in placental endothelial
cells. This study did not fully explore the regulation of intracellular trafficking and dynamics of FCGR2B2 compartments
by RAB proteins. Further functional studies of the FCGR2B2
compartment-associated RAB proteins are required to elucidate the detailed mechanisms of maternal-fetal IgG transport
in the human placental endothelium.
In conclusion, in this study, we investigated the dynamics
and properties of FCGR2B2 compartments for IgG trafficking
using an in vitro model of placental endothelial cells. Our datasuggest the involvement of FCGR2B2 in IgG transcytosis of thehuman placental endothelium, thus providing new insight into
the mechanism through which IgG crosses the human placenta.
Acknowledgements
We would like to thank Akira Katayama for assisting in LC-MS/
MS and Takuji Kosuge for providing technical assistance. This
study was supported in part by Grants-in-Aid for the Scientific

1288

ISHIKAWA et al: FCGR2B IN HUMAN PLACENTAL ENDOTHELIAL CELLS

Research from the Ministry of Education, Culture, Sports,
Science and Technology/Japan Society for the Promotionof Science (MEXT), Japan and by a grant from the MEXTSupported Program for the Strategic Research Foundation atPrivate Universities (2013-2017).
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