The Fc Receptor for IgG Expressed in the
Villus Endothelium of Human Placenta Is Fc
γRIIb2
This information is current as
of June 14, 2017.
Timothy W. Lyden, John M. Robinson, Susheela
Tridandapani, Jean-Luc Teillaud, Stacey A. Garber, Jeanne
M. Osborne, Jürgen Frey, Petra Budde and Clark L.
Anderson
References
Subscription
Permissions
Email Alerts
This article cites 41 articles, 13 of which you can access for free at:
http://www.jimmunol.org/content/166/6/3882.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
J Immunol 2001; 166:3882-3889; ;
doi: 10.4049/jimmunol.166.6.3882
http://www.jimmunol.org/content/166/6/3882
The Fc Receptor for IgG Expressed in the Villus Endothelium
of Human Placenta Is Fc␥RIIb21
Timothy W. Lyden,* John M. Robinson,† Susheela Tridandapani,* Jean-Luc Teillaud,‡
Stacey A. Garber,* Jeanne M. Osborne,* Jürgen Frey,§ Petra Budde,§ and Clark L. Anderson2*
T
he human placenta transports maternal IgG to an otherwise Ab-deficient fetus. This transport mechanism is
thought to be an active process on the basis of two considerations: first, on the establishment late in gestation of a significant fetal-to-maternal IgG concentration gradient; and second, on
the selectivity of the process for IgG but not the other Ig molecules
(1–3). The placental barrier between maternal and fetal circulatory
systems, across which IgG must pass, consists of two cell layers
and an intervening stroma (4). The first of these cell layers is the
epithelial syncytiotrophoblast, which completely covers chorionic
villi and constitutes the point of direct fetal contact with circulating
maternal blood. IgG transport across the syncytiotrophoblast is
thought to include a combination of bulk phase uptake and receptor-mediated sorting. Such sorting is generally accepted to involve
the MHC class I-like Fc receptor, FcRn, in a manner analogous to
IgG transport across the rat neonatal gut epithelium (5). Once
across the syncytiotrophoblast, IgG appears to transit the villus
interstitium via bulk fluid flow (6, 7). How IgG crosses the fetal
villus endothelial cell (EC)3 layer is not known.
The villus EC layer, transporting materials arriving from the
syncytiotrophoblast, is marked by continuous tight junctions,
highly attenuated cell bodies, and abundant intracellular transport
structures called caveolae (8). These caveolae have a characteristic
Departments of *Internal Medicine and †Physiology and Cell Biology, Ohio State
University, Columbus, OH 43210; ‡Institut Curie, Paris, France; and §University of
Bielefeld, Bielefeld, Germany
Received for publication October 4, 2000. Accepted for publication January 10, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This study was supported in part by U.S. Public Health Service Grants R01
HD38764, R01 HD35121, R01 CA44983, and P30 CA16058, and by a Pharmacia
Upjohn grant. S.T. is a Leukemia Society of America Fellow.
2
Address correspondence and reprint requests to Dr. Clark L. Anderson, 473 West
12th Avenue, Room 415, Columbus, OH 43210. E-mail address:
[email protected]
3
Abbreviations used in this paper: EC, endothelial cell(s): TVT, terminal villus tips;
MV, mixed villus/villi; Fc␥R, Fc receptor for IgG; RCF, relative centrifugal force;
TVC, terminal villus capillary/ies; CV, cord vessel(s).
Copyright © 2001 by The American Association of Immunologists
flask shape when associated with the cell membrane and a consistent size ranging from ⬃50 to 100 nm (9). Caveolae appear to
function in cellular transport pathways in a manner similar to, but
distinct from, clathrin-coated vesicles (10) and likely form the
structural basis for IgG transit across the fetal villus EC. Early
studies using aggregated IgG and IgG-coated RBC (11,12) showed
that the villus EC expressed receptors specific for IgG (Fc␥R).
With the later development of Abs against the classical Fc␥R, one
of the three groups of Fc␥R, namely Fc␥RII, was shown to be
present (13–15). This finding is remarkable because as a rule (with
one exception showing Fc␥RII expression in skin; Ref. 16) endothelium in the body does not express Fc␥R. Whether Fc␥RII is
associated with caveolae has not been established.
Fc␥RII (CD32) is the most abundant and widely distributed
group within the Fc␥R family, which includes Fc␥RI (CD64) and
Fc␥RIII (CD16). In humans, the Fc␥RII group consists of at least
six different proteins encoded by three distinct genes (A, B, and C)
(17, 18). Of these several proteins there are two that are likely
candidates for expression on the villus EC, namely, Fc␥RIIa and
Fc␥RIIb, major products of genes A and B, respectively. As a rule
these two receptors when expressed elsewhere mediate opposing
signals. Fc␥RIIa initiates such functions as endocytosis, inflammatory mediator release, gene transcription, and others. Fc␥RIIb
displays an essentially antagonistic character by transducing inhibitory signals that down-regulate several immune functions (19 –
23). The molecular basis for this dichotomy resides in different
phosphotyrosyl-based sequence motifs in the cytoplasmic tails of
these otherwise very similar receptors. These different motifs serve
as docking sites for different sets of SH2 domain-containing enzymes, which in turn distinguish the receptor responses (24).
In the mouse there is only a single Fc␥RII gene, and it resembles
the human B gene. As a rule, its products mediate inhibitory responses, as in the human. However, distinct from the situation with
the human receptors, the capacity of its products to mediate endocytosis has been studied in detail, but only by means of transfection experiments. Only one of the two products of the murine
Fc␥RII gene, namely, Fc␥RIIb2, is capable of mediating rapid IgG
endocytosis and transcytosis by means of clathrin-coated vesicles
0022-1767/01/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
To evaluate the potential role of human placental endothelial cells in the transport of IgG from maternal to fetal circulation, we
studied Fc␥ receptor (Fc␥R) expression by immunohistology and immunoblotting. Several pan-Fc␥RII Abs that label the placental endothelium displayed a distribution pattern that correlated well with transport functions, being intense in the terminal
villus and nil in the cord. In contrast, the MHC class 1-like IgG transporter, FcRn, and the classical Fc␥RIIa were not expressed
in transport-related endothelium of the placenta. Our inference, that Fc␥RIIb was the likely receptor, we confirmed by analyzing
purified placental villi, enriched in endothelium, by immunoblotting with a new Ab specific for the cytoplasmic tail of Fc␥RIIb.
These experiments showed that the Fc␥RII expressed in villus endothelium was the b2 isoform whose cytoplasmic tail is known
to include a phosphotyrosyl-based motif that inhibits a variety of immune responses. We suggest that this receptor is perfectly
positioned to transport IgG although as well it may scavenge immune complexes. The Journal of Immunology, 2001, 166:
3882–3889.
The Journal of Immunology
(25). The other product, Fc␥RIIb1, expresses a 47-aa insert in its
cytoplasmic tail that appears to inhibit receptor inclusion in coated
pits and thus to inhibit endocytic capacity (26, 27). A comparable
insert (19 aa) appears in the human homolog, Fc␥RIIb1, although
similar studies of endocytic capacity have not been performed in
humans. There is no Fc␥RIIa in the mouse.
In the studies reported here we have used a variety of approaches to determine that Fc␥RIIb2 is the predominant receptor
expressed by the terminal villus EC. These results suggest that
villus Fc␥RIIb2 may be functioning as an IgG transporter as well
as perhaps an immune complex scavenging receptor.
Materials and Methods
Antibodies
Tissue procurement and handling
Placental tissue samples were supplied by the regional tissue procurement
facility of The Ohio State University Medical Center. These were randomly
selected from normal full-term Caesarian deliveries and were processed
within 30 – 45 min of delivery. Individual lobes were dissected out and
washed with fresh cold PBS (0.15 M PBS, pH 7.2). Tissue pieces ⬃1 cm3
were excised, rinsed by immersion in cold PBS, and fixed in 4% paraformaldehyde/PBS at 4°C for 12 h. Samples were washed with four changes
of PBS (⬃10⫻ volume) for a total of 16 h at 4°C. Tissues were then
suffused by immersion in 2.5 M sucrose/PBS for 8 –16 h (until sample sank
following saturation). Next, the samples were rinsed with PBS and placed
in TBS freeze medium (Triangle Biomedical Sciences, Durham, NC)
within CMS Tissue Path molds (Fisher Scientific, Pittsburgh, PA) and
snap-frozen by immersion in liquid nitrogen. Resultant blocks were stored
at ⫺70°C until sectioning.
Immunohistology
Tissue sample blocks were warmed to ⫺20°C for 2 h before 5-␮m sections
were cut. These were air-dried onto Superfrost microscope slides (Fisher
Scientific) and stored at ⫺20°C.
To label, slides with sections were thawed 5 min at room temperature,
submerged in PBS for 5 min to solubilize the TBS medium, and washed in
PBS for 5 min. Sections were then blocked with 5% goat serum in PBS for
1 h at room temperature in a humidified chamber. Blocking solution was
replaced with 50 ␮l of primary Abs in 5% goat serum/PBS, and sections
were incubated 2 h at room temperature (anti-Fc␥R) or 16 h at 4°C (antiFcRn) in a humid chamber. Next, slides were washed with three changes
of PBS for 15 min each. Sections were then incubated with 50 ␮l of appropriate FITC-conjugated secondary Abs in 5% goat serum/PBS for 1 h.
Finally, these were washed again in PBS three times for 15 min, and coverslips were mounted with Gelmount (Fisher Scientific).
Densitometry
After labeling and mounting, sections were examined within 24 – 48 h with
a Nikon Optiphot Epifluorescent microscope and photographed on TMax
400 film rated at EI 1600. The resultant negatives were then digitally
scanned with a Polaroid SprintScan 35 slide scanner. Full frame grayscale
scans were made of each negative at 1024 DPI resolution. These files were
imported into Sigma-Image analysis software (Jandel Scientific, Corte
Madera, CA), and density measurements performed. Three linear measurements of pixel intensity were taken at 60° angles to each other across
selected vessels. In any given image, vessels with clear cross-sections were
measured, whereas tangentially cut vessels were excluded. In addition, at
least three background measurements were also collected in each villus
cross-section, which included the syncytiotrophoblast layer and avascular
stroma. All observations were transferred to Excel 97 (Microsoft, Seattle,
WA), and calculations were performed to subtract average background
values from observed intensities. Vessels were grouped according to the
morphologic nature of the villi into terminal, intermediate, or stem villi and
cord vessels (CV). Then the average areas under the peak were calculated
and plotted for each grouping (n ⬎ 15 independent observations for each
grouping).
Immunoblotting
Tissue lysates were prepared by selective dissection and isolation of villus
tips (a modification of methods by Kacemi et al., Ref. 32) from samples
collected as described above. In this procedure, following extensive washing with PBS, fresh 2 cm3 blocks of tissue were placed in cold PBS and
progressively diced by cross-cutting with paired razor blades, which released large numbers of very small villus pieces into suspension. After
dissection, the suspended fragments were transferred to a 50-ml conical
centrifuge tube, and large pieces were allowed to settle. Chorionic villus
tips remained suspended and were removed to a 15-ml centrifuge tube after
5 min and were pelleted at 1000 RCF in a Beckman model GPR centrifuge.
This produced samples enriched for terminal villus tips (TVT) from the
suspension as well as mixed villus (MV) samples from the initial sunken
material. Relative sample contents were confirmed by phase contrast microscopic examination.
Immediately following harvest, ⬃0.1 g of pelleted material was resuspended (working volumes ⬃30 ␮l), and 100 ␮l of lysis buffer (25 mM
HEPES, 20 mM Na4P2O7.10 H2O, 100 mM NaF, 4 mM EDTA, 2 mM
Na3VO4, 1% Triton X-100, 0.34 mg/ml PMSF, 0.01 mg/ml aprotinin, and
0.01 mg/ml leupeptin) was added. Following a 30-min incubation on ice,
debris was pelleted in a refrigerated Eppendorf 5415 microfuge at 16,000
relative centrifugal force (RCF), and the lysate was stored at ⫺20°C (typically for 18 h). Control cells (U937, Raji and Daudi; American Type
Culture Collection (ATCC), Manassas, VA) were grown to an approximate
density of 1–2 ⫻ 106 cells/ml in RPMI 1640 medium (Life Technologies,
Rockville, MD) containing 15% FBS (HyClone, Logan, UT), 100 U/ml of
penicillin, and 100 ␮g/ml streptomycin (Life Technologies). These were
then pelleted at 1000 RCF, washed three times in cold PBS, and resuspended to ⬃1 ⫻ 107 cells/tube. These were then pelleted and subjected to
lysis with 500 ␮l of buffer as above. Additional isoform controls in these
experiments included transfected COS-7 fibroblast cells (ATCC). In each
case, pCEXV-3 Fc␥RIIa, IIb1, IIb2 constructs (provided by Dr. J. Ravetch,
Rockefeller University, New York, NY) or empty vectors were transfected
into cells using LipofectAMINE reagent (Life Technologies) as previously
described (33).
Samples were next thawed, and 250 ␮l of each was subjected to immunoprecipitation with 50 ␮l of anti-Fc␥RII mAb [KB61 (1 ␮l ascites),
AT10 (1 ␮l ascites), and IV3 (1 ␮g IgG)]-coated F(ab⬘)2 goat anti-mouse
IgG (Pierce, Rockford, IL)-conjugated Sepharose beads (Amersham Pharmacia Biotech). Resulting samples were then subjected to SDS-PAGE on
10% gels as described (29). Proteins were transferred to nitrocellulose
membranes (Hybond ECL; Amersham Pharmacia Biotech), blocked, and
probed with test Abs in 5% low fat milk in 10 mM TBS/0.1% Tween 20.
The signal was then detected by addition of ECL substrate reagent (Amersham Pharmacia Biotech) and visualized on Kodak X-OMAT AR film
(Eastman Kodak, Rochester, NY).
Flow cytometry
The relative blotting efficiency of the isoform-specific Fc␥RII antisera (Ab
260 and Ab 163.96) was quantified in the following manner. We compared
Fc␥RII expression on cultured U937 cells (expressing virtually only the
Fc␥RIIa isoform) and Raji cells (expressing only the Fc␥RIIb isoforms) by
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
The following anti-Fc␥R mAbs were used for immunolabeling; KB61
(IgG1; D. Mason, Radcliffe Hospital, Oxford, U.K.), KU79 (IgG2b; T.
Mohanakumar, St. Louis, MO), 41H16 (IgG2a; T. Zipf, Houston, TX),
CIKM5 (IgG1; G. Pilkington, Melbourne, Australia), 2E1 (IgG2a; Immunotech, Westbrook, ME), IV3 (IgG2b; Medarex, Lebanon, NH), 32.2
(IgG1; Medarex), and 3G8 (IgG1; Medarex) (28). All were applied at a
working concentration of 10 ␮g/ml as defined below. Anti-FcRn Abs in
these studies were directed against the cytoplasmic tail (anti-CT) and the
extracellular region (anti-H2) of the molecule (29); both were used at 20
␮g/ml. Additional control Abs were anti-cytokeratin, anti-placental alkaline phosphatase (prediluted mAb and rabbit sera, respectively; both obtained from Zymed, San Francisco, CA), and anti-CD31 (10 ␮g/ml; Sigma,
St. Louis, MO). Isotype controls included IgG fractions of myeloma proteins MOPC-21, MOPC-141, and HOPC-1 (10 ␮g/ml; Sigma).
For immunoblotting studies, isoform-specific rabbit polyclonal antiserum Ab 260 (30) was used to detect Fc␥RIIa while a recently developed
rabbit polyclonal antiserum (Ab 163.96) directed against GST fused to the
cytoplasmic portion of Fc␥RIIb1 at its N terminus (GST-ALPGY; produced by J.-L.T.) was used to detect both b1 and b2. Both antisera were
used at a 1:2000 dilution. In additional studies anti-Fc␥RII mAbs II8D2 (1
␮g/ml) and II1A5 (supernatant at 1:1000) (31) were used to confirm isoform-specific reactions in control cells and placental samples.
Secondary Abs for immunohistology were FITC-conjugated goat
F(ab⬘)2 anti-mouse IgG and FITC-conjugated goat F(ab⬘)2 anti-rabbit IgG
used at 1:50 dilution (Caltag, South San Francisco, CA). For immunoblotting, HRP-conjugated sheep anti-mouse and anti-rabbit IgG Abs were used
at a 1:5000 dilution (Amersham Pharmacia Biotech, Piscataway, NJ).
3883
Fc␥RIIb2 RECEPTOR EXPRESSION IN VILLUS ENDOTHELIAL CELLS
3884
flow cytometry using pan-Fc␥RII mAb KB61 as previously described (34).
We found the two cells to express roughly equal numbers of Fc␥RII. Simultaneously, we assessed the Fc␥RII band density of immunoblots labeled with Ab 260 and Ab 163.96 of whole cell lysates of varying numbers
of U937 and Raji cells. For an equal number of Fc␥RII from the two cell
types, we found the Ab 163.96 signal to be 2.5 times brighter than the Ab
260 signal.
Endoglycosidase assay
To evaluate the glycosylation of placental Fc␥RII proteins, Fc␥RII was
immunoadsorbed from detergent lysates as above, then submitted to Nglycosidase F (Boehringer Mannheim, Indianapolis, IN) treatment as described (35). Briefly, after three washings with PBS, adsorbed samples
were denatured with 0.1 M 2-ME/0.1% SDS and heated to 90°C for 5 min.
Then 10 ␮l of each sample was combined with 3 ␮l of 0.5 M Tris-Cl, pH
8.6; 5 ␮l of H2O; 2 ␮l of 10% Triton X-100, and 5 ␮l of 250 mU/ml
N-glycosidase F or 5 ␮l of 0.5 M Tris-Cl, and these were incubated for 16 h
at 37°C. These samples were analyzed by SDS-PAGE and immunoblotting
as above.
Results
Anti-Fc␥RII mAb labeling of placental villus vasculature
Distribution of Fc␥RII in placental vascular tree
Our observations with the anti-Fc␥RII mAbs that label the villus
endothelium most intensely (pattern 1 summarized in Table I) suggested that the fluorescence signal in the placental vascular tree
was brightest in TVC, virtually absent in vessels of the umbilical
cord, and that the change in signal intensity between these two
extremes along the vascular branch was gradual. This point is illustrated microscopically in Fig. 2, where the three panels show
the entire intensity spectrum of endothelial labeling. Panel A shows
a TVT labeled with anti-Fc␥RII mAb KB61 in which all capillary
endothelium is intensely bright (arrows). Panel B shows intermediate villi where the large vessel is only weakly fluorescent (arrowhead), whereas peripheral capillaries are intensely positive (arrows). Panel C shows the lack of endothelial labeling of
umbilical CV.
Table I. Distribution of placental villus labeling with anti-Fc␥R mAbs by immunofluorescence microscopya
Villus Vascular Endothelium
mAb
Clone
Isotype
Specificity
Reactivityb
KB61
41H16
Ku79
CIKM 5
2E1
IV3
32.2
3G8
IgG1
IgG2a
IgG2b
IgG1
IgG2a
IgG2b
IgG1
IgG1
Fc␥RII
Fc␥RII
Fc␥RII
Fc␥RII
Fc␥RII
Fc␥RII
Fc␥RI
Fc␥RIII
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
Pattern
1
1
1
2
2
3
4
4
Nonendothelial
Terminal
villus
Intermediate
villus
Stem
villus
CV
Stromal
cellsc
Trophoblast
layer
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹/⫺
⫹/⫺
⫺/⫹
⫺
⫺
⫹⫹
⫹⫹
⫹⫹
⫹/⫺
⫹/⫺
⫺/⫹
⫺
⫺
⫹/⫺
⫹/⫺
⫹/⫺
⫺/⫹
⫺/⫹
⫺/⫹
⫺
⫺
⫺/⫹
⫺/⫹
⫺/⫹
⫺/⫹
⫺/⫹
⫺/⫹
⫺
⫺
⫺/⫹
⫺/⫹
⫺/⫹
⫹/⫺
⫹/⫺
⫹⫹
⫹⫹
⫹⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹⫹⫹, Intensely positive; ⫹⫹, diminished intensity; ⫹/⫺, extensive but weak positive; ⫺/⫹, negative with occasional positive cells; —–, negative labeling.
Four distinctive morphologic anti-Fc␥R patterns of Ab reactivity were noted by immunofluorescence microscopy.
Based on morphology and relative distribution villus stromal cells are principally considered to be fetal tissue macrophages (Hofbauer cells), but these may also include
pericytes, fibroblasts, and vascular smooth muscle cells in the larger villi.
a
b
c
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
Using a library of mAbs, we evaluated the distribution of Fc␥RII
by indirect immunofluorescence of paraformaldehyde-fixed frozen
sections of villus tissue from six randomly selected normal placentas. Table I presents an overview of observations made
throughout the placental vasculature. It can be seen from these data
that three patterns of cell-specific reactivity were detected with this
anti-Fc␥RII mAb library. Pattern 1 was seen with the mAbs KB61,
41H16, and Ku79. With these three Abs, the endothelium of all
capillaries within terminal villi, as well as those of the intermediate
villus peripheral capillary network, displayed an intense positive
fluorescence signal. Larger vessels within the placental vascular
tree (intermediate and stem villi) showed a decreasing signal as
distance from the capillaries increased. In the umbilical CV, little
or no signal was seen in EC. Non-EC within the stroma of larger
villi and cord tissue also labeled with a positive signal. Based on
distribution and overall morphology, these positive stromal cells
were identified as fetal tissue macrophages (Hofbauer cells).
Fig. 1, A and B, illustrates pattern 1 reactivity seen with KB61.
Several types of chorionic villi from terminal (marked with “T”) to
intermediate (marked with “I”) are shown in A. All terminal villus
capillaries (TVC) are labeled with anti-Fc␥RII Ab, as are peripheral capillaries of intermediate villi (arrows). In B, an intermediate
villus (center) shows both peripheral capillaries (arrows) and
larger vessels (arrowhead) labeled with positive fluorescence signal. As indicated in Table I, mAbs 41H16 and Ku79 produced
virtually identical labeling throughout the vasculature as that seen
with mAb KB61.
The second type of reactivity (pattern 2) was observed with
mAbs CIKM 5 and 2E1. As can be seen in Table I, these mAbs
showed a low level of EC labeling, whereas stromal cells presented
a strong positive signal. The third type of reactivity (pattern 3) was
observed with mAb IV3 and was characterized by a strong positive
signal in the stromal Hofbauer cells with very little or no endothelial labeling. Fig. 1, C and D, shows chorionic villi labeled with
mAb IV3. In many of the larger villi positive stromal cells (arrows)
can be seen. Based on location and relative distribution within the
tissue, these cells were considered to represent the Hofbauer cells
commonly reported in larger villi.
In addition to anti-Fc␥RII mAbs, we also examined placental
sections with Abs to Fc␥RI and Fc␥RIII (mAbs 32.2 and 3G8,
respectively). In both cases, stromal cells were positive but no
endothelial or trophoblast signal was detected (Table I). Although
both of these Abs presented similar signals (pattern 4), it should be
noted that differences in signal distribution were observed between
each of these mAbs and those signals seen with mAb IV3 or
CIKM5/2E1 (above). These data suggest the possible presence of
different Fc␥R expression patterns within villus Hofbauer cells at
term, although further pursuit of this question is beyond the scope
of this report.
Labeling with anti-cytokeratin mAb was used in these studies to
identify the trophoblast layer (Fig. 1E, arrows). None of the antiFc␥R mAbs used in these studies labeled normal trophoblast. Myeloma proteins MOPC-21, MOPC-141, and HOPC-1 were all applied to test for nonspecific Fc␥R binding of mouse Ab classes
IgG1, IgG2a, and IgG2b, respectively. Fig. 1F, showing MOPC-21
labeling, illustrates that no endothelial or other villus labeling was
detected with these reagents.
The Journal of Immunology
3885
Quantification of Fc␥RII distribution
To validate our qualitative impressions of the anti-Fc␥RII signal
gradation illustrated in Fig. 2, we measured pixel intensities of
representative digitized micrographs of fluorescence-labeled vessels along the length of the vascular tree. Fig. 3, A and B, illustrates
this approach. Following digitization of the micrograph, pixel intensity measurements were taken of lines drawn at 60° angles to
each other across randomly selected vessels of each type. These
lines record both individual pixel intensities and length of the measurement. For simplicity, Panel A shows a single line of the trio
drawn across each of two vessels, one a capillary and one a larger
vessel. Plots of the pixel intensity vs distance for these two vessels
(B) indicate that in this case the thickness of the endothelial layers
are fairly equal (⬃5.2 ␮m) despite the marked difference in vessel
diameters (ratio of ⬃4:1) and that the smaller vessel is ⬃3 times
brighter than the larger vessel (area beneath the curve).
We applied the measurement technique illustrated in B to four
groups of vessels along the villus vascular tree: TVC, intermediate
villus vessels (IVV), stem villus vessels (SVV), and CV. These
measurements were then used to calculate the mean areas of observed peaks, and the results were plotted for each group of vessels. Panel C shows the results obtained for Fc␥RII signals. The
resultant data clearly documented our qualitative impression that
Fc␥RII signal intensity decreased as the size of the vessel increased and as location moved away from the capillaries and toward the cord. In distinct contrast, the distribution of FcRn in
endothelium displayed a reciprocal pattern of expression, with
brighter signal being seen in the cord and minimal signal in the
TVC (D). Overall, it can be seen from these data that the Fc␥RII
signal detected in terminal capillaries has an ⬃4.8-fold higher intensity than that detected in CV. In contrast, FcRn can be seen to
have an inverse signal differential of ⬃5.1-fold from CV to capillaries. It should be noted that with both anti-Fc␥RII and antiFcRn, very low background pixel intensities were recorded within
the cord and capillaries, respectively. This background represents
nonspecific autofluorescence, which is commonly seen in placental
tissue samples. In these studies, such background is considered to
be negative for specific Ab binding.
The predominant Fc␥RII isoform in placental EC is b2
In light of published reports of specificity of the anti-Fc␥RII mAbs
(36 – 40), the cell-specific reactivity patterns presented in Table I
suggest that villus endothelium may express predominantly the
Fc␥RIIb isoform. However, none of these mAbs is known to label
unequivocally only Fc␥RIIa or Fc␥RIIb. Therefore, a different approach was taken to define which isoform is actually expressed in
the TVC. In these studies, immunoblot analysis using isoformspecific rabbit anti-sera (anti-Fc␥RIIa Ab 260 and anti-Fc␥RIIb1
Ab 163.96) was combined with a specific tissue dissection technique to evaluate the relative distribution of Fc␥RIIa and Fc␥RIIb
within the villus branch. This was accomplished by fine dissection
followed by isolation of villus branches based on relative size and
resulted in two samples, one enriched for TVT and the other for
MV containing both terminal and intermediate villi. Based on villus morphology (41), samples containing primarily small terminal
villi (TVT) are greatly enriched for EC relative to the core tissue
samples (MV). Fc␥RII was immunoadsorbed with anti-Fc␥RII
mAbs from detergent lysates of the two villus samples, separated
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
FIGURE 1. Photomicrographs illustrating the
distribution of immunofluorescent anti-Fc␥RII
signal within placental villi. A and B, Extensive
EC labeling of the terminal and peripheral capillary networks with anti-Fc␥RII mAb KB61 (arrows). In A, a terminal villus (marked with a T)
and an intermediate villus (marked with an I) are
indicated for reference. B, Intermediate villus
with both peripheral capillary (arrows) and larger
vessel (arrowhead) labeling. C and D, Little or no
endothelial but extensive stromal cell labeling
with the anti-Fc␥RII mAb IV3 (arrows). E, Positive control labeling of trophoblast (arrows) but
not endothelium with anti-cytokeratin. F, No EC
binding of MOPC-21, an IgG1 isotype control
(asterisk indicates low autofluorescent background); IgG2a and IgG2b isotype control proteins (HOPC-1 and MOPC-141) were similarly
negative. Original magnifications: A and C,
⫻200; B, D, E, and F, ⫻400.
3886
Fc␥RIIb2 RECEPTOR EXPRESSION IN VILLUS ENDOTHELIAL CELLS
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
FIGURE 2. Vascular distribution of EC labeling observed with antiFc␥RII mAbs. This composite figure illustrates the vascular distribution of
labeling with mAb KB61, which is representative of that seen with mAbs
41H16 and Ku79 as well. A, Extensive reactivity of endothelium throughout the TVC network (arrows). B, Mixed signal intensities detected within
intermediate villi, capillaries of the peripheral network being intensely labeled (arrows), whereas larger vessels display a weaker signal (arrowhead). C, Lack of EC signal observed in umbilical CV (arrowhead). Original magnifications: ⫻400 (A) and ⫻200 (B and C).
by SDS-PAGE, and identified by immunoblotting with antiFc␥RIIa Ab 260 or anti-Fc␥RIIb Ab 163.96. Cell lines were included as controls for Fc␥RIIa (U937) and Fc␥RIIb (Raji and
Daudi), as were COS 7 cells transfected with expression vectors
containing Fc␥RIIa, Fc␥RIIb1, and Fc␥RIIb2 cDNA.
Placental TVT and MV samples probed with Ab 163.96 (Fig. 4,
top) show an intense band at ⬃35 kDa characteristic of Fc␥RIIb2
(lower arrow). This band comigrates with the major band observed
in the Fc␥RIIb2-transfected COS 7 cells and moves faster than the
major band (⬃38 kDa) in Fc␥RIIb1 transfectant or Raji/Daudi
control lanes (upper arrow). No such bands were seen in samples
of Fc␥RIIa transfectants or of U937 cells, which express abundant
Fc␥RIIa. In addition to these major bands, Ab 163.96 also detected
a somewhat weaker and slower moving band in the MV sample at
⬃50 kDa. Similar bands are also seen in Raji (⬃55 kDa) and both
the Fc␥RIIb-transfected COS 7 cells (⬃55 and 50 kDa). Although
there are minor differences in the position of these slower moving
bands, the degree of similarity and distribution within all positive
controls suggests that these are different forms of Fc␥RIIb, perhaps
resulting from differential posttranslational modifications. In additional experiments immunoadsorbing with Ab 163.96 and blotting
with the anti-Fc␥RIIb mAb II8D2 or pan-Fc␥RII mAb II1A5, both
the 35-kDa placental bands and slower moving Raji/Daudi bands
FIGURE 3. Quantification of signal intensity distribution observed
within the placental vascular tree. A and B, Approach taken to quantify
signal intensities in these studies; linear pixel intensity measurements were
made across vessels of representative morphology for each villus vascular
branch (A, original magnification, ⫻200). In all cases, three intersecting
lines, only one of which is shown for each of these vessels, were drawn at
60° angles to randomize observations. In this example a capillary and
larger vein have been measured and plotted. B, Basic EC height (lumenalto-basal aspect) is very similar at ⬃5.2 ␮m for both vessels. In contrast, the
signal intensity ratio is ⬃3:1 (capillary/vessel). C and D, Mean area intensity data for ⬎200 independent observations labeled with anti-FcRII
(KB61, C) or anti-FcRn (CT/H2, D). An inverse relationship is evident for
these two FcR. IVV, Intermediate villus vessels; SVV, stem villus vessels.
were observed (data not shown), which supports the interpretation
of these bands as Fc␥RIIb.
In contrast, Ab 260 (Fig. 4, lower panel) failed to detect any
Fc␥RIIa signal in the placental TVT sample in this experiment.
The MV sample did show a minor band at ⬃43 kDa, which corresponded with the major Fc␥RIIa band detected in U937 cells as
The Journal of Immunology
3887
well as a specific band in the Fc␥RIIa-transfected COS 7 sample.
Thus, very little Fc␥RIIa was seen in our placental samples and
none in the TVT.
This experiment was repeated on eight separate placental samples. In all eight experiments, we saw robust Fc␥RIIb2 bands in
both TVT and MV lanes. Moreover, in six of eight, there was little
or no Fc␥RIIa signal in the TVT and either a weak or no signal in
MV. However, in two of the eight experiments, the Fc␥RIIa signal
was present in both TVT and MV lanes. Therefore, we quantified
the efficiency by which each of these Abs recognizes its target,
determining stoichiometric relationships by flow cytometry, as described in Materials and Methods. We found that in both of these
experiments the number of Fc␥RIIb found in TVT was at least
2.5-fold the number of Fc␥RIIa detected. Our experiments, in sum,
indicate that Fc␥RIIb2 is the predominant isoform in placental
TVT and, by inference, in the terminal villus endothelium.
sizes of Fc␥RIIb2 and Fc␥RIIb1, the relative difference corresponding to the 19-aa cytoplasmic tail insert of Fc␥RIIb1. No
Fc␥RIIb signal was detectable in U937.
Discussion
We interpret our experiments above to indicate that the Fc␥RII
expressed in the endothelium of placental villi is predominantly
the b2 isoform. Our strongest and most direct evidence comes from
Fc␥RII glycosylation
We next examined the glycosylation of placental Fc␥RII to assess
the size of the core protein. TVT and MV along with U937 and
Raji cell lysates were immunoadsorbed on beads conjugated with
anti-Fc␥RII mAbs, and the immunoadsorbed receptor was incubated with and without N-glycosidase F. Both glycosylated and
deglycosylated samples were then immunoblotted with Ab 163.96.
Fig. 5 shows that the ⬃35-kDa Fc␥RIIb band, detected by Ab
163.96 in both TVT- and MV-glycosylated samples (upper arrow), was replaced by a major band at ⬃29 kDa after deglycosylation. Raji cell lysates showed the same pattern (arrowheads),
although the respective MWs were ⬃3 kDa higher in both cases
than the placental bands (at ⬃38 and 31 kDa). The deglycosylated
sizes of these proteins correspond to the expected core protein
FIGURE 5. Mobility of deglycosylated Fc␥RII purified from placental
villi. As described in Fig. 4, Fc␥RII was purified by immunoadsorption
from detergent lysates of TVT and MV of human placenta and from two
Fc␥RII-expressing cell lines, U937 and Raji. After equilibration with the
appropriate buffer the immunadsorbed samples were incubated with (⫹) or
without (⫺) N-glycosidase F and then were analyzed by immunoblotting as
in Fig. 4. The arrows mark native and deglycosylated Fc␥RII from placenta. The arrowheads mark more slowly moving Fc␥RIIb1 from Raji
cells.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
FIGURE 4. Immunoblot analysis of Fc␥RII immunoadsorbed from placental villi. Nonionic lysates were prepared from two regions of human placental chorionic villus branch tissue, namely, TVT and MV; from three cell
lines known to express specific isoforms of Fc␥RII (U937,
Raji, and Daudi); and from COS 7 cells transfected with
expression vectors containing the cDNAs of each of three
Fc␥RII isoforms (IIa, IIb1, or IIb2) and the vector alone
(VO). Fc␥RII was immunoadsorbed from these lysates
with a mixture of anti-Fc␥RII mAbs (KB61, AT10, IV3),
was separated by SDS-PAGE under reducing conditions,
and was analyzed by immunoblotting with rabbit antiFc␥RIIb Ab 163.96 and rabbit anti-Fc␥RIIa Ab 260. Double arrows indicate Fc␥RIIb1 (top) and Fc␥RIIb2 (bottom). Note that the apparent mobility of Fc␥RIIb2 is
partially retarded in the transfected IIb2 lane by a blotting
artifact. The single arrow marks Fc␥RIIa. Asterisks indicate nonspecific high molecular mass bands (see Results;
the predominant Fc␥RII isoform in placental EC13b2).
Molecular mass markers are indicated in kilodaltons.
3888
Fc␥RIIb2 RECEPTOR EXPRESSION IN VILLUS ENDOTHELIAL CELLS
receptor molecules and a single IgG ligand (46, 47). Although the
details of these two models differ, it seems reasonable to postulate
that in such trimolecular structures the receptor may be capable of
two different affinities for ligand, a high and a low affinity. A high
affinity would be seen when two receptors bind a single ligand, and
a low affinity would result from a single receptor binding a single
ligand. Certainly, it is conceivable that the switch from one configuration to the other might be mediated by phosphorylation of the
tyrosine-based signal motif in the cytoplasmic tail of Fc␥RIIb2.
Furthermore, it is possible that an association with specialized
membrane microdomains such as caveolae could facilitate both the
ligand/receptor interaction and the postulated shift in the phosphorylation/affinity state of the receptor.
Whether endothelial Fc␥RIIb2 serves to scavenge immune complexes is also a consideration, although the endothelium is not
generally thought of as a scavenging cell type, and other cells,
namely Hofbauer cells, perform this function in the villus. However, Fc␥RIIb2, when expressed by transfection in fibroblasts and
B cells, has been shown to mediate the endocytosis of immune
complexes via clathrin-coated pits and vesicles (25–27). Resolution of the precise function of endothelial Fc␥RIIb will await additional experiments.
References
1. Malek, A., R. Sager, and H. Schneider. 1998. Transport of proteins across the
human placenta. Am. J. Reprod. Immunol. 40:347.
2. Malek, A., R. Sager, P. Kuhn, K. H. Nicolaides, and H. Schneider. 1996. Evolution of maternofetal transport of immunoglobulins during human pregnancy.
Am. J. Reprod. Immunol. 36:248.
3. Malek, A., R. Sager, A. B. Lang, and H. Schneider. 1997. Protein transport across
the in vitro perfused human placenta. Am. J. Reprod. Immunol. 38:263.
4. Benirschke, K. 1998. Remarkable placenta. Clin. Anat. 11:194.
5. Junghans, R. P. 1997. The brambell receptor (FcRB): mediator of transmission of
immunity and protection from catabolism for IgG. Immunol. Res. 16:29.
6. Stulc, J. 1997. Placental transfer of inorganic ions and water. Physiol. Rev. 77:
805.
7. Schneider, H. 1991. The role of the placenta in nutrition of the human fetus.
Am. J. Obstet. Gynecol. 164:967.
8. Leach, L., and J. A. Firth. 1997. Structure and permeability of human placental
microvasculature. Microsc. Res. Tech. 38:137.
9. Kurzchalia, T. V., and R. G. Parton. 1999. Membrane microdomains and caveolae. Curr. Opin. Cell Biol. 11:424.
10. Anderson, R. G. W. 1998. The caveolae membrane system. Annu. Rev. Biochem.
67:199.
11. Johnson, P. M., and R. Matre. 1979. Membrane receptors for IgG in the human
placenta. In Protein Transmission through Living Membranes. W. A. Hemmings,
ed. Elsevier/North-Holland Biomedical Press, Amsterdam, p. 45.
12. Johnson, P. M., and P. J. Brown. 1981. Fc ␥ receptors in the human placenta.
Placenta 2:355.
13. Sedmak, D. D., D. H. Davis, U. Singh, J. G. van de Winkel, and C. L. Anderson.
1991. Expression of IgG Fc receptor antigens in placenta and on endothelial cells
in humans: an immunohistochemical study. Am. J. Pathol. 138:175.
14. Micklem, K. J., W. P. Stross, A. C. Willis, J. L. Cordell, M. Jones, and
D. Y. Mason. 1990. Different isoforms of human FcRII distinguished by CDw32
antibodies. J. Immunol. 144:2295.
15. Kristoffersen, E. K., E. Ulvestad, C. A. Vedeler, and R. Matre. 1990. Fc␥ receptor
heterogeneity in the human placenta. Scand. J. Immunol. 32:561.
16. Groger, M., G. Sarmay, E. Fiebiger, K. Wolff, and P. Petzelbauer. 1996. Dermal
microvascular endothelial cells express CD32 receptors in vivo and in vitro.
J. Immunol. 156:1549.
17. Ravetch, J. V., and J. P. Kinet. 1991. Fc receptors. Annu. Rev. Immunol. 9:457.
18. Hulett, M. D., and P. M. Hogarth. 1994. Molecular basis of Fc receptor function.
Adv. Immunol. 57:1.
19. Phillips, N. E., and D. C. Parker. 1984. Cross-linking of B lymphocyte Fc ␥
receptors and membrane immunoglobulin inhibits anti-immunoglobulin-induced
blastogenesis. J. Immunol. 132:627.
20. Sinclair, N. R., and A. Panoskaltsis. 1988. Antibody response and its regulation.
Curr. Opin. Immunol. 1:228.
21. Takai, T., M. Ono, M. Hikida, H. Ohmori, and J. V. Ravetch. 1996. Augmented
humoral and anaphylactic responses in Fc␥RII-deficient mice. Nature 379:346.
22. Clynes, R., J. S. Maizes, R. Guinamard, M. Ono, T. Takai, and J. V. Ravetch.
1999. Modulation of immune complex-induced inflammation in vivo by the coordinate expression of activation and inhibitory Fc receptors. J. Exp. Med. 189:
179.
23. Hunter, S., Z. K. Indik, M. K. Kim, M. D. Cauley, J. G. Park, and A. D. Schreiber.
Inhibition of Fc␥ receptor-mediated phagocytosis by a nonphagocytic Fc␥ receptor. Blood 91:1762.
24. Daeron, M. 1997. Fc receptor biology. Annu. Rev. Immunol. 15:203.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
immunoblotting experiments using a new rabbit polyclonal Ab directed toward the entire cytoplasmic tail of Fc␥RIIb1. The data in
Fig. 4 show that this Ab recognizes the b1 isoform produced by
transfected fibroblasts and human B cell lines (Daudi and Raji) as
well as the b2 isoform (lacking the 19-aa exonic insert) produced
in transfected cells, but it does not cross-react with Fc␥RIIa
present in COS 7 transfectants or in the human monocyte line
U937. So, we judge that it is specific for the two forms of Fc␥RIIb.
Parenthetically, we would add that recent studies indicate that
U937 cells express low levels of Fc␥RIIb (S. Tridandapani,
K. Siefken, J.-L. Teilland, J. E. Carter, M. D. Wewers, and C. L.
Anderson, manuscript in preparation).
For the immunoblotting experiments with this Ab, we partially
purified villus tips from placenta such that the cells of our tissue
lysate were predominantly EC and syncytiotrophoblast, whereas
Hofbauer cells were rarely present. Thus, the major Fc␥RII-expressing cell in this preparation, according to our immunofluorescence studies (Table I), was endothelium. Lysates of these villus
tips, in the majority of our experiments, expressed only the IIb
form of Fc␥RII. This conclusion was supported by additional experiments (data not shown) in which Fc␥RII was immunoadsorbed
from villus tips with Ab 163.96 or anti-Fc␥RII mAbs and identified by immunoblotting with the anti-Fc␥RIIb-specific mAb II8D2
or pan-Fc␥RII mAb II1A5.
Furthermore, the mobility in sizing gels of the Fc␥RIIb band
from villus tips was identical with that expressed by the Fc␥RIIb2
transfectant and faster than the band derived from the Fc␥RIIb1
transfectant and from both human B cell lines. Although B cells
express both b1 and b2 isoforms (42), b1 appears to be the predominant form (31). The mobilities of the deglycosylated receptors
also confirmed these observations (Fig. 5). Thus, we conclude that
villus endothelium predominantly expresses the Fc␥RIIb2
isoform.
The occasional presence of Fc␥RIIa, seen in two of eight immunoblotting experiments (where it represented ⬍30% of total
Fc␥RII molecules), we suspect was due to villus tip preparations
with greater numbers of blood leukocytes or Hofbauer cells, which
are known to express high concentrations of the Fc␥RIIa isoform
(Table I) (34). This conclusion is supported by immunofluorescence studies (Table I), which indicated that mAb IV3, known to
be relatively specific for Fc␥RIIa, bound to only occasional EC in
the same placental sections where Hofbauer cells were positive,
whereas three pan-anti-Fc␥RII mAbs bound well to endothelium.
The possibility that a product of the C gene of Fc␥RII might be
expressed is eliminated by the negative immunoblotting results
with Ab 260 directed toward the cytoplasmic tail of Fc␥RIIa,
which is identical with the cytoplasmic tail of Fc␥RIIc (43).
Whether the b2 isoform of Fc␥RII participates in the movement
of IgG across the villus endothelium is currently a matter of conjecture. It would appear that the large amount of IgG that traverses
the endothelium moves in transport vesicles, possibly in the abundant caveolae (44, 45). A transport function for the receptor is
definitely compatible with our observation that expression varies
with location in the placental vascular tree. Expression is highest
in the villus tips and nil in the cord, correlating with transport
functions in general. As well, the murine form of this receptor has
been studied by transfection in Madin-Darby canine kidney cells
and has been shown to transport Fab of anti-Fc␥RII Ab from one
pole of the cell to the other, suggesting its capacity to mediate IgG
transport (27). Were such a postulated transport process to be active, the receptor would require more than a single affinity state,
which has not been described. However, recent studies of the crystal structures of both Fc␥RIIa and Fc␥RIIb have suggested models
in which the receptors bind ligand in a complex consisting of two
The Journal of Immunology
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
antigen which is selectively expressed on a subpopulation of human B lymphocytes. Immunology 57:71.
Budde, P., V. Weinrich, P. Sondermann, N. Bewarder, A. Kilian, O. Schulzeck,
and J. Frey. 1995. Specifity of CD32 mAB for FcyRIIa, FcyRIIb1, and FcyRIIb2
expressed in transfected mouse B cells and BHK-21 cells. In Leucocyte Typing V:
White Cell Differentiation Antigens. S. F. Schlossman, L. Boumsell, W. Gilks,
J.M. Harlan, and T. Kishimoto, eds. Oxford University Press, New York, p. 828.
Looney, R. J., G. N. Abraham, and C. L. Anderson. 1986. Human monocytes and
U937 cells bear two distinct Fc receptors for IgG. J. Immunol. 136:1641.
van de Winkel, J. G. J., and C. L. Anderson. 1995. Cluster report: CD32. In
Leukocyte Typing V: White Cell Differentiation Antigens. S. F. Schlossman,
L. Boumsell, W. Gilks, J. M. Harlan, and T. Kishimoto, eds. Oxford University
Press, New York, p. 823.
Sondermann, P., U. Jacob, C. Kutscher, and J. Frey. 1999. Characterization and
crystallization of soluble human Fc ␥ receptor II (CD32) isoforms produced in
insect cells. Biochemistry 38:8469.
Novak, R. F. 1991. A brief review of the anatomy, histology, and ultrastructure
of the full term placenta. Arch. Pathol. Lab. Med. 115:654.
Cassel, D. L., M. A. Keller, S. Surrey, E. Schwartz, A. D. Schreiber,
E. F. Rappaport, and S. E. McKenzie. 1993. Differential expression of Fc␥RIIA,
Fc␥RIIB, and Fc␥RIIC in hematopoietic cells: analysis of transcripts. Mol. Immunol. 30:451.
Warmerdam, P. A. M., N. M. J .M. Nabben, S. A. R. van de Graaf,
J. G. J. van de Winkel, and P. J. A. Capel. 1993. The human low affinity IgG Fc
receptor IIC gene is a result of an unequal crossover event. J. Biol. Chem. 268:
7346.
King, B. F. 1990. Absorption of macromolecules by the placenta-some morphological perspectives. Trophoblast Res. 5:1.
Leach, L., B. M. Eaton, J. A. Firth, and S. F. Contractor. 1991. Immunocytochemical and labelled tracer approaches to uptake and intracellular routing of
immunoglobulin-G (IgG) in the human placenta. Histochem. J. 23:444.
Sondermann, P., R. Huber, and U. Jacob. 1999. Crystal structure of the soluble
form of the human Fc␥-receptor IIb: a new member of the immunoglobulin
superfamily at 1.7 Å resolution. EMBO J. 18:1095.
Maxwell, K. F., M. S. Powell, M. D. Hulett, P. A. Barton, I. F. C. McKenzie,
T. P. J. Garrett, and P. M. Hogarth. 1999. Crystal structure of the human leukocyte Fc receptor, FcRIIa. Nat. Struct. Biol. 6:437.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
25. Miettinen, H. M., J. K. Rose, and I. Mellman. 1989. Fc receptor isoforms exhibit
distinct abilities for coated pit localization as a result of cytoplasmic domain
heterogeneity. Cell 58:317.
26. Miettinen, H. M., K. Matter, W. Hunziker, J. K. Rose, and I. Mellman. 1992. Fc
receptor endocytosis is controlled by a cytoplasmic domain determinant that actively prevents coated pit localization. J. Cell Biol. 116:875.
27. Hunziker, W., and I. Mellman. 1989. Expression of macrophage-lymphocyte Fc
receptors in Madin-Darby canine kidney cells: polarity and transcytosis differ for
isoforms with or without coated pit localization domains. J. Cell Biol. 109:3291.
28. van de Winkel, J. G., and P. J. A. Capel. 1996. Yellow pages. In Human IgG Fc
Receptors. J. G. Van de Winkel and P. J. A. Capel, eds. R. G. Landes Company,
Georgetown TX, p. 227.
29. Leach, J. L., D. D. Sedmak, J. M. Osborne, B. Rahill, M. D. Lairmore, and
C. L. Anderson. 1996. Isolation from human placenta of the IgG transporter,
FcRn, and localization to the syncytiotrophoblast: implications for maternal-fetal
antibody transport. J. Immunol. 157:3317.
30. Vely, F., N. Gruel, J. Moncuit, O. Cochet, H. Rouard, S. Dare, J. Galon,
C. Sautes, W. H. Fridman, and J.-L. Teillaud. 1997. A new set of monoclonal
antibodies against human Fc␥RII (CD32) and Fc␥RIII (CD16): characterization
and use in various assays. Hybridoma 16:519.
31. Weinrich, V., P. Sondermann, N. Bewarder, K. Wissel, and J. Frey. 1996. Epitope
mapping of new monoclonal antibodies recognizing distinct human FcRII
(CD32) isoforms. Hybridoma 15:109.
32. Kacemi, A., J. C. Challier, M. Galtier, and G. Olive. 1996. Culture of endothelial
cells from human placental microvessels. Cell Tissue Res. 283:183.
33. Tridandapani, S., T. W. Lyden, J. L. Smith, J. E. Carter, K. M. Coggeshall, and
C. L. Anderson. 2000. The adapter protein LAT enhances Fc␥ receptor-mediated
signal transduction in myeloid cells. J. Biol. Chem. 275:20480.
34. Anderson, C. L., R. J. Looney, D. J. Culp, D. H. Ryan, H. B. Fleit, M. J. Utell,
M. W. Frampton, P. Manganiello, and P. M. Guyre. 1990. Human alveolar and
peritoneal macrophages bear three distinct classes of Fc receptors for IgG. J. Immunol. 145:196.
35. Freeze, H. H. 1999. Endoglycosidase and glycoamidase release of N-linked oligosaccarides. In Current Protocols in Molecular Biology, F. M. Ausubel,
R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl,
eds. Wiley, New York, p.17.13A12.
36. Pulford, K., E. Ralfkiaer, S. M. Macdonald, W. N. Erber, B. Falini, K. C. Gatter,
and D. Y. Mason. 1986. A new monoclonal antibody (KB61) recognizing a novel
3889