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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1762-1768
Inhibition of Fc Receptor-Mediated Phagocytosis by a
Nonphagocytic Fc Receptor
By
Sharon Hunter,
Zena K. Indik,
Moo-Kyung Kim,
M. Danielle Cauley,
Jong-Gu Park, and
Alan D. Schreiber
From the Hematology and Oncology Division, University of Pennsylvania
School of Medicine, Philadelphia.
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ABSTRACT |
There are three major classes of human Fc receptors (FcRI,
FcRII, and FcRIII) and various isoforms of each class are capable of mediating phagocytosis. FcRIIA is an unusual Fc receptor in
that it transmits a phagocytic signal in the absence of an additional
receptor subunit. The cytoplasmic domain of FcRIIA contains a
conserved motif containing two copies of the sequence YXXL. The
tyrosines (Y) within the motif are phosphorylated after receptor
crosslinking and the integrity of these conserved sequences is required
for efficient phagocytosis. The FcRIIB receptors, FcRIIB1 and
FcRIIB2, contain one copy of the cytoplasmic YXXL sequence and do
not transmit a phagocytic signal. In B cells, FcRIIB negatively
regulates B-cell activation by the B-cell antigen receptor. Human
macrophages express both FcRIIA and FcRIIB and while FcRIIA
mediates phagocytosis, the function of FcRIIB in these cells is
unknown. Using the epithelial/fibroblast-like cell line COS-1 as a
model to examine the molecular events that regulate the phagocytosis of
IgG-coated cells (EA), we investigated the effect of FcRIIB on
FcRIIA signaling. FcRIIB inhibited phagocytosis mediated both by
FcRIIA and by a chimeric FcRIIA receptor containing the
extracellular domain of FcRI and the transmembrane and cytoplasmic domains of FcRIIA. This inhibition occurred at an early signaling stage because tyrosine phosphorylation of the FcRIIA cytoplasmic domain was inhibited after concurrent stimulation of these receptors with EA. FcRIIB mutations showed the importance of the FcRIIB YXXL for inhibition of FcRIIA-mediated phagocytosis. Deletion of the
FcRIIB YXXL or conservative replacement of the YXXL tyrosine substantially reduced the inhibitory signal. FcRIIB had a lesser inhibitory effect on phagocytosis by the Fc receptor FcRIIIA, which requires a subunit to mediate a phagocytic signal. These results show that FcRIIB negatively regulates phagocytic signaling by FcRIIA and suggests that FcRIIB plays a role in modulating FcRIIA function in vivo.
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INTRODUCTION |
PHAGOCYTOSIS PLAYS an important role in
host defense against microbial infection and is a major function of
monocytes and macrophages. Monocytes and macrophages induce a
phagocytic signal through their cell-surface Fc receptors (FcR)
which detect and bind IgG coated cells through the constant (Fc) region
of IgG.1-3 Human macrophages express receptors from all
three FcR classes: FcRI, FcRII, and FcRIII. Although Fc
receptors share similar structures, including homologous extracellular
IgG binding domains, the individual Fc receptors differ
significantly in their cytoplasmic regions. The divergence in the
structures of the Fc receptor cytoplasmic domains largely accounts
for their distinct functions.
The FcRII class of receptors is encoded by three genes: FcRIIA,
FcRIIB, and FcRIIC.4 FcRIIA is expressed on
phagocytic cells such as monocytes and macrophages and is the only
FcR present on platelets. FcRIIB receptors are expressed on
monocytes and macrophages as well as on lymphoid cells (B cells and
some subpopulations of T cells) and mast cells.5 The two
major isoforms of FcRIIB, FcRIIB1, and FcRIIB2 are identical
except for a 19-amino acid insertion in the FcRIIB1 cytoplasmic
domain resulting from alternative splicing of the FcRIIB
gene.6 FcRIIB isoforms are homologous to FcRIIA in
their extracellular and transmembrane regions, but the cytoplasmic
domains of the FcRIIB receptors share little homology with
FcRIIA. Because of the difficulty associated with examining
individual Fc receptors in hematopoietic cells where several
different FcR classes may be expressed, we have used the
fibroblast/epithelial-like cell line COS-1 as a model system to
evaluate individual FcR function. FcRIIA, when transfected into
these cells, can efficiently phagocytose antibody coated erythrocytes
(EA).7
Much attention has focused on the presence of the pair of tyrosine
containing sequences (YXXL) found within the cytoplasmic domains of
most receptors of the Ig gene superfamily or their associated
subunits.8-10 This immunoreceptor tyrosine-based activation motif (ITAM) is required for signal transduction through these receptors. FcRIIA contains an ITAM-like consensus motif consisting of two YXXL sequences separated by 12 amino acids. Phosphorylation of
the tyrosines (Y) within this motif is essential for FcRIIA-mediated phagocytosis and the importance of the ITAM-like region in
FcRIIA-mediated signaling has been established by extensive mutation
and deletion studies.10 Although FcRIIB receptors do not
contain a consensus ITAM in their cytoplasmic domain, they do contain
one YXXL sequence (YSLL). This YSLL is contained within an ITIM
sequence (for immunoreceptor tyrosine-based inhibitory motif). The ITIM
sequence found in FcRIIB was first studied in B lymphocytes where it
inhibits B-cell receptor-mediated Ig production.11,12 When
transfected into COS-1 cells, FcRIIB isoforms do not mediate
phagocytosis of opsonized cells although they bind EA
avidly.13 These studies suggest that although FcRIIB is
expressed in cells of myeloid origin, it does not play a direct role in
mediating phagocytosis in vivo.
Since FcRIIB contains an ITIM sequence, we used our COS-1 cell model
to examine the hypothesis that FcRIIB may regulate FcRIIA-mediated phagocytosis. Both FcRIIB1 and FcRIIB2
inhibited FcRIIA-mediated phagocytosis whereas phagocytosis by the
Fc receptor, FcRIIIA, was only minimally reduced. Using a
chimeric FcRIIA receptor containing the cytoplasmic domain of
FcRIIA, we further showed that the FcRIIB YSLL tyrosine is
important for the inhibitory effect. Signaling through the FcRIIA
cytoplasmic domain and tyrosine phosphorylation of the FcRIIA
chimeric receptor are decreased after costimulation of the FcRIIA
chimera and FcRIIB. These data indicate that one Fc receptor
isoform, FcRIIB, can regulate phagocytosis mediated by another
monocyte/macrophage Fc receptor isoform, FcRIIA.
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MATERIALS AND METHODS |
Construction of FcRIIB mutants.
All cDNAs were expressed in the eukaryotic expression vector PRC/CMV
(Invitrogen, San Diego, CA). Two-step overlap-extension polymerase
chain reaction (PCR) using the appropriate oligonucleotide primers was
used to construct the mutant cDNAs. Clones were subjected to DNA
sequencing to verify the mutations. The following FcRIIB mutants
were constructed: Trun-B2 (FcRIIB2 with the cytoplasmic domain
deleted), B1-YF and B2-YF (FcRIIB1 and FcRIIB2 isoforms in which
the tyrosine [Y] of the YSLL sequence has been mutated to
phenylalanine [F]), B1 YSLL and B2YSLL (FcRIIB1 and
FcRIIB2 isoforms in which the YSLL sequence has been deleted), and
B2-YETL and B2-YMTL (FcRIIB2 in which the YSLL has been changed to
YETL or YMTL, respectively).
Cell culture and COS-1 cell transfection.
COS-1 cells were cultured and maintained in Dulbecco's modified
Eagle's medium (DMEM) containing glucose (4.5 mg/mL), glutamine (2 mmol/L), streptomycin (100 U/mL), penicillin (100 mg/mL), and 10%
heat-inactivated fetal bovine serum. COS-1 cells were transiently transfected at 80% confluency (3 × 105 cells/well in a
six-well plate) in DMEM culture medium containing diethylaminoethyl
(DEAE)-dextran (750 µg/mL), chloroquine chloride (100 µmol/L), and 4.0 µg plasmid DNA per milliliter. After 3.5 hours of
incubation at 37°C the transfection medium was removed and the cells
incubated in a 10% solution of dimethyl sulfoxide in
phosphate-buffered saline (PBS) for 2 minutes at room temperature. The
cells were then washed twice with DMEM, overlaid with fresh medium, and
incubated for 48 hours before analysis.
Binding and phagocytosis of IgG sensitized red blood cells (RBCs).
Antibody-coated sheep erythrocytes (Rockland, Gilbertsville, PA) (EA)
were prepared in magnesium- and calcium-free PBS by incubating
109/mL sheep RBCs with an equal volume of the highest
subagglutinating concentration of IgG rabbit-anti-sheep RBC antibody
(Cappel Laboratories, West Chester, PA) as previously
described.7 The medium was removed from the COS-1
transfectants and the cells overlaid with a large excess of EA
(108 EA in 1.0 mL for 3 × 105 COS-1 cells)
and incubated at 37°C for 30 minutes. Unbound EA were washed away
with PBS, and EA binding to the transfected cells was determined.
Extracellularly bound EA was then removed by a short (20 seconds) wash
with 0.25% PBS. The cells were stained with Wright-Giemsa and the
number of COS-1 cells with more than one internalized EA was determined
in a blinded fashion by light microscopy. At least 300 cells were
counted per experiment. Phagocytosis was expressed as phagocytic index
(PI), the number of EA ingested per 100 COS-1 cells. The percent cells
phagocytosing at least 1 EA was also determined. Levels of expression
of the phagocytic chimeric receptors (I-IIA-IIA or  --) were
determined by flow cytometry.
Flow cytometry.
A total of 105 cells was incubated on ice for 30 minutes
with anti-FcRII monoclonal antibody (MoAb) IV.3 (for FcRIIB
staining) or anti-FcRI MoAb 32.2 (for staining the chimeric
FcRIIA receptor containing the extracellular [EC] domain of
FcRI, I-IIA-IIA [EC-TM-CYT]). The cells were washed and labeled
with fluorescein isothiocyanate-conjugated goat-anti-mouse
F(ab )2 IgG (TAGO Inc, Burlingame, CA) for 30 minutes on ice. After washing, the cells were fixed in a solution of
4% paraformaldehyde. Isotype controls were used for all antibodies and
fluorescence was measured on a FACSTAR cytometer (Becton Dickinson, Mountain View, CA).
Immunoprecipitation and Western blotting.
After stimulation of FcR-transfected COS-1 cells with EA at 37°C
for 30 minutes, the cells were placed on ice to stop further phagocytosis. Externally bound EA was removed by hypotonic lysis and
the COS-1 cell lysate obtained by the addition of 1.0 mL RIPA buffer
(1% Triton X-100 [Sigma, St Louis, MO], 1% deoxycholate, 0.1%
sodium dodecyl sulfate [SDS], 158 mmol/L NaCl, 10 mmol/L Tris pH 7.2, 5 mmol/L NaEGTA, 1 mmol/L phenylmethylsulphonyl fluoride [PMSF], 1 mmol/L Na3VO4, 50 µg/mL leupeptin, and 10 µg/mL aprotinin) followed by incubation on ice for 30 minutes.
Cleared lysates were immunoprecipitated with the appropriate antibody
(10 µg/mL antiphosphotyrosine polyclonal antibody [PharMingen,
San Diego, CA; cat. no. 14201A]). Immunoprecipitates were analyzed by
SDS-polyacrylamide gel electrophoresis and immunoblots were performed
with antiphosphotyrosine MoAb 4G10 (UBI, Lake Placid, NY). Immunoblots
were developed with horseradish peroxidase-conjugated goat-anti-mouse
IgG (BioRad, Richmond, VA) and visualized by enhanced chemiluminescence
(Amersham Corp, Arlington Heights, IL).
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RESULTS |
FcRIIB inhibits FcRIIA-mediated
phagocytosis.
When transfected into COS-1 cells, the Fc receptor, FcRIIA,
mediates the phagocytosis of IgG-coated RBCs (EA).7 In
contrast, two other members of this Fc receptor class, FcRIIB1
and FcRIIB2, do not phagocytose EA when expressed in COS-1
cells.13 In other cell types such as B cells, FcRIIB
receptors are known to inhibit certain signaling
pathways.11,12 Therefore, we examined the possibility that
the FcRIIB receptors might inhibit the FcRIIA signal for
phagocytosis.
The FcRIIB isoforms FcRIIB1 or FcRIIB2 were cotransfected with
FcRIIA into COS-1 cells. The cotransfected cells were incubated with
EA and the extent of phagocytosis was compared with cells expressing
FcRIIA alone. Cotransfection of an FcRIIB receptor had a marked
inhibitory effect on the phagocytosis of EA by FcRIIA. We observed a
reduction of approximately 60% in both the phagocytic index and the % transfectants phagocytosing EA.
In these transfection experiments, MoAb 41H16, reported to
differentiate between the His 131 variant of FcRIIA and FcRIIB, which has Arg at 131, did not clearly distinguish between FcRIIA and
FcRIIB. Therefore, it was difficult using FcRII MoAbs and flow
cytometry to measure the expression of FcRIIA in the presence of
FcRIIB. However, we have previously shown the importance of the
cytoplasmic domain in transmitting the FcRIIA phagocytic signal.10 Truncation of the FcRIIA cytoplasmic domain
eliminates its phagocytic function and disruption of the FcRIIA
cytoplasmic ITAM sequence, by substituting the tyrosines
with phenylalanine, severely reduces or eliminates the
receptor's phagocytic signal.10 To clearly determine the
expression of FcRIIA in the presence of FcRIIB in our
cotransfection experiments and to confirm that the decrease in
phagocytosis was not caused by a change in FcRIIA expression, we
used a chimeric FcRII receptor I-IIA-IIA (EC-TM-CYT). I-IIA-IIA
contains both the transmembrane (TM) and cytoplasmic (CYT) domains of
wild-type (WT) FcRIIA but the extracellular (EC) domain of FcRI
and is therefore recognized by the anti-FcRI MoAb 32.2. When
expressed in COS-1 cells the I-IIA-IIA chimera mediated the
phagocytosis of EA with an efficiency comparable to WT
FcRIIA.1,14,15 Therefore, the chimeric receptor
I-IIA-IIA serves as a model for FcRIIA, allowing examination of
phagocytosis by the FcRIIA cytoplasmic domain.
Similar to the observation with WT FcRIIA, cotransfection of COS-1
cells with I-IIA-IIA and FcRIIB2 resulted in a decrease in
phagocytosis compared to COS-1 cells transfected with I-IIA-IIA alone
(Fig 1A). In the three representative
independent experiments shown, flow cytometry histograms demonstrate
that similar levels of I-IIA-IIA were expressed in both the presence
and absence of FcRIIB (Fig 1B). Thus, the decrease in I-IIA-IIA
phagocytosis observed in cells cotransfected with FcRIIB is not
caused by a change in the expression of the I-IIA-IIA receptor.
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| Fig 1.
Phagocytosis of EA mediated by the chimeric receptor
I-IIA-IIA in COS-1 cells expressing I-IIA-IIA alone or I-IIA-IIA plus FcRIIB2. (A) The percent of cells that phagocytose EA and the phagocytic index (PI, number of ingested EA/100 transfected COS-1 cells) for three representative experiments are shown. (B) Expression of the chimeric receptor as determined by flow cytometry. Cells were
stained with MoAb 32.2 to measure I-IIA-IIA expression in the presence
(-----) or absence (.........) of FcRIIB2. (. . . .), Indicates transfectants stained with isotype control antibody. The
expression of FcRIIB was determined by flow cytometry after staining
with MoAb IV.3. Mean fluorescence intensity (MFI) for FcRIIB2 in
experiments 1, 2, and 3 was 104, 91, and 80, respectively. The
fluorescence histogram shows that the expression of I-IIA-IIA (MFI) in
the presence or absence of FcRIIB2 was virtually the same. Thus,
FcRIIB2 decreased phagocytosis by I-IIA-IIA without changing the
cell-surface expression of I-IIA-IIA.
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We constructed a mutant FcRIIB, Trun-B2, which contains the WT
extracellular and transmembrane regions of FcRIIB2 but lacks the
entire FcRIIB cytoplasmic domain. This truncated FcRIIB bound EA
as efficiently as WT FcRIIB. However, in contrast to cotransfection
of WT FcRIIB and I-IIA-IIA, cotransfection of Trun-B2 and I-IIA-IIA
did not inhibit phagocytosis (Fig 2). These data indicate that the inhibition of phagocytosis mediated through the
cytoplasmic domain of FcRIIA by cotransfected FcRIIB requires the
FcRIIB receptor cytoplasmic domain.
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| Fig 2.
Phagocytosis mediated by the chimeric receptor I-IIA-IIA
in COS-1 cells expressing the I-IIA-IIA chimera alone or the I-IIA-IIA chimera plus the FcRIIB mutant lacking a cytoplasmic domain, Trun-B2. (A) The percent of cells phagocytosing EA and the phagocytic index for three representative experiments are shown. (B) Expression of
the chimeric receptor I-IIA-IIA as determined by flow cytometry. Cells
were stained with MoAb 32.2 to measure I-IIA-IIA expression in the
presence (-----) or absence (.........) of Trun-B2. (. . . .), Indicates transfectants stained with isotype control antibody. The
expression (MFI) of I-IIA-IIA was the same in the presence or absence
of Trun-B2 in each experiment. The expression of Trun-B2 was determined
by staining with anti-FcRII MoAb IV.3. MFI for Trun-B2 in
experiments 1, 2, and 3 was 55, 44, and 60, respectively.
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The FcRIIB YSLL sequence is required for the
inhibition of FcRIIA-mediated phagocytosis.
A series of experiments was performed to examine the importance of
FcRIIB cytoplasmic sequences for inhibiting FcRIIA-mediated phagocytosis. The FcRIIB cytoplasmic domain contains a 13-amino acid
region, the ITIM, which includes a YSLL sequence required for
regulation of receptor signaling in B lymphocytes.11 That this sequence is also required for FcRIIB-mediated inhibition of
FcRIIA phagocytic signaling was indicated in experiments using a
deletion mutant of FcRIIB (Table 1). Deletion of
the FcRIIB YSLL region (B2YSLL)
reduced the ability of FcRIIB2 to inhibit phagocytosis by I-IIA-IIA.
Replacement of the YSLL tyrosine (Y) with phenylalanine (F) (B2-YF)
also reduced the inhibitory activity of FcRIIB2. Flow cytometry
analysis showed that the cell-surface expression of the I-IIA-IIA
chimeric receptor was similar within each experiment and that the
cell-surface expression of the FcRIIB2 mutants and WT FcRIIB2 was
also similar within each experiment. These data indicate that
FcRIIB2 inhibition of the phagocytic signal mediated by the chimeric
receptor I-IIA-IIA directly involves the FcRIIB2 cytoplasmic YSLL
sequence. The results of experiments with FcRIIB1 were similar to
those with FcRIIB2. Costimulation with EA of I-IIA-IIA and
FcRIIB1 also resulted in inhibition of phagocytosis by I-IIA-IIA and
deletion of the FcRIIB1 YSLL (B1YSLL) or mutation of the tyrosine
(Y) to phenylalanine (F) (B1-YF) reduced the inhibitory effect of
FcRIIB1 (data not shown).
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Table 1.
The Tyrosine-Containing Sequence Within the FcRIIB
Cytoplasmic Domain Is Required for the Inhibition of Phagocytosis by
the Chimeric Receptor I-IIA-IIA
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Another Fc receptor that mediates a phagocytic signal is FcRIIIA.
We previously showed that the efficiency of FcRIIIA-mediated signal
transduction is due in part to the sequence of internal (XX) amino
acids within the YXXL motifs of its associated -chain.16 Therefore, we studied the role of the internal amino acids (XX) within
the FcRIIB YXXL cytoplasmic sequence. We observed that the specific
YXXL internal amino acids (SL) of FcRIIB do not appear to be
important for regulating the FcRIIA phagocytic signal. Changing the
FcRIIB YSLL to either YMTL or YETL (B2-YETL and B2-YMTL), thus
creating YXXL sequences identical to those found in FcRIIA or the
Fc receptor -chain, did not alter the inhibitory activity of
FcRIIB (data not shown). Thus, the cytoplasmic tyrosine rather than
the sequence of the internal XX amino acids within the YXXL of the ITIM
is of primary importance for FcRIIB inhibition of FcRIIA-mediated
phagocytosis.
FcRIIB reduces tyrosine phosphorylation of
FcRIIA.
The cytoplasmic ITAM-like sequence is important for phagocytic
signaling by FcRIIA. FcRIIA is tyrosine phosphorylated after receptor stimulation and this early signaling event is required for
efficient phagocytosis.10 Disruption of the tyrosines
within the ITAM-like region significantly inhibits Fc
receptor-mediated phagocytosis in FcRIIA-transfected COS-1 cells. In
addition, treatment with inhibitors of tyrosine phosphorylation reduces FcRIIA-mediated phagocytosis in monocytes and macrophages as well as
in transfected COS-1 cells.10 Costimulation of the chimeric Fc receptor I-IIA-IIA and FcRIIB2 in COS-1 cell transfectants resulted in decreased tyrosine phosphorylation of I-IIA-IIA (lane 6)
compared with I-IIA-IIA alone (lane 2) (Fig
3).
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| Fig 3.
Antiphosphotyrosine immunoblot of the chimeric I-IIA-IIA
receptor in COS-1 cells transfected with I-IIA-IIA alone, I-IIA-IIA plus FcRIIB2, and I-IIA-IIA plus Trun-B2. Lanes 1 and 2, I-IIA-IIA transfectants; lanes 3 and 4, I-IIA-IIA plus Trun-B2 cotransfectants; lanes 5 and 6, I-IIA-IIA plus FcRIIB2 cotransfectants. Transfected cells were either not stimulated ( ) or stimulated (+) with EA. The
arrow shows the position of the tyrosine phosphorylated I-IIA-IIA chimera (60 kD). The expression of I-IIA-IIA was similar
in each transfection. FcRIIB and Trun-B2 were also expressed at
similar levels (MFI for FcRIIB was 114 and 104 for Trun-B2).
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The antiphosphotyrosine blot in Fig 3 indicates that it is the
contribution of the FcRIIB2 cytoplasmic domain that is responsible for the inhibition of I-IIA-IIA tyrosine phosphorylation. In this blot
we show that costimulation of I-IIA-IIA and the FcRIIB2 mutant that
is missing the cytoplasmic domain (Trun-B2) does not decrease tyrosine
phosphorylation of I-IIA-IIA (lane 4). In contrast, costimulation of WT
FcRIIB2 markedly reduces tyrosine phosphorylation of I-IIA-IIA. In
this experiment there is no significant difference between the
expression levels of the WT and mutant (tailless) FcRIIB receptors.
I-IIA-IIA expression also does not change with the coexpression of the
additional FcRIIB receptors. Thus, stimulating FcRIIB2 leads to
both decreased tyrosine phosphorylation of I-IIA-IIA and decreased
I-IIA-IIA-mediated phagocytosis.
Inhibition by FcRIIB of phagocytosis by the
Fc receptor FcRIIIA.
The Fc receptor FcRIIIA, which is expressed in such myeloid cells
as macrophages, is also capable of mediating the phagocytosis of IgG
coated cells. Unlike FcRIIA-mediated phagocytosis, FcRIIIA phagocytic signaling is mediated through an associated -chain subunit. In these experiments we made use of a chimeric molecule consisting of the EC domain of FcRIIIA and the transmembrane (TM)
and cytoplasmic (CYT) domains of the -chain, --
(EC-TM-CYT). In contrast to I-IIA-IIA-mediated phagocytosis,
phagocytosis of EA by -- was only minimally decreased by
costimulating FcRIIB and -- in COS-1 cell transfectants
(Table 2).
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Table 2.
The Effect of FcRIIB1(B1) and FcRIIB2 (B2) on
Phagocytosis by the Chimeric Receptor FcRIIIA--
(EC-TM-CYT)
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DISCUSSION |
We have observed that FcRIIB inhibits the phagocytosis of IgG-coated
cells mediated by the FcRIIA cytoplasmic domain. In these studies we
used a COS-1 cell-model system and a chimeric receptor, I-IIA-IIA, to
examine phagocytosis mediated by the FcRIIA cytoplasmic domain
sequences. We previously determined that the phagocytic signals
mediated by FcRIIA and the chimeric receptor I-IIA-IIA are similar.
This approach, using FcRIIA in the form of a chimera, allows the
expression of FcRIIA to be distinguished from the expression of
FcRIIB in co-transfectants using MoAbs 32.2 and IV.3. MoAb 32.2 recognizes the EC of FcRI and does not crossreact with the EC of
FcRIIB, and MoAb IV.3 recognizes the EC of FcRII but not the EC
of FcRI in the chimera I-IIA-IIA.
The data indicate that the decrease in I-IIA-IIA-mediated phagocytosis
is not caused by decreased expression of the phagocytic receptor
I-IIA-IIA. In addition, WT FcRIIB2 inhibits I-IIA-IIA phagocytosis
whereas FcRIIB2 cytoplasmic domain mutants, which are expressed at
similar levels as WT FcRIIB, do not inhibit phagocytosis by
I-IIA-IIA (Table 1). Furthermore, because a large excess of ligand
(>300 EA/COS-1 cell) was used to overlay the cells and because the
extracellular domain of FcRI (and I-IIA-IIA) has a higher affinity
for IgG ligand than does the extracellular domain of FcRIIB,
competition for available EA cannot explain our findings.
Studies with FcRIIB mutants showed that inhibition of phagocytosis
requires an FcRIIB cytoplasmic sequence, YSLL, within the 13-amino
acid ITIM region. Of primary importance for the inhibitory signal is
the presence of the YSLL tyrosine because its replacement with
phenylalanine reduced the ability of FcRIIB to inhibit phagocytosis mediated by the chimeric receptor I-IIA-IIA. We observed that the
internal SL amino acids of the YSLL sequence are not critical for the
inhibition because their replacement had no effect on the phagocytic
signal.
Because I-IIA-IIA contains the complete FcRIIA cytoplasmic domain
responsible for mediating the phagocytic signal, the observations with
I-IIA-IIA are pertinent for WT FcRIIA. Costimulation of FcRIIA
and FcRIIB likely alters signal transduction mediated by the
FcRIIA cytoplasmic domain. The mechanism of this interaction has not
as yet been completely delineated, although in other signal transduction pathways, a role has been implicated for tyrosine phosphatases.17,18 It is likely that a cytosolic factor,
such as a phosphatase, mediates the inhibition of phagocytosis by
FcRIIB. Inhibition by FcRIIB of phagocytosis mediated by the
FcRIIA cytoplasmic domain occurs at an early stage after receptor
activation, because tyrosine phosphorylation of the FcRIIA
cytoplasmic domain was reduced after costimulation of both the
I-IIA-IIA chimera and FcRIIB2. Receptor tyrosine phosphorylation,
including FcRIIA tyrosine phosphorylation, is an early step in
signal transduction and is required for efficient FcRIIA-mediated
phagocytosis both in our COS-1 cell model system and in human monocytes
and macrophages.7,10
FcRIIB is known to play a role in inhibiting the proliferation of B
cells.11,12 Recent work in B-cell and mast cell model systems has shown that this phenomenon is not restricted to B cells and
has led to further understanding of the mechanism of the FcRIIB
inhibitory process.19-22 One observation emerging from work
with rat basophilic leukemia (RBL) cells and B-lymphoma cell lines is
that the FcRIIB inhibitory signal may affect many receptors of the
Ig gene superfamily that signal through ITAM sequences. The studies
show that FcRIIB inhibits serotonin release induced by costimulating
Fc RI (a member of the Ig gene family) in mast cells. Serotonin
release in RBL cells by chimeric receptors containing the cytoplasmic
domain of the T-cell receptor  -chain or by FcRIIA is also
inhibited by FcRIIB.22 Here we have shown that FcRIIB can also inhibit an important FcRIIA function of monocytes and macrophages.
Our observations suggest that a novel mechanism exists for the
regulation of phagocytosis in phagocytic cells such as monocytes and
macrophages, which express both FcRIIA and FcRIIB. Both receptors
are members of the same Ig receptor gene family, recognize the same
ligand, and are members of the same Fc receptor class (FcRII).
The extensive homology of the class II Fc receptor extracellular
domains suggests that similar IgG ligands may both activate and inhibit
FcRIIA-mediated phagocytosis and it is likely that
FcRIIA-mediated phagocytosis is regulated by FcRIIB in monocytes
and macrophages. The extent of the phagocytic signal may depend on the
relative expression of FcRIIA and FcRIIB and/or their
affinity for IgG ligand. Both FcRIIA and FcRIIB recognize the Fc
domain of most IgG molecules. However, it should be noted that there
are some differences. For example, an FcRIIA isoform (FcRIIA-His-131)23 has high affinity for the IgG
subclass IgG2. Thus, it is unlikely that FcRIIA-mediated
phagocytosis induced by IgG2 antibodies is regulated by FcRIIB
because IgG2 does not bind FcRIIB.
A similar phenomenon has recently been observed in T cells. For
example, T cells express two distinct receptors for recognizing major
histocompatibility complex class 1 proteins, one that mediates a
positive signal, the T-cell receptor (TCR), and a second receptor, NKB1, that mediates an inhibitory signal.24,25 The studies indicate that the negative NKB1 signal may inhibit tyrosine
phosphorylation of the TCR CD3 -chain. A similar situation also
exists for another TCR system. The costimulatory receptor CD28 and the
CTLA-4 receptor bind the same ligand and CTLA-4 appears to play a
negative regulatory role in CD3/CD28-mediated T-cell
activation.26-28 Also, we have recently observed that in
polymorphonuclear leukocytes the Fc receptor FcRIIIB, a glycan
phosphoinositol-linked receptor protein, similarly can also negatively
regulate signaling by FcRIIA.29 This evidence for
positive and inhibitory signals initiated by the same ligand through
two distinct cell-surface receptors is similar to our observations with
FcRIIA and FcRIIB and suggests that such regulation of receptor
signaling may be operable in several cell systems.
We also examined the ability of FcRIIB to regulate
FcRIIIA-mediated phagocytosis using the FcRIIIA chain and chain chimeric receptor -- (EC-TM-CYT). Our results indicate
that FcRIIB inhibition of FcRIIIA phagocytosis is less pronounced than that of FcRIIA phagocytosis. This observation is of interest because FcRIIIA mediates phagocytosis through a -chain subunit which is common to both FcRIIIA and FcRI.30 These
data support other studies which suggest that FcRIIA and FcRIIIA
differ in their requirements for phagocytosis and that they transmit a
phagocytic signal through pathways that differ at some
point.15,30 Taken together, the results suggest that not
only do FcRIIA and FcRIIIA differ in their requirements for
phagocytosis, but that their phagocytic signal is differentially
regulated.
Thus, the data indicate that the FcRIIB receptor can regulate
phagocytosis transmitted by the FcRIIA receptor. Because tyrosine phosphorylation of FcRIIA is important for the phagocytic signal by
FcRIIA,2,10,13 decreased tyrosine phosphorylation
induced by FcRIIB is likely responsible for the inhibition of
FcRIIA-mediated phagocytosis.
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FOOTNOTES |
Submitted August 29, 1996;
accepted October 17, 1997.
Supported by National Institutes of Health Grants No. HL-27068 and
AI-22193.
Address reprint requests to Sharon Hunter, PhD, 7 Silverstein,
University of Pennsylvania School of Medicine, 3400 Spruce St,
Philadelphia, PA 19104.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract