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J. Biol. Chem., Vol. 277, Issue 7, 5082-5089, February 15, 2002
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From the Department of Internal Medicine, The Ohio State
University, Columbus, Ohio 43210 and § INSERM U.255, 75270 Paris Cedex 06, Paris, France
Received for publication, October 25, 2001, and in revised form, December 6, 2001
Human monocytes/macrophages express three classes
of receptors for IgG: Fc Clustering of the Fc In humans, Fc Fc In this report we demonstrate for the first time the presence of
Fc Antibodies and Reagents--
Anti-Fc Cells and Cell Culture--
THP-1, U937 (monocytic cell lines),
and Raji B cells were obtained from ATCC. IIA1.6 cells were a
kind gift from Dr. Ira Mellman. The IIA1.6+IIa were obtained
from Dr. Jan G. J. Van de Winkel. All cells were maintained at
37 °C in RPMI supplemented with 10% heat-inactivated fetal bovine
serum and 5% CO2.
Peripheral blood monocytes (PBM) were purified from buffy coats of
healthy donors as described previously (18). Briefly, peripheral blood
mononuclear cells (PBMCs) were first isolated by density gradient
centrifugation over Histopaque (Sigma). Monocytes were then purified
from the PBMCs by negative selection using the MACs Monocyte Isolation
Kit (Miltenyi Biotech). PBMCs were then treated with FcR blocking
reagent (hIgG), followed by a hapten-antibody mixture (mixture of
monoclonal hapten-conjugated CD3, CD7, CD19, CD45RA, CD56, and anti-IgE
antibodies). The labeled cells were further treated with MACS
anti-hapten magnetic microbeads that were conjugated to a monoclonal
anti-hapten antibody. The cells were then passed over a MACS column,
and the effluent was collected as the negative fraction representing
enriched monocytes. The monocytes thus purified were subsequently
analyzed for purity by double labeling with CD14-phycoerthrin
and CD45-fluorescein isothiocyanate antibodies followed by flow
cytometry. Data from 10,000 cells indicated that the isolated monocytes
were >99% CD14 positive.
Cell Stimulation and Lysis--
For activation, 107
cells per sample were resuspended in 100 µl of HBSS, incubated with
10 µg/ml IV.3 Fab, IV.3, or FL18 for 25 min at 4 °C. The unbound
antibody was washed off, cells were resuspended again in 100 µl of
HBSS and treated with F(ab')2 goat anti-mouse IgG for the
desired time periods at 37 °C. Resting and activated cells were
lysed in TN1 lysis buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 10 mM
Na4P2O7, 10 mM NaF, 1%
Triton X-100, 125 mM NaCl, 3 mM
Na3VO4, 10 µg/ml each aprotinin and
leupeptin, and 2 mM phenylmethylsulfonyl fluoride) for 30 min on ice.
Immunoprecipitation and Immunoblotting--
Postnuclear lysates
were incubated overnight with the antibody of interest and protein
G-agarose beads (Invitrogen) or goat anti-mouse Ig covalently linked to
agarose, depending on the immunoprecipitating antibody. Immune
complexes bound to beads were washed in TN1 and boiled in SDS sample
buffer (60 mM Tris, pH 6.8, 2.3% SDS, 10% glycerol,
0.01% bromphenol blue, and 1% 2-mercaptoethanol) for 5 min. Proteins
were separated by SDS-PAGE, transferred to nitrocellulose filters,
probed with the antibody of interest, and developed by enhanced chemiluminescence.
Immunoblot Data Quantitation--
The ECL signal was quantitated
using a scanner and a densitometry program (Scion Image). The
non-linearity of ECL signal and the low dynamic range of the film used
was corrected by generating a calibration curve for the experiments by
serial dilution of a control sample, and film exposure time was varied
to include the entire range of data. To quantitate the phosphotyrosine
signal in the activated samples, we first subtracted background,
normalized the signal to the amount of precipitated protein, and
plotted the values obtained by subtracting the value in unstimulated samples.
Deglycosylation--
Fc Preparation of IgG-coated sheep RBCs--
Sheep RBCs (Colorado
Serum, Denver, CO) were washed in PBS, and labeled overnight with 0.1 mg/ml fluorescein isothiocyanate in PBS at 4 °C. Fluorescein
isothiocyanate-labeled cells were then washed in PBS and incubated with
a subagglutinating dose of rabbit anti-sheep RBC IgG (Diamedix, Miami,
FL) at 37 °C for 1 h. Unbound IgG was removed by washing the
cells with PBS.
Phagocytosis Assay--
IgG-coated SRBCs described above were
added to THP-1 cells in suspension, and the cells were pelleted by low
speed centrifugation to increase contact between SRBCs and phagocytes.
The samples were prepared in duplicate and incubated for 1 h at
either 4 °C to study binding, or 37 °C to study phagocytosis. All
cells were fixed in 1% paraformaldehyde and mounted on slides to be
viewed under a fluorescence microscope. For the phagocytosis assay,
cells were subjected to brief hypotonic lysis with water to get rid of
externally bound RBCs prior to fixation in paraformaldehyde. The
ability of the THP-1 cells to bind IgG-coated targets was expressed as
the percentage of cells that each bound three or more SRBCs (rosetting
activity, Fig. 5A). That the binding was via the Fc
receptors was confirmed by the lack of binding observed with
non-IgG-coated SRBCs. No binding or phagocytosis was seen in any of the
samples treated with non-opsonized RBCs. Phagocytosis was measured by
counting the total number of RBCs ingested by 200 THP-1 cells
(phagocytic index, Fig. 5A). The experiment was performed twice.
Phagocytosis via Fc Fc Both b1 and b2 Isoforms of Fc Expression of Fc
During the course of our investigations we observed that the expression
of Fc Co-clustering Fc
First, tyrosine phosphorylation of SHIP was examined in THP-1 cells
that had been activated with IV.3 Fab, IV.3 intact IgG, or with
FLI8.26. Results show that co-clustering Fc
We next asked whether co-clustering Fc Co-clustering Fc Up-regulation of Fc
To test whether the decrease in phagocytic efficency displayed by THP-1
cells cultured in the presence of IL-4 was indeed due to the inhibitory
effects of Fc Our data show that Fc The function of Fc Using a combination of antibodies that recognize either Fc It must be noted, however, that it is not clear whether the inhibitory
effects are mediated by Fc We have analyzed the role of IL-4 in Fc The expression of Fc Finally, an understanding of factors that regulate the expression of
Fc Similar findings regarding the effect of IL-4 on the expression on
Fc We thank Drs. T. W. Lyden and J. M. Robinson for many helpful discussions.
*
This work was supported by National Institutes of Health
Grants CA44983, HD35121, and HO38764.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: The Ohio State
University College of Medicine, Rm. 430, Heart & Lung Research Institute (HLRI), 473 West Twelfth Ave., Columbus, OH 43210. Tel.: 614-247-7650; Fax: 614-247-7669; E-mail: anderson.48@osu.edu.
Published, JBC Papers in Press, December 7, 2001, DOI 10.1074/jbc.M110277200
The abbreviations used are:
Fc
Regulated Expression and Inhibitory Function of Fc
RIIb in
Human Monocytic Cells*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RI, FcRII, and FcRIII. The expression
and function of these receptors has been extensively studied with the
exception of one, FcRIIb. While the mRNA for FcRIIb has been
detected in human monocytes, the protein has remained elusive. Studies in mouse models indicated that the macrophage FcRIIb serves to down-regulate FcR-mediated phagocytosis and immune complex-induced inflammation. FcRIIb has also been shown to modulate the action of
cytotoxic antibodies against tumors in mouse models. Hence, an
understanding of how FcRIIb expression is regulated is of great
importance. Here we demonstrate for the first time FcRIIb protein
expression and function in human monocytes. We also report that the
expression of FcRIIb is highly up-regulated by interleukin-4, a Th2 cytokine, and that the up-regulation of FcRIIb results in a
decrease in the phagocytic efficiency of interleukin-4-treated THP-1
cells. Furthermore co-clustering FcRIIb with FcRIIa resulted in
enhanced phosphorylation of the inositol phosphatase SHIP, association
of SHIP with Shc, and phosphorylation of additional proteins around 120 and 60-65 kDa, with a concomitant attenuation of Akt activation. We,
therefore, propose that FcRIIb serves to inhibit
FcRI/IIa-mediated macrophage activation using SHIP as its effector.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptors
(FcR)1 on
monocytes/macrophages by immune complexes initiates a series of
intracellular biochemical events that are necessary for induction of
phagocytosis. The phagocytic process itself is accompanied by the
generation of tissue-damaging products such as inflammatory cytokines,
reactive oxygen species, and lysosomal enzymes. Thus, like all immune
responses the phagocytic response must be subject to homeostatic
control exerted by inhibitory receptors and/or inhibitory enzymes and
resulting in a tempered immune response. Indeed in mouse models it has
been established that expression of FcRIIb, an inhibitory receptor,
results in down-regulation of FcR-mediated phagocytosis (1). That a
similar regulation might occur in human macrophages has been speculated but not confirmed. Human macrophages but not murine macrophages express
the ITAM-bearing FcRIIa whose extracellular and transmembrane domains are similar to FcRIIb (2), thereby complicating the detection and analysis of expression and function of FcRIIb in these cells.
RIIb are expressed as two alternatively spliced
products, FcRIIb1 and -b2 (3). A 13-amino acid motif within the
cytoplasmic tail of FcRIIb termed ITIM (immunoreceptor
tyrosine-based inhibitory motif),
confers the ability to inhibit cellular activation mediated by
ITAM-bearing immunoreceptors (4-6). Inhibition occurs only when
FcRIIb is co-clustered with an ITAM-bearing receptor. The role of
FcRIIb as a negative regulator of immune cell function is
demonstrated in mice genetically altered to be deficient in the
expression of this receptor. Thus, FcRIIb knockout mice display hypergammaglobulinemia and augmented IgG-mediated anaphylaxis in
response to antigenic challenge (1, 7). The inhibitory function of
FcRIIb is mediated by the inositol phosphatase SHIP (8), which
associates with the phosphorylated ITIM of FcRIIb via the SHIP SH2
domain (9-11). Association of SHIP with FcRIIb results in the
tyrosine phosphorylation and recruitment of SHIP to the cell membrane
where it subsequently hydrolyzes PtdIns(3,4,5)P3 to
PtdIns(3,4)P2 (9). PtdIns-P3 is required for
binding and activation of plextrin homology domain containing
molecules such as Btk, a Tec family tyrosine kinase (12), Vav, a
guanine exchange factor for the low molecular weight GTP-binding
proteins of the Rho family (13), and Akt, a serine/threonine kinase
involved in the protection of cells from apoptosis (14, 15). SHIP
consumption of PtdIns-P3 thus leads to the down-regulation
of the above plextrin homology domain containing enzymes, and blocks
the ensuing biologic responses.
RIIb has also been shown to associate with the hematopoetic
cell-specific protein-tyrosine phosphatase SHP-1 both in in vitro analyses using synthetic phosphopeptides corresponding to the ITIM of FcRIIb (16) and in in vivo analyses by
co-immunoprecipitation experiments under special conditions of cell
stimulation (17). However, no functional role for SHP-1 in
FcRIIb-mediated inhibition has thus far been identified. Indeed,
experiments in B cells expressing chimeric receptors with the
extracellular domain of FcRIIb fused to either SHIP or SHP-1
indicated that SHP-1 plays no role in FcRIIb-mediated inhibition of
the B cell antigen receptor signaling (8).
RIIb in human monocytes using a novel anti-FcRIIb rabbit polyclonal antibody. Interestingly, the expression of FcRIIb in
human monocytes is not constant, but is highly regulated by factors
such as density of cell culture and the presence of the inflammatory
cytokines such as interleukin-4 in the surrounding milieu. We have
further characterized the function of FcRIIb in human monocytes and
provide evidence that this receptor plays an inhibitory role in
FcR-mediated monocyte/macrophage function. Thus, co-clustering
FcRIIb with the ITAM-bearing FcRIIa resulted in enhanced
phosphorylation of SHIP, association of SHIP with Shc along with a
concomitant decrease in Akt activation. Finally, we report that
IL-4-induced up-regulation of FcRIIb results in a loss of phagocytic
efficiency of THP-1 cells, strongly supporting an inhibitory role for
this receptor.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RII mAb IV.3
Fab and IV.3 intact IgG were obtained from Medarex (Annandale, NJ).
Anti-CD32 mAb FLI8.26 was from Pharmingen (San Diego, CA).
Anti-phosphotyrosine antibody 4G10 was from UBI (Lake Placid, NY).
Anti-SHIP rabbit polyclonal antibody was a kind gift from Dr. K. Mark
Coggeshall (Oklahoma Medical Research Foundation, Oklahoma City, OK).
Anti-Akt and Anti-pAkt were from New England Biolabs (Beverley, MA).
Goat F(ab')2 anti-mouse IgG was from Pierce. Protein
G-agarose beads were from Invitrogen (Rockville, MD).
N-Glycosidase F was purchased from Roche Molecular Biochemicals. Anti-FcRII mAb KB61 was obtained from Dr. D. Mason, Oxford, UK. Anti-FcRIIb rabbit polyclonal antibody, Ab163, was from
Dr. Jean-Luc Teillaud, and was raised against a glutathione S-transferase fusion protein of the cytoplasmic tail of
FcRIIb1. All FcRII antibodies used have been previously described
(40).
RII were immunoprecipitated from
THP-1, U937, PBM, and Raji cells with a mixture of anti-FcRII
antibodies (equal amounts of AT10, KB61, and IV.3), washed in TN1 lysis
buffer, and eluted by boiling in 30 µl of 0.7% SDS for 5 min. The
eluates were treated with either the enzyme diluent alone or with
N-Glycosidase F at 37 °C overnight. The enzyme reaction
was stopped by boiling in SDS sample buffer.
RIIa alone or via FcRIIa and FcRIIb was
performed as described previously (39). Briefly, THP-1 cells were
labeled with either IV.3Fab or FLI8 antibodies for 25 min on ice.
Unbound antibody was washed off in PBS and the cells were resuspended
in PBS. SRBC were first fluoresceinated as described above and
subsequently biotinylated with
n-hydroxysuccinimidi-LC-biotin. The biotinylated SRBC were
then incubated with 200 µg/ml streptavidin and washed in PBS before
adding 40 µg/ml biotinylated F(ab')2 of goat anti-mouse
IgG. The SRBCs thus prepared were then mixed with mAb-labeled THP-1
cells and the phagocytosis assay was performed as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RIIb Is Expressed in Human Monocytes and Monocyte-like Cell
Lines--
To assess the presence of FcRIIb protein in human PBMs
and in THP-1 and U937 monocyte-like cell lines we used a novel rabbit polyclonal antibody, Ab163, raised against the cytoplasmic tail of
FcRIIb (40). Although the extracellular and transmembrane domains
are similar, the cytoplasmic tail of FcRIIa and FcRIIb are
largely divergent with the exception of the first 8 amino acid residues
thus allowing the production of FcRIIb-specific antibody. In these
experiments FcRII was immunoprecipitated with a mixture of
anti-FcRII mAbs, the immune complexes were separated by SDS-PAGE and
subjected to immunoblotting with either an FcRIIa-specific antibody
(Ab260) (Fig. 1A, upper
panel), or the anti-FcRIIb antibody (Ab163) (Fig. 1A,
lower panel). Immunoprecipitates from Raji B cells were used as a
positive control for the expression of FcRIIb. A murine B cell line
that lacks endogenous Fc receptors, IIAI.6, and its stably transfected
derivative that expresses the human FcRIIa, IIA1.6+IIa, were used as
additional controls. That Ab163 does not cross-react with FcRIIa is
evident from the reactivity pattern of FcRII immunoprecipitates from
IIA1.6+IIa cells with Ab260 and Ab163; i.e. FcRIIa in
IIA1.6-IIa cells is not detected by Ab 163, while being readily
recognized by Ab 260. The detection of FcRIIa required the use of
far fewer cells than did that of FcRIIb. Hence in all experiments,
immunoprecipitates from only 107 cells were used
immunoblotting with Ab260, whereas 4 × 107 cells were
used for immunoblotting with Ab163. Results indicated the presence of
FcRIIb in PBMs, THP-1, and U937 cells. The amount of IIb present in
U937 cells appeared much lower than that in PBMs and THP-1 cells.
Additionally we noted the presence of a doublet in the PBMs and Raji
cells that reacted with Ab163, perhaps representing either
differentially glycosylated forms and/or the b1 and b2 isoforms of the
receptor; b1 migrates more slowly owing to a 19-amino acid insertion in
its cytoplasmic tail (3). THP-1 and U937 cells also exhibited the
doublet after deglycosylation (see below).
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Fig. 1.
Fc RIIb1 and -b2 are
both expressed in human monocytes and monocytic cell lines.
A, FcRII receptors were immunoprecipitated from lysates
of 107 (upper panel) and 4 × 107 cells (lower panel) per sample with a
mixture of anti-FcRII mAbs, and immunoblotted with anti-FcRIIa
antibody 260 (upper panel) and anti-FcRIIb antibody 163 (lower panel). B, FcRII immunoprecipitates
were obtained as described above and incubated with either
N-glycosidase F (N Gly F) (+) or the enzyme
diluent (
) as indicated in the figure. The membrane in the
upper panel was probed with Ab260 and that in the
lower panel with Ab163. Molecular weight markers are
indicated as kDa on the left of each panel.
RIIb Are Expressed in Human
Monocytes--
In the absence of glycosylation, the mobility of
FcRIIb1 differs from that of b2 in SDS-PAGE (3). Thus, to determine
which of the isoforms of FcRIIb are present in monocytes, FcRII
immunoprecipitates were deglycosylated with N-glycosidase F,
separated by SDS-PAGE, and immunoblotted with Ab260 (Fig. 1B,
upper panel), or with Ab163 (Fig. 1B, lower panel).
Deglycosylation reduced both FcRIIa and IIb to around 30-35 kDa,
the reported size of the core proteins. Results indicated that both the
b1 and b2 isoforms of FcRIIb are present in all cells tested. The
finding that both isoforms are present in U937 and Raji cells is
consistent with an earlier report that demonstrated the presence of
these isoforms by RT-PCR (2). Our data (Fig. 1B, lower
panel) also indicated that b2 is the predominant isoform expressed
in PBMs and U937 cells, whereas b1 is the major isoform expressed in
Raji B cells; these observations are consistent with earlier reports
analyzing FcRIIb1 and b2 mRNA levels in these cells (2, 19).
Such a distinction between the levels of expression of the FcRIIb1
and b2 was not so evident in THP-1 cells.
RIIb in Human Monocytes Is
Regulated--
Previous studies have shown that Fc receptor expression
is regulated by cytokines (20-22). However, no information exists
regarding the regulation of FcRIIb expression. Based on the fact
that Th1 cytokines enhance macrophage responses while Th2 cytokines
inhibit the same, we hypothesized that the above effect could be a
reflection of the influence of these cytokines on the expression of
activating versus inhibitory FcR. Consistent with this
notion it has long been known that treatment of monocytes with IFN,
a Th1 cytokine, results in a significant up-regulation of the
expression of the ITAM-associated FcRI receptors (20). Here, we
undertook to analyze the effect of IL-4, a Th2 cytokine, on the
expression of FcRIIb and FcRIIa. Thus, U937 cells were cultured
in increasing concentrations of recombinant hIL-4 for 24 h (Fig.
2A). The expression of
FcRIIb and IIa in these cells was then analyzed by immunoblotting. U937 cells, which express the least amount of FcRIIb among the monocyte-like cells tested, were chosen for this experiment as we
reasoned that a regulatory effect on the expression of FcRIIb would
be more evident on a background of low level expression of the
receptor. Results indicated that the presence of IL-4 had no effect on
the expression of FcRIIa (Fig. 2A, lower panel). In
contrast, the expression of FcRIIb was significantly enhanced by all
doses of IL-4 (Fig. 2A, upper panel). The enhancing effect of IL-4 on FcRIIb expression was also observed in PBMs (Fig. 2B) and in THP-1 cells (Fig. 5B).
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Fig. 2.
Expression of Fc RIIb
is altered significantly by IL-4 and culture conditions.
A, U937 cells were cultured for 24 h in the presence of
varying doses of IL-4 as indicated in the figure. FcRII was
immunoprecipitated with a mixture of anti-FcRII mAbs, separated by
SDS-PAGE and immunoblotted with Ab163 (upper panel) or Ab260
(lower panel). B, PBM were cultured in the
presence of IL-4 as indicated in the figure and analyzed for the
expression of FcRIIb in the upper panel and FcRIIa in
the lower panel. C, FcRII was
immunoprecipitated from U937 cells cultured for 24 h at the
densities indicated, and immunoblotted with Ab163 (upper
panel) or Ab260 (lower panel).
RIIb varied based on the density of the cell culture. To
formally address this, U937 cells were seeded at the 3 densities
indicated in Fig. 2C and cultured for 24 h. FcRII receptors were immunoprecipitated from lysates of equal number of cells
from the three different cultures with the anti-FcRII mixture
described above, and probed by immunoblotting with either Ab163
(upper panel) or Ab260 (lower panel). Results
indicated that as the cell density decreased, the amount of FcRIIb
decreased dramatically. Densitometry measurements of band intensities
indicated that the amount of protein reactive with Ab163 was 70%
reduced in lane 2, and 95% reduced in lane 3 when compared with lane 1 (Fig. 2C). In contrast,
in the duplicate blot probed with Ab260 the amount of FcRIIa
remained unaltered verifying, at the very least, that all lanes were
loaded with equal numbers of cells. This trend was consistently
observed in three other experiments.
RIIb with FcRIIa Enhances the SHIP
Phosphorylation, SHIP-Shc Association, and the Phosphorylation of 120- and 60-65-kDa Molecules--
Next we examined the function of
FcRIIb in human monocytes. FcRIIb has been shown to serve as an
inhibitory receptor when co-clustered with the B cell antigen receptor
(23). The inhibitory influence of FcRIIb has been demonstrated to
work via the phosphorylation and activation of the inositol phosphatase
SHIP (8). In B cells, co-clustering B cell antigen receptor and
FcRIIb up-regulates the tyrosine phosphorylation of SHIP, the
association of SHIP with the adapter protein Shc (24), as well as the
tyrosine phosphorylation of Shc itself (25). While a direct role for
FcRIIb in human monocytes has not been established, co-transfection
experiments of FcRIIa and IIb in COS-7 cells revealed that FcRIIb
may serve to inhibit the phagocytic process initiated by FcRIIa
(26). Hence we wished to examine the signaling processes induced by co-clustering FcRIIb with IIa. In these experiments we used either the Fab fragments of mAb IV.3 and GAM to specifically cluster FcRIIa
or IV.3 intact IgG and GAM to co-cluster FcRIIa and IIb. The
rationale for this usage is that IV.3 is of the murine IgG2b isotype
that is reported to have a fairly high capability of serving as a
ligand for FcRIIb (27) and could therefore potentially recruit
FcRIIb. In addition to these antibodies we also used the pan
FcRII mAb FL18.26, which interacts equally well with both FcRIIa
and IIb (28). Thus, THP-1 cells were activated by clustering the
FcRII receptors with the above antibodies, and the ensuing signaling
events were analyzed.
RIIb with IIa enhanced
SHIP phosphorylation (Fig. 3A,
upper panel, lanes 4 and 5). A reprobe of the same
membrane with anti-SHIP antibody showed equal loading of SHIP in all
lanes (lower panel). Asking whether the enhancement of SHIP
phosphorylation by IV.3 intact IgG and FLI8.26 was a consequence of
FcRIIb-IIa co-clustering, or simply a reflection of these antibodies
clustering more FcRIIa receptors, we employed a supplementary
approach clustering IIa in cells expressing no IIb. For this we used
the IIA1.6 mouse B cells that lack FcRIIb but have been stably
transfected to express human FcRIIa. These cells were activated by
methods described above and the resultant SHIP phosphorylation was
analyzed by immunoblotting with anti-phosphotyrosine antibody. As seen
in Fig. 3B, upper panel, in the absence of FcRIIb, SHIP
phosphorylation induced by all three antibodies was equivalent. These
results strongly suggest that the enhancement of SHIP phosphorylation
observed in THP-1 cells activated with IV.3 intact IgG or FLI8.26 is
not a consequence of differences in the capacities of the
anti-FcRIIa antibodies to cluster FcRIIa, but rather is likely
the consequence of recruiting FcRIIb into a complex with
FcRIIa.
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Fig. 3.
Co-clustering Fc RIIb
with FcRIIa leads to an enhanced SHIP
phosphorylation, SHIP-Shc association and phosphorylation of 120- and
60-65-kDa proteins. A, 107 THP-1 cells per
sample were activated for 3 min by clustering either FcRIIa alone
with IV.3 Fab + GAM or co-clustering FcRIIa and FcRIIb with
either IV.3 intact + GAM or FLI8.26 + GAM. 1 µg of primary antibody
was used in lanes 3-5, whereas only 0.3 µg of IV.3 Fab
was used in lane 2 as a molar equivalent of the antibody
used in lanes 4 and 5. SHIP was
immunoprecipitated from detergent lysates of unstimulated
(R) and activated cells, and immunoblotted with
anti-phosphotyrosine antibody (upper panel). The membrane
was subsequently re-probed with anti-SHIP antibody to ensure equal
loading in all lanes. The last lane is an
immunoprecipitation with normal rabbit Ig. B, IIA1.6 cells
stably transfected to express human FcRIIa were activated as in
A by receptor clustering with antibodies indicated in the
figure. SHIP proteins were immunoprecipitated with anti-SHIP antibody,
and immunoblotted with anti-phosphotyrosine antibody (upper
panel). The same membrane was reprobed with anti-SHIP antibody
(lower panel). C, detergent lysates of THP-1
cells activated as indicated above were subjected to
immunoprecipitation with anti-Shc antibody and analyzed by
immunoblotting with anti-phosphotyrosine antibody (upper
panel) followed by a re-probe with anti-Shc antibody (lower
panel). D, whole cell lysates (WCL) from
106 THP-1 cells activated for 1 and 3 min as above were
separated by SDS-PAGE and immunoblotted with anti-phosphotyrosine
antibody. Arrowhead indicates the proteins that are
maximally phosphorylated under conditions that co-cluster FcRIIa
with FcRIIb. These figures are representative of four independent
experiments.
RIIb-IIa would also enhance
Shc phosphorylation (25) and SHIP-Shc association (24, 29), as reported
in other cell systems following antigen receptor co-clustering with
FcRIIb. Here, we immunoprecipitated Shc from lysates of THP-1 cells
activated as described above and probed the membrane with
anti-phosphotyrosine antibody. Results indicated that both Shc
phosphorylation and SHIP-Shc association are indeed enhanced under
conditions of FcRIIa-IIb co-clustering (Fig. 3C, upper
panel, lanes 3 and 4). The same membrane was
reprobed with anti-Shc antibody to ensure equal loading of Shc in all
lanes (lower panel). Interestingly, an anti-phosphotyrosine
blot of whole cell lysates from THP-1 cells activated as above, by
clustering either FcRIIa alone or by co-clustering FcRIIa with
IIb, revealed additional molecules that displayed enhanced
phosphorylation under conditions of co-clustering (Fig. 3D).
Specifically, proteins in the molecular weight range of 120,000 and
60,000-65,000 were apparently phosphorylated more efficiently by
FcRIIb-IIa co-clustering than by FcRIIa clustering alone.
Although the identity of these proteins is not known, we speculate that
the 120-kDa protein is probably Cb1. The band seen around 60-65 kDa
could represent the protein-tyrosine phosphatase SHP-1 and/or the
RasGAP-binding protein p62dok. All of the above proteins serve
inhibitory roles in other cell types (30-32). Studies are underway to
determine the identity of these proteins.
RIIb with FcRIIa Down-regulates Akt
Phosphorylation and Concomitantly Increases SHIP
Phosphorylation--
We next examined the influence of FcRIIb-IIa
co-clustering on the activation of the Akt. Akt is a serine/threonine
kinase that serves to protect cells from apoptosis. Activation of Akt requires the binding of the plextrin homology domain of Akt to PtdIns(3,4,5)P3 and the phosphorylation of Akt on
serine/threonine residues (33). Recent studies indicated that
hydrolysis of PtdIns(3,4,5)P3 by SHIP attenuates Akt
activation in B cells (14, 15). Since co-clustering FcRIIa-IIb
correlates with SHIP activation in human monocytes we undertook to
determine whether Akt activation was down-regulated under the same
conditions. To do this we first examined the ability of FcRIIa to
activate Akt. Activation of Akt by FcRIIa clustering in neutrophils,
but not monocytes, has been previously reported (34). Thus, THP-1 cells
were activated for various time periods as indicated in Fig.
4A, and whole cell lysates
were probed with anti-phospho-Akt antibodies (upper panel). The relative intensities of the Akt bands in the several lanes indicated that FcRIIa clustering induced phosphorylation of Akt as
early as 1 min and that this activation peaked at 5 min and began to
decline after 10 min. Based on the results of this experiment, we
modified our protocol to include co-clustering of FcRIIa and FcRIIb. We activated THP-1 cells for 3 min by clustering either FcRIIa alone with IV.3 Fab and GAM, or by co-clustering
FcRIIb-IIa with IV.3 intact IgG and GAM or FLI8.26 and GAM. Akt
phosphorylation in THP-1 cells thus treated was assessed by probing
whole cell lysates with anti-pAkt antibody. In a parallel experiment
the concomitant SHIP phosphorylation was analyzed by immunoblotting anti-SHIP immunoprecipitates with anti-phosphotyrosine antibody. Phosphorylation levels of Akt and SHIP, quantified by laser
densitometry, are illustrated graphically as fold increases over that
observed in unstimulated cells (Fig. 4B). Remarkably,
phosphorylation of Akt declined under conditions of FcRIIb-IIa
co-clustering while phosphorylation of SHIP increased under the same
conditions.
View larger version (17K):
[in a new window]
Fig. 4.
Co-clustering Fc RIIb
with FcRIIa results in a decrease in Akt
phosphorylation with a concomitant increase in SHIP
phosphorylation. A, THP-1 cells were activated by
clustering FcRIIa with IV.3 Fab + GAM. Whole cell lysates were
probed anti-pAkt antibodies to detect phosphorylated Akt (upper
panel). The same membrane was reprobed with anti-Akt antibody
(middle panel). Akt band intensities were quantitated by
laser densitometry, and phosphorylation levels were expressed as
increase over the unstimulated sample (lower panel).
B, THP-1 cells were activated for 3 min by clustering
FcRIIa receptors alone with IV.3 Fab + GAM, or FcRIIa-IIb were
co-clustered with either IV.3 intact IgG + GAM or with FLI8.26 + GAM.
Akt phosphorylation was measured as described above. SHIP
phosphorylation in the same samples was assessed by
anti-phosphotyrosine immunoblots of SHIP immunoprecipitates.
Phosphorylation levels are expressed as increase over the resting
samples. The graph represents the mean and standard
deviation of four separate experiments.
RIIb Expression Decreases Phagocytic
Efficiency in THP-1 Cells--
FcRIIb is reported to attenuate
phagocytic efficiency of murine macrophages (1). To determine whether
FcRIIb might similarly decrease the ability of human macrophages to
phagocytose IgG-opsonized particles, we tested the FcR-mediated
phagocytic capacity of monocytic cells cultured with and without IL-4.
THP-1 cells were first cultured in IL-4 for 24 h. Expression of
FcRIIb in these cells was up-regulated as confirmed by
immunoblotting, while no significant effect was seen in the expression
of FcRIIa (Fig. 5B). We
then measured the ability of THP-1 cells, cultured in the presence or
absence of IL-4, to bind and phagocytose fluoresceinated IgG-opsonized
sheep red blood cells (EA) by methods described previously by our
laboratory (18). Results indicated that the overall percent of THP-1
cells that bound 3 or more SRBC (rosetting activitiy) were equivalent,
regardless of whether the cells were cultured in IL-4 (Fig. 5A,
top panel). However, the THP-1 cells that were cultured in IL-4
consistently bound a greater number of RBC (adherence index) than the
THP-1 cells that were cultured without IL-4, consistent with the
up-regulated expression of FcRIIb in these cells (middle
panel). In contrast, the phagocytic capacity of THP-1 cells
cultured in the presence of IL-4 was diminished by 40% in comparison
to the THP-1 cells that were not cultured with IL-4. These results
suggest an inhibitory role for FcRIIb.
View larger version (21K):
[in a new window]
Fig. 5.
IL-4 treatment of THP-1 cells decreases
phagocytic efficiency while increasing the expression of
Fc RIIb receptors. A, THP-1
cells cultured in the presence or absence of IL-4 were examined for
rosetting activity, i.e. the number of THP-1 cells binding 3 or more IgG-coated SRBC (upper panel; adherence index,
i.e. the total number of SRBC bound to 100 THP-1 cells
(middle panel), and their ability to phagocytose IgG-coated
SRBC (lower panel)). The graph represents the
mean of two independent experiments; error bars indicate
deviation from the mean. B, THP-1 cells cultured in the
presence or absence of IL-4 were analyzed for the expression of
FcRIIb by immunoblotting anti-FcRII immunoprecipitates with Ab163
(upper panel) and for FcRIIa with Ab260 (lower
panel). C, THP-1 cells cultured with or without IL-4
were analyzed for phagocytosis via FcRIIa alone (marked as IV.3
Fab)or via FcRIIa-IIb (marked as FLI8). The graph
represents the mean of two independent experiments; error
bars indicate deviation from the mean.
RIIb, we measured phagocytosis via either FcRIIa
alone or via FcRIIa and FcRIIb as described under "Experimental
Procedures." Fig. 5C is an average of two independent
experiments, each time analyzing 200 cells. The results indicate that
phagocytosis via FcRIIa is unaffected by IL-4 treatment (88 ± 4 SRBC ingested by non treated THP-1 cells and 85 ± 3 SRBC ingested by IL-4-treated THP-1). In contrast, phagocytosis via FcRIIa-IIb is diminished by about 42% in cells treated with IL-4 when compared with cells that were not cultured in IL-4 (58 ± 3 SRBC ingested by nontreated THP-1 and 34 ± 2 by IL-4-treated THP-1). These results strongly support the notion that the diminished phagocytic efficiency of THP-1 cells cultured in IL-4 is directly due
to the up-regulation of the expression of FcRIIb on these cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RIIb is expressed in human monocytic cells
and that it serves to down-regulate immune complex-mediated activation
of monocytic cell function. Our data also indicate that the expression
of FcRIIb in human monocytic cells is highly regulated. Taken
together these observations suggest that FcRIIb serves as a
modulator of monocyte response such that the level of expression of
FcRIIb is inversely proportional to the magnitude of the response.
The identification of this receptor reveals a regulatory mechanism that
has thus far not been demonstrated in human monocytic cells.
RIIb in B cells (11, 23) and mast cells (35),
where it is the only FcR expressed, has been extensively studied.
However, in monocytic cells the presence of multiple FcR has
prevented the functional analysis of FcRIIb in isolation. In human
monocytes the expression of FcRIIa whose extracellular and
transmembrane domains are virtually identical to FcRIIb has further
complicated the study of this receptor. In this study we took advantage
of a novel FcRIIb-specific antibody, Ab163, to first identify the
receptor in human monocytic cells. Earlier work from our laboratory
reported the inability to detect FcRIIb in U937 cells using the only
available FcRIIb-specific mAb, II8D2 (36). With the use of Ab163 we
demonstrate here the presence of FcRIIb in PBMs as well as in the
monocyte-like cell lines U937 and THP-1. It is noteworthy, however,
that the detection of this receptor required the use of detergent
lysates from four times the number of cells used to detect FcRIIa,
suggesting that FcRIIb is present in very low levels in these cells.
RIIa alone
or share a common epitope on FcRIIa and IIb, we have further
characterized the function of this receptor in FcR-mediated signaling. Fab fragments of mAb IV.3 were used to specifically cluster
FcRIIa alone. To co-cluster FcRIIa with IIb we employed two
approaches: first, IV.3 intact IgG was chosen since it could recruit
FcRIIb by a ligand interaction owing to the fact that this antibody
is of the murine IgG2b isotype, which has a measurable affinity for the
otherwise low affinity FcRIIb receptor (27). It is unlikely that the
IV.3 intact IgG could have served as a ligand for FcRI since this
receptor has very low affinity for mIgG2b (37). As a second approach,
we used mAb FLI8.26, which is also of the murine IgG2b isotype, and
recognizes both FcRIIa and IIb equally well as antigen. The latter
method of co-clustering was more effective at inducing negative
signaling events in monocytic cells. That IV.3 intact IgG and FLI8.26
do indeed recruit FcRIIb is demonstrated in that receptor clustering
with these antibodies in cells expressing FcRIIa but not IIb
(IIA1.6+IIa) did not induce enhanced SHIP phosphorylation. Furthermore,
activation of THP-1 cells with IV.3 intact or FLI8.26 led to decreased
Akt phosphorylation while concomitantly leading to an enhancement of
SHIP phosphorylation under the same conditions of activation. Maximal
SHIP phosphorylation and inhibition of Akt phosphorylation was achieved
with FLI8.26, confirming that the use of this antibody is a more
effective way to co-cluster FcRIIa-IIb, in comparison to
co-clustering with IV.3 intact IgG. Thus these experiments provide
compelling evidence that in monocytic cells FcRIIb functions to
down-regulate ITAM-FcR mediated signaling events.
RIIb1, b2, or by both. Although the b1 and
b2 forms of FcRIIb differ in that b1 has a 19-amino acid insertion
in its cytoplasmic tail, they both express the ITIM and are, therefore,
likely to function in a similar manner with respect to the induction of
SHIP activation and other associated negative signaling events.
However, this point requires formal testing.
R-mediated phagocytosis. Our
results indicated that THP-1 cells cultured in the presence of IL-4
display enhanced expression of FcRIIb with an associated decrease in
phagocytic efficiency. We suggest that the reduced phagocytic
efficiency is not the result of a decrease in the expression of
ITAM-FcR in the THP-1 cells cultured with IL-4 since in our hands
there was no detectable decrease in the expression of FcRIIa or
FcRI. To determine whether the IL-4-mediated increase in the negative regulation of FcR activation was directly due to the up-regulation of FcRIIb, we analyzed the phagocytosis via FcRIIa alone or via FcRIIa-IIb in THP-1 cells cultured in the presence or
absence of IL-4. As seen in Fig. 5C phagocytosis by
FcRIIa clustering is not significantly different in cells cultured
with or without IL-4, indicating that IL-4 has no effect on FcRIIa signaling. In contrast, the inhibition of phagocytosis by FcRIIa-IIb co-clustering is enhanced in cells cultured with IL-4. We interpret these results to indicate that the enhanced inhibition of
FcR-mediated activation in cells cultured with IL-4 is directly due
to the up-regulation of FcRIIb.
RIIb appears to be very highly regulated not
only by the presence of inflammatory cytokines, such as IL-4, but also
by culture conditions, such as density of cell culture and passage
number of the culture. Thus, in our hands, expression of FcRIIb
increased as cell density was increased. Likewise, Fc RIIb
expression was also up-regulated in later passages of the cell culture
(data not shown). Studies to identify specific factors in the culture
medium that relate to modulation of FcRIIb expression are currently
in progress.
RIIb is of considerable importance based on the recent observations of Clynes et al. (38) analyzing the influence
of FcR on the efficacy of therapeutic anti-tumor antibodies. Using mice that were genetically engineered to be deficient in the expression of either the FcR -chain or FcRIIb they elegantly demonstrated that the presence of FcRIIb down-regulates the efficacy of the therapeutic antibodies. These observations suggest that the ratio of
ITAM-FcR to ITIM-FcR is critical to the magnitude of any IgG-mediated immune response. Identification of factors that influence the expression of these receptors can, therefore, potentially allow us
to vary the levels of Fc receptor expression to achieve the desired
immune response.
RIIb in human monocytes were reported while our manuscript was in
review (41). These studies, however, in contrast to our observations
were unable to detect the expression of FcRIIb in U937 cells.
ACKNOWLEDGEMENTS
FOOTNOTES
Fellow of the Leukemia and Lymphoma Society (formerly Leukemia
Society of America).
ABBREVIATIONS
R, Fc receptor
for IgG;
ITIM, immunoreceptor tyrosine-based inhibition motif;
ITAM, immunoreceptor tyrosine-based activation motif;
SH2, Src homology
domain 2;
PI 3-kinase, phosphatidylinositol 3-kinase;
PBM, peripheral
blood monocyte;
SHIP, SH2 domain-containing inositol phosphatase;
SHP-1, SH2 domain-containing protein tyrosine phosphatase;
SRBC, sheep
red blood cell;
IL, interleukin;
mAb, monoclonal antibody;
PBS, phosphate-buffered saline;
PBMC, peripheral blood monocyte cells.
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 T. Avril, S. D. Freeman, H. Attrill, R. G. Clarke, and P. R. Crocker Siglec-5 (CD170) Can Mediate Inhibitory Signaling in the Absence of Immunoreceptor Tyrosine-based Inhibitory Motif Phosphorylation J. Biol. Chem., May 20, 2005; 280(20): 19843 - 19851. [Abstract] [Full Text] [PDF]
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Y. Liu, E. Masuda, M. C. Blank, K. A. Kirou, X. Gao, M.-S. Park, and L. Pricop Cytokine-mediated regulation of activating and inhibitory Fc{gamma} receptors in human monocytes J. Leukoc. Biol., May 1, 2005; 77(5): 767 - 776. [Abstract] [Full Text] [PDF]
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J. N. Samsom, L. A. van Berkel, J. M. L. M. van Helvoort, W. W. J. Unger, W. Jansen, T. Thepen, R. E. Mebius, S. S. Verbeek, and G. Kraal Fc{gamma}RIIB Regulates Nasal and Oral Tolerance: A Role for Dendritic Cells J. Immunol., May 1, 2005; 174(9): 5279 - 5287. [Abstract] [Full Text] [PDF]
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L. P. Ganesan, G. Wei, R. A. Pengal, L. Moldovan, N. Moldovan, M. C. Ostrowski, and S. Tridandapani The Serine/Threonine Kinase Akt Promotes Fc{gamma} Receptor-mediated Phagocytosis in Murine Macrophages through the Activation of p70S6 Kinase J. Biol. Chem., December 24, 2004; 279(52): 54416 - 54425. [Abstract] [Full Text] [PDF]
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K. Su, J. Wu, J. C. Edberg, X. Li, P. Ferguson, G. S. Cooper, C. D. Langefeld, and R. P. Kimberly A Promoter Haplotype of the Immunoreceptor Tyrosine-Based Inhibitory Motif-Bearing Fc{gamma}RIIb Alters Receptor Expression and Associates with Autoimmunity. I. Regulatory FCGR2B Polymorphisms and Their Association with Systemic Lupus Erythematosus J. Immunol., June 1, 2004; 172(11): 7186 - 7191. [Abstract] [Full Text] [PDF]
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L. P. Ganesan, H. Fang, C. B. Marsh, and S. Tridandapani The Protein-tyrosine Phosphatase SHP-1 Associates with the Phosphorylated Immunoreceptor Tyrosine-based Activation Motif of Fc{gamma}RIIa to Modulate Signaling Events in Myeloid Cells J. Biol. Chem., September 12, 2003; 278(37): 35710 - 35717. [Abstract] [Full Text] [PDF]
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Z.-Y. Huang, S. Hunter, M.-K. Kim, Z. K. Indik, and A. D. Schreiber The effect of phosphatases SHP-1 and SHIP-1 on signaling by the ITIM- and ITAM-containing Fc{gamma} receptors Fc{gamma}RIIB and Fc{gamma}RIIA J. Leukoc. Biol., June 1, 2003; 73(6): 823 - 829. [Abstract] [Full Text] [PDF]
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C. Chaudhury, S. Mehnaz, J. M. Robinson, W. L. Hayton, D. K. Pearl, D. C. Roopenian, and C. L. Anderson The Major Histocompatibility Complex-related Fc Receptor for IgG (FcRn) Binds Albumin and Prolongs Its Lifespan J. Exp. Med., February 3, 2003; 197(3): 315 - 322. [Abstract] [Full Text] [PDF]
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E. Garcia-Garcia and C. Rosales Signal transduction during Fc receptor-mediated phagocytosis J. Leukoc. Biol., December 1, 2002; 72(6): 1092 - 1108. [Abstract] [Full Text] [PDF]
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S. Tridandapani, Y. Wang, C. B. Marsh, and C. L. Anderson Src Homology 2 Domain-Containing Inositol Polyphosphate Phosphatase Regulates NF-{kappa}B-Mediated Gene Transcription by Phagocytic Fc{gamma}Rs in Human Myeloid Cells J. Immunol., October 15, 2002; 169(8): 4370 - 4378. [Abstract] [Full Text] [PDF]
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