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Published online before print May 22, 2003
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receptors FcRIIB and FcRIIA
University of Pennsylvania School of Medicine, Philadelphia
Correspondence: Dr. Alan D. Schreiber, University of Pennsylvania School of Medicine, Hematology/Oncology Division, Biomedical Research Building II/III, Room 705, 421 Curie Blvd., Philadelphia, PA 19104. E-mail: schreibr{at}mail.med.upenn.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RIIB, in B cells, mast cells, and monocytes. Here, we propose a role for the Src homology 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1) in FcRIIB-mediated inhibition of FcR signaling. Coexpression of SHP-1 enhances FcRIIB-mediated inhibition of FcRIIA phagocytosis in COS-1 cells. SHP-1 also enhances the reduction in FcRIIA tyrosine phosphorylation that accompanies this inhibition. Significantly, tyrosine phosphorylation of Syk kinase is substantially inhibited by SHP-1. Furthermore, the activation of SHP-1 tyrosine phosphorylation is observed following stimulation of FcRII in COS-1 cells and in human monocytes. The SH2 domain containing inositol phosphatase (SHIP), SHIP-1 also enhances FcRIIB-mediated inhibition of FcRIIA, indicating that FcRIIB can use more than one pathway for its inhibitory action. In addition, SHP-1 and SHIP-1 can inhibit FcRIIA phagocytosis and signal transduction in the absence of FcRIIB. The data support emerging evidence that SH2-containing phosphatases, such as SHP-1 and SHIP-1, can modulate signaling by "activating" receptors.
Key Words: phagocytosis • tyrosine phosphorylation • inhibition
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R) family, the phagocytosis of immunoglobulin (IgG)-coated particles, the endocytosis of immune complexes, and the release of inflammatory mediators.
Inhibitory receptors, which negatively regulate signal-transduction events, have been identified in a wide variety of cell types [4
, 5
]. They include the FcR, FcRIIB, expressed in B cells, mast cells, and monocytes; killer cell Ig-like receptors (KIR), expressed in natural killer and T cells; and the paired Ig-like receptor PiR-B, expressed in mast and B cells [6
7
8
9
]. These inhibitory receptors have one or more tyrosine-containing immunoreceptor tyrosine-based inhibition motif (ITIM) sequences in their cytoplasmic domains. The ITIM resembles the ITAM in that it contains a tyrosine/leucine motif (YXXL/I). However, in inhibitory receptors, a single YXXL sequence is embedded within a 13 amino acid sequence that typically contains a hydrophobic residue at the -2 position [10
].
Coaggregation of ITAM- and ITIM-containing receptors by an extracellular ligand is required to trigger ITIM-mediated inhibition of cellular signaling responses. Following coaggregation, the cytoplasmic ITIMs undergo tyrosine phosphorylation and may recruit SH2-containing proteins. The inhibitory action of ITIM-containing receptors has been linked to the recruitment of proteins such as the SH2-containing tyrosine phosphatases (SHP) SHP-1 and SHP–2 and the SH2 domain containing inositol phosphatases (SHIP) SHIP-1 and SHIP-2 [11 ].
We have demonstrated that FcRIIB can inhibit FcR-mediated phagocytic pathways [12
]. In COS-1 cells, coligation of human FcRIIA and FcRIIB results in a dramatic reduction in the efficiency of FcRIIA-mediated phagocytosis. The inhibitory action of FcRIIB on FcRIIA-mediated signaling is of interest, considering the homology of the extracellular domains of these two FcRII receptors [13
]. Both receptors bind the Fc portion of most IgGs and therefore are stimulated by the same ligands. Thus, in cells in which FcRIIA and FcRIIB are coexpressed, such as human monocytes and macrophages [6
], stimulation by IgG complexes could potentially elicit both positive and negative responses.
The inhibitory action of FcRIIB in B cells, mast cells, and monocytes has been linked to the activation of the inositol phosphatase SHIP-1 [14
]. However, our observation that the reduction in phagocytic efficiency following coclustering of FcRIIA and FcRIIB in COS-1 cells is accompanied by a dramatic reduction in the tyrosine phosphorylation of FcRIIA suggested a role for a tyrosine phosphatase such as SHP-1 in modulating the FcRIIA phagocytic response [12
]. A recent report that overexpression of SHP-1 in a macrophage cell line reduces phagocytic efficiency supports this theory [15
]. Since tyrosine phosphorylation of FcRIIA is a crucial, early activation step in the FcRIIA-mediated signaling pathway [1
], we have now examined the ability of SHP-1 to inhibit FcRIIA-mediated phagocytosis. Our present studies indicate that both tyrosine (SHP-1) and inositol (SHIP-1) phosphatases enhance FcRIIB-mediated inhibition of phagocytosis by FcRIIA and that SHP-1 enhances the inhibition of FcRIIA tyrosine phosphorylation. We also observed that overexpression of SHP-1 has a dramatic effect on the tyrosine phosphorylation of Syk kinase, a crucial downstream activator of FcRIIA signaling. In addition, tyrosine phosphorylation of SHP-1 is enhanced following stimulation of FcRIIA and FcRIIB in COS-1 cells as well as in peripheral blood monocytes.
It is interesting that even in the absence of the inhibitory FcRIIB receptor, overexpression of SHP-1 or SHIP-1 in COS-1 cells significantly inhibits FcRIIA-mediated phagocytosis, and that SHP-1 also inhibits FcRIIA tyrosine phosphorylation. In addition, we demonstrate that FcRIIA itself can activate SHP-1 in the absence of an inhibitory receptor. These observations support the thesis that the tyrosine and inositol phosphatases SHP-1 and SHIP-1 have the ability to modulate ITAM-containing receptors in the absence of ITIM-containing inhibitory receptors [14
, 15
].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RII mAb (IV.3) was from Medarex (Princeton, NJ), and the goat affinity-purified F(ab')2 fragment to mouse IgG was purchased from ICN Biomedicals (Aurora, OH). Rabbit anti-sheep red blood cell (RBC) antibody was from Cappel Laboratories (West Chester, PA). All cDNAs were subcloned into the mammalian expression vector pcDNA3.1. Human FcRIIA was tagged with the Myc/histidine epitope. FcRIIB was tagged with the HA epitope. SHIP-1 cDNA was also tagged with the HA epitope. Dr. Benjamin Neel (Harvard Institute of Medicine, Boston, MA) kindly provided the pcDNA3.1/SHP-1 plasmid. The phosphatase-negative mutant of SHP-1, which lacks the phosphatase domain (residues 220–595), was generated by polymerase chain reaction and inserted into pcDNA3.
Cell culture and transfection
COS-1 cells were maintained in Dulbecco’s modified Eagle’s medium containing glucose (4.5 mg/ml), glutamine (2 mmol/L), streptomycin (100 U/ml), penicillin (100 mg/ml), and 10% heat-inactivated fetal calf serum (FCS). Transient transfection of cells at 70–80% confluence was performed in complete media containing diethylaminoethyl–dextran (0.6 mg/ml), chloroquine chloride (100 mmol/L), and 4 µg plasmid DNA per milliliter of transfection media. After 4 h at 37°C, the transfection medium was replaced with 10% dimethyl sulfoxide in phosphate-buffered saline (PBS) for 90 s at room temperature. The cells were then washed, overlaid with fresh media for further incubation, and analyzed after 48 h. The expression of transfected receptors was analyzed by flow cytometry and/or by immunoblotting aliquots of cell lysates.
Flow cytometry
Cell samples incubated with anti-FcRII mAb IV.3 for 30 min at 4°C were washed, labeled with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (G
M) F(ab')2 (Tago, Inc., Burlingame, CA) for 30 min at 4°C, and then washed and fixed with 4% paraformaldehyde. Isotype controls were used for all reactions, and fluorescence was measured on a Becton Dickinson FACScan (Mansfield, MA). For all samples, 5000 events were recorded on a logarithmic fluorescence scale.
Immunoprecipitation and Western blotting
Transfected cells were lysed directly on culture plates with Brij 96 lysis buffer (1% Brij 96, 5 mmol/L Hepes–KOH, pH 7.4) in the presence of the protease and phosphatase inhibitors, 5 mM EGTA, 3 mM Na orthovanadate, 2 mM phenylmethylsulfonyl fluoride, and 10 mg/L aprotinin and leupeptin (Sigma Chemical Co., St. Louis, MO). Following centrifugation at 12,000 rpm for 30 min at 4°C, the lysates were precleared by incubation with protein G or A plus agarose (Santa Cruz Biotechnology) and then incubated overnight at 4°C with appropriate antibodies. Immune complexes were bound to protein G or A plus agarose in lysis buffer. Pellets were washed three times in lysis buffer and adsorbed proteins, eluted into reducing or nonreducing sample buffer, and resolved on 7.5% or 10% sodium dodecyl sulfate-polyacrylamide gels. Following electrophoretic transfer to nitrocellulose, proteins were immunoblotted with appropriate blotting antibodies. Blots were developed with horseradish peroxidase-conjugated GM or goat anti-rabbit IgG (Santa Cruz Biotechnology). The specific bands were detected by enhanced chemiluminescence (Amersham, Arlington Heights, IL).
Preparation of antibody-sensitized sheep erythrocytes and analysis of phagocytosis
Antibody-sensitized sheep erythrocytes (EA) were prepared in magnesium- and calcium-free PBS by incubating 109/ml sheep RBCs (Rockland, Gilbertsville, PA) with an equal volume of the highest subagglutinating concentration of rabbit anti-sheep RBC antibody (Cappel Laboratories). COS-1 cells were incubated with EA at 37°C for 30 min, and unbound EA were removed by washing with PBS. To assess phagocytosis, externally bound RBCs were removed by brief hypotonic shock. The cells were then stained with Wright’s–Giemsa, and the number of COS-1 cells with one or more internalized EA was determined in a blinded manner. The phagocytic index (PI) is calculated as the number of ingested RBCs/100 cells and is adjusted to reflect expression of the transfected DNA determined by flow cytometry.
Monocyte isolation and culture
Peripheral blood mononuclear cells from healthy individuals were isolated as described previously [16
]. Briefly, the heparinized blood was centrifuged on Ficoll-Hypaque (Lymphocyte Separation Medium; Organon Teknika, Durham, NC), and interface cells were washed twice in PBS. Mononuclear cells were resuspended in complete medium containing RPMI 1640 (Life Technologies, Grand Island, NY) with 10% heat-inactivated FCS and 2 mM L-glutamine. Cells were allowed to adhere at 37°C onto tissue-culture flasks precoated with FCS. After 45–60 min, nonadherent cells were removed by extensive washing in PBS. Cells were harvested by vigorous agitation. Isolated monocytes were maintained in RPMI 1640 supplemented with L-glutamine (2 mM) and 10% heat-inactivated FCS at 37°C in 5% CO2.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RIIB-mediated inhibition of phagocytosis by FcRIIARIIB is a nonphagocytic member of the FcR family expressed on a variety of cells of the immune system including monocytes [6
]. It has been classified as an inhibitory receptor as a result of the presence of the ITIM sequence in its cytoplasmic domain, and it has been shown to attenuate immune responses in B cells, mast cells, and monocytes following coaggregation with ITAM-containing immunoreceptors [5
]. In a previous study, we demonstrated that FcRIIB inhibits FcRIIA-mediated phagocytosis of IgG-coated EA by up to 50% in transfected COS-1 cells [12
]. To determine whether the phosphatases SHP-1 and SHIP-1 augment the inhibitory effect of FcRIIB on FcRIIA function, we examined FcRIIA-mediated phagocytosis in COS-1 cells cotransfected with the FcRII receptors and these phosphatases.
As we previously reported, treatment with EA coaggregates FcRIIB and FcRIIA and reduces FcRIIA-mediated phagocytosis by 
50% (Table 1 ) [12
]. In cells cotransfected with SHP-1 or SHIP-1, the inhibitory effect of FcRIIB increases from 50% to 85%. The reduction in phagocytosis was not a result of differences in the levels of FcRIIA expression, since Western blot analysis determined that receptor expression within each experiment was constant (not shown). Although a role for the inositol phosphatase SHIP-1 in signaling by the inhibitory FcRIIB receptor has been described in B cells and in monocytes [17
18
19
20
], we were interested in investigating whether SHP-1 plays a role in FcRIIB-mediated inhibition of FcR signaling in monocytes. The enhancement of inhibition observed when SHP-1 was coexpressed with FcRIIB suggests that this tyrosine phosphatase interacts with FcRIIB to modulate FcRIIA-mediated phagocytosis.
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RIIA-mediated phagocytic signaling even in the absence of FcRIIB (Table 1)
. Coexpression of either phosphatase with FcRIIA alone decreased FcRIIA-mediated phagocytosis by >50%. We determined by flow cytometry and by Western blot that the decrease in phagocytosis was not a result of a decrease in the cell-surface expression or level of FcRIIA protein (not shown). This observation suggests that both SHP-1 and SHIP-1 can interact with the FcRIIA signaling process directly, and although the effect on phagocytosis was less potent than that of FcRIIB and the phosphatases combined, the phosphatases do not necessarily require an ITIM-containing receptor to be recruited to the signaling machinery following stimulation of an "activating" receptor.
The effect of SHP-1 on tyrosine phosphorylation of FcRIIA
Tyrosine phosphorylation of the FcRIIA ITAM is an important and necessary step in signal-transduction pathways stemming from the stimulation of FcRIIA and is required to mediate the phagocytic signal [1
]. Our previous studies indicated that inhibition of FcRIIA-mediated phagocytosis by FcRIIB is accompanied by a decrease in tyrosine phosphorylation of the FcRIIA receptor itself in transfected COS-1 cells [12
]. Coexpression of the tyrosine phosphatase SHP-1 with FcRIIA and FcRIIB results in further reduction of FcRIIA tyrosine phosphorylation in transfected cells incubated with EA (Fig. 1
). The observation that overexpression of SHP-1 enhances the inhibition of FcRIIA tyrosine phosphorylation by FcRIIB suggests that SHP-1 can be recruited by the FcRIIA/FcRIIB receptor complex in these cells, resulting in the dephosphorylation of FcRIIA.
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RIIA in the absence of FcRIIB expression (Fig. 1
, compare lanes 1 and 4). Although the decrease in tyrosine phosphorylation of FcRIIA is not as dramatic as that observed when FcRIIB is also present (lane 3), these data suggest that the activating FcRIIA receptor itself interacts with SHP-1 and has the potential to modulate its own signaling pathways. The interaction of FcRIIA and SHP-1 that results in decreased tyrosine phosphorylation of FcRIIA may involve direct interaction of the SHP-1 SH2 domains with the ITAM itself or may involve an intermediary substrate in the receptor complex, such as a Src-related tyrosine kinase (SRTK) or Syk, a key molecule in the FcRIIA-mediated pathway leading to phagocytosis.
In contrast to SHP-1, the inositol phosphatase SHIP-1 (although dramatically reducing FcRIIA-mediated phagocytosis) had no effect on FcRIIA tyrosine phosphorylation (not shown). This contrast highlights the distinct pathways that these tyrosine and inositol phosphatases use to attenuate signaling cascades.
The effect of the tyrosine phosphatase SHP-1 on the activation of Syk kinase
Syk kinase plays an essential role in the activation pathway leading to FcR-mediated phagocytosis in monocytes, and overexpression of Syk enhances FcR-mediated phagocytosis in transfected epithelial cells [1
]. Having demonstrated that SHP-1 expression results in a decrease in FcRIIA-mediated phagocytosis and FcRIIA tyrosine phosphorylation, we examined whether overexpression of SHP-1 also affects the activation of Syk. Cross-linking FcRIIA with anti-FcRII mAb IV.3 (whole IgG) and GM F(ab')2 in COS-1 cells expressing FcRIIA and Syk kinase produced a dramatic increase in the tyrosine phosphorylation of Syk (1 and Fig. 2
, top, lane 1 vs. 5). FcRII cross-linking in cells coexpressing FcRIIB with FcRIIA and Syk did not noticeably affect the tyrosine phosphorylation of Syk (Fig. 2
, top, lane 4). However, overexpression of SHP-1 in the presence and absence of FcRIIB (Fig. 2
, top, lanes 2 and 3) reduced Syk tyrosine phosphorylation to a level comparable with that in unstimulated cells (Fig. 2
, top, lane 1). We also observed that SHP-1 coimmunoprecipitates with Syk (Fig. 2
, middle, lanes 2 and 3).
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R stimulation of COS-1 cells expressing FcRIIA, FcRIIB, SHP-1, and Syk (Fig. 3A
, lane 4). SHP-1 also decreased the tyrosine phosphorylation of Syk in the absence of FcRIIB expression (Fig. 3A
, lane 5), and phosphatase-negative SHP-1 also had no effect on Syk tyrosine phosphorylation in the absence of FcRIIB expression (Fig. 3A
, lane 6). In similar experiments, coexpression of the inositol phosphatase SHIP-1 had no effect on the tyrosine phosphorylation of Syk kinase (Fig. 3B
, lane 4).
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R cross-linking on the tyrosine phosphorylation of SHP-1Rs affects tyrosine phosphorylation (activation) of SHP-1 in transiently transfected COS-1 cells and in human monocytes. COS-1 cells transfected with SHP-1 and only the FcRIIB inhibitory receptor were incubated with anti-FcRII mAb IV.3 (whole IgG) and cross-linked with GM F(ab')2 at 37°C for 1 and 5 min (Fig. 4A
). SHP-1 was strongly phosphorylated on tyrosine following 1 and 5 min stimulation of FcRIIB (lanes 2 and 3). In the absence of FcRIIB stimulation, tyrosine phosphorylation of SHP-1 was not detected (lane 1). This not only demonstrates the ability of FcRIIB to stimulate the tyrosine phosphorylation of SHP-1 but also demonstrates the ability of mAb IV.3 to bind and stimulate FcRIIB. SHP-1 was also tyrosine-phosphorylated following FcRIIA stimulation in cells cotransfected with SHP-1 and only FcRIIA (Fig. 4A
, lanes 5 and 6). Again, in the absence of FcRIIA stimulation with mAb IV.3, there was no tyrosine phosphorylation of SHP-1 (lane 4).
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RIIA, FcRIIB, and SHP-1 (Fig. 4B)
. The FcRs were costimulated with mAb IV.3 (whole IgG) for 1, 5, 15, and 30 min. SHP-1 was heavily tyrosine-phosphorylated after 1 min (Fig. 4B
, lane 4). After 30 min stimulation, tyrosine phosphorylation of SHP-1 diminished (lane 7). In unstimulated cells (cells incubated at 0, 5, or 30 min in the absence of the stimulating mAb IV.3), there was no detectable activation of SHP-1 tyrosine phosphorylation (lanes 1–3). In each case (A and B), the amount of SHP-1 in the immunoprecipitates does not account for the differences in tyrosine phosphorylation of SHP-1.
To determine whether SHP-1 is involved in the inhibitory action of FcRIIB in monocytes/macrophages, we examined SHP-1 tyrosine phosphorylation in human monocytes (Fig. 4C)
, which express FcRIIA and FcRIIB [6
, 19
, 20
]. As we have demonstrated, mAb IV.3 recognizes both of these FcRII receptors (Fig. 4A)
. Following stimulation of FcRII with mAb IV.3 (whole IgG), SHP-1 tyrosine phosphorylation was clearly detectable on immunoblots after 1 min (Fig. 4C
, lane 2).
We have also observed the association of SHP-1 with FcRIIB (Fig. 5
). We immunoprecipitated SHP-1 from COS-1 cell lysates expressing SHP-1 and FcRIIB (Fig. 5)
. Following 1 min of stimulation with mAb IV.3, a substantial amount of FcRIIB coprecipitated with SHP-1 (lane 1). By 30 min stimulation, the amount of FcRIIB associated with SHP-1 had declined (lane 2), and in the absence of FcRIIB stimulation, the FcRIIB associated with SHP-1 was barely detectable (lane 3). In similar experiments, we were unable to detect coprecipitation of FcRIIA with SHP-1 (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RIIB-mediated attenuation of B cell and mast cell responses, and in mouse macrophages, both phosphatases have been associated with the inhibition of FcR phagocytosis [15
, 20
21
22
23
24
]. Following recruitment to the receptor complex at the cell membrane, SHP-1 is thought to inhibit cellular responses by dephosphorylating tyrosines on substrates crucial for signaling. Possible substrates include the ITAM of the activating receptor itself, Syk family kinases, phospholipase C (PLC), and adaptor proteins such as SLP76 [25
, 26
]. Our previous data suggested that recruitment of a tyrosine phosphatase is associated with the suppression of FcRIIA-mediated phagocytosis by FcRIIB. Our present data demonstrate that overexpression of the tyrosine phosphatase SHP-1 in COS-1 cells enhances the ability of FcRIIB to inhibit FcRIIA-mediated phagocytosis and suggest that the tyrosine phosphatase SHP-1 interacts with FcRIIB in vivo (Table 1)
.
The phosphorylated FcRIIB ITIM is capable of binding SHP-1 and SHIP-1 phosphatases [23
, 24
]. However, studies on FcRIIB-mediated modulation of FcR signaling in monocytes and macrophages have focused on the role of the inositol phosphatase SHIP-1. For example, in macrophages from SHIP-1-/- mice, FcR-mediated phagocytosis is enhanced compared with macrophages from SHIP-1+/+ mice [20
], and in human monocytes, coclustering of FcRIIB and FcRIIA or FcRI receptors results in the stimulation of SHIP-1 tyrosine phosphorylation [14
, 19
]. SHIP-1 inhibits signaling pathways through the dephosphorylation of phosphatidylinositol triphosphate (PIP3) [3
, 4
, 5
], a product of the phosphatidylinositol-3 kinase signaling pathway. A consequence of SHIP-1 activation is the attenuation of PIP3-dependent events, e.g., the translocation of Tec family kinases such as Akt and Btk (required for the activation of PLC) to the plasma membrane [27
, 28
]. These findings suggest a role for inositol phosphatases in the FcRIIB-mediated inhibition of FcR signal transduction in monocytic cells. Our data also support a role for SHIP-1 in this inhibitory process, since coexpression of SHIP-1 with FcRIIA and FcRIIB enhances FcRIIB-mediated inhibition of phagocytosis by FcRIIA (Table 1) .
Our data provide evidence that the tyrosine phosphatase SHP-1 is also involved in the FcRIIB-mediated inhibition of FcRIIA signaling in phagocytic cells. Coexpression of SHP-1 enhances the FcRIIB-mediated inhibition of FcRIIA phagocytosis and the FcRIIB-mediated decrease in tyrosine phosphorylation of FcRIIA in transfected COS-1 cells (Table 1
and Fig. 1
). Furthermore, activation of FcRII receptors results in the stimulation of SHP-1 tyrosine phosphorylation in COS-1 cells and in human monocytes (Fig. 4C)
. Both FcRIIA and FcRIIB are expressed in human monocytes [6
, 19
, 29
], and the extent to which SHP-1 interacts with FcRIIB in monocytes is difficult to quantify. (mAb IV.3 is capable of binding to both of these FcRII receptors; unpublished results and Fig. 4A
.) The recent demonstration that FcRIIB expression in monocytes can be regulated by cytokines suggests that the mechanisms used by FcRIIB to down-regulate FcRIIA signal transduction may depend on the relative expression of the two FcRII receptors [19
, 29
]. However, even if the relative expression of FcRIIB is low compared with the expression of FcRIIA, our data clearly demonstrate that the tyrosine phosphatase SHP-1 is activated in human monocytes following FcRII stimulation and is likely to play a role in the inhibition of FcR-mediated signaling in vivo.
Recent studies, using peptides containing the phosphorylated FcRIIB ITIM sequence (pITIM), have suggested that FcRIIB binds more avidly to SHIP-1 than to SHP-1 and consequently, preferentially uses SHIP-1 in vivo [21
]. In these studies, coprecipitation of the FcRIIB pITIM and SHP-1 was detected by immunoblot only if there were a high level of tyrosine phosphorylation of the ITIM peptides (e.g., following in vitro phosphorylation of the ITIM by an SRTK). The binding of SHP-1 to the FcRIIB ITIM was also detectable on immunoblot in pervanadate-treated cells [21
], where phosphatases are inhibited, and tyrosine phosphorylation levels consequently remain relatively high. We have observed coprecipitation of FcRIIB and SHP-1 in our COS-1 cell model (Fig. 5)
. It is possible that the relatively high level of FcRIIB and SHP-1 expression in our transfected COS-1 cells facilitates detection of the interaction of SHP-1 and FcRIIB.
Tyrosine phosphatases such as SHP-1 are recruited to pITIMs via their SH2 domains, and it has been suggested that to be recruited to receptor complexes, tyrosine phosphatases may require both SH2 domains to be engaged [21
]. Such a requirement is consistent with reports that SHP-1 is involved in the inhibitory responses mediated by such receptors as the KIR, PIR-B, and the platelet-endothelial cell adhesion molecule receptor, all of which contain more than one ITIM. Although FcRIIB contains a single ITIM sequence, one possibility is that SHP-1 may be recruited to the FcRIIA/FcIIB receptor complex by interacting with phosphorylated ITIM sequences on two adjacent FcRIIB receptors.
Our data also suggest that phosphatases do not necessarily require an ITIM for their recruitment to activating receptors. This thesis is derived from our observations that SHP-1 and SHIP-1 in the absence of FcRIIB can inhibit FcRIIA-mediated phagocytosis in transfected COS-1 cells (Table 1)
, and that SHP-1 inhibits tyrosine phosphorylation of FcRIIA (Fig. 1)
. Furthermore the phosphatase itself becomes tyrosine-phosphorylated following FcRIIA stimulation in COS-1 cells in the absence of FcRIIB (Fig. 4A , lanes 5 and 6). It is not clear from these studies whether SHP-1, in the absence of an ITIM-containing receptor, interacts directly with FcRIIA or whether SHP-1 is recruited to the receptor complex through an intermediary molecule. We have not observed the coimmunoprecipitation of FcRIIA and SHP-1 in our experiments. Although such an interaction cannot be ruled out (others have detected an association between the inositol phosphatase SHIP-1 and the -chain ITAM sequence in vitro; ref. [30
]), it is possible that a protein such as the Syk kinase may recruit SHP-1 following FcRIIA activation.
Syk kinase is a key activator of the FcRIIA signaling pathway leading to phagocytosis. Our data indicate that tyrosine phosphorylation of Syk in COS-1 cells following FcRIIA/FcRIIB coaggregation is dramatically reduced in the presence of cotransfected SHP-1 (Figs. 2
and 3A)
. SHP-1 also inhibited the activation of Syk tyrosine phosphorylation when Syk was coexpressed with FcRIIA and SHP-1 in the absence of FcRIIB (Fig. 3A)
. A phosphatase-negative mutant of SHP-1 did not affect Syk tyrosine phosphorylation, suggesting that Syk is a substrate for SHP-1 (Fig. 3)
. Phosphorylation of ITAM sequences and the activation of Syk kinase are regarded as the initial steps in the signaling cascade initiated by the activation of Syk and Src-related tyrosine kinases. That Syk kinase activation is inhibited in the presence of overexpressed SHP-1 confirms that SHP-1 is acting at an early stage of the FcRIIA signaling cascade. We have shown that Syk and SHP-1 coimmunoprecipitate both in the presence and absence of FcRIIB (Fig. 2)
. Taken together with our previous observations that Syk associates with FcRIIA following FcRIIA stimulation [1
], the data suggest that Syk kinase may play a role in the recruitment of SHP-1.
In summary, our data indicate that SHP-1 enhances FcRIIB-mediated inhibition of FcRIIA phagocytosis as well as the reduction in FcRIIA tyrosine phosphorylation that accompanies this inhibition. SHP-1 also modulates the tyrosine phosphorylation of the Syk kinase. We also observed that SHP-1 inhibits FcRIIA phagocytosis and signal transduction in the absence of FcRIIB. That the activating FcRIIA receptor itself may interact with SHP-1 suggests that FcRIIA has the potential to modulate its own signaling pathways. Finally, it is likely that FcRIIB can use more than one pathway for its inhibitory action, since the inositol phosphatase SHIP-1 and the tyrosine phosphatase SHP-1 enhance FcRIIB-mediated inhibition of FcRIIA.
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ACKNOWLEDGEMENTS |
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Received September 12, 2002; revised November 12, 2002; accepted November 22, 2002.
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
receptor mediated phagocytosis Blood 86,4389-4399 receptor-mediated phagocytosis as a model system Semin. Immunol. 13,339-345[CrossRef][Medline]
RIIB, regulates negatively BCR-, TCR- and FcR-dependent cell activation Immunity 3,635-646[CrossRef][Medline]
RIIA, FcRIIB and FcRIIC in hematopoietic cells: analysis of transcripts Mol. Immunol. 30,451-460[CrossRef][Medline]
RIIB modulates B-cell receptor signaling Nature 368,70-73[CrossRef][Medline]
receptor-mediated phagocytosis by a nonphagocytic Fc receptor Blood 91,1762-1768 receptor clustering in monocytes J. Immunol. 162,6458-6465 receptor-mediated phagocytosis and the activation of RAC Blood 100,1852-1858RIIIA activates the receptor-associated protein tyrosine kinase Syk and induces phosphorylation of multiple proteins including p95Vav and p62/GAP-associated protein J. Immunol. 152,5429-5437[Abstract]
RIIb in human monocytic cells J. Biol. Chem. 277,5082-5089 receptors and complement receptor 3 (alpha(M)beta(2); CD11b/CD18) J. Exp. Med. 193,61-71
RIIB from recruiting the SH2 domain-containing protein-tyrosine phosphatase SHP-1 J. Biol. Chem. 276,6327-6336RIIB Nature 383,263-266[CrossRef][Medline]
receptors on human monocytes by Th1 and Th2 cytokines J. Immunol. 166,531-537This article has been cited by other articles:
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S. F. Bairstow, K. Ling, and R. A. Anderson Phosphatidylinositol Phosphate Kinase Type I{gamma} Directly Associates with and Regulates Shp-1 Tyrosine Phosphatase J. Biol. Chem., June 24, 2005; 280(25): 23884 - 23891. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ghansah, K. H. T. Paraiso, S. Highfill, C. Desponts, S. May, J. K. McIntosh, J.-W. Wang, J. Ninos, J. Brayer, F. Cheng, et al. Expansion of Myeloid Suppressor Cells in SHIP-Deficient Mice Represses Allogeneic T Cell Responses J. Immunol., December 15, 2004; 173(12): 7324 - 7330. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Swanson and A. D. Hoppe The coordination of signaling during Fc receptor-mediated phagocytosis J. Leukoc. Biol., December 1, 2004; 76(6): 1093 - 1103. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. J. Marshall, J. A. Willment, H.-H. Lin, D. L. Williams, S. Gordon, and G. D. Brown Identification and Characterization of a Novel Human Myeloid Inhibitory C-type Lectin-like Receptor (MICL) That Is Predominantly Expressed on Granulocytes and Monocytes J. Biol. Chem., April 9, 2004; 279(15): 14792 - 14802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. van Mirre, A. van Royen, C. E. Hack, A. R. Crow, S. Song, J. Freedman, C. D. Helgason, R. K. Humphries, K. A. Siminovitch, and A. H. Lazarus IVIg-mediated amelioration of murine ITP via Fc{gamma}RIIb is not necessarily independent of SHIP-1 and SHP-1 activity Blood, March 1, 2004; 103(5): 1973 - 1974. [Full Text] [PDF]
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