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The Journal of Immunology, 2005, 174: 2004-2011.
Copyright © 2005 by The American Association of Immunologists

Negative Regulation of Phagocytosis in Macrophages by the CD47-SHPS-1 System1

Hideki Okazawa*, Sei-ichiro Motegi*,{dagger}, Naoko Ohyama*,{dagger}, Hiroshi Ohnishi*, Takeshi Tomizawa{ddagger}, Yoriaki Kaneko{ddagger}, Per-Arne Oldenborg§, Osamu Ishikawa{dagger} and Takashi Matozaki2,*

* Biosignal Research Center, Institute for Molecular and Cellular Regulation, and Departments of {dagger} Dermatology and {ddagger} Medicine and Clinical Science, Graduate School of Medicine, Gunma University, Showa-Machi, Maebashi, Gunma, Japan; and § Department of Integrative Medical Biology, Section for Histology and Cell Biology, Umeå University, Umeå, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Src homology 2 domain-containing protein tyrosine phosphatase (SHP) substrate-1 (SHPS-1) is a transmembrane protein that is expressed predominantly in macrophages. Its extracellular region interacts with the transmembrane ligand CD47 expressed on the surface of adjacent cells, and its cytoplasmic region binds the protein tyrosine phosphatases SHP-1 and SHP-2. Phagocytosis of IgG- or complement-opsonized RBCs by peritoneal macrophages derived from mice that express a mutant SHPS-1 protein that lacks most of the cytoplasmic region was markedly enhanced compared with that apparent with wild-type macrophages. This effect was not observed either with CD47-deficient RBCs as the phagocytic target or in the presence of blocking Abs to SHPS-1. Depletion of SHPS-1 from wild-type macrophages by RNA interference also promoted Fc{gamma}R-mediated phagocytosis of wild-type RBCs. Ligation of SHPS-1 on macrophages by CD47 on RBCs promoted tyrosine phosphorylation of SHPS-1 and its association with SHP-1, whereas tyrosine phosphorylation of SHPS-1 was markedly reduced in response to cross-linking of Fc{gamma}Rs. Treatment with inhibitors of PI3K or of Syk, but not with those of MEK or Src family kinases, abolished the enhancement of Fc{gamma}R-mediated phagocytosis apparent in macrophages from SHPS-1 mutant mice. In contrast, Fc{gamma}R-mediated tyrosine phosphorylation of Syk, Cbl, or the {gamma} subunit of FcR was similar in macrophages from wild-type and SHPS-1 mutant mice. These results suggest that ligation of SHPS-1 on macrophages by CD47 promotes the tyrosine phosphorylation of SHPS-1 and thereby prevents the Fc{gamma}R-mediated disruption of the SHPS-1-SHP-1 complex, resulting in inhibition of phagocytosis. The inhibition of phagocytosis by the SHPS-1-SHP-1 complex may be mediated at the level of Syk or PI3K signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Macrophages are professional phagocytes that play an important role in innate and acquired immunity as a result of their ability to internalize and degrade pathogens (1, 2, 3). These cells also contribute to preservation of tissue integrity and function by engulfing apoptotic bodies and toxic particles (1, 2, 4). Phagocytosis occurs by extension of the plasma membrane around an extracellular object and subsequent internalization of the latter within a membrane-bounded intracellular phagosome (5). The mechanisms by which phagocytosis is triggered by the interaction of specific receptors on the macrophage surface, including Fc{gamma}Rs (for IgG-coated particles), complement receptors (CRs)3 (for C3bi-opsonized particles), scavenger receptors, and phosphatidylserine receptors (for apoptotic cells), with their ligands have been relatively well characterized (2, 4, 5, 6). In contrast, the molecular mechanisms for negative regulation of phagocytosis in macrophages remain largely unknown.

Src homology 2 domain-containing protein tyrosine phosphatase (SHP) substrate-1 (SHPS-1) (7, 8), also known as signal regulatory protein {alpha} (9), brain Ig-like molecule with tyrosine-based activation motifs (10), macrophage fusion receptor (11), and p84 neural adhesion molecule (12), is a member of the Ig superfamily of proteins. It was initially discovered as a tyrosine-phosphorylated transmembrane protein that binds and serves as a substrate for SHP-1 or SHP-2 (7, 13), both of which are SHPs. The putative extracellular region of SHPS-1 comprises three Ig-like domains with multiple N-linked glycosylation sites, whereas its cytoplasmic region contains four YXX(L/V/I) motifs, which are putative tyrosine phosphorylation sites and binding sites for the Src homology 2 domains of SHP-1 and SHP-2 (Fig. 1A) (7, 9, 14). SHPS-1 is especially abundant in macrophages and neurons (15, 16, 17), although it is also expressed in other cell types, including fibroblasts (7).



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  		FIGURE 1. Expression of a mutant form of SHPS-1 that lacks most of the cytoplasmic region in PEMs. A, Schematic representation of the interactions of SHPS-1 both with its ligand CD47 and with the protein tyrosine phosphatases SHP-1 and SHP-2. CD47 on RBCs binds to the extracellular region of SHPS-1 on macrophages. The cytoplasmic region of SHPS-1 contains four putative tyrosine phosphorylation sites that are also potential binding sites for SHP-1 and SHP-2. Mice used in the present study express a mutant version of SHPS-1 (SHPS-1-{Delta} cyto) that lacks most of the cytoplasmic region and thus is not tyrosine phosphorylated and does not form a complex with SHP-1 or SHP-2. mAbs specific for the extracellular region of SHPS-1 (anti-p84, FG2, and 15D9) block the binding of CD47 to SHPS-1, whereas pAbs (anti-SHPS-1) recognize the cytoplasmic region of the protein. B, Lysates of PEMs derived from wild-type (WT) or homozygous SHPS-1 mutant (knockout (KO)) mice were subjected to immunoblot analysis with a mAb to the extracellular region of SHPS-1 (anti-p84), pAbs to the cytoplasmic region of SHPS-1 (anti-SHPS-1), pAbs to SHP-1 (anti-SHP-1), or a mAb to SHP-2 (anti-SHP-2), as indicated. The positions of wild-type and mutant (SHPS-1-{Delta} cyto) forms of SHPS-1, of SHP-1, of SHP-2, and of molecular size standards (in kDa) are indicated. C, Surface expression of SHPS-1 in PEMs from wild-type and SHPS-1 mutant mice was evaluated by flow cytometry, as described in Materials and Methods (thick trace). The specific mAb (anti-p84) was replaced with normal rat IgG as a negative control (thin trace). D, Serum-deprived PEMs from wild-type or SHPS-1 mutant mice were incubated for 10 min at 37°C in the absence (–) or presence (+) of 2 mM pervanadate, after which cell lysates were subjected to immunoprecipitation (IP) with the anti-p84 mAb to SHPS-1. The resulting precipitates were subjected to immunoblot analysis with either HRP-conjugated mAb PY20 to phosphotyrosine (anti-pY), anti-p84, pAbs to SHP-1, or a mAb to SHP-2. All results are representative of three separate experiments.

 
CD47 has been implicated as a ligand for SHPS-1 (Fig. 1A) (16, 18). This protein, which was originally identified in association with {alpha}v{beta}3 integrin (19), is also a member of the Ig superfamily, possessing an Ig-V-like extracellular domain, five putative membrane-spanning segments, and a short cytoplasmic tail (20). CD47 and SHPS-1 appear to constitute a cell-cell communication system (the CD47-SHPS-1 system) that plays important roles in hemopoietic cells and other cell types. Indeed, the rate of clearance of CD47-deficient RBCs from the bloodstream was found to be markedly increased compared with that apparent for wild-type cells (21, 22). Furthermore, the phagocytosis of CD47-deficient RBCs by splenic or bone marrow-derived macrophages was markedly enhanced in an in vitro assay (21, 22). The binding of CD47 on RBCs to SHPS-1 on macrophages is thus thought to inhibit the phagocytosis of the former cells by the latter. In addition, SHP-1, which forms a complex with SHPS-1, has been suggested to participate in such regulation, given that the phagocytic response is enhanced in motheaten variable mice, in which the activity of SHP-1 is reduced, compared with that apparent in wild-type mice (22). It remains unclear, however, whether SHPS-1 and SHP-1 indeed participate in such negative regulation of phagocytosis in macrophages. Moreover, the molecular mechanism whereby the binding of CD47 to SHPS-1 might inhibit phagocytosis by these cells is unknown.

We previously generated mice that express a mutant version of SHPS-1 that lacks most of the cytoplasmic region (Fig. 1A) (23, 24). This mutant protein is not tyrosine phosphorylated, nor does it form a complex with SHP-1 or SHP-2. The mutant mice manifest thrombocytopenia as a result of a reduced circulation time of platelets in the bloodstream (24). Moreover, the preliminary observations indicated that peritoneal macrophages (PEMs) from the mutant mice phagocytose RBCs more effectively than do those from wild-type animals. With the use of PEMs derived from these SHPS-1 mutant mice, we have now investigated the role of SHPS-1, through its interaction with CD47, in the regulation of Fc{gamma}R- or CR-mediated phagocytosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and reagents

Rat (anti-p84) and hamster (FG2, 15D9) mAbs to SHPS-1 were kindly provided by C. Lagenaur (University of Pittsburgh, Pittsburgh, PA) and S. Nagata (Osaka University, Osaka, Japan), respectively. Rabbit polyclonal Abs (pAbs) to the {gamma} subunit of FcR and to Gab-2 were kindly provided by T. Takai (Tohoku University, Miyagi, Japan) and T. Hirano (Osaka University, Osaka, Japan), respectively. Rabbit pAbs to SHPS-1 were obtained from ProSci. Rabbit pAbs to SHP-1, to Syk (N-19), and to Cbl; an HRP-conjugated mouse mAb (PY20) to phosphotyrosine; and a mouse mAb to SHP-2 were from Santa Cruz Biotechnology. A mouse mAb to phosphotyrosine (PY100) was from Cell Signaling Technology. Normal rat, mouse, and hamster IgG as well as HRP-conjugated secondary Abs were from Jackson ImmunoResearch Laboratories. Streptavidin-PE was from BD Pharmingen, and rabbit pAbs to mouse RBCs were from Cedarlane Laboratories. A rat IgM mAb to mouse CD24 (J11d) and a rat mAb to Fc{gamma}RIII/II (2.4G2) were prepared from the culture supernatants of hybridoma cells. The anti-p84 mAb was conjugated to sulfosuccinimidyl-6-(biotinamido)hexanoate (sulfo-NHS-LC biotin) from Pierce. C5-deficient mouse serum was obtained from Charles River Laboratories Japan. Wortmannin, LY294002, and piceatannol were from Sigma-Aldrich; PD98059, PP2, and PP3 were from Calbiochem; and U0126 was from Promega. Glutaraldehyde-stabilized IgG-opsonized SRBCs were obtained from InterCell Technologies. A mouse SHPS-1-Fc fusion protein was prepared, as described previously (25).

Mice

The generation of mice that express a mutant version of SHPS-1 that lacks most of the cytoplasmic region was described previously (23). Mice were bred and maintained in the Institute of Experimental Animal Research of Gunma University under specific pathogen-free conditions. The mice were backcrossed onto the C57BL/6 background for five generations. Genotyping of mice was performed by PCR, as described (23). Mice were handled in accordance with the animal care guidelines of Gunma University.

Isolation of PEMs

PEMs were isolated essentially as described (26). In brief, the peritoneum of mice was flushed with ice-cold PBS 3 days after i.p. injection with 3 ml of 3% thioglycolate broth (Nissui Pharmaceutical). The exudate cells were separated by centrifugation at 400 x g for 5 min at 4°C, washed with ice-cold RPMI 1640 medium (Sigma-Aldrich), and then resuspended in RPMI 1640 supplemented with 10% FBS. The cells were transferred to culture dishes and were maintained at 37°C under a humidified atmosphere of 5% CO2 in air. After incubation for 24 h, nonadherent cells, which include neutrophils, B cells, and T cells, were washed away. The remaining adherent cells (~99%) were identified as PEMs (26).

Flow cytometry

The surface expression of SHPS-1 on PEMs was examined by flow cytometry, as described (27). In brief, cells were detached from culture dishes by treatment with 0.01% EDTA and then washed with ice-cold PBS. They (0.5 x 106 to 1 x 106) were then incubated with rat mAb 2.4G2 to Fc{gamma}R (1 µg/ml) for 30 min on ice, washed twice with ice-cold PBS, and incubated for 30 min on ice with a biotinylated mAb (anti-p84) to SHPS-1 (2 µg/ml). After two washes with ice-cold PBS, the cells were incubated with streptavidin-PE (1/2000 dilution), washed twice with PBS, suspended in 1 ml of PBS, and analyzed with a FACSCalibur flow cytometer (BD Biosciences). Data were processed with CellQuest software (BD Biosciences).

Opsonization of mouse RBCs

IgG-opsonized mouse RBCs were prepared by incubation of washed RBCs with rabbit polyclonal IgG to mouse RBCs (3 µg/ml) for 20 min at 37°C. Complement-opsonized mouse RBCs were prepared, as described (22). In brief, washed RBCs were incubated first for 30 min at 37°C with a subagglutination dilution of a rat IgM mAb to CD24 and then for 60 min at 37°C with 80% C5-deficient mouse serum. Opsonized cells were finally washed with and resuspended in Ringer’s solution (155 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM Na2HPO4, 10 mM glucose, 10 mM HEPES-NaOH (pH 7.2), 0.5 mg/ml BSA) before assays.

In vitro phagocytosis assays

Isolated PEMs were plated at a density of 1.25 x 105 cells/well in 24-well plates and cultured for 1–2 days. Opsonized or nonopsonized RBCs from wild-type or CD47-deficient mice, or IgG-opsonized SRBCs (InterCell Technologies) were then overlaid on the adherent cells at a density of 1.25 x 107 cells in 200 µl of Ringer’s solution per well. After incubation for the indicated times, the cells were washed with PBS and then incubated for 5 min at room temperature with hemolysis buffer (154 mM NH4Cl (pH 7.3), 10 mM KHCO3, 0.1 mM EDTA) to remove attached, but not phagocytosed, RBCs. After fixation of the cells with 4% paraformaldehyde in PBS, phagocytosis was detected by phase-contrast light microscopy and quantified as a phagocytic index (the number of engulfed RBCs per 100 macrophages). For treatment of PEMs with mAbs, the cells were incubated for 30 min at 4°C with mAbs (100 µg/ml) to SHPS-1 (anti-p84, FG2, or 15D9) or with corresponding control Abs before phagocytosis assays.

Immunoprecipitation and immunoblot analysis

PEMs cultured were incubated in serum-free medium for 12 h before stimulation. For Fc{gamma}R cross-linking, cells (2 x 106) were placed on ice and incubated for 30 min with rat mAb 2.4G2 to Fc{gamma}R (10 µg/ml). After washing with PBS, the cells were exposed to secondary cross-linking Abs (15 µg/ml) in PBS and immediately placed at 37°C. For stimulation with IgG-opsonized RBCs, PEMs (~2 x 106) were incubated with the RBCs (2.0 x 108 cells in 200 µl) for the indicated times at 37°C. Stimulated cells were washed with ice-cold PBS and then lysed on ice in 400 µl of lysis buffer (20 mM Tris-HCl (pH 7.6), 140 mM NaCl, 1 mM EDTA, 1% Nonidet P-40) containing 1 mM PMSF, aprotinin (10 µg/ml), and 1 mM sodium vanadate. The lysates were centrifuged at 10,000 x g for 15 min at 4°C, and the resulting supernatants were subjected to immunoprecipitation and immunoblot analysis.

For immunoprecipitation, the supernatants were incubated for 3 h at 4°C with Ab-coupled protein G-Sepharose beads (20 µl of beads) (Amersham Biosciences). The beads were then washed three times with 1 ml of wash buffer (50 mM HEPES-NaOH (pH 7.6), 150 mM NaCl, 0.1% Triton X-100), suspended in Laemmli sample buffer, boiled for 5 min, and subjected to SDS-PAGE, followed by immunoblot analysis with various Abs and an ECL detection system (Amersham Biosciences).

RNA interference

A short interfering RNA (siRNA) specific for mouse SHPS-1 mRNA was designed according to the criteria of Elbashir et al. (28). Oligonucleotides corresponding to residues 105–123 (relative to the translation start codon) of mouse SHPS-1 cDNA (5'-GGUGACUCAGCCUGAGAAAdTdT-3') and to the complementary sequence (5'-UUUCUCAGGCUGAGUCACCdTdT-3') were synthesized (Nippon Bioservice) and annealed to form a short dsRNA with 3'-dithymidine overhangs. An irrelevant siRNA duplex with a random nucleotide sequence (Qiagen) was used as a control. RNA interference (RNAi) was performed, as described (28), with minor modifications. In brief, PEMs at 50% confluency (5.0 x 105 cells per 35-mm dish) were transfected for 24 h with 100 pmol siRNA with the use of OligoFectamine (Invitrogen Life Technologies) in complete medium without antibiotics. The cells were then transfected again for 24 h with another batch of siRNA before assays.

Statistical analysis

Data are presented as means ± SE. Statistical analysis was performed by Student’s t test with the use of Stat View 5.0 software (SAS Institute). A p value of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of a mutant SHPS-1 protein in mouse PEMs

To investigate the role of SHPS-1 in regulation of phagocytosis in macrophages, we backcrossed mice of mixed C57BL/6 x 129Sv genetic background that were heterozygous for a mutant SHPS-1 allele (23) to the C57BL/6 background for five generations. Wild-type and homozygous mutant littermates were then used in the present study.

The mutant protein lacks most of the cytoplasmic region of SHPS-1, and its expression was first examined in lysates of PEMs by immunoblot analysis with either a mAb that recognizes the extracellular region of SHPS-1 (anti-p84) or pAbs that recognize the cytoplasmic region of the protein (anti-SHPS-1) (Fig. 1A). Immunoblot analysis with anti-p84 revealed the presence of full-length SHPS-1 (~120 kDa) in lysates of PEMs from wild-type mice, but only the mutant form of SHPS-1 (~100 kDa) was detected in the lysates of PEMs from homozygous mutant animals; the abundance of the mutant protein in these latter cells was also markedly reduced compared with that of the full-length protein in wild-type PEMs (Fig. 1B). In contrast, immunoblot analysis with the pAbs to SHPS-1 yielded no signal with PEMs from the homozygous mutant mice. The amounts of SHP-1 and SHP-2 in PEMs from the SHPS-1 mutant mice were similar to those in PEMs from wild-type animals (Fig. 1B).

Consistent with these observations, flow cytometric analysis with anti-p84 revealed the presence of SHPS-1 immunoreactivity on the surface of PEMs from homozygous mutant mice, although its abundance was substantially reduced compared with that on the surface of wild-type cells (Fig. 1C). Exposure of PEMs from wild-type mice to pervanadate induced a marked increase in the extent of tyrosine phosphorylation of SHPS-1 as well as in that of its binding to SHP-1 or SHP-2, whereas the mutant SHPS-1 neither underwent tyrosine phosphorylation nor associated with SHP-1 or SHP-2 in response to pervanadate (Fig. 1D).

Enhanced phagocytosis of IgG- or C3bi-opsonized RBCs by PEMs from SHPS-1 mutant mice

We next examined the effect of the loss of wild-type SHPS-1 expression on phagocytosis by PEMs in an in vitro assay. Little phagocytosis of nonopsonized RBCs from wild-type donor mice was apparent with PEMs from either wild-type or SHPS-1 mutant mice (Fig. 2A). In contrast, IgG-opsonized mouse RBCs underwent phagocytosis to a marked extent by PEMs from wild-type mice. Furthermore, the phagocytic response of PEMs from SHPS-1 mutant mice was even greater than that apparent with PEMs from wild-type animals. Similar results were obtained with C3bi-opsonized mouse RBCs (Fig. 2B), although the extent of the phagocytic response in both wild-type and mutant PEMs was greatly reduced compared with that for Fc{gamma}R-mediated phagocytosis (Fig. 2A). We also examined the phagocytosis of IgG-opsonized SRBCs by PEMs. RBCs exhibit a high level of surface expression of CD47 (21, 29). Indeed, a mouse SHPS-1-Fc fusion protein bound to a marked extent to the surface of mouse RBCs. This fusion protein failed to bind to SRBCs, however (data not shown), suggesting that mouse SHPS-1 is not able to bind to CD47 expressed on the surface of SRBCs. The phagocytic response of mutant PEMs with IgG-opsonized SRBCs was still greater than that apparent for wild-type PEMs (Fig. 2C), but the difference in these responses was greatly reduced compared with that observed with IgG-opsonized mouse RBCs (Fig. 2A). These results suggested that SHPS-1 on the surface of PEMs negatively regulates Fc{gamma}R- or CR-mediated phagocytosis of opsonized RBCs.



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  		FIGURE 2. Phagocytosis of opsonized RBCs by PEMs derived from wild-type or SHPS-1 mutant mice. PEMs isolated from wild-type (WT) or homozygous SHPS-1 mutant (KO) mice were assayed for their phagocytic responses in vitro with nonopsonized ((IgG(–) or C3(–)), IgG-opsonized (A), or C3bi-opsonized (B) RBCs from wild-type donor mice or with IgG-opsonized SRBCs (C). The phagocytic index was determined after incubation of cells for the indicated times at 37°C. Data are means ± SE of values from three independent experiments.

 
Effects of CD47 ablation in RBCs or of mAbs to SHPS-1 on Fc{gamma}R-mediated phagocytosis by PEMs

The enhanced phagocytosis observed in PEMs from SHPS-1 mutant mice might have been attributable either to a lack of functional interaction of SHPS-1 (on PEMs) with CD47 (on RBCs) or to a loss of SHPS-1 function independent of CD47. To distinguish between these possibilities, we performed in vitro phagocytosis assays with RBCs from CD47-deficient mice (21, 30). Nonopsonized CD47–/– RBCs underwent only a low level of phagocytosis by PEMs from either wild-type or SHPS-1 mutant mice even after incubation for 120 min (Fig. 3A). The phagocytosis of IgG-opsonized CD47–/– RBCs by PEMs from wild-type mice was markedly increased, however, compared with that of IgG-opsonized CD47+/+ RBCs (Fig. 3B), consistent with previous observations with bone marrow-derived macrophages (22). In contrast, the phagocytosis of IgG-opsonized CD47–/– RBCs by PEMs from SHPS-1 mutant mice was similar to that of IgG-opsonized CD47+/+ RBCs by the mutant PEMs as well as to that of IgG-opsonized CD47–/– RBCs by wild-type PEMs. These results thus support the notion that the inhibition by SHPS-1 of phagocytosis by macrophages is almost entirely dependent on interaction of SHPS-1 on PEMs with CD47 on RBCs.



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  		FIGURE 3. Effects of CD47 ablation in RBCs or of mAbs to SHPS-1 on phagocytosis by PEMs from wild-type or SHPS-1 mutant mice. A, Nonopsonized CD47–/– mouse RBCs were incubated with PEMs derived from wild-type (WT M{phi}) or SHPS-1 mutant (KO M{phi}) mice for the indicated times, after which the phagocytic index was determined. B, PEMs from wild-type or SHPS-1 mutant mice were assayed for their ability to phagocytose IgG-opsonized RBCs from wild-type (dashed lines) or CD47–/– (solid lines) mice. C, PEMs from wild-type or SHPS-1 mutant mice were treated for 30 min at 4°C with rat mAb anti-p84 to SHPS-1, hamster mAbs FG2 or 15D9 to SHPS-1, or control normal rat or hamster IgG, all at 100 µg/ml, before subjection to the in vitro phagocytosis assay for 60 min at 37°C with nonopsonized or IgG-opsonized RBCs from wild-type mice. All data are means ± SE of values from three independent experiments.

 
We also examined the effects of mAbs to SHPS-1 (anti-p84, FG2, 15D9) on Fc{gamma}R-mediated phagocytosis of RBCs by PEMs. These mAbs block the interaction between CD47 and SHPS-1 (Fig. 1A) (18, 31). Treatment of PEMs from either wild-type or SHPS-1 mutant mice with these mAbs did not affect the phagocytosis of nonopsonized mouse RBCs (Fig. 3C). In contrast, treatment with either of the three mAbs to SHPS-1 markedly enhanced the phagocytosis of IgG-opsonized mouse RBCs by wild-type PEMs. This increased level of phagocytosis in wild-type PEMs was similar to that apparent in mutant PEMs treated with either the SHPS-1-specific mAbs or control IgG.

Effect of RNAi-mediated SHPS-1 depletion on Fc{gamma}R-mediated phagocytosis in PEMs

It was possible that expression of the SHPS-1 mutant protein enhanced the phagocytic response of PEMs as a result of an unexpected gain of function rather than of a loss of function. To exclude this possibility, we examined the effect of SHPS-1 depletion by RNAi on Fc{gamma}R-mediated phagocytosis. Transfection with an siRNA specific for SHPS-1 mRNA resulted in a marked decrease in the amount of SHPS-1 protein in PEMs from wild-type mice, without an effect on the abundance of SHP-1 or SHP-2 (Fig. 4A). Transfection of wild-type PEMs with the SHPS-1 siRNA also promoted Fc{gamma}R-mediated phagocytosis of RBCs (Fig. 4B), suggesting that the observed phenotype of the SHPS-1 mutant cells was due to a loss of function of SHPS-1 and not to a gain of function of the mutant.



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  		FIGURE 4. Effect of SHPS-1 depletion by RNAi on phagocytosis of IgG-opsonized mouse RBCs by PEMs. A, PEMs from wild-type mice were transfected with an siRNA specific for SHPS-1 mRNA or with a control siRNA of random sequence. They were subsequently lysed and subjected to immunoblot analysis with pAbs to SHPS-1 and to SHP-1 and with a mAb to SHP-2. B, PEMs transfected as in A were incubated with IgG-opsonized mouse RBCs for 30 min, after which the phagocytic index was determined. Data are means ± SE of values from three independent experiments.

 
Tyrosine dephosphorylation of SHPS-1 in response to Fc{gamma}R cross-linking and its prevention by engagement of SHPS-1 by CD47

Our results indicated that the interaction of CD47 on RBCs with SHPS-1 on PEMs regulates Fc{gamma}R- or CR-meditated phagocytosis by PEMs in a negative manner. To investigate the molecular mechanism of such regulation, we first examined the effects of cross-linking of Fc{gamma}Rs on tyrosine phosphorylation of SHPS-1 and on its formation of a complex with SHP-1. Only a low level of tyrosine phosphorylation of SHPS-1 was apparent in immunoprecipitates prepared from serum-deprived wild-type PEMs with the mAb anti-p84 (Fig. 1C). In contrast, tyrosine phosphorylation of a ~120-kDa protein was prominent in immunoprecipitates prepared from serum-deprived PEMs with pAbs to SHP-1 (Fig. 5A). Reprobing of the same blot with anti-p84 showed that this ~120-kDa protein was indeed SHPS-1. Cross-linking of Fc{gamma}Rs with the mAb 2.4G2 in wild-type PEMs resulted in an initial small increase (apparent at 1 min) in the extent of tyrosine phosphorylation of SHPS-1 associated with SHP-1, but the amount of tyrosine-phosphorylated SHPS-1 associated with SHP-1 decreased thereafter (Fig. 5A).



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  		FIGURE 5. Effects of Fc{gamma}R cross-linking or engagement of SHPS-1 by CD47 on tyrosine phosphorylation of SHPS-1 and its interaction with SHP-1 in PEMs. A, PEMs from wild-type (WT) mice were deprived of serum and then incubated for the indicated times with mAb 2.4G2 and secondary cross-linking Abs. Cell lysates were then prepared and subjected to immunoprecipitation with pAbs to SHP-1, and the resulting precipitates were subjected to immunoblot analysis with mAb PY100 to phosphotyrosine; the membrane was reprobed with mAb anti-p84 to SHPS-1. Duplicate precipitates were subjected to immunoblot analysis with pAbs to SHP-1. B, Serum-deprived wild-type PEMs were incubated for the indicated times with IgG-opsonized RBCs from wild-type (CD47+/+) or CD47–/– mice, after which cell lysates were subjected to immunoprecipitation and immunoblot analysis, as in A. Results are representative of three independent experiments.

 
Incubation of wild-type PEMs with IgG-opsonized RBCs from wild-type mice resulted in a transient increase apparent between 5 and 20 min in the amount of tyrosine-phosphorylated SHPS-1 associated with SHP-1 (Fig. 5B). In contrast, no such effect was apparent on incubation of wild-type PEMs with IgG-opsonized RBCs from CD47–/– mice. These results suggest that engagement of surface SHPS-1 on PEMs by CD47 on RBCs promotes the tyrosine phosphorylation of SHPS-1 and prevents the dephosphorylation of SHPS-1 induced by Fc{gamma}R cross-linking.

Effects of inhibitors of Src family kinases, Syk, PI3K, or MEK on the enhancement of Fc{gamma}R-mediated phagocytosis in PEMs from SHPS-1 mutant mice

The cross-linking of Fc{gamma}Rs induces tyrosine phosphorylation by a Src family kinase of the receptors themselves and of associated proteins that contain an ITAM (32, 33, 34). The phosphorylated ITAMs then serve as docking sites for the tyrosine kinase Syk. Downstream signaling mediated by PI3K, MAPK, and small GTPases of the Rho family eventually triggers phagocytosis of IgG-opsonized particles (32, 33, 34, 35, 36). We investigated the effect of an inhibitor of Src family kinases, PP2 (37), on the enhancement of Fc{gamma}R-mediated phagocytosis apparent in PEMs from SHPS-1 mutant mice. Treatment with PP2 resulted in slight inhibition of Fc{gamma}R-mediated phagocytosis in PEMs from either wild-type or SHPS-1 mutant mice, but it did not abolish the enhancement of this process induced by loss of SHPS-1 function (Fig. 6A). The inactive analog PP3 had no effect on Fc{gamma}R-mediated phagocytosis in wild-type or mutant PEMs. In contrast, the Syk inhibitor piceatannol (26, 38) inhibited Fc{gamma}R-mediated phagocytosis in wild-type PEMs and abolished the enhancement of this process apparent in PEMs from SHPS-1 mutant mice (Fig. 6B). The PI3K inhibitors wortmannin and LY294002 (39, 40) exerted effects similar to those of piceatannol (Fig. 6C). Two different inhibitors of MAPK kinase MEK, PD98059 (41) and U0126 (42), slightly inhibited Fc{gamma}R-mediated phagocytosis by PEMs from wild-type mice, but they had minimal effects on the enhancement of this process apparent in PEMs from SHPS-1 mutant mice (Fig. 6D).



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  		FIGURE 6. Effects of inhibitors of Src family kinases, Syk, PI3K, or MEK on the enhancement of Fc{gamma}R-mediated phagocytosis apparent in PEMs from SHPS-1 mutant mice. PEMs from wild-type or SHPS-1 mutant mice were incubated for 30 min at 37°C in the absence or presence of 10 µM PP2 or PP3 (A), 10 µM piceatannol (B), 50 nM wortmannin or 50 µM LY294002 (C), or 100 µM PD98059 or 2 µM U0126 (D). They were then assayed for phagocytic activity by incubation for 30 min with IgG-opsonized mouse RBCs in the continued absence or presence of inhibitor. Data are means ± SE of values from three independent experiments. NS, not significant; all other corresponding differences between wild-type and mutant PEMs were statistically significant (p < 0.05).

 
Tyrosine phosphorylation of signaling molecules downstream of Fc{gamma}R in PEMs from SHPS-1 mutant mice

We further investigated the effect of loss of SHPS-1 function on signaling downstream of Fc{gamma}R by examining the tyrosine phosphorylation of signaling molecules in PEMs from wild-type and SHPS-1 mutant mice. However, neither the tyrosine phosphorylation of the {gamma} subunit of FcR nor that of Syk induced by Fc{gamma}R cross-linking differed substantially between PEMs from wild-type mice and those from SHPS-1 mutant mice (Fig. 7, A and B). Tyrosine phosphorylation of Cbl is implicated in the Fc{gamma}R-mediated activation of PI3K and phagocytosis (43). However, tyrosine phosphorylation of Cbl induced by Fc{gamma}R cross-linking was similar in PEMs from wild-type mice and those from SHPS-1 mutant mice (Fig. 7C).



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  		FIGURE 7. Tyrosine phosphorylation of signaling molecules downstream of Fc{gamma}R in PEMs from wild-type (WT) and SHPS-1 mutant mice (KO). Serum-deprived PEMs were stimulated with the 2.4G2 mAb and secondary cross-linking Abs for the indicated times, after which cell lysates were subjected to immunoprecipitation with pAbs to the {gamma} subunit of FcR (anti-FcR{gamma}) (A), with pAbs to Syk (anti-Syk) (B), or with pAbs to Cbl (anti-Cbl) (C). The resulting precipitates were subjected to immunoblot analysis with mAb PY100 to phosphotyrosine. Duplicate samples were subjected to immunoblot analysis with the corresponding pAbs used for immunoprecipitation. All results are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that phagocytosis of IgG- or C3bi-opsonized RBCs by PEMs derived from mice expressing a mutant SHPS-1 protein that lacks most of the cytoplasmic region was markedly increased compared with that apparent with wild-type PEMs. Depletion of SHPS-1 from wild-type PEMs by RNAi also promoted Fc{gamma}R-mediated phagocytosis. These data thus suggest that SHPS-1 negatively regulates Fc{gamma}R- or CR-mediated phagocytosis by PEMs, and that the cytoplasmic region of SHPS-1 is required for such negative regulation. SHP-1 is a protein tyrosine phosphatase that negatively regulates multiple functions of hemopoietic cells, including macrophages (44, 45). SHPS-1 is implicated in the recruitment and activation of SHP-1 at sites near the plasma membrane in response to cytokines or integrin-mediated cell adhesion in macrophages (46, 47, 48). It is therefore likely that SHPS-1 negatively regulates phagocytosis in macrophages through its formation of a complex with SHP-1.

The rate of clearance of transfused CD47-deficient RBCs from the bloodstream is greater than that for wild-type cells (21, 22). In addition, the phagocytosis of CD47-deficient RBCs by splenic or bone marrow-derived macrophages was found to be markedly enhanced in an in vitro assay (21, 22). However, it was unclear whether such enhancement was mediated by loss of the interaction of CD47 on RBCs with SHPS-1 on macrophages. We have now shown that the enhancement of the phagocytic response in PEMs from SHPS-1 mutant mice was greatly reduced with IgG-opsonized SRBCs, which failed to bind mouse SHPS-1, compared with that observed with mouse RBCs. In addition, the difference in the phagocytic responses between PEMs from wild-type mice and those from SHPS-1 mutant animals was also abolished with CD47–/– mouse RBCs as the target. Furthermore, the phagocytic response of wild-type PEMs with wild-type mouse RBCs was potentiated by the presence of blocking mAbs to SHPS-1, whereas the difference in the responses between PEMs from wild-type mice and those from SHPS-1 mutant animals was no longer apparent in the presence of these Abs. We have also shown that transfused RBCs from wild-type donor mice were cleared more efficiently from the peripheral circulation in SHPS-1 mutant mice than in wild-type mice (T. Ishikawa, Y. Kaneko, and T. Matozaki, unpublished data). Together, these results thus suggest that negative regulation of phagocytosis by SHPS-1 is attributable predominantly to its interaction with CD47 on target RBCs.

We found that SHPS-1 is tyrosine phosphorylated and present in a complex with SHP-1 in serum-deprived wild-type PEMs. Integrin-mediated cell adhesion most likely contributes to this tyrosine phosphorylation of SHPS-1, as previously shown in fibroblasts and bone marrow macrophages (7, 46, 47, 49). In contrast, the extent of tyrosine phosphorylation of SHPS-1 was markedly reduced by cross-linking of Fc{gamma}Rs with the mAb 2.4G2. Dephosphorylation of SHPS-1 in response to Fc{gamma}R cross-linking was previously observed in the macrophage cell line BAC1.2F5 (50). These observations suggest that ligation of Fc{gamma}R on macrophages promotes phagocytosis not only through activation of downstream signaling molecules such as PI3K or small GTPases of the Rho family (5, 34, 51), but also through dephosphorylation of SHPS-1 and subsequent disruption of its interaction with SHP-1.

Incubation of PEMs from wild-type mice with IgG-opsonized wild-type RBCs increased the level of tyrosine phosphorylation of SHPS-1 and of its association with SHP-1. The dephosphorylation of SHPS-1 induced by ligation of Fc{gamma}Rs was thus prevented under these conditions. In contrast, incubation of PEMs with IgG-opsonized CD47–/– RBCs failed to induce such a response. Exposure of bone marrow-derived macrophages to wild-type (but not CD47-deficient) RBCs was previously shown to promote tyrosine phosphorylation of SHPS-1 in the presence of pervanadate (21). Our results therefore suggest that ligation of SHPS-1 (on macrophages) by CD47 (on RBCs) inhibits Fc{gamma}R-mediated phagocytosis by promoting tyrosine phosphorylation of SHPS-1 and its interaction with SHP-1 and thereby preventing SHPS-1 dephosphorylation in response to Fc{gamma}R activation. The mechanism responsible for the tyrosine phosphorylation of SHPS-1 induced by CD47 ligation is unknown. It is possible that its engagement by CD47 induces translocation of SHPS-1 to a cellular compartment that facilitates its interaction with tyrosine kinases (such as Src family kinases) responsible for its tyrosine phosphorylation (49).

We found that PEMs from either wild-type or SHPS-1 mutant mice phagocytosed nonopsonized RBCs at only a low level. The enhancement of phagocytosis in PEMs from SHPS-1 mutant mice was thus observed only with opsonized RBCs. Signaling downstream of the Fc{gamma}R includes protein tyrosine phosphorylation (5, 6, 43). Ligation of SHPS-1 by CD47 may promote the recruitment and activation of SHP-1 at a site near the plasma membrane, where it has access to proteins that have undergone tyrosine phosphorylation in response to Fc{gamma}R activation. An inhibitor of Src family kinases, PP2, did not affect the enhancement of Fc{gamma}R-mediated phagocytosis apparent in PEMs from SHPS-1 mutant mice, however. In addition, the Fc{gamma}R-induced tyrosine phosphorylation of the {gamma} subunit of FcR, which is thought to be mediated by the Src family kinase Lyn (6, 52), was not enhanced in PEMs from SHPS-1 mutant mice. In contrast, the Syk inhibitor piceatannol abolished the enhancement of Fc{gamma}R-mediated phagocytosis apparent in PEMs from SHPS-1 mutant mice. Although the Fc{gamma}R-mediated tyrosine phosphorylation of Syk was not enhanced in these cells, it is possible that the tyrosine phosphorylation by Syk of adapter proteins, such as SH2 domain-containing leukocyte phosphoprotein of 76 kDa or B cell linker protein (26, 53), is regulated by the SHPS-1-SHP-1 complex.

Inhibitors of PI3K, but not those of MEK, also abolished the enhancement of Fc{gamma}R-mediated phagocytosis in PEMs from SHPS-1 mutant mice. Potentiation of the Fc{gamma}R-mediated activation of PI3K may thus contribute to the increased level of phagocytosis in PEMs from SHPS-1 mutant mice. In contrast, the Fc{gamma}R-stimulated tyrosine phosphorylation of Cbl, which binds to the p85 subunit of PI3K in response to Fc{gamma}R ligation (43), was not enhanced in PEMs from SHPS-1 mutant mice. In addition, the Fc{gamma}R-mediated tyrosine phosphorylation of Gab-2, which also binds to the p85 subunit of PI3K in response to Fc{gamma}R ligation (54, 55), was not increased in PEMs from the mutant mice (H. Okazawa, S. Motegi, N. Ohyama, and T. Matozaki, unpublished data). Together, these results thus suggest that the SHPS-1-SHP-1 complex might inhibit PI3K-mediated signaling downstream of the Fc{gamma}R, presumably through tyrosine dephosphorylation of an unidentified protein (or proteins).


   Acknowledgments
 
We thank S. Nagata, T. Hirano, C. F. Lagenaur, and T. Takai for reagents, as well as H. Kobayashi, K. Tomizawa, Y. Hayashi, and Y. Niwayama for technical assistance.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas Cancer, a Grant-in-Aid for Scientific Research (B), a Grant-in-Aid for Young Scientists, and a 21st Century Center of Excellence Program grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, as well as by grants from the Swedish Research Council (06P-14098, 31X-14286), National Institutes of Health (GM57573-06), the Swedish Society of Medicine, the Faculty of Medicine at Umeå University, the Uehara Foundation, the Nakatomi Foundation, and the Kato Memorial Foundation. Back

2 Address correspondence and reprint requests to Dr. Takashi Matozaki, Biosignal Research Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-Machi, Maebashi, Gunma 371-8512, Japan. E-mail address: matozaki{at}showa.gunma-u.ac.jp Back

3 Abbreviations used in this paper: CR, complement receptor; pAb, polyclonal Ab; PEM, peritoneal macrophage; RNAi, RNA interference; SHP, Src homology 2 domain-containing protein tyrosine phosphatase; SHPS-1, SHP substrate-1; siRNA, short interfering RNA. Back

Received for publication September 14, 2004. Accepted for publication November 23, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Savill, J., V. Fadok. 2000. Corpse clearance defines the meaning of cell death. Nature 407:784.[Medline]
  2. Greenberg, S., S. Grinstein. 2002. Phagocytosis and innate immunity. Curr. Opin. Immunol. 14:136.[Medline]
  3. Medzhitov, R., C. A. Janeway, Jr. 2002. Decoding the patterns of self and nonself by the innate immune system. Science 296:298.[Abstract/Free Full Text]
  4. Platt, N., R. P. da Silva, S. Gordon. 1998. Recognizing death: the phagocytosis of apoptotic cells. Trends Cell Biol. 8:365.[Medline]
  5. Aderem, A., D. M. Underhill. 1999. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17:593.[Medline]
  6. Ravetch, J. V., S. Bolland. 2001. IgG Fc receptors. Annu. Rev. Immunol. 19:275.[Medline]
  7. Fujioka, Y., T. Matozaki, T. Noguchi, A. Iwamatsu, T. Yamao, N. Takahashi, M. Tsuda, T. Takada, M. Kasuga. 1996. A novel membrane glycoprotein, SHPS-1, that binds the SH2-domain-containing protein tyrosine phosphatase SHP-2 in response to mitogens and cell adhesion. Mol. Cell. Biol. 16:6887.[Abstract]
  8. Yamao, T., T. Matozaki, K. Amano, Y. Matsuda, N. Takahashi, F. Ochi, Y. Fujioka, M. Kasuga. 1997. Mouse and human SHPS-1: molecular cloning of cDNAs and chromosomal localization of genes. Biochem. Biophys. Res. Commun. 231:61.[Medline]
  9. Kharitonenkov, A., Z. Chen, I. Sures, H. Wang, J. Schilling, A. Ullrich. 1997. A family of proteins that inhibit signalling through tyrosine kinase receptors. Nature 386:181.[Medline]
  10. Ohnishi, H., M. Kubota, A. Ohtake, K. Sato, S. Sano. 1996. Activation of protein-tyrosine phosphatase SH-PTP2 by a tyrosine-based activation motif of a novel brain molecule. J. Biol. Chem. 271:25569.[Abstract/Free Full Text]
  11. Saginario, C., H. Sterling, C. Beckers, R. Kobayashi, M. Solimena, E. Ullu, A. Vignery. 1998. MFR, a putative receptor mediating the fusion of macrophages. Mol. Cell. Biol. 18:6213.[Abstract/Free Full Text]
  12. Comu, S., W. Weng, S. Olinsky, P. Ishwad, Z. Mi, J. Hempel, S. Watkins, C. F. Lagenaur, V. Narayanan. 1997. The murine P84 neural adhesion molecule is SHPS-1, a member of the phosphatase-binding protein family. J. Neurosci. 17:8702.[Abstract/Free Full Text]
  13. Noguchi, T., T. Matozaki, Y. Fujioka, T. Yamao, M. Tsuda, T. Takada, M. Kasuga. 1996. Role of SH-PTP2, a protein-tyrosine phosphatase with Src homology 2 domains, in insulin-stimulated Ras activation. J. Biol. Chem. 271:27652.[Abstract/Free Full Text]
  14. Cant, C. A., A. Ullrich. 2001. Signal regulation by family conspiracy. Cell Mol. Life Sci. 58:117.[Medline]
  15. Veillette, A., E. Thibaudeau, S. Latour. 1998. High expression of inhibitory receptor SHPS-1 and its association with protein-tyrosine phosphatase SHP-1 in macrophages. J. Biol. Chem. 273:22719.[Abstract/Free Full Text]
  16. Seiffert, M., P. Brossart, C. Cant, M. Cella, M. Colonna, W. Brugger, L. Kanz, A. Ullrich, H. J. Bühring. 2001. Human signal-regulatory protein is expressed on normal, but not on subsets of leukemic myeloid cells and mediates cellular adhesion involving its counterreceptor CD47. Blood 97:2741.[Abstract/Free Full Text]
  17. Chuang, W., C. F. Lagenaur. 1990. Central nervous system antigen P84 can serve as a substrate for neurite outgrowth. Dev. Biol. 137:219.[Medline]
  18. Jiang, P., C. F. Lagenaur, V. Narayanan. 1999. Integrin-associated protein is a ligand for the P84 neural adhesion molecule. J. Biol. Chem. 274:559.[Abstract/Free Full Text]
  19. Brown, E., L. Hooper, T. Ho, H. Gresham. 1990. Integrin-associated protein: a 50-kD plasma membrane antigen physically and functionally associated with integrins. J. Cell Biol. 111:2785.[Abstract/Free Full Text]
  20. Brown, E. J., W. A. Frazier. 2001. Integrin-associated protein (CD47) and its ligands. Trends Cell Biol. 11:130.[Medline]
  21. Oldenborg, P. A., A. Zheleznyak, Y. F. Fang, C. F. Lagenaur, H. D. Gresham, F. P. Lindberg. 2000. Role of CD47 as a marker of self on RBCs. Science 288:2051.[Abstract/Free Full Text]
  22. Oldenborg, P. A., H. D. Gresham, F. P. Lindberg. 2001. CD47-signal regulatory protein {alpha} (SIRP{alpha}) regulates Fc{gamma} and complement receptor-mediated phagocytosis. J. Exp. Med. 193:855.[Abstract/Free Full Text]
  23. Inagaki, K., T. Yamao, T. Noguchi, T. Matozaki, K. Fukunaga, T. Takada, T. Hosooka, S. Akira, M. Kasuga. 2000. SHPS-1 regulates integrin-mediated cytoskeletal reorganization and cell motility. EMBO J. 19:6721.[Medline]
  24. Yamao, T., T. Noguchi, O. Takeuchi, U. Nishiyama, H. Morita, T. Hagiwara, H. Akahori, T. Kato, K. Inagaki, H. Okazawa, et al 2002. Negative reguration of platelet clearance and of the macrophage phagocytic response by the transmembrane glycoprotein SHPS-1. J. Biol. Chem. 277:39833.[Abstract/Free Full Text]
  25. Miyashita, M., H. Ohnishi, H. Okazawa, H. Tomonaga, A. Hayashi, T. T. Fujimoto, N. Furuya, T. Matozaki. 2004. Promotion of neurite and filopodium formation by CD47: roles of integrins, Rac, and Cdc42. Mol. Biol. Cell 15:3950.[Abstract/Free Full Text]
  26. Hayashi, A., H. Ohnishi, H. Okazawa, S. Nakazawa, H. Ikeda, S. Motegi, N. Aoki, S. Kimura, M. Mikuni, T. Matozaki. 2004. Positive regulation of phagocytosis by SIRP{beta} and its signaling mechanism in macrophages. J. Biol. Chem. 279:29450.[Abstract/Free Full Text]
  27. Motegi, S., H. Okazawa, H. Ohnishi, R. Sato, Y. Kaneko, H. Kobayashi, K. Tomizawa, T. Ito, N. Honma, H. J. Bühring, et al 2003. Role of the CD47–SHPS-1 system in regulation of cell migration. EMBO J. 22:2634.[Medline]
  28. Elbashir, S. M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, T. Tuschl. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494.[Medline]
  29. Lindberg, F. P., D. M. Lublin, M. J. Telen, R. A. Veile, Y. E. Miller, H. Donis-Keller, E. J. Brown. 1994. Rh-related antigen CD47 is the signal-transducer integrin-associated protein. J. Biol. Chem. 269:1567.[Abstract/Free Full Text]
  30. Lindberg, F. P., D. C. Bullard, T. E. Caver, H. D. Gresham, A. L. Beaudet, E. J. Brown. 1996. Decreased resistance to bacterial infection and granulocyte defects in IAP-deficient mice. Science 274:795.[Abstract/Free Full Text]
  31. Tada, K., M. Tanaka, R. Hanayama, K. Miwa, A. Shinohara, A. Iwamatsu, S. Nagata. 2003. Tethering of apoptotic cells to phagocytes through binding of CD47 to Src homology 2 domain-bearing protein tyrosine phosphatase substrate-1. J. Immunol. 171:5718.[Abstract/Free Full Text]
  32. Strzelecka, A., K. Kwiatkowska, A. Sobota. 1997. Tyrosine phosphorylation and Fc{gamma} receptor-mediated phagocytosis. FEBS Lett. 400:11.[Medline]
  33. Ninomiya, N., K. Hazeki, Y. Fukui, T. Seya, T. Okada, O. Hazeki, M. Ui. 1994. Involvement of phosphatidylinositol 3-kinase in Fc{gamma} receptor signaling. J. Biol. Chem. 269:22732.[Abstract/Free Full Text]
  34. Araki, N., M. T. Johnson, J. A. Swanson. 1996. A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages. J. Cell Biol. 135:1249.[Abstract/Free Full Text]
  35. Karimi, K., M. R. Lennartz. 1998. Mitogen-activated protein kinase is activated during IgG-mediated phagocytosis, but is not required for target ingestion. Inflammation 22:67.[Medline]
  36. Mansfield, P. J., J. A. Shayman, L. A. Boxer. 2000. Regulation of polymorphonuclear leukocyte phagocytosis by myosin light chain kinase after activation of mitogen-activated protein kinase. Blood 95:2407.[Abstract/Free Full Text]
  37. Hanke, J. H., J. P. Gardner, R. L. Dow, P. S. Changelian, W. H. Brissette, E. J. Weringer, B. A. Pollok, P. A. Connelly. 1996. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor: study of Lck- and FynT-dependent T cell activation. J. Biol. Chem. 271:695.[Abstract/Free Full Text]
  38. Oliver, J. M., D. L. Burg, B. S. Wilson, J. L. McLaughlin, R. L. McLaughlin. 1994. Inhibition of mast cell Fc{epsilon}R1-mediated signaling and effector function by the Syk-selective inhibitor, piceatannol. J. Biol. Chem. 269:29697.[Abstract/Free Full Text]
  39. Yano, H., S. Nakanishi, K. Kimura, N. Hanai, Y. Saitoh, Y. Fukui, Y. Nonomura, Y. Matsuda. 1993. Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells. J. Biol. Chem. 268:25846.[Abstract/Free Full Text]
  40. Vlahos, C. J., W. F. Matter, K. Y. Hui, R. F. Brown. 1994. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269:5241.[Abstract/Free Full Text]
  41. Dudley, D. T., L. Pang, S. J. Decker, A. J. Bridges, A. R. Saltiel. 1995. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad. Sci. USA 92:7686.[Abstract/Free Full Text]
  42. Favata, M. F., K. Y. Horiuchi, E. J. Manos, A. J. Daulerio, D. A. Stradley, W. S. Feeser, D. E. Van Dyk, W. J. Pitts, R. A. Earl, F. Hobbs, et al 1998. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem. 273:18623.[Abstract/Free Full Text]
  43. Sato, N., M. K. Kim, A. D. Schreiber. 1999. Enhancement of Fc{gamma} receptor-mediated phagocytosis by transforming mutants of Cbl. J. Immunol. 163:6123.[Abstract/Free Full Text]
  44. Shultz, L. D., P. A. Schweitzer, T. V. Rajan, T. Yi, J. N. Ihle, R. J. Matthews, M. L. Thomas, D. R. Beier. 1993. Mutations at the murine motheaten locus are within the hematopoietic cell protein-tyrosine phosphatase (Hcph) gene. Cell 73:1445.[Medline]
  45. Zhang, J., A. K. Somani, K. A. Siminovitch. 2000. Roles of the SHP-1 tyrosine phosphatase in the negative regulation of cell signalling. Semin. Immunol. 12:361.[Medline]
  46. Timms, J. F., K. Carlberg, H. Gu, H. Chen, S. Kamatkar, M. J. Nadler, L. R. Rohrschneider, B. G. Neel. 1998. Identification of major binding proteins and substrates for the SH2-containing protein tyrosine phosphatase SHP-1 in macrophages. Mol. Cell. Biol. 18:3838.[Abstract/Free Full Text]
  47. Timms, J. F., K. D. Swanson, A. Marie-Cardine, M. Raab, C. E. Rudd, B. Schraven, B. G. Neel. 1999. SHPS-1 is a scaffold for assembling distinct adhesion-regulated multi-protein complexes in macrophages. Curr. Biol. 9:927.[Medline]
  48. Oshima, K., A. R. Ruhul Amin, A. Suzuki, M. Hamaguchi, S. Matsuda. 2002. SHPS-1, a multifunctional transmembrane glycoprotein. FEBS Lett. 519:1.[Medline]
  49. Tsuda, M., T. Matozaki, K. Fukunaga, Y. Fujioka, A. Imamoto, T. Noguchi, T. Takada, T. Yamao, H. Takeda, F. Ochi, et al 1998. Integrin-mediated tyrosine phosphorylation of SHPS-1 and its association with SHP-2: roles of Fak and Src family kinases. J. Biol. Chem. 273:13223.[Abstract/Free Full Text]
  50. Gresham, H. D., B. M. Dale, J. W. Potter, P. W. Chang, C. M. Vines, C. A. Lowell, C. F. Lagenaur, C. L. Willman. 2000. Negative regulation of phagocytosis in murine macrophages by the Src kinase family member, Fgr. J. Exp. Med. 192:515.
  51. Cox, D., P. Chang, Q. Zhang, P. G. Reddy, G. M. Bokoch, S. Greenberg. 1997. Requirements for both Rac1 and Cdc42 in membrane ruffling and phagocytosis in leukocytes. J. Exp. Med. 186:1487.[Abstract/Free Full Text]
  52. Suzuki, T., H. Kono, N. Hirose, M. Okada, T. Yamamoto, K. Yamamoto, Z. Honda. 2000. Differential involvement of Src family kinases in Fc{gamma} receptor-mediated phagocytosis. J. Immunol. 165:473.[Abstract/Free Full Text]
  53. Bonilla, F. A., R. M. Fujita, V. I. Pivniouk, A. C. Chan, R. S. Geha. 2000. Adapter proteins SLP-76 and BLNK both are expressed by murine macrophages and are linked to signaling via Fc{gamma} receptors I and II/III. Proc. Natl. Acad. Sci. USA 97:1725.[Abstract/Free Full Text]
  54. Gu, H., R. J. Botelho, M. Yu, S. Grinstein, B. G. Neel. 2003. Critical role for scaffolding adapter Gab2 in Fc{gamma}R-mediated phagocytosis. J. Cell Biol. 161:1151.[Abstract/Free Full Text]
  55. Momose, H., H. Kurosu, N. Tsujimoto, K. Kontani, K. Tsujita, H. Nishina, T. Katada. 2003. Dual phosphorylation of phosphoinositide 3-kinase adaptor Grb2-associated binder 2 is responsible for superoxide formation synergistically stimulated by Fc{gamma} and formyl-methionyl-leucyl-phenylalanine receptors in differentiated THP-1 cells. J. Immunol. 171:4227.[Abstract/Free Full Text]



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