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Prepublished online as a Blood First Edition Paper on July 12, 2002; DOI 10.1182/blood-2002-03-0787. This Article Abstract Full Text (PDF) All Versions of this Article: 2002-03-0787v1 100/9/3374    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Nakamura, K. Articles by Coggeshall, K. M. Search for Related Content PubMed PubMed Citation Articles by Nakamura, K. Articles by Coggeshall, K. M. Related Collections Phagocytes Signal Transduction Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 November 2002, Vol. 100, No. 9, pp. 3374-3382 PHAGOCYTES The Src homology 2 domain-containing inositol 5-phosphatase negatively regulates Fc receptor-mediated phagocytosis through immunoreceptor tyrosine-based activation motif-bearing phagocytic receptors Koji Nakamura, Alexander Malykhin, and K. Mark Coggeshall From The Oklahoma Medical Research Foundation, Program in Immunobiology and Cancer, Oklahoma City, OK.     Abstract Top Abstract Introduction Materials and methods Results Discussion References Molecular mechanisms by which the Src homology 2 domain-containing inositol 5-phosphatase (SHIP) negatively regulates phagocytosis in macrophages are unclear. We addressed the issue using bone marrow-derived macrophages from FcR- or SHIP-deficient mice. Phagocytic activities of macrophages from FcRII(b)/ and SHIP/ mice were enhanced to a similar extent, relative to those from wild type. However, calcium influx was only marginally affected in FcRII(b)/, but greatly enhanced in SHIP/ macrophages. Furthermore, SHIP was phosphorylated on tyrosine residues upon FcR aggregation even in macrophages from FcRII(b)/ mice or upon clustering of a chimeric receptor containing CD8 and the immunoreceptor tyrosine-based activation motif (ITAM)-bearing -chain or human-restricted FcRIIa. These findings indicate that, unlike B cells, SHIP is efficiently phosphorylated in the absence of an immunoreceptor tyrosine-based inhibition motif (ITIM)-bearing receptor. We further demonstrate that SHIP directly bound to phosphorylated peptides derived from FcRIIa with a high affinity, comparable to that of FcRII(b). Lastly, FcRIIa-mediated phagocytosis was significantly enhanced in THP-1 cells overexpressing dominant-negative form of SHIP in the absence of FcRII(b). These results indicate that SHIP negatively regulates FcR-mediated phagocytosis through all ITAM-containing IgG receptors using a molecular mechanism distinct from that in B cells. (Blood. 2002;100:3374-3382) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References Phagocytosis of IgG-coated particles is initiated by clustering of the phagocytic receptors for the Fc moiety of IgG (FcRs). There are 3 murine forms of FcRs, encoded by 3 distinct genes.1 Two of these forms, FcRI and FcRIII, consist of ligand-binding -chain and common -chain, and are able to promote phagocytosis. The -chain contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic region, a motif shared among all immunoreceptors. There are a total of 8 human FcR genes: 3 for FcRI (A-C); 3 for FcRII (A-C); and 2 for FcRIII (A and B). FcRIA-C and FcRIIIA are the respective equivalents of murine FcRI and FcRIII, whereas FcRIIA and FcRIIIB are unique to human and absent in mouse. The other class of murine and human FcR, FcRII(b), is a single-chain receptor containing an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic region and does not promote phagocytosis. The term FcRII(b) will be used to indicate murine FcRII and human FcRIIb. FcRII(b) functions as a negative regulator in B cells and mast cells by recruiting inhibitory molecules such as SH2 domain-containing inositol 5-phosphatase (SHIP) and SH2 domain-containing protein tyrosine phosphatase-1 (SHP-1).2,3 SHIP is only recruited and activated when FcRII(b) is coclustered with B-cell receptor (BCR)4,5 or FcRI.6-8 Hence, coclustering of the ITAM-bearing BCR with the ITIM-bearing FcRII(b) provokes efficient SHIP phosphorylation and a block in cell activation. Indeed, all of the ITIM-bearing inhibitory receptors provoke inhibitory functions only upon their coclustering with an activating receptor. The signal transduction process following such coclustering was termed coinhibition,9,10 or negative signaling.3 Other examples of coinhibition include natural killer (NK) cell-mediated lysis, a process blocked when killer cell inhibitory receptor (KIR) is coclustered with the NK cell-activating receptors (reviewed in Taylor et al11). Likewise, the paired immunoglobulinlike receptor B (PIR-B) in mast cells or B cells blocks signal transduction only when coclustered with an antigen or Ig receptor.12 Lastly, the gp49 inhibitory receptor blocks mast cell secretion of mediators when coclustered with IgE receptors.13 The block of cell activation in each of these examples is completely dominant over the activation signal provided by the ITAM-bearing receptor. Clustering of -chain-containing FcRs by particles opsonized with IgG triggers intracellular events such as activation of protein tyrosine kinases of the Src family14 and Syk,15 calcium mobilization,16 and actin polymerization,17 leading to internalization of the particles. The molecular mechanisms of signal transduction for FcR-mediated phagocytosis is similar to that of antigen receptors in lymphocytes. In addition to Src-family and Syk protein tyrosine kinases, phosphatidylinositol 3-kinase (PtdIns 3-kinase) activity is required for FcR-mediated phagocytosis.18,19 After FcRs are clustered by binding of immune complexes, these molecules are recruited to the phosphorylated tyrosines of the -chain of FcRI/III and are sequentially activated to transduce the signal to downstream events such as actin polymerization and particle internalization.20 Recently, it was reported that FcRII(b)-deficient macrophages show greater phagocytic and calcium mobilization responses upon FcRIII engagement, indicating the FcRII(b) inhibits -chain-containing IgG receptor function.21 Similar findings were made using macrophages of SHIP/ mice.22 In B cells, FcRII(b) imparts a negative signal by plasma membrane recruitment of SHIP,23 and the SH2 domain of SHIP is required for its recruitment by ITIM receptors.5 Thus, the functional experiments of macrophages from FcRII(b)/21 and SHIP/22 mice suggest that, like the B cell, coclustering the ITAM-containing -chain FcR and the ITIM-containing FcRII(b) inhibits macrophage activation and function by recruitment and phosphorylation of SHIP. Recent experiments in human macrophages and neutrophils support the notion that coclustering FcRs containing ITAM and ITIM sequences regulate cellular function.24 In contrast to this model, other studies suggest that SHIP is efficiently phosphorylated upon clustering of ITAM-bearing FcR25,26 and that an ITIM receptor is not necessary. Thus, it is unclear whether SHIP is involved in the regulation of macrophage activation through ITIM-containing receptors like FcRII(b). Likewise, it is unclear whether FcRII(b)-mediated inhibition of macrophage activation involves SHIP. Here, we explored the requirement for the ITIM-bearing FcRII(b) to induce SHIP phosphorylation and to regulate phagocytosis in bone marrow-derived macrophages from wild-type or gene-targeted mice and in cell lines expressing chimeric receptors of FcRI/III -chain with extracellular region of CD8. We report that, in contrast to the B-cell model, SHIP phosphorylation is efficiently induced in FcRII(b)-deficient macrophages, and can be elicited by an ITAM-containing receptor chimera through direct binding to ITAM-containing phagocytic receptors. Furthermore, the SH2 domain of SHIP has an affinity for phosphorylated ITAM tyrosines of human FcRIIa comparable to the affinity for phosphotyrosines of the ITIM. Thus, SHIP is able to regulate FcR-mediated phagocytosis independently of FcRII(b).     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Animals The FcRII(b)/ or -chain/ single-deficient and FcRII(b)// -chain/ double-deficient mice were purchased from Taconic Farms (Westminster, NY). The SHIP/ mice were kindly provided by Dr G. Krystal, Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada. All gene-targeted mice were of C57Bl/6 background. C57Bl/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and used as wild-type controls. Antibodies We purchased 2.4G2 (anti-mouse FcRII/III),27 antigen-presenting cell (APC)-conjugated anti-Mac-1 (IgG2b), fluorescein isothiocyanate (FITC)-conjugated anti-CD8, and APC-conjugated IgG2b from Pharmingen (San Diego, CA). The monoclonal IgG2a (UPC10) was from Caltag (Burlingame, CA). The FITC-conjugated F(ab')2 fragment of rabbit anti-mouse IgG was from Jackson ImmunoResearch (West Grove, PA). The polyclonal anti-sheep red blood cells antibody was from Sigma (St Louis, MO). The polyclonal mouse IgG was from Pierce (Rockford, IL). The rabbit anti-SHIP antibody was described previously and used for immunoprecipitation.5 Antiphosphotyrosine monoclonal antibody 4G10 was purchased from Upstate Biotechnology (Lake Placid, NY). Anti-CD8 monoclonal antibody was purified from culture supernatant of hybridoma, OKT8 (American Type Culture Collection, Manassas, VA), and used as a F(ab')2 fragment. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody and HRP-conjugated sheep anti-rabbit Ig antibody were obtained from Kappel (West Chester, PA) and Amersham Pharmacia (Piscataway, NJ), respectively. Anti-myc (9E10) monoclonal antibody was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Cell culture and transfection RAW264.7 and THP-1 were obtained from American Type Culture Collection. The cells were maintained in complete medium (RPMI supplemented with 10% fetal calf serum [FCS], 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin). Transfection of cDNA was performed by electroporation at 310 V, 975 µF by Gene Pulser (Bio-Rad, Hercules, CA). Stable transfectants were selected and maintained in complete medium containing 1 mg/mL G418 (Invitrogen, Carlsbad, CA). CD8+ cells stained with FITC-conjugated anti-CD8 antibody or green fluorescence protein (GFP)-positive cells were sorted by Moflo Cytometer (Cytomation, Fort Collins, CO). Bone marrow-derived macrophages Bone marrow-derived macrophages (BMMs) were prepared by standard methods from gene-targeted mice. Briefly, the bone marrow cells were isolated by flushing femurs and tibias and cultured overnight in 10 cm2 dishes with complete medium containing 20% L cell-conditioned medium at 37°C in 5% CO2. Nonadherent cells were transferred to new dishes and cultured for an additional 5 days at 37°C in 5% CO2 for experiments. Flow cytometry BMMs and RAW264.7 were harvested from plates using Cell Dissociation Medium (Sigma). Staining and flow cytometry were performed according to standard methods and analyzed by FACSCalibur and CELLQUEST software (Becton Dickinson, San Jose, CA). Phagocytosis assay Phagocytic index was measured as previously described.20 Briefly, sheep red blood cells (RBCs) were labeled by fluorescent dye (PKH26; Sigma) according to manufacturer's instruction. The RBCs were opsonized by polyclonal anti-sheep RBC (IgG-RBC) and used as targets for phagocytosis. For FcRIIa-restricted phagocytosis, RBCs were biotinylated and treated with streptavidin. The streptavidin-labeled RBCs were then coupled with biotinylated Fab fragments of IV.3 antibody. Phagocytes were plated on 24-well plates at 2 × 105 cells per well and incubated overnight at 37°C in 5% CO2. Opsonized RBCs (4 × 106) were added to the prechilled 24-well plates and incubated on ice for 10 minutes to be formed rosettes. The cells were warmed to 37°C to initiate phagocytosis. Uninternalized RBCs were removed by incubation with ammonium chloride potassium (ACK) buffer (10 mM HEPES, pH 7.3, 150 mM NH4Cl). Internalized RBCs were visualized under fluorescence microscope and counted. Phagocytic index was defined as a number of internalized RBCs per 100 phagocytes. Calcium mobilization measurements Heat-aggregated IgG (IgG) was prepared by heating 10 mg/mL normal mouse IgG at 64°C for 30 minutes. Cells were incubated in complete medium containing 2.5 µM Indo-1 AM (Molecular Probe, Eugene, OR) for 30 minutes at 37°C. The cells were stimulated with 40 µg/mL IgG and monitored by spectrofluorometry (Perkin-Elmer, Norwalk, CT). The Indo-1 fluorescence emission was converted to Ca++i according to the manufacturer's instructions. Immunoprecipitation and immunoblot All procedures were essentially as described earlier.20 Briefly, cells were lysed in TN-1 buffer (50 mM Tris-HCl, pH 8.0, 125 mM NaCl, 10 mM ethylenediaminetetraacetic acid [EDTA], 1% Nonidet P-40, 10 mM NaF, 3 mM Na3VO4, 10 mM Na4P2O7, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 100 µg/mL phenylmethylsulfonyl fluoride) and centrifuged at 16 000g for 10 minutes at 4°C to remove insoluble materials. The resulting supernatants were subjected to immunoprecipitation using the indicated antibodies followed by protein A- or protein G-agarose (Invitrogen). The beads were extensively washed with TN-1 and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were electrophoretically transferred to nitrocellulose membranes, blotted with appropriate antibodies, and visualized by enhanced chemiluminescence (ECL) system (Pierce). Reverse transcriptase-polymerase chain reaction and construction of plasmids Total RNAs were isolated from RAW264.7 cells and reverse-transcribed to cDNAs by standard methods. The intracellular portion of -chain, corresponding to amino acids 47-86, were obtained by polymerase chain reaction (PCR) and the product was fused to extracellular and transmembrane regions of human CD8. The resulting cDNA was cloned into pEF/myc/cyto (Invitrogen). The intracellular portion of human FcRIIa, corresponding to the amino acids 285-307, were also fused to the extracellular and transmembrane portions of human CD8, and cloned into pEF/myc/cyto. The substitution of tyrosine residues within ITAM of FcRIIa with phenylalanine was performed based on PCR technique using a CD8/IIa chimera as a template. For GFP-SH2-SHIP expression vector, the cDNA fragment of SH2-SHIP, corresponding to amino acids 1-114 of murine SHIP,28 was generated by PCR and ligated into pEGFP-N1 (Clontech). The materials were confirmed by sequencing. In vitro peptide binding assay Whole-cell lysates were incubated with biotinylated peptides described elsewhere.5,29 Peptides were collected with Neutravidin-Sepharose (Pierce) after 5 washes with TN1 lysis buffer. The proteins associated with the peptides were analyzed by immunoblot. The identical protocol was done in experiments using the purified, recombinant GST-SHIP SH2 domain, as earlier described.5,30,31 The purified, recombinant GST-SHIP SH2 domain fusion protein showed a single band on SDS-PAGE analysis corresponding to the fusion protein. Affinity measurements of the SH2 domain of SHIP to phosphopeptides Affinities of SH2 domain of SHIP to phosphopeptides were determined by BIAcore system (BIAcore, Uppsala, Sweden) according to the manufacturer's instructions. In this system, the amount of analytes (GST-SH2-SHIP) bound to the sensor chip via phosphopeptides was correlated with the response unit (RU) observed. Biotinylated peptides were immobilized to streptavidin-coated chips. No direct binding of GST-SH2-SHIP to the streptavidin-coated sensor chip was observed. The GST-SH2-SHIP in the binding buffer (phosphate-buffered saline [PBS] containing 0.05% Tween-20) was injected at a flow rate of 30 µL/min for 5 minutes at 25°C. Binding was monitored and the chip was continuously washed with the binding buffer for another 5 minutes at 25°C. The chip was regenerated by washing with PBS containing 0.05% SDS. The kinetic parameters were calculated by the BIAevaluation 3.0 software (BIAcore) according to data from at least 5 different concentrations of the analytes injected.     Results Top Abstract Introduction Materials and methods Results Discussion References FcR-mediated phagocytosis is enhanced in bone marrow-derived macrophages isolated from FcRII(b)/ and SHIP/ knockout mice To examine the roles of FcRII(b) and SHIP on FcR-mediated phagocytosis, the phagocytic abilities of the BMMs from C57Bl/6 wild-type, FcRII(b)/, FcR -chain/ (-chain/), FcRII(b)// -chain/, and SHIP/ mice were compared using IgG-opsonized sheep red blood cells (IgG-RBCs) as phagocytic targets (Figure 1). BMMs from either -chain/ or FcRII(b)// -chain/ double-deficient mice were incapable of phagocytosis, due to the lack of phagocytic receptors FcRI and FcRIII, as reported previously.32 However, phagocytic activities of BMMs from FcRII(b)/ and SHIP/ were greatly enhanced, compared with that of wild-type BMMs These observations indicate that both FcRII(b) and SHIP negatively regulate FcR-mediated phagocytosis. The data are consistent with the possibility that, like B cells, paired coclustering an ITAM- and an ITIM-containing receptor with an IgG-coated particle blocks cell activation. View larger version (12K): [in this window] [in a new window]   Figure 1. Phagocytosis assay using BMMs from gene-targeted mice. Fluorescent IgG-opsonized RBCs were incubated with BMMs from gene-targeted mice indicated at a ratio of 20:1. Internalized IgG-RBCs were counted under a fluorescence microscope. The results were expressed as the number of the internalized IgG-RBCs per 100 BMMs (phagocytic index). Results shown are the average of duplication and are representative of 2 independent experiments. Calcium mobilization is enhanced in BMMs of SHIP/ but not those of FcRII(b)/ animals Clustering of phagocytic receptors, like all ITAM-containing receptors, is accompanied by an increase of intracellular calcium.33 In B cells, the calcium mobilization induced by clustering of B-cell receptors is inhibited by coclustering of B-cell receptor with the ITIM-bearing FcRII(b),34,35 which promotes SHIP recruitment.5,31 To explore the possibility that the inhibitory effect of SHIP on phagocytosis is associated with FcRII(b) like the B-cell model, we compared the intracellular calcium mobilization in BMMs from gene-targeted mice upon stimulation with IgG. Stimulation of macrophages with IgG engages all mouse FcR, including phagocytic receptors FcRI and FcRIII, and inhibitory receptor FcRII(b). This model enables us to measure the calcium mobilization through both activating receptors FcRI and FcRIII, and investigate the contribution of FcRII(b) by using cells from gene-targeted animals. The model is an improvement over earlier studies that used 2.4G2 monoclonal antibody (mAb) to stimulate peritoneal macrophages21 because 2.4G2 does not recognize FcRI. Calcium mobilization upon stimulation of IgG was only marginally increased in BMMs derived from FcRII(b)-deficient mice, relative to BMMs from wild-type mice (Figure 2). However, calcium influx was greatly enhanced in BMMs from SHIP/ compared with those of wild-type, or FcRII(b)/. Minimal calcium mobilization was observed in BMMs from -chain/ or from double-deficient BMMs, as reported elsewhere.21 These findings indicate that the negative signal induced by FcRII(b) coclustering minimally affects IgG receptor-triggered intracellular calcium, whereas SHIP has a more pronounced influence. These observations suggest that SHIP functions through other receptors besides FcRII(b), unlike the B-cell model. View larger version (21K): [in this window] [in a new window]   Figure 2. Calcium mobilization upon FcR stimulation in BMMs from gene-targeted mice. BMMs (5 × 105) from gene-targeted mice indicated were loaded with Indo-1 AM and stimulated with 40 µg/mL IgG. The intracellular Ca++ was monitored by spectrofluorometry. The bar indicates intracellular Ca++ as a reference. The arrow indicates the time when IgG was added. SHIP is efficiently phosphorylated by FcR clustering in the absence of FcRII(b) B-cell lines show efficient SHIP phosphorylation when the cells express FcRII(b), but less SHIP phosphorylation when FcRII(b) is absent.5 To test whether SHIP phosphorylation in macrophages likewise requires expression of FcRII(b), we determined the tyrosine phosphorylation of SHIP in BMMs from the various gene-targeted mice using IgG as a stimulus. We found that SHIP phosphorylation was increased upon stimulation with IgG in BMMs from wild-type mice, but not in BMMs from mice lacking FcR -chain (Figure 3A). These data show that SHIP phosphorylation minimally requires clustering of ITAM-bearing receptors, FcRI and/or FcRIII, associated with the -chain. Surprisingly, SHIP was significantly phosphorylated in BMMs from FcRII(b)/ mice. The stoichiometry of SHIP phosphorylation was estimated from several identical experiments by quantitating the ratio of phosphorylated SHIP to total immunoprecipitated SHIP in BMMs from wild-type or the gene-targeted animals. The data are expressed as fold increase and presented in Figure 3B. These data show that SHIP phosphorylation does not require the inhibitory receptor FcRII(b), unlike the B-cell model.5 View larger version (19K): [in this window] [in a new window]   Figure 3. Tyrosine phosphorylation of SHIP upon FcR stimulation in BMMs from gene-targeted mice. (A) BMMs (4 × 106) from gene-targeted mice indicated were stimulated with 40 µg/mL IgG for indicated minutes, lysed, and immunoprecipitated with anti-SHIP antibody. The immunoprecipitates were blotted with antiphosphotyrosine antibody (upper panel; anti-pTyr) or anti-SHIP antibody (lower panel). (B) The amount of tyrosine-phosphorylated SHIP to total SHIP shown in panel A was quantified and expressed as fold increase of the ratio. The results were shown as relative values of the time-zero controls and as averages from 2 independent experiments. Bars represent standard errors of duplicate measurements. Clustering of the -chain or FcRIIa is sufficient to induce SHIP phosphorylation in macrophages The efficient phosphorylation of SHIP in the BMMs from FcRII(b)/ mice raises the possibility that clustering of activating, phagocytic FcRs is sufficient for SHIP activation. To directly address this issue, and to eliminate any potential contribution from FcRII(b), we transfected the RAW264.7 mouse macrophage cell line with a chimeric receptor containing the intracellular region of FcR -chain fused to the unrelated extracellular region of human CD8 (CD8/). Because the ITAM sequence of the -chain is sufficient to trigger phagocytosis,36 the receptor chimera enables us to discriminate signaling through the -chain associated with phagocytic FcRI/III from that of FcRII(b), and to examine whether SHIP is phosphorylated upon clustering of ITAM-bearing -chain alone. The transfected RAW264.7 macrophages were sorted based on CD8 expression levels to derive stable transfectants expressing high or low levels of CD8/ and CD8 alone (Figure 4A). The stable transfectants were stimulated with biotinylated F(ab')2 fragment of anti-CD8 (OKT8) and the receptor was clustered by the addition of streptavidin. This stimulation protocol using F(ab')2 fragments was applied to avoid stimulation of any endogenous IgG receptors of RAW264.7. We confirmed by flow cytometry that the F(ab')2 fragment of OKT8 failed to recognize untransfected cells (Figure 4A, untransfected), indicating that the endogenous FcRs are not engaged by this stimulation protocol. Stimulation with biotinylated F(ab')2 fragment of OKT8 followed by streptavidin revealed tyrosine phosphorylation appearing in whole-cell lysates (Figure 4B) in CD8/ transfectants, but not in untransfected RAW264.7 cells, or in the transfectants expressing CD8 alone. Likewise, we found that SHIP tyrosine phosphorylation was greatly increased after OKT8 stimulation in cells expressing either high or low levels of the chimeric receptor, depending on the expression of chimeras (Figure 4C). However, SHIP phosphorylation was absent in cells transfected with CD8 only. These data indicate that clustering of the -chain ITAM is sufficient for SHIP phosphorylation and that participation of FcRII(b) is not necessary. View larger version (30K): [in this window] [in a new window]   Figure 4. Clustering of FcRIIa or the -chain of FcRs is sufficient for SHIP phosphorylation. (A) The expression of CD8 chimeras in stable RAW264.7 transfectants was examined by fluorescence-activating cell sorter (FACS) analysis. The cells were stained with biotinylated F(ab')2 fragments of OKT8 followed by FITC-conjugated streptavidin and analyzed by FACS. Dotted lines indicate fluorescence of unstained cells. (B,C) The RAW264.7 transfectants were stimulated with biotinylated F(ab')2 fragments of OKT8 followed by streptavidin. Whole-cell lysates (B) or SHIP immunoprecipitates (C) were separated by SDS-PAGE and blotted with antiphosphotyrosine (anti-pTyr). The filter in C was reprobed with anti-SHIP antibody (lower panel). (D) The expression of CD8 chimeras in stable THP-1 transfectants was examined by FACS analysis using biotinylated F(ab')2 fragments of OKT8 followed by FITC-conjugated streptavidin. (E,F) THP-1 transfectants were stimulated with biotinylated F(ab')2 fragments of OKT8 followed by streptavidin. Whole-cell lysates (E) or SHIP immunoprecipitates (F) were blotted with anti-pTyr. In panel F, untransfected cells were also stimulated with Fab fragments of IV.3 antibody followed by F(ab')2 fragments of goat anti-mouse antibody (2 lanes on the farthest right). The membrane was reprobed with anti-SHIP antibody (lower panel). The human-restricted FcRIIa is unique among immunoreceptors and consists of a single polypeptide chain containing ITAM in its cytoplasmic tail. We therefore asked whether the ITAM of the human-restricted FcRIIa was also sufficient to induce SHIP phosphorylation, like the murine -chain ITAM. To test this possibility, we expressed in human THP-1 monocytes a chimera of the ITAM of human-restricted FcRIIa fused to the extracellular region of CD8 (CD8/IIa). Flow cytometry analysis of CD8 expression shows that the cells express CD8 or the CD8/IIa chimera (Figure 4D). Stimulation of the transfected CD8+ THP-1 cells with biotinylated F(ab')2 fragments of OKT8 followed by streptavidin as above revealed tyrosine phosphorylation appearing in whole-cell lysates (Figure 4E) in the cells expressing CD8IIa, but not in the untransfected population or the cells expressing CD8 alone. SHIP tyrosine phosphorylation was similarly increased in cells expressing the CD8/IIa chimera (Figure 4F). As an additional control, we stimulated endogenous FcRIIa in THP-1 cells with specific monoclonal antibody IV.3 (Looney et al37; Figure 4F; untransfected). Earlier studies showed the IV.3 mAb recognizes FcRIIa and not FcRIIb in hematopoietic cells.38 These data clearly demonstrate that the ITAM of the -chain associated with activating FcRs or the ITAM of FcRIIa in macrophages is capable of efficiently promoting SHIP phosphorylation. SHIP directly binds to the ITAM-containing receptor, FcRIIa, with an affinity comparable to the ITIM-containing receptor, FcRII(b) Although SHIP was phosphorylated upon clustering of -chain or FcRIIa in the absence of FcRII(b), the mechanism by which SHIP is recruited to the ITAM-containing phagocytic receptors is unclear. To begin to address this issue, we tested the in vitro binding of SHIP to doubly phosphorylated peptide derived from ITAM of FcRIIa (P4; Figure 5A) in the cell lysates of THP-1 cells with or without stimulation of IV.3 antibody. SHIP bound to P4 as well as to phosphopeptides of FcRII(b) ITIM (pITIM), but not unphosphorylated peptide of FcRIIa ITAM (P1) in vitro (Figure 5B). Because the binding of SHIP to P4 or pITIM did not require prior cell stimulation, binding in this case indicated that SHIP is capable of direct association to the ITAM of FcRIIa and did not involve a phosphorylated adapter molecule(s). We examined the binding between FcRIIa peptides and purified, recombinant GST-SH2-SHIP fusion protein by in vitro peptide binding assay. The GST-SH2-SHIP fusion protein bound to P4 as well as pITIM, but not to P1 or to P4 after dephosphorylation by alkaline phosphatase (Figure 5C). To explore whether SHIP could associate with FcRIIa in cells, we examined coimmunoprecipitation of endogenous SHIP with the ITAM of FcRIIa in THP-1 cells expressing CD8/IIa. We found (Figure 5D) that SHIP coimmunoprecipitated with CD8/IIa in an activation-dependent manner. Equal amounts of the immunoprecipitated CD8/IIa was verified by immunoblot with anti-myc antibody. These data demonstrate that SHIP is capable of binding to ITAM-containing FcRIIa in cells and in vitro. View larger version (14K): [in this window] [in a new window]   Figure 5. SHIP binds directly to the ITAM of FcRIIa in vitro and in vivo. (A) Sequences of peptides used are shown and described previously.29 (B) THP-1 cells were stimulated with Fab fragments of IV.3 antibody followed by F(ab')2 fragments of goat anti-mouse antibody. The lysates were incubated with biotinylated peptides indicated and purified by Neutravidin beads. The precipitates were resolved by SDS-PAGE, and blotted with anti-SHIP antibody. (C) The recombinant GST-SH2-SHIP fusion protein (right 5 lanes) or GST protein (left 5 lanes) was incubated with biotinylated peptides indicated, precipitated with Neutravidin beads, resolved by SDS-PAGE, and blotted with anti-GST antibody. The phosphorylated peptides were pretreated with calf intestinal alkaline phosphatase (CIAP) before the incubation with recombinant proteins as indicated. The position of GST-SH2-SHIP was indicated at right. (D) THP-1 transfectants were stimulated with biotinylated F(ab')2 fragments of OKT8 followed by streptavidin and lysates were immunoprecipitated with anti-myc. The immunoprecipitates (ip) were separated by SDS-PAGE and probed with antibody to SHIP (upper panel) and reprobed with anti-myc (lower panel). To measure the affinity between SHIP and FcRIIa ITAM, surface plasmon resonance measurement was performed using purified GST-SH2-SHIP protein and phosphopeptides immobilized on sensor chips, listed in Figure 5A. GST-SH2-SHIP bound to P4 with a slow association rate and a comparable dissociation rate compared with pITIM (Figure 6). We found no significant binding of GST-SH2-SHIP to P1 or pIg-, derived from ITAM of the Ig- chain of BCR. Kinetic parameters for the interaction of GST-SH2-SHIP with singly phosphorylated peptides (P2 and P3), P4, pITIM, and pIg- were determined by sensor measurements using at least 5 different concentrations of GST-SH2-SHIP (Table 1). Although GST-SH2-SHIP associated with P4 at slower rate (association constant [kon], 2616 M1s1) than pITIM (kon, 3370 M1s1), it dissociated from P4 at a rate (dissociation constant [koff], 0.188 ms1) similar to that of pITIM (koff, 0.154 ms1). These binding kinetics translate into comparable affinities of P4 (affinity constant [KD], 71.0 nM) compared with pITIM (KD, 47.2 nM). Singly phosphorylated peptides P2 and P3 also showed moderate affinities to GST-SH2-SHIP, 149 nM and 75.1 nM of KD, respectively. The pIg- ITAM peptide showed an approximately 10-fold lower affinity (KD, 402 nM) than P4 or pITIM, which may account for earlier findings that SHIP is phosphorylated only when BCR is coclustered with FcRII(b).4 View larger version (18K): [in this window] [in a new window]   Figure 6. Measurements of affinities between GST-SH2-SHIP fusion protein and phosphopeptides by surface plasmon resonance. Biotinylated phosphopeptides indicated were captured on a streptavidin-coated sensor chip, and GST-SH2-SHIP was injected for 5 minutes at a flow rate of 30 µL/min at 25°C. The chip was washed with binding buffer for a further 5 minutes to examine dissociation rates.                                View this table: [in this window] [in a new window]   Table 1. Kinetic parameters for the interaction of GST-SH2-SHIP with synthetic peptides Because SHIP has been found to bind to both P2 and P3 in vitro with moderate affinities, we also examined SHIP phosphorylation upon stimulation of OKT8 in THP-1 cells expressing CD8/IIa. In these experiments, we used a mutant construct in which the ITAM tyrosine residues positioned at amino acid 288 (CD8/Y1F), or at amino acid 304 (CD8/Y2F), or at both (CD8/Y1FY2F) were substituted with phenylalanines. The mutant receptor chimeras were used to test which tyrosine residues within the ITAM of FcRIIa are responsible for SHIP phosphorylation in vivo. FACS analysis showed comparable expressions of CD8 chimeras in THP-1 cells (Figure 7A). SHIP phosphorylation was induced upon stimulation of OKT8 in THP-1 expressing CD8/Y1F or CD8/Y2F, but the level was less than that induced by the wild-type chimera. However, the cells expressing CD8/Y1FY2F were incapable of phosphorylating SHIP (Figure 7B). These results indicate that either single tyrosine residue within ITAM of FcRIIa are capable of recruiting SHIP. This finding is consistent with the in vitro data showing that GST-SH2-SHIP is able to bind P2 and P3 (Table 1). View larger version (26K): [in this window] [in a new window]   Figure 7. Both tyrosine residues in FcRIIa ITAM are responsible for SHIP phosphorylation in vivo. (A) The expression of CD8 chimeras with substitutions of tyrosine residues with phenylalanine was examined by FACS analysis using biotinylated F(ab')2 fragments of OKT8 followed by FITC-conjugated streptavidin. Dotted lines indicate fluorescence of unstained cells. (B) The THP-1 transfectants were stimulated with biotinylated F(ab')2 fragments of OKT8 followed by streptavidin. The lysates from the cells were immunoprecipitated with anti-SHIP antibody, separated on SDS-PAGE gels, and blotted with antiphosphotyrosine (anti-pTyr) antibody. The filter was reprobed with anti-SHIP antibody. SHIP negatively regulates FcR-mediated phagocytosis independently of ITIM-containing FcRII(b) The findings shown above demonstrate that SHIP inhibits macrophage function through ITAM tyrosines in activating FcRs. To address the contribution of SHIP to inhibition of FcRIIa-triggered macrophage function, we transiently introduced GFP-SH2-SHIP into THP-1 cells. The SH2 domain has been shown to function as a dominant-negative form by inhibiting endogenous SHIP in B cells.39 Thus, cells expressing the SH2 domain should show enhanced function in FcRIIa-stimulated macrophages. The cells expressing GFP or GFP-SH2-SHIP were isolated by cell sorting and used for phagocytosis assay. After sorting, the population of THP-1 cells expressing GFP-SH2-SHIP or GFP alone was 95% or 98%, respectively (Figure 8A). For the phagocytosis assay, we used RBCs coated with Fab fragment of IV.3 which binds to only FcRIIa on THP-1, and not to FcRII(b).38 This assay system allows us to direct phagocytosis to FcRIIa and thereby exclude a contribution by FcRII(b). The average of duplicate samples of 2 separate experiments is shown in Figure 8B. We found that the phagocytic ability of THP-1 cells expressing GFP-SH2-SHIP was significantly enhanced compared with control transfectants. However, the extent of increase was less than that seen in either SHIP/ or FcRII/. These data indicate that SHIP is able to function as a negative regulator directly through ITAM-containing phagocytic receptors and independently of FcRII(b). Additionally, SHIP might have functions in macrophages that are induced independently of its SH2 domain. View larger version (18K): [in this window] [in a new window]   Figure 8. The introduction of SH2-SHIP enhances the phagocytic abilities in the absence of FcRII(b). (A) THP-1 cells were transiently transfected with GFP or GFP-SH2-SHIP. The GFP-positive cells were sorted and analyzed by FACS analysis. Dashed lines indicate untransfected THP-1 cells. (B) The sorted GFP-positive cells were incubated with RBCs coated with Fab fragments of IV.3 antibody for 20 minutes at 37°C. The results were expressed as the number of the internalized RBCs per 100 cells which phagocytosed at least one RBC (phagocytic index). Open and closed bars represent phagocytic indexes for GFP-expressing cells and GFP-SH2-SHIP-expressing cells, respectively. Results are shown as the averages of duplication and are representative of 2 independent experiments. Bars represent standard errors of duplicate samples.     Discussion Top Abstract Introduction Materials and methods Results Discussion References Recent studies indicate that FcR-mediated phagocytosis is negatively regulated by FcRII(b)21 and SHIP.22 However, the molecular mechanism of the negative regulation for phagocytosis has not been elucidated. In this report, we directly compared phagocytic rates and signal transduction events of macrophages from FcRII(b)/ and SHIP/ mice, as well as wild-type mice. We found that macrophages of both FcRII(b)/ mice and SHIP/ mice displayed a similar elevated phenotype regarding phagocytic potential, suggesting that FcRII(b) contributes to SHIP function in macrophages as it does in B cells. However, in contrast to this possibility, macrophages of SHIP/ but not FcRII(b)/ showed elevated intracellular Ca++ influx. Additionally, macrophages of FcRII(b)/ mice displayed efficient tyrosine phosphorylation of SHIP. These findings suggest that FcRII(b) and SHIP function independently of each other, but both inhibit phagocytosis. In support of the notion that SHIP functions independently of FcRII(b), we found that clustering a receptor chimera containing the ITAM tyrosine residues of the FcR -chain or human-restricted FcRIIa and without coengagement of FcRII(b) is sufficient for SHIP phosphorylation in a macrophage cell line. In vitro studies using phosphopeptides indicated that the SH2 domain of SHIP is able to interact with phosphorylated ITAM peptides, such as -, -, -, and -chains of T-cell receptor (TCR)/CD3 complex.40 It is unclear whether phosphopeptide binding reflects an event occurring in stimulated cells. Previous experiments in T cells showed minimal SHIP phosphorylation under stimulation conditions promoting efficient phosphorylation of ITAM tyrosines in TCR/CD3,41 suggesting that SHIP has low affinity for these ITAMs in live cells. We demonstrated efficient SHIP phosphorylation caused by clustering ITAM-bearing molecules, and that SHIP directly binds to the ITAM of FcRIIa with a comparable affinity to that of FcRII(b). Furthermore, we show using dominant-negative mutants, that SHIP negatively regulates FcRIIa-mediated phagocytosis independently of FcRII(b). Our findings are consistent with the possibility that the direct binding of SHIP to the phosphorylated ITAM-containing receptor, followed by SHIP phosphorylation, leads to the negative regulation of FcR-mediated phagocytosis. Although direct binding of SHIP to phosphorylated FcRIIa ITAM tyrosines represents the most simple explanation for our findings, we do not rule out the possibility of indirect binding through an adapter molecule. SHIP is known to interact with phosphorylated Shc42 and with Grb2.30,31 Either of these proteins might play an important role in SHIP recruitment to phosphorylated FcRIIa. The phosphopeptide pull-down experiment shown in Figure 5 indicated that SHIP was able to bind FcRIIa phospho-ITAM without cell stimulation. Although this observation argues against the need for an activation-induced adapter protein, the finding is based on synthetic peptides, which might disclose nonphysiologic interactions. Nevertheless, whether direct or indirect, our findings show that SHIP in macrophages engages activating receptors. This situation is in contrast to that in other hematopoietic cells where inhibitory phosphatases require pairing of ITIM- and ITAM-bearing receptors. Macrophages from SHIP- and FcRII(b)-deficient mice show similar enhanced phagocytic abilities. Our data show that SHIP is a mediator of ITAM-stimulated signal transduction and biology. It is unclear what enzyme is the mediator of FcRII(b)-induced inhibition. Analysis of synthetic phosphopeptides corresponding to the phosphorylated ITIM of FcRII(b) revealed binding to the protein phosphatases SHP-1 and SHP-2.43 It is possible that the negative regulation of phagocytosis by FcRII(b) is mediated by SHP-1. Indeed, FcR-triggered functions are enhanced in macrophages from motheaten viable mice, which are deficient in expression of SHP-1.44 However, FcRII(b) is expressed on monocytes/macrophages, and SHIP binds to the phosphorylated ITIM of FcRII(b) with 2-fold higher affinity than that of FcRIIa. Therefore, it is still possible that SHIP contributes to negative regulation for FcR-mediated phagocytosis by FcRII(b). Our observations indicate that negative signaling involving SHIP is concomitant with the positive, phagocytic signaling; that is, both positive and negative effects are induced through the identical ITAM-containing receptors. The situation is therefore distinct from other paired inhibitory receptors that function only under conditions of receptor coclustering. Based on these and previous findings, we propose that hematopoietic cells employ the 2 suppressor phosphatases SHP-1 and SHIP in very different ways, and that they are induced under very different conditions. One manner of employment is a general one which regulates but does not abort signal transduction and biology. The other manner of employment is a specific one that critically involves coclustering of an ITIM-bearing receptor, and completely blocks cell activation. Regulator phosphatases are induced by directly or indirectly engaging the ITAM of the activating receptor. Regulator phosphatases act to control the biochemical and biologic output, but cannot completely abort the response. These features are consistent with SHIP behavior in myeloid cells where it appears to be directly recruited to the receptor, does not require the ITIM-containing FcRII(b), and controls but does not block biologic function. Our earlier findings in B cells suggest that SHP-1 behaves similarly: SHP-1 is indirectly recruited to the activating receptor, its recruitment does not require the ITIM-containing FcRII(b), and SHP-1 controls but does not block biologic function.45 Lymphoid or myeloid cells can also employ phosphatases as specific inhibitors. Phosphatases working in this way act only under conditions of paired ITAM/ITIM coclustering, as in the cases of BCR/FcRII,4 BCR/PIR-B,12 FcR/gp49,13 or any of several activating NK cell receptors coclustered with KIR (reviewed in Taylor et al11). Thus, the specific inhibitory phosphatases require an ITIM-containing receptor for their induction. The mechanism for such exclusivity is phosphorylation of the ITIM tyrosine residue, and the selectivity of the SH2 domain of SHIP for phosphorylated tyrosines in an ITIM rather than the B-cell ITAM configuration, as shown in the affinity measurements summarized in Table 1. However, phosphatases working in this way are more potent and can completely block cell activation. These features are consistent with SHIP behavior in B lymphocytes. Experiments of signal transduction by granulocyte-colony-stimulating factor reveal a situation more like macrophage FcRs where SHIP behaves as a regulator phosphatase. PtdIns 3-kinase and SHIP are recruited by distinct positive and negative growth regulatory domains in the cytokine receptor and coordinately regulate growth signaling.46 SHIP appears to function as a general regulator phosphatase in other situations of cytokine-induced biology47,48 (reviewed in Liu et al49). Thus, SHIP appears to play a regulatory role to prevent hyperinflammation. This notion is consistent with the SHIP/ animal, which displays a pronounced hyperinflammatory phenotype,47,48 but a mild B-cell phenotype.50     Footnotes Submitted March 13, 2002; accepted June 17, 2002. Prepublished online as Blood First Edition Paper, July 12, 2002; DOI 10.1182/blood-2002-03-0787. Supported by National Institutes of Health grants CA64268 and AI41447. K.M.C. is a scholar of the Leukemia and Lymphoma Society. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: K. M. Coggeshall, The Oklahoma Medical Research Foundation, Program in Immunobiology and Cancer, 825 NE 13th St, Oklahoma City, OK 73104; e-mail: mark-coggeshall{at}omrf.ouhsc.edu.     References Top Abstract Introduction Materials and methods Results Discussion References 1. Daeron M. Fc receptor biology. 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