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The Journal of Immunology, 2001, 167: 844-854.
Copyright © 2001 by The American Association of Immunologists

Signal Transduction by Human-Restricted Fc{gamma}RIIa Involves Three Distinct Cytoplasmic Kinase Families Leading to Phagocytosis1

Damon S. Cooney*,{dagger}, Hyewon Phee*,{ddagger}, Anand Jacob*,§ and K. Mark Coggeshall2,*

* Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104; {dagger} Molecular, Cellular and Developmental Biology Program and Departments of {ddagger} Biochemistry and § Microbiology, Ohio State University, Columbus, OH 43210


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent experiments indicate an important role for Src family and Syk protein tyrosine kinases and phosphatidylinositol 3-kinase in the signal transduction process initiated by mouse receptors for IgG and leading to phagocytosis. Considerably less is known regarding signal transduction by the human-restricted IgG receptor, Fc{gamma}RIIa. Furthermore, the relationship among the Src family, Syk, and phosphatidylinositol 3-kinase in phagocytosis is not understood. Here, we show that Fc{gamma}RIIa is phosphorylated by an Src family member, which results in recruitment and concomitant activation of the distal enzymes Syk and phosphatidylinositol 3-kinase. Using a Fc{gamma}RI-p85 receptor chimera cotransfected with kinase-inactive mutants of Syk or application of a pharmacological inhibitor of Syk, we show that Syk acts in parallel with phosphatidylinositol 3-kinase. Our results indicate that Fc{gamma}RIIa-initiated monocyte or neutrophil phagocytosis proceeds from the clustered IgG receptor to Src to phosphatidylinositol 3-kinase and Syk.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IgG receptors for the Fc portion of IgG are expressed on cells of myeloid and lymphoid lineage and function in the innate immune system by providing phagocytic function to clear IgG-opsonized particulate Ags (reviewed in Refs. 1 and 2). The amino acid sequences of the various Fc{gamma}R extracellular domains are highly conserved; major differences are found in the proteins with which they associate or within the primary amino acid sequence of their cytoplasmic tails.

The mouse has three distinct Fc{gamma}R genes (I, II, and III). Fc{gamma}RI and -III associate with an immunoreceptor tyrosine-based activation motif (ITAM)3 -bearing {gamma}-chain, which mediates signal transduction events leading to phagocytosis. The ITAM sequence, found in all Ag and IgG receptors, is characterized by dual YxxI/L amino acid motifs separated by a 6- to 12-residue spacer (3). The cytoplasmic tail of murine Fc{gamma}RII contains the inhibitory immunoreceptor tyrosine-based inhibition motif (ITIM) sequence represented by a single YxxI/L sequence (reviewed in Refs. 4 5). Murine Fc{gamma}RII blocks cell activation when coclustered with an activating receptor, but does not provide phagocytic function. Thus, only {gamma}-chain-containing Fc{gamma}R of the mouse elicit phagocytosis.

The arrangement and structure of Fc{gamma}Rs in humans is more complex. A total of eight Fc{gamma}R genes are present: three for Fc{gamma}RI, three for Fc{gamma}RII, and two for Fc{gamma}RIII. Human Fc{gamma}RIa and Fc{gamma}RIIIa are the respective functional and structural equivalents of murine Fc{gamma}RI and Fc{gamma}RIII, mediating phagocytosis by interacting with ITAM-bearing {gamma}-chain. Human Fc{gamma}RIIb is the ITIM-bearing equivalent of the inhibitory murine Fc{gamma}RII and functions to block cell activation. Fc{gamma}RIIa is unique to humans and absent in mice. The remaining human-restricted Fc{gamma}R include Fc{gamma}RIIIb, which is a GPI-linked receptor, and three others (Fc{gamma}RIb, Fc{gamma}RIc, and Fc{gamma}RIIc), for which no distinct function has been assigned and that otherwise appear to operate as the a isoform within the same family.

Besides being unique to humans, Fc{gamma}RIIa is unique in other ways. First, Fc{gamma}RIIa is the only immunoreceptor in which the ITAM is present within the receptor itself rather than found on an associated molecule. Second, Fc{gamma}RIIa contains 12 amino acid residues separating the dual YxxI/L motifs within the ITAM and thus presents the longest spacer region of all ITAMs. Third, Fc{gamma}RIIa is the most widely expressed human IgG receptor, found on nearly all hemopoietically derived cells, including platelets, monocytes, macrophages, neutrophils, and various populations of lymphoid cells. When transfected into T cells (6) or COS fibroblasts (7, 8), Fc{gamma}RIIa expression converts these into phagocytic cells, capable of internalizing IgG-coated targets.

Signal transduction events associated with Fc{gamma}R-mediated phagocytosis have been studied largely in animal models or in Fc{gamma}R-transfected fibroblasts. For the murine {gamma}-chain-associated IgG receptors, the tyrosines of the ITAM are phosphorylated by an Src family protein tyrosine kinase (PTK). Thus, recent experiments using macrophages derived from animals deficient in expression of the Src family PTKs Lyn, Fgr, and Hck showed that the Fc{gamma}RI- and Fc{gamma}RIII-associated {gamma}-chain ITAM tyrosines were Src family PTK substrates, and that the Src PTKs greatly enhanced phagocytosis but were not absolutely essential for the process (9).

It is unclear which PTK family carries out phosphorylation of the ITAM tyrosines within the human-restricted Fc{gamma}RIIa. Earlier experiments showed Fc{gamma}RIIa stimulated the activity of two structurally and functionally distinct PTK classes, members of the Src family (10, 11) and Syk (12). Furthermore, both Src and Syk kinase family members were capable of phosphorylating the ITAM tyrosines of Fc{gamma}RIIa when applied to an in vitro kinase assay using the cytoplasmic tail of Fc{gamma}RIIa as a GST fusion protein (13) or upon coexpression of Fc{gamma}RIIa and Syk or Src family PTKs in a murine B cell line (14). Thus, either kinase family is conceptually capable of phosphorylating the Fc{gamma}RIIa ITAM. However, Src-deficient fibroblasts transfected with Fc{gamma}RIIa were capable of efficient phagocytosis only upon cotransfection with a Src family PTK (15), indicating that this PTK family is essential to Fc{gamma}RIIa-mediated phagocytosis, as it is for the murine {gamma}-chain-associated IgG receptors (9). In contrast, experiments with Syk-deficient mice (16, 17) indicated an essential role for this PTK in particle uptake by all mouse IgG receptors. Furthermore, earlier reports from our laboratory (18, 19) indicate that the Src homology (SH)2 domains of Syk were able to directly engage the phosphorylated ITAM of Fc{gamma}RIIa. Thus, although both Src- and Syk-PTK families play an important role in IgG receptor-mediated signaling and biology of both human and murine phagocytes, the relationship between these two kinase families in the signal transduction process triggered by the human-restricted Fc{gamma}RIIa is unresolved.

Once phosphorylated, the ITAM tyrosines of Fc{gamma}R and other immunoreceptors recruit SH2 domains of additional enzymes and adapter proteins that participate in the signaling process. ITAM recruitment of SH2 domain-containing adapter proteins and signaling enzymes is often sufficient for induction of the distal biological events. The p85 adapter and the p110 catalytic subunits of phosphatidylinositol 3-kinase (PtdIns 3-kinase) coimmunoprecipitate with Fc{gamma}RIIa (18, 19), indicating that PtdIns 3-kinase is one enzyme so recruited to the receptor. It is not known whether this association is ITAM-mediated and direct or indirect and through an adapter molecule. Other reports show that pharmacological inhibition of PtdIns 3-kinase with wortmannin (17, 20, 21) or LY294002 (our unpublished observations) prevents particle uptake. Thus, although PtdIns 3-kinase activity is important in phagocytosis, the precise molecular role and distal effectors of PtdIns 3-kinase in the process are unknown.

Previous experiments have established that an IgG receptor chimera composed of an extracellular domain of Fc{gamma}RI and an intracellular domain containing the p85 subunit of PtdIns 3-kinase is sufficient to induce particle uptake in transfected fibroblasts (22). The p85 regulatory subunit constitutively associates with the p110 catalytic subunit of PtdIns 3-kinase, and the resultant membrane-targeted p110 bound to the chimeric receptor will exhibit high and unrestricted PtdIns 3-kinase activity (reviewed in Ref. 23). However, similar findings regarding phagocytosis function were reported using fibroblasts that express an Fc{gamma}RI chimera with Syk as the intracellular domain (24). These observations of Syk and PtdIns 3-kinase indicate that either signaling enzyme appears to function as both necessary and sufficient to confer phagocytic function. Although it is conceivable that Syk and PtdIns 3-kinase are necessary for phagocytosis, it is paradoxical that both enzymes should appear sufficient by these measurements.

Our earlier studies of Fc{gamma}RIIa in human platelets indicated that the SH2 domain(s) of Syk directly bound the phosphorylated tyrosines within the ITAM and that the p85 subunit of PtdIns 3-kinase indirectly bound the receptor after activation (18, 19). Based on these findings, we proposed that Syk might act as an adapter protein to recruit the SH2 domain(s) of p85 following Syk tyrosine phosphorylation and association with the phosphorylated Fc{gamma}RIIa ITAM. According to this model, Syk would act proximal to PtdIns 3-kinase by directly facilitating its recruitment to activated Fc{gamma}RIIa.

We have investigated these issues in peripheral blood neutrophils and in the monocytic cell lines THP-1 (human) and RAW 264.7 (murine). We show that phosphorylation of the ITAM tyrosines within Fc{gamma}RIIa is probably due to an Src family PTK. First, phosphorylation was sensitive to the specific Src family PTK inhibitor PP2 (25), but not to the specific Syk inhibitor, piceatannol (26). Second, ITAM recruitment of SH2 domain-containing downstream effector molecules was sensitive to PP2 but not to piceatannol. Third, cotransfection of fibroblasts with the Src family member Lyn, but not Syk, induced potent phosphorylation of the Fc{gamma}RIIa ITAM tyrosine residues. Additionally, we found that the phosphorylated ITAM tyrosines of Fc{gamma}RIIa bound the SH2 domains of both Syk, as previously reported (19), and the p85 subunit of PtdIns 3-kinase.

We used pharmacological inhibitors and kinase-inactivated Syk to investigate the roles of PtdIns 3-kinase and Syk. In mouse monocytes transfected with a chimeric receptor composed of Fc{gamma}RI-p85 subunit of PtdIns 3-kinase, we found that phagocytosis was sensitive to the Syk inhibitor piceatannol or cotransfection with a kinase-inactive mutant of Syk. Thus, despite the fact that PtdIns 3-kinase is permanently recruited to the chimeric receptor, the phagocytic process still required Syk catalytic activity. These findings reveal a model in which the receptor is phosphorylated by an Src family PTK to concomitantly recruit the SH2 domains of Syk and PtdIns 3-kinase. Both these latter enzymes act in concert and are required for subsequent particle uptake by the Fc{gamma}R-bearing cell.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

THP-1 and RAW 264.7 monocytes were obtained from American Type Culture Collection (Manassas, VA). Human peripheral blood neutrophils were isolated from heparinized whole blood by density gradient centrifugation using Ficoll-Paque according to the manufacturer’s instructions (Cellgro, Herndon, VA). Briefly, whole blood was diluted with 6% dextran in saline, and erythrocytes were allowed to settle by gravity. The supernatant was underlaid with Ficoll-Paque with a density of 1.077 g/ml and centrifuged at 500 x g for 30 min. The neutrophils, present in the layer beneath the Ficoll, were collected and washed by centrifugation five times with PBS to remove Fc{gamma}RIIa+ platelets. The resulting cells were >98% neutrophils and were free of monocytes, platelets, and lymphocytes, as assessed by flow cytometry (data not shown).

Reagents

Anti-Lyn and anti-Syk Ab were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-mouse IgG F(ab')2 (GaM) was purchased from Pierce (Rockford, IL). The Fc{gamma}RIIa-specific mAb IV.3 was purchased from Medarex (Anandale, NJ); murine Fc{gamma}RII- and Fc{gamma}RIII-specific mAb 2.4G2 (27) and PE-labeled goat anti-rat IgG were purchased from BD PharMingen (San Diego, CA). Anti-phosphotyrosine (4G10) mAb was obtained from Upstate Biotechnology (Lake Placid, NY). Anti-p85 antiserum was prepared in rabbits and used as described previously (28, 29). PP2 was purchased from Calbiochem (La Jolla, CA); phosphoinositides and piceatannol were obtained from Sigma (St. Louis, MO). Biotinylated peptides were purchased from Quality Control Biochemicals (Hopkinton, MA). Fc{gamma}RIIa peptides used were P1–P4, corresponding to the unphosphorylated (P1), N-terminal (P2), or C-terminal (P3) monophosphorylated or bis-phosphorylated (P4) ITAM of Fc{gamma}RIIa and described previously (19), a peptide corresponding to Y292 of human Fc{gamma}RIIb (AENTITpYSLLMH), to Y239 of Shc (DHQYpYNDFPGKE), to Y493 of human CD66a (NEVTpYSTLNFEA), and to Y931 of murine SH2 domain containing inositol phosphatase (LNEMINPNpYIGM). These latter phosphopeptides were described and used previously in a similar capacity (30). cDNA encoding murine Lyn was obtained from Dr. A. DeFranco (University of California, San Francisco, CA); cDNA encoding Syk was obtained from Dr. K. Zoller (Ariad Pharmaceuticals, Cambridge, MA).

Lysis, precipitation, and immunoblotting

Cell lysis, immunoprecipitation, and immunoblotting were performed as previously described (18, 19). Briefly, monocytes were incubated with 10 µg/ml F(ab')2 of IV.3 (Medarex), washed in PBS, and stimulated by the addition of 30 µg/ml GaM to cluster the receptor. The cells were incubated at 37°C and lysed with TN1 buffer (50 mM Tris (pH 8.0), 10 mM EDTA, 10 mM Na4P2O7, 10 mM NaF, 1% Nonidet P-40, 125 mM NaCl, 3 mM Na3VO4, and 10 µg/ml each aprotinin and leupeptin). Postnuclear extracts were incubated overnight with the Ab of interest or with 1 µM biotinylated phosphopeptides, followed by the addition of protein G-agarose or neutravidin-agarose (Pierce). Samples were washed five times with 1 ml lysis buffer, resuspended in SDS sample buffer (60 mM Tris (pH 6.8), 2.3% SDS, 10% glycerol, and 0.01% bromophenol blue) and boiled at 95°C for 5 min. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose filters, probed with the Ab of interest, and developed by chemiluminescence using secondary Abs labeled with HRP. The immunoblotted material was viewed and quantitated using a LumiImager with LumiAnalyst software supplied by the manufacturer (Roche, Indianapolis, IN). In some cases filters were stripped of primary Ab with 2% SDS-0.1 M 2-ME in 0.1 M Tris (pH 6.8), washed, and reprobed. Total cell lysates were prepared by lysing 2 x 106 cells in 50 µl lysis buffer. SDS sample buffer was added to postnuclear extracts, and samples were boiled at 95°C for 5 min.

The Far Western analysis shown in Fig. 2Go used a p85 immunoprecipitate from 10 x 106 unstimulated THP-1 monocytes. The immunoprecipitated material was separated by SDS-PAGE and transferred to a nitrocellulose filter. The filter was cut into strips of equal size and probed with 10 µM P1–P4 biotinylated phosphopeptides overnight at 4°C. After washing to remove unbound peptide, the filters were developed by incubation with streptavidin-HRP and developed as described above.



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  		FIGURE 2. The p85 subunit of PtdIns 3-kinase directly binds to the phosphorylated ITAM of Fc{gamma}RIIa. Lysates of unstimulated THP-1 were immunoprecipitated with a polyclonal p85 antiserum. The immunoprecipitates were separated on SDS-PAGE and transferred to a nitrocellulose filter. The filter was cut into equal strips and incubated with the indicated biotinylated Fc{gamma}RIIa phosphopeptide or with no peptide (right strip). The filters were developed with streptavidin-HRP. The position of p85 is indicated on the right; the lower band is an unknown, endogenously biotinylated protein in THP-1 monocytes. Similar results were obtained in three separate experiments.

 
Measurements of cytoplasmic Ca2+

Monocytes were incubated with 5 µM Indo-1 acetoxymethyl ester in PBS for 30–40 min at 37°C. The cells were washed twice by centrifugation and resuspended to 1 x 106/ml in PBS containing 1 mM CaCl2 and 1 mg/ml BSA. The labeled cells were added to quartz cuvettes and placed in a thermostatically controlled chamber of a Perkin-Elmer (Norwalk, CT) LS50B spectrofluorometer. The samples were excited at 355 nm, and emission of Ca2+-bound indo-1 was recorded at 380 nm over a period of 15 min. After the baseline was established (3–4 min), F(ab')2 of IV.3 mAb were added to the cuvette to a final concentration of 10 µg/ml. After an additional 3–4 min, the receptor was clustered by the addition of F(ab')2 of GaM to a final concentration of 30 µg/ml. Indo-1 fluorescence was recorded for an additional 10–15 min.

In vitro kinase assays

Lyn and Syk from pervanadate (1 mM sodium orthovanadate and 0.6% H2O2)-stimulated THP-1 monocytic cells were immunoprecipitated as described previously (31) and resuspended in kinase buffer (20 mM HEPES (pH 7.4), 10 mM MgCl2, and 5 mM MnCl2). To each reaction, 10 µM ATP and 2–4 µCi [{gamma}-32P]ATP (3000 Ci/mmol) were incubated with the immunoprecipitated kinases for 10 min at 30°C in a total volume of 25 µl. The reaction was stopped by adding 5x SDS sample buffer (0.6 M Tris (pH 6.8), 50% glycerol, and 12% SDS) and incubating the samples at 95°C for 5 min. The phosphorylated products, including the autophosphorylated kinases themselves, were analyzed by 7.5% SDS-PAGE and identified by autoradiography. PtdIns 3-kinase activity of peptide-bound samples shown in Fig. 1Go was measured as described previously (28).



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  		FIGURE 1. PtdIns 3-kinase binds to the phosphorylated ITAM of Fc{gamma}RIIa. A, Lysates of resting (-) or anti-Fc{gamma}RIIa/GaM-stimulated (+) THP-1 human monocytes were incubated with 1 µM biotinylated P1–P4 for 2 h at 4°C, followed by streptavidin-Sepharose. The precipitated material was separated by SDS-PAGE, transferred to nitrocellulose filters, and probed with Abs to p85. The position of p85 is indicated on the right. The result is representative of six similar trials. B, In parallel, resting (-) or Fc{gamma}RIIa-stimulated (+) THP-1 whole-cell lysates were separated by SDS-PAGE, transferred to nitrocellulose filters, and probed with Abs to phosphotyrosine. The positions of nascent phosphorylated proteins are indicated on the right. The results are representative of two identical experiments. C, Lysates of resting (-) or Fc{gamma}RIIa-stimulated (3, 7, time in minutes) THP-1 monocytes were immunoprecipitated with Abs to Fc{gamma}RIIa. The immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose filters, and probed with Abs to phosphotyrosine ({alpha}pTyr). The filter was stripped and reprobed with Abs to Syk or p85, as indicated beneath the panels. The position of the target protein is indicated on the right. The results are representative of two trials. D, Phosphopeptide-bound proteins (P1, P4, M-pep) or an anti-p85 PtdIns 3-kinase immunoprecipitate were assessed for PtdIns 3-kinase activity by incubation with commercial PtdIns and radiolabeled ATP. The reaction products were separated by TLC. The migration of the PtdIns 3-kinase product, PtdIns-3P, is indicated at the right. Similar results were obtained in two separate experiments. E, The indicated biotinylated phosphopeptides (1 µM) were incubated with lysates of THP-1 monocytes, and bound proteins were collected with streptavidin-Sepharose. The precipitated material was separated by SDS-PAGE, transferred to nitrocellulose filters, and probed with Abs to p85. The position of p85 is indicated on the right. Similar results were obtained in two separate experiments.

 
Phagocytosis assays

The assay was essentially as previously described (8, 22). Phagocytosis initiated by the Fc{gamma}RI-p85 chimera used SRBC (Colorado Serum, Denver, CO) labeled with FITC (Molecular Probes, Eugene, OR) and opsonized by rabbit anti-SRBC stroma (Sigma). The opsonized RBC targets were added at a ratio of 50:1 to RAW 264.7 cells that had been incubated with 1% trypsin for 10 min at 37°C. Monocytes and targets were incubated at 37°C for 10–20 min in 50 µl PBS. The mixture was centrifuged at 500 x g for 5 min, and the supernatant was removed. To eliminate unincorporated RBCs, 900 µl water was added, and the cells were held for 45 s. The lysis reaction was stopped by the addition of 100 µl of 10x PBS, and the samples were washed twice in PBS by centrifugation. After washing, the samples were fixed with 2% paraformaldehyde and viewed with a Zeiss (New York, NY) fluorescent microscope. Uptake of RBC particles was quantitated by counting ~500 monocytes. The data are expressed as the percentage of cells that had internalized at least one RBC (percent phagocytic) as well as by the number of internalized RBCs per 100 phagocytes (phagocytic index).

Phagocytosis initiated by endogenous Fc{gamma}RIIa was assayed by labeling THP-1 monocytes or peripheral blood neutrophils with IV.3 mAb for 10 min on ice. Unbound mAb was removed by washing, and the cells were resuspended in PBS. SRBCs were labeled with PKH26 (Sigma) and biotinylated with N-hydroxysuccinimido-LC-biotin (Pierce). The RBCs were incubated with 200 µg/ml purified streptavidin (Sigma) and washed in PBS before the addition of 40 µg/ml biotinylated F(ab')2 of GaM. The GaM- and PKH26-labeled RBCs were mixed with the IV.3-labeled monocytes or neutrophils at a 50:1 ratio, and the phagocytosis assay was performed as described above.

Representative photomicrographs of cells that have bound and/or internalized the labeled RBC targets are shown in Fig. 6Go. Controls using RBCs lacking GaM, monocytes lacking the transfected Fc{gamma}R gene, or monocytes or neutrophils lacking the primary mAb IV.3 showed minimal particle uptake (see data in Figs. 6Go and 7Go). These controls indicate that despite the fact that the monocytes and neutrophils express other IgG receptors, particle uptake is directed toward Fc{gamma}RIIa, as designed.



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  		FIGURE 6. Phagocytosis by the human-restricted Fc{gamma}RIIa requires Syk kinase activity. A and B, Photomicrographs showing THP-1 monocytes before (A) and after (B) the water lysis step. The monocytes were incubated with IV.3 mAb against Fc{gamma}RIIa for 30 min on ice and washed, and presented RBCs were labeled with FITC and GaM. The cells incubated at 4°C form rosettes (A) due to Fc{gamma}RIIa binding. After incubation at 37°C, external RBCs are removed by brief exposure to water (B). Phagocytes that have bound RBCs but failed to internalize them appear with a diffuse label on their surface (cell labeled 1 in B). Internalized RBCs appear intact within phagocytes (cell labeled 2 in B). C and D, Human peripheral blood neutrophils (C) or THP-1 monocytes (D) were labeled with IV.3 mAb against Fc{gamma}RIIa and incubated with 25 µg/ml piceatannol or were left untreated. The phagocytes were presented PKH26-labeled RBC targets containing GaM to cluster the bound IV.3 (IV.3/GaM). Phagocytosis was quantitated as described in Materials and Methods and is expressed as the percentage of cells with at least one internalized RBC or as the average number of internalized RBCs per 100 phagocytes ({blacksquare}). Control samples were incubated at 4°C. This result is representative of five independent and identical trials.

 


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  		FIGURE 7. Monocyte phagocytosis induced by the Fc{gamma}R/p85 chimera requires Syk kinase activity. A, Murine RAW 264.7 monocytes were left untreated (0) or were incubated with 1% trypsin for the indicated time in minutes. The trypsinized and untreated cells were presented anti-sheep IgG-opsonized RBCs, and the percentage of uptake was determined by fluorescence microscopy. The results show the percentage of RAW 264.7 cells that internalized at least one RBC. These results are representative of at least four similar experiments. B, Murine RAW 264.7 monocytes were transfected with vector or human Fc{gamma}RI-p85, as indicated. After 24 h, the cells were incubated with 1% trypsin to remove the endogenous murine Fc receptors. The monocytes were then incubated in the presence or the absence of 25 µg/ml piceatannol as indicated and presented FITC-labeled RBCs that had been coated with rabbit anti-sheep RBC Abs. Phagocytosis was quantitated as described in Materials and Methods and is expressed as the percentage of cells with at least one internalized RBC or as the number of internalized RBCs per 100 phagocytes ({blacksquare}). Similar results were obtained in three separate experiments. C, RAW 264.7 cells were transfected with vector (lane 1) or with Fc{gamma}RI-p85 (lanes 2 and 3) and cotransfected with vector (lane 2) or kinase-inactive Syk (lane 3). After 24 h, to permit expression of the transfected genes, the cells were trypsinized and presented FITC-labeled RBCs that had been coated with rabbit anti-sheep RBC Abs. Phagocytosis was quantitated after counting at least 500 monocytes/sample. The data shown are the average and SE of three separate transfections and are expressed as the increase above the control value. The percentages of phagocytosis of 500 cells counted in the mock-transfected control for each of the three experiments were 1.7, 3.5, and 32. The phagocytic indexes in the same samples were 2.3, 5.5, and 58%, respectively. Inset, The identical experiment was conducted with RBC targets labeled with anti-human Fc{gamma}RI (10.1 mAb). The lane assignments are described above.

 
Cell transfections

COS-7 cells were transfected with 10 µg cDNA encoding vector, Lyn, or Syk and 2 µg cDNA encoding Fc{gamma}RIIa using effectene (Qiagen, Valencia, CA). Transfectants were analyzed after 24 h. RAW 264.7 monocytes were transfected by electroporation with 1 or 10 µg cDNA for the Fc{gamma}RI/p85 chimera; in some experiments, the cells were cotransfected with 5 µg cDNA for kinase inactive Syk or vector. Transfections were performed at room temperature in a Bio-Rad Gene Pulser (Bio-Rad, Hercules, CA) at 310 mV in 4-mm gap cuvette. The transfected cells were diluted in warm medium and cultured for 24–36 h before analysis. The cells were routinely treated with 1% trypsin (Sigma) for 10 min at 37°C to remove endogenous murine Fc{gamma}Rs. Generally, ~10% of transfected RAW 264.7 expressed the human Fc{gamma}RI/p85 chimera, based on analysis by flow cytometry using 10.1 mAb (PharMingen) specific for human Fc{gamma}RI. The efficiency of trypsin-mediated removal of the endogenous murine Fc{gamma}RI from RAW 264.7 ranged from 80–95%, based on their ability to form rosettes with and internalize IgG-opsonized RBCs. RAW 264.7 binding of free IgG (MOPC 141 mAb), indicating the presence of the high affinity Fc{gamma}RI, declined by about 90% after trypsinization (data not shown). Representative data on removal of the endogenous receptor by trypsin is shown in Fig. 7Go. The trypsinized RAW 264.7 cells still express murine Fc{gamma}RII/III, indicated by staining with 2.4G2 mAb (data not shown).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PtdIns 3-kinase p85 regulatory subunit is recruited directly to the phosphorylated ITAM of Fc{gamma}RIIa

The relationship between Syk and PtdIns 3-kinase in IgG receptor-mediated signal transduction is unclear. This is particularly true of the IgG receptors restricted to humans, such as Fc{gamma}RIIa, where studies of gene-targeted animals are not revealing. Based on earlier findings of Fc{gamma}RIIa signaling events in human platelets, Syk might act proximal to PtdIns 3-kinase by acting to recruit p85 subunit of PtdIns 3-kinase. In this model, Syk associates with the phosphorylated Fc{gamma}RIIa ITAM, becomes phosphorylated itself, and is then positioned to bind and recruit the SH2 domain(s) of the p85 subunit (19).

To test the alternate possibility that the SH2 domain(s) of the p85 subunit will directly engage the phosphorylated Fc{gamma}RIIa ITAM, we incubated lysates of resting or Fc{gamma}RIIa-activated THP-1 monocytes with biotinylated peptides corresponding to the Fc{gamma}RIIa ITAM. The peptides were completely unphosphorylated (P1), phosphorylated in the N- or C-terminal YxxL (P2 and P3, respectively), or bis-phosphorylated (P4). After collecting the peptides with streptavidin-agarose, the bound proteins were analyzed by immunoblot with antiserum against the p85 subunit of PtdIns 3-kinase. The results (Fig. 1GoA) demonstrate that p85 bound either monophosphorylated (P2 or P3) peptide equally well and regardless of prior monocyte activation. p85 bound the bis-phosphorylated ITAM (P4) ~2-fold more than either monophosphorylated peptide; binding was likewise independent of cell activation. The unphosphorylated peptide (P1) failed to bind p85. Anti-phosphotyrosine immunoblots of whole-cell lysates of the Fc{gamma}RIIa-activated monocytes (Fig. 1GoB) indicated that the cells were indeed activated. Thus, we detected tyrosine-phosphorylated proteins of 145, 72, and 42 kDa in cells that had been stimulated through Fc{gamma}RIIa and GaM, but not in unstimulated cells.

Peptide-binding studies show potential interactions, whereas results of coimmunoprecipitation analyses reveal actual interactions. We explored the kinetics of p85 and Syk association to phosphorylated Fc{gamma}RIIa by coimmunoprecipitation; the results are shown in Fig. 1GoC. For these experiments, Fc{gamma}RIIa immunoprecipitates from resting or IV.3- plus GaM-stimulated THP-1 monocytes were separated by SDS-PAGE. The filters were immunoblotted with anti-phosphotyrosine, stripped, and re-probed with anti-Syk and anti-p85 Abs. We observed that both Syk and the p85 subunit of PtdIns 3-kinase became associated with the receptor in activated, but not resting, monocytes, as we observed in the platelet system (19). Receptor association of both p85 and Syk was rapid and stable for up to 7 min after receptor clustering.

The p85 constituent of PtdIns 3-kinase represents the adapter subunit of the complex, while the catalytic portion is the p110 subunit. To test whether p85-receptor interactions represent recruitment of PtdIns 3-kinase enzymatic activity, we applied the material from the peptide pull-down experiments to an in vitro PtdIns 3-kinase assay; an anti-p85 immunoprecipitate from resting THP-1 monocytes was used as a positive control. We found (Fig. 1GoD) that the bis-phosphorylated Fc{gamma}RIIa ITAM peptide, but not the unphosphorylated peptide, was able to recover PtdIns 3-kinase activity from lysates of unstimulated THP-1 monocytes, as was a control phosphopeptide containing a pYMxM motif (M-pep), optimal for engaging the p85 SH2 domains (32). To control for phosphopeptide specificity, we tested a number of other biotinylated phosphopeptides for their ability to engage p85 by incubation in lysates of resting THP-1 monocytes. When compared with P4, none of these control peptides bound p85 (Fig. 1GoE), although they were competent in binding other SH2 domain-containing proteins (30) (not shown). These data reveal that the SH2 domain(s) of the p85 subunit of PtdIns 3-kinase is capable of binding either phosphorylated YxxL motif within the Fc{gamma}RIIa ITAM. Because p85 binding to the phosphorylated ITAM peptides is independent of monocyte activation and, hence, Syk phosphorylation, the data argue that p85 SH2 domain engagement to either YxxL motif is direct, rather than through a phosphorylated adapter protein such as Syk. However, these findings do not completely exclude the involvement of an unidentified adapter protein in p85/PtdIns 3-kinase recruitment.

To better address the issue of direct vs indirect p85 binding to ITAM tyrosines, we tested the ability of the Fc{gamma}RIIa peptides to bind the p85 subunit that was immobilized on filters. For these experiments, p85 was immunoprecipitated from resting THP-1 monocytes, separated by SDS-PAGE, and transferred to nitrocellulose filters. The filters were then probed with the various biotinylated Fc{gamma}RIIa peptides, washed, and probed with HRP-streptavidin. The results (Fig. 2Go) indicated that the unphosphorylated P1 peptide failed to bind p85, while P4 bound ~5-fold greater than either P2 or P3. We also observed an ~80-kDa protein that bound the secondary reagent in the absence of any biotinylated peptide, as revealed in the control (right lane), in which no peptide was added. These findings reveal that the p85 subunit of PtdIns 3-kinase is capable of direct binding to the receptor. Hence, phosphorylation of the ITAM tyrosines within Fc{gamma}RIIa is the only limiting feature of p85 SH2 domain engagement and PtdIns 3-kinase recruitment to the Fc{gamma}RIIa. In contrast to platelets, Syk does not play an adapter role in the ITAM recruitment process of p85/p110 in human monocytes.

Phosphorylation of ITAM tyrosines of Fc{gamma}RIIa is mediated by an Src family PTK

Although Syk does not seem to recruit PtdIns 3-kinase to Fc{gamma}RIIa, it may act proximal to PtdIns 3-kinase by phosphorylating the tyrosines of the ITAM and thus indirectly serving to recruit PtdIns 3-kinase. Previous studies using immunopurified Syk and recombinant forms of Fc{gamma}RIIa (13) or using a murine B cell line transfected with Syk and Fc{gamma}RIIa (14) indicated that Syk was capable of phosphorylating tyrosines within the ITAM. To resolve the potential of Syk vs Src family PTKs to phosphorylate the tyrosines within the ITAM of Fc{gamma}RIIa, we used pharmacological inhibitors of the two PTK families: PP2, a selective inhibitor of the Src family (25), and piceatannol, which inhibits Syk (26). We first tested the efficacy of two PTK inhibitors in their ability to block Fc{gamma}RIIa-mediated Ca2+ influx in human monocytes. Signaling events leading to increased cytoplasmic Ca2+ should be blocked by both inhibitors, because Src and Syk kinases families are proximal to increased Ca2+ induced by other immunoreceptors. Changes in indo-1 fluorescence due to increased cytosolic Ca2+ in THP-1 monocytes stimulated with Abs to Fc{gamma}RIIa are shown in Fig. 3GoA. The cells were treated with IV.3 mAb for 3 min, then the receptor was clustered by the addition of GaM polyclonal F(ab')2 Ab. The data show a rapid increase in indo-1 fluorescence only upon Fc{gamma}RIIa clustering with GaM.



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  		FIGURE 3. Selectivity of PP2 and piceatannol for Src-family and Syk PTKs, respectively. A, THP-1 monocytes were labeled with 5 µM indo-1/AM for 40 min and washed in PBS. Cells (1 x 106) were placed in a thermostatic cuvette; indo-1 fluorescence was excited at 355 nM, and Ca2+-bound indo-1 was recorded at 380 nM. After a baseline was established, the cells were treated with IV.3 mAb, followed by GaM to cluster the receptor. B and C, Indo-1-labeled THP-1 monocytes were incubated with the indicated concentrations of PP2 or piceatannol and stimulated as described in A. Shown is the percentage of maximal Ca2+ influx, indicated by peak indo-1 fluorescence, relative to that of untreated, stimulated THP-1 monocytes. Similar results were obtained in two separate experiments. D, THP-1 monocytes were activated by pervanadate stimulation, and lysates were immunoprecipitated with Abs against Lyn or Syk. The active kinases were incubated with radiolabeled ATP in the presence of the solvent (DMSO) or the indicated amounts of PP2 or piceatannol. The in vitro kinase reaction was stopped after 10 min by the addition of SDS sample buffer, separated by SDS-PAGE, and exposed to film. Similar results were obtained in three separate experiments.

 
Using the peak height as an indicator, we applied PP2 and piceatannol in dose-response experiments to determine the optimal inhibitor concentration. The data (Fig. 3Go, B and C) showed that Fc{gamma}RIIa-stimulated Ca2+ influx was completely blocked by 25 µg/ml piceatannol and 2 µg/ml PP2. We then applied these inhibitor doses to an in vitro kinase reaction using immunoprecipitated Lyn as a representative Src family kinase and Syk. The kinases were incubated in the presence of [{gamma}-32P]ATP, with or without the optimal doses of the inhibitors. The level of activity was determined by the extent of kinase autophosphorylation. The results shown in Fig. 3GoD indicated that PP2 blocked 90% of Lyn autophosphorylation at 2 µg/ml, but this dose of PP2 did not affect Syk autophosphorylation. Likewise, 25 µg/ml piceatannol reduced Syk autophosphorylation by >90%, but did not affect Lyn autophosphorylation. Thus, Syk, but not Src, kinase activity is blocked by 25 µg/ml piceatannol, and Src, but not Syk, kinase activity is blocked by 2 µg/ml PP2. These inhibitor concentrations were chosen to selectively inhibit the two PTK families.

To identify the PTK family responsible for phosphorylation of Fc{gamma}RIIa ITAM tyrosines, THP-1 monocytes were preincubated with 2 µg/ml PP2, 25 µg/ml piceatannol, or an equivalent amount of DMSO, the solvent for both inhibitors, as a control. The cells were coated with the anti-Fc{gamma}RIIa mAb IV.3, and the receptor was clustered by the subsequent addition of GaM. Fc{gamma}RIIa was immunoprecipitated from the resulting lysed samples, and the immunoprecipitated material was analyzed by anti-phosphotyrosine immunoblot. The data (Fig. 4GoA, upper panel) revealed that receptor phosphorylation was completely blocked by inclusion of the Src family PTK inhibitor PP2, and receptor phosphorylation was insensitive to the Syk inhibitor piceatannol. Reprobing the same filter with Abs to Fc{gamma}RIIa (Fig. 4GoA, lower panel) showed essentially equal amounts of the target protein. The chemiluminescent signal from these two filters was quantitated and determined as a ratio of phosphorylated Fc{gamma}RIIa to total immunoprecipitated Fc{gamma}RIIa to eliminate any variation in the amount of immunoprecipitated Fc{gamma}RIIa. The ratio increase from 8 to 53 in DMSO-treated samples, from 9 to 55 in piceatannol-treated samples, and from 9 to 12 in PP2-treated samples upon Fc{gamma}RIIa stimulation. These findings indicate a role for Src family PTKs in phosphorylation of the ITAM tyrosines of Fc{gamma}RIIa in monocytes and exclude a role for Syk.



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  		FIGURE 4. The Src inhibitor PP2, but not the Syk inhibitor piceatannol, blocks phosphorylation of the tyrosines within the ITAM of Fc{gamma}RIIa. A, Upper panel, THP-1 monocytes were stimulated (+) or not (-) with anti-Fc{gamma}RIIa/GaM in the presence of 2 µg/ml PP2 or 25 µg/ml piceatannol (Pic). Lysates were subjected to immunoprecipitation with IV.3 mAb, and the precipitates were analyzed by anti-phosphotyrosine immunoblot. The position of the receptor is indicated with an arrowhead. Similar results were obtained in three separate experiments. Lower panel, The same filter was stripped and reprobed with a polyclonal Fc{gamma}RIIa antiserum. The position of the receptor is indicated with an arrowhead. B, The signal corresponding to Fc{gamma}RIIa from the anti-pY and anti-Fc{gamma}RIIa blots in A was quantitated by LumiImager software and expressed as a ratio. The conditions were as described in A. C, Fc{gamma}RIIa-stimulated (+) or resting (-) THP-1 monocytes pretreated with 2 µg/ml PP2 or 25 µg/ml piceatannol (Pic) were lysed and subjected to immunoprecipitation with IV.3 mAb. The receptor immunoprecipitates were analyzed by immunoblot using a polyclonal antiserum against p85. The positions of the target proteins are indicated with arrowheads. Similar results were obtained in two separate experiments. D, The signal corresponding to p85 from the blot in C were quantitated by LumiImager software and expressed as the total amount. The conditions were as described in C.

 
The data shown in Figs. 1Go and 2Go indicated that ITAM phosphorylation is the only signaling event limiting p85/PtdIns 3-kinase recruitment to the Fc{gamma}RIIa. Thus, if the Fc{gamma}RIIa ITAM is phosphorylated by an Src family kinase and not Syk, recruitment of the p85 adapter subunit of PtdIns 3-kinase should be blocked by PP2 and not piceatannol. To test this prediction, we stimulated THP-1 monocytes with Abs to Fc{gamma}RIIa in the presence or the absence of 2 µg/ml PP2 or 25 µg/ml piceatannol. The immunoprecipitated receptor was then analyzed by immunoblotting with anti-p85 Abs (Fig. 4GoC) to test for recruitment of these proteins to the phosphorylated receptor. The amount of p85 coimmunoprecipitating with the receptor was quantitated, and the results are shown in Fig. 4GoD. We observed an interaction between Fc{gamma}RIIa and p85 only in Fc{gamma}RIIa-stimulated monocytes, consistent with the idea that receptor engagement by both these SH2 domain-containing proteins is limited by ITAM phosphorylation. Furthermore, the interactions of both SH2 domain-containing proteins to the phosphorylated ITAM were blocked by the inclusion of PP2, but not piceatannol. These findings further support a role for Src family and not Syk in phosphorylation of the ITAM tyrosines of Fc{gamma}RIIa in monocytes.

To confirm a role for Src family PTKs in this initial signaling event, we transfected the human Fc{gamma}RIIa gene into COS-7 fibroblasts and cotransfected the cells with cDNA encoding p72syk, p55lyn as a representative Src family PTK, or both kinases. The transfected or untransfected control fibroblasts were stimulated with pervanadate, and the transfected Fc{gamma}RIIa was immunoprecipitated. An anti-phosphotyrosine immunoblot analysis (Fig. 5GoA, upper panel) revealed potent Fc{gamma}RIIa tyrosine phosphorylation in the cells cotransfected with Lyn, but not in those cells cotransfected with Syk, despite the fact that the receptor was present and expressed in all transfected cells (Fig. 5GoA, lower panel). Receptor phosphorylation was not improved by transfection of receptor with both Lyn and Syk. Indeed, in three separate experiments, Lyn-mediated Fc{gamma}RIIa phosphorylation was substantially reduced by the presence of Syk. Both transfected kinases were expressed in the transfected COS cells, as detected in immunoblots of whole cell lysates using anti-Syk (Fig. 5GoB, upper panel) and anti-Lyn (Fig. 5GoB, lower panel). Finally, both transfected kinases exhibited enzymatic activity, as measured in an in vitro kinase reaction after immunoprecipitation (Fig. 5GoC). These observations of human Fc{gamma}RIIa are consistent with earlier findings showing a role for Src family PTKs in phosphorylation of the ITAM tyrosines in the {gamma}-chain associated with murine Fc{gamma}RI and -III (9). Thus, by these three different criteria the ITAM of the human-restricted Fc{gamma}RIIa is phosphorylated by an Src family PTK, and not by Syk. Furthermore, the data also show that Syk does not act upstream of PtdIns 3-kinase by affecting its recruitment to the receptor.



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  		FIGURE 5. Transfection of Fc{gamma}RIIa and Lyn, but not Syk, into fibroblasts permits receptor phosphorylation. A, COS-7 fibroblasts were transfected with nothing (mock) or cDNA encoding Fc{gamma}RIIa. The Fc{gamma}RIIa-transfected COS-7 cells were cotransfected with an equal amount of cDNA of vector, wild-type Syk or Lyn, or both kinases as indicated. The cells were treated with pervanadate, and lysates were subjected to immunoprecipitation with IV.3. The immunoprecipitated receptor was analyzed by anti-phosphotyrosine immunoblots (upper panel) or anti-Fc{gamma}RIIa immunoblots (lower panel). The position of the receptor is indicated with an arrowhead. Identical results were obtained in three separate experiments. B, Whole-cell lysates of the transfected cells were separated by SDS-PAGE, transferred to filters, and immunoblotted (IB) with anti-Syk (upper panel) or anti-Lyn (lower panel). C, Lysates of cotransfected COS-7 cells were immunoprecipitated (IP) with normal rabbit sera (NRS), normal mouse Ig (NMIg), or Abs to Lyn or Syk as indicated. The immunoprecipitates were incubated with radiolabeled ATP, and the reaction products were separated by SDS-PAGE. The autophosphorylated kinases were detected by autoradiography. Similar results were obtained in two separate experiments.

 
Syk and PtdIns 3-kinase act concomitantly during phagocytosis by Fc{gamma}RIIa

The data shown above indicate that Syk does not participate in Fc{gamma}RIIa ITAM phosphorylation, nor does it contribute to p85/PtdIns 3-kinase recruitment to the receptor. Hence, Syk does not act upstream of PtdIns 3-kinase in Fc{gamma}RIIa signal transduction by any of these measurements. However, Syk might act concomitant with PtdIns 3-kinase following ITAM phosphorylation by an Src family PTK.

To investigate this possibility, we treated with IV.3 mAb human neutrophils (which express Fc{gamma}RIIa and Fc{gamma}RIIIb) or human THP-1 monocytes (which express Fc{gamma}RIIa and Fc{gamma}RIa). The cells were then incubated in the presence or the absence of the Syk inhibitor 25 µg/ml piceatannol and provided fluoresceinated target RBCs coated with GaM as a F(ab')2 to cluster the IV.3-bound receptor. Phagocytosis was allowed to proceed in vitro for 10 min (for the neutrophils) or 40 min (for the monocytes). The cells were then exposed to an osmotic shock sufficient to lyse all external RBCs, but not those that had been phagocytosed. Particle uptake was quantitated by counting neutrophils or monocytes that had internalized at least one RBC (percent phagocytic) and by counting the number of internalized RBCs per 100 monocytes or neutrophils (phagocytic index). A photomicrograph of THP-1 monocytes before (Fig. 6GoA) and after (Fig. 6GoB) the osmotic shock shows the appearance of RBC rosettes, consisting of the labeled RBCs engaged to the Ab-targeted IgG receptor of the phagocyte. Phagocytes that internalized RBCs, and thus protected them from the water lysis, show intracellular labeled, intact RBCs (the cell labeled 2 in Fig. 6GoB). Phagocytes that expressed the Ab-targeted receptor but failed to internalize the labeled RBCs show externally bound fluorescence, derived from water-lysed RBCs (the cell labeled 1 in Fig. 6GoB).

This assay was used to explore the sensitivity of Fc{gamma}RIIa-mediated phagocytosis by ex vivo neutrophils or the monocytic cell line THP-1. We observed that neutrophils (Fig. 6GoC) or monocytes (Fig. 6GoD) preincubated with IV.3 were capable of phagocytosis (IV.3/GaM). Cells that were not labeled with IV.3 failed to phagocytose targets (control), indicating that the phagocytic signal is dependent on the primary IV.3 Ab and is thus a measurement of phagocytosis initiated by Fc{gamma}RIIa, but not other IgG receptors expressed in these cells. Neutrophils or monocytes that were labeled with IV.3 and treated with 25 µg/ml piceatannol displayed a severe block of particle internalization (IV.3/GaM+piceatannol). Thus, like the murine IgG receptors associated with the ITAM-containing {gamma}-chain (Fc{gamma}RI and Fc{gamma}RIII) (16, 17), phagocytosis by the human-restricted Fc{gamma}RIIa requires Syk catalytic activity.

To examine the ability of PtdIns 3-kinase to initiate phagocytosis in the absence of Syk, we made use of the Fc{gamma}RI-p85 chimeric molecule (22), consisting of the extracellular and transmembrane regions of the human Fc{gamma}RI molecule fused with the p85 regulatory subunit of PtdIns 3-kinase. When expressed in cells, the p85 molecule of the chimera will associate with the catalytic p110 subunit and generate the PtdIns 3-kinase product, phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5P3), by virtue of its proximity to the plasma membrane (33). Hence, PtdIns 3-kinase activity is not limiting for the Fc{gamma}RI-p85 receptor chimera. For these experiments the murine cell line RAW 264.7 was transfected with the chimeric Fc{gamma}RI-p85 receptor. Twenty-four hours after transfection the cells were treated with trypsin to remove the extracellular portion of the endogenous murine Fc{gamma}RI (34); the transfected human molecule lacks the trypsin site and hence is not removed by this treatment.

We quantitated the phagocytic potential of the trypsinized or untrypsinized RAW cells using the assay described above. The trypsinized RAW 264.7 cells displayed a progressive decrease in their ability to internalize IgG-coated RBCs with increasing time of exposure to trypsin (Fig. 7GoA). Thus, without trypsin essentially all RAW 264.7 monocytes were able to internalize at least one IgG-opsonized target. After exposure to trypsin for 10 min, <5% of the cells took up any target. These data indicate that trypsinization efficiently removes any functional murine IgG receptor(s) on the surface of RAW 264.7 monocytes capable of eliciting phagocytosis. Flow cytometric analysis of RAW 264.7 indicated that trypsinization caused an ~90% reduction in binding to free IgG (MOPC 141 mAb), which reveals the high affinity Fc{gamma}RI. However, staining the cells with 2.4G2 mAb, which has specificity for murine Fc{gamma}RII and Fc{gamma}RIII (27), revealed no change after trypsinization. Murine Fc{gamma}RII and Fc{gamma}RIII lack the trypsin site present in Fc{gamma}RI. Thus, although the trypsinized cells have no IgG receptor capable of eliciting phagocytosis, they express Fc{gamma}RII, Fc{gamma}RIII, or both, but are greatly reduced in Fc{gamma}RI expression.

RAW 264.7 monocytes were then transfected by electroporation with 10 µg cDNA of Fc{gamma}RI/p85 chimera or 10 µg control, vector-only cDNA. Twenty-four hours later the cells were presented IgG-coated fluorescent RBC targets in the presence or the absence of piceatannol, and the number of internalized particles was counted by fluorescence microscopy. The results of this analysis (Fig. 7GoB) indicated that control, vector-only transfected cells failed to internalize a significant number of particles due to the trypsin-mediated removal of the endogenous receptors. However, RAW 264.7 monocytes that were transfected and expressed the Fc{gamma}RI/p85 chimera internalized 7-fold more RBC targets than the vector-transfected control samples, indicating that transfection of the receptor chimera reconstitutes phagocytic function in the trypsinized cells. Hence, phagocytosis in this system is entirely dependent on the transfected human-restricted receptors that are trypsin resistant. We further observed that piceatannol treatment efficiently blocked phagocytosis of the targets when they were internalized by the Fc{gamma}RI/p85 chimera. These findings indicate that Syk enzymatic activity is indeed required for phagocytosis, even in the presence of activated PtdIns 3-kinase.

As an alternative approach to this issue, we transfected RAW 264.7 with 1 µg cDNA of Fc{gamma}RI/p85 chimera and 5 µg control, vector-only or 5 µg kinase-inactive Syk cDNA. Twenty-four hours later the cells were presented IgG-coated fluorescent RBC targets, and the number of internalized particles was quantitated by fluorescence microscopy. In this cotransfection model, the total number of internalized particles is much less than that shown in Fig. 7GoB because of the amount of transfected receptor chimera cDNA (10 vs 1 µg). The lower amount was chosen for cotransfection to ensure that receptor-expressing cells likewise coexpressed kinase-inactive Syk. The results of three separate experiments are shown in Fig. 7GoC. The data indicated that cells transfected with the chimeric receptor and cotransfected with vector were phagocytosis-competent. In contrast, and like piceatannol-treated cells, the RAW 264.7 monocytes transfected with the receptor chimera and cotransfected with kinase-inactive Syk failed to undergo phagocytosis of IgG-opsonized particles. To ensure that the low level phagocytosis was not due to residual amounts of the trypsinized murine receptor, we conducted the identical experiment using RBC targets labeled with anti-human Fc{gamma}RI (10.1 mAb). The results are shown in the inset to Fig. 7GoC and reveal an identical pattern of phagocytosis. These observations are consistent with the those described above using the Syk inhibitor piceatannol. Together, the findings demonstrate a requirement for Syk enzymatic activity in phagocytosis initiated by the wild-type IgG receptor of neutrophils and monocytes or an IgG receptor chimera composed of membrane-targeted PtdIns 3-kinase.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Biochemical analysis of signal transduction events induced by Fc{gamma}Rs indicate an important role for three kinase families: protein tyrosine kinases of the Src family and Syk, and the lipid kinase, PtdIns 3-kinase. However, the relationship between these kinase families is unclear, especially regarding the human-restricted Fc{gamma}R gene products. Because these genes are absent in animals, they are not amenable to analysis in gene-targeted mice.

We have analyzed the sequential involvement of these protein and lipid kinases in early signal transduction events induced in monocytes by Fc{gamma}RIIa, because they clearly play important roles for phagocytosis induced by IgG receptors of the mouse. By a combination of pharmacological and genetic approaches, we found that the ITAM tyrosines of the human-restricted Fc{gamma}RIIa are phosphorylated by an Src family kinase. Secondly, using phosphopeptides, Far Western analysis, and receptor immunoprecipitations, we observed that both Syk (18, 19) and the p85 subunit of PtdIns 3-kinase (this report) bind the phosphorylated ITAM of Fc{gamma}RIIa. However, phagocytosis by a receptor chimera harboring unregulated PtdIns 3-kinase activity was still sensitive to the Syk inhibitor piceatannol and by cotransfection with a kinase-inactive mutant of Syk. This finding indicates that Syk and PtdIns 3-kinase activities are jointly required for particle internalization by Fc{gamma}RIIa.

Our findings reported here place the action of the Src family PTK upstream of both Syk and PtdIns 3-kinase, because it is required to phosphorylate the tyrosines of Fc{gamma}RIIa and thereby promote recruitment of the distal enzymes. However, our data do not permit us to discern a proximal/distal relationship between Syk and PtdIns 3-kinase. Although our model proposes that both enzymes are concomitantly activated, it may be that one of these acts upstream and is required for activation of the other. In B cells, Syk kinase activity acts proximal to that of PtdIns 3-kinase (35). Likewise, in neutrophils stimulated through their entire complement of IgG receptors, piceatannol treatment blocked the activation of PtdIns 3-kinase, indicating that Syk acts proximal to PtdIns 3-kinase (36). Nevertheless, our data indicate that neither enzyme is sufficient to promote phagocytosis, and both enzymes are required. This idea is in contrast to earlier findings using fibroblasts as a model for phagocytosis (22, 24).

These findings suggest that, upon receptor clustering, the ITAM tyrosines of Fc{gamma}RIIa are phosphorylated by a member of the Src family. ITAM phosphorylation then elicits concomitant recruitment of Syk and the p85 adapter subunit of PtdIns 3-kinase via their tandem SH2 domains. Activated PtdIns 3-kinase generates PtdIns-3,4,5P3, which promotes signal transduction by binding to proteins through their pleckstrin homology (PH) domain, a conserved motif of amino acids found in numerous enzymes implicated in receptor-mediated signaling (2, 37). Upon PtdIns-3,4,5P3 binding, PH domain-containing enzymes translocate from the cytoplasm to the plasma membrane, where they carry out various functions. PH domain-containing enzymes distal to and dependent on PtdIns 3-kinase include Vav, Tec family PTKs, and phospholipase C{gamma}.

Vav probably plays an important role in phagocytosis, because it activates Rac, a member of the Rho family of GTPases, in a PTK-dependent manner (38, 39, 40). Several observations indicate a role for Rac in phagocytosis. First, dominant-negative versions of Rac block Fc{gamma}R-mediated phagocytosis in monocytes (41). Second, Rac-induced formation of pseudopods during phagocytosis is dependent on PtdIns 3-kinase (21), as is Vav-stimulated activation of Rac in fibroblasts (42). Third, inactivation of Rho family GTPases in the mouse J774 macrophage cell line completely blocks phagocytosis (43). The involvement of Vav itself in neutrophil or monocyte phagocytosis has not been studied, although Vav plays a role in target lysis by Fc{gamma}RIIIa in NK cells (40, 44). Tyrosine phosphorylation by Syk (45, 46) or an Src family (42) PTK is essential for stimulation of Vav activity. Thus, by extension to other models, PtdIns 3-kinase and Syk/Src-mediated activation of Vav probably plays an important role in Fc{gamma}R-stimulated phagocytosis in monocytes and neutrophils.

Bmx/Etk, expressed in monocytic cells (47), is a member of the Tec family of PTKs (reviewed in Ref. 48). Tec kinases contain a regulatory PH domain that binds PtdIns-3,4,5P3 with high affinity (49, 50). Like Vav, tyrosine phosphorylation of Tec family PTKs is obligatory for their activation (51, 52, 53). Although their precise role in ITAM-mediated signaling is unclear, evidence from other models indicates that Tec kinases contribute to Ag receptor-triggered increased cytoplasmic Ca2+ (54, 55) and support activation of phospholipase C{gamma} (54, 56). Induction of Bmx/Etk, the Tec family PTK present in human neutrophils and macrophages (47), has not been studied. However, clustering of human neutrophil IgG receptors induces Ca2+ influx that is dependent on PtdIns 3-kinase (57, 58). This observation is similar to lymphocyte immunoreceptors, which also show a Tec kinase and PtdIns 3-kinase-dependent Ca2+ influx (54, 55). An increase in cytosolic Ca2+ is essential for phagocytosis initiated by Fc{gamma}RIIa (59, 60).

The idea that Syk and PtdIns 3-kinase are concomitantly required for phagocytosis in monocytes is in contrast to earlier reports of fibroblasts transfected with IgG receptor chimeras containing Syk or the p85 subunit of PtdIns 3-kinase as the intracellular domain and indicating that either enzyme alone was sufficient to support phagocytosis. Clustering these receptor chimeras probably activates the respective enzymes to initiate phagocytosis in the fibroblast host, although this has not been documented. In any case, activation of Syk or of PtdIns 3-kinase by chimera receptor clustering does not preclude activation of other enzymes present in the host cell. That is, clustered Syk might activate PtdIns 3-kinase, and clustered PtdIns 3-kinase might activate a Syk homologue in fibroblasts. In addition, the earlier studies using receptor chimeras expressed the constructs in fibroblasts and measured fibroblast phagocytosis using IgG-opsonized RBCs. Thus, an alternative explanation for the discrepancy might be that particle uptake in fibroblasts uses a signaling pathway distinct from that in monocytes or neutrophils. Certainly, fibroblasts lack hemopoietically restricted proteins such as Syk, although they might express unidentified functional homologues of such proteins involved in IgG receptor signal transduction. Our findings are therefore consistent with an alternative model of IgG receptor-initiated particle uptake in which both Syk and PtdIns 3-kinase are required for phagocytosis and in which neither enzyme alone is sufficient.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants CA64268 and AI41447. K.M.C. is a Scholar of the Leukemia and Lymphoma Society (formerly Leukemia Society of America). Back

2 Address correspondence and reprint requests to Dr. K. Mark Coggeshall, Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, 825 Northeast 13th Street, Oklahoma City, OK 73104. E-mail address: mark-coggeshall{at}omrf.ouhsc.edu Back

3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibition motif; GaM, goat anti-mouse IgG; PH, pleckstrin homology; PtdIns, phosphatidylinositol; PtdIns 3,4,5P3, phosphatidylinositol-3,4,5-trisphosphate; PTK, protein tyrosine kinase; SH, Src homology. Back

Received for publication November 9, 2000. Accepted for publication May 10, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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