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Originally published online as doi:10.1189/jlb.0106019 on September 11, 2006

Published online before print September 11, 2006
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(Journal of Leukocyte Biology. 2006;80:1553-1562.)
© 2006 by Society for Leukocyte Biology

Differential kinase requirements in human and mouse Fc-gamma receptor phagocytosis and endocytosis

Zhen-Yu Huang1, Daniel R. Barreda1, Randall G. Worth2, Zena K. Indik, Moo-Kyung Kim, Paul Chien and Alan D. Schreiber3

University of Pennsylvania School of Medicine, Hematology and Oncology Division, Philadelphia, Pennsylvania, USA

3 Correspondence: University of Pennsylvania School of Medicine, Hematology and Oncology Division, Rm. 705 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104, USA. E-mail: schreibr{at}mail.med.upenn.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Fc gamma receptors (Fc{gamma}Rs) contribute to the internalization of large and small immune complexes through phagocytosis and endocytosis, respectively. The molecular processes underlying these internalization mechanisms differ dramatically and have distinct outcomes in immune clearance and modulation of cell function. However, it is unclear how the same receptors (Fc{gamma}R) binding to identical ligands (IgG) can elicit such distinct responses. We and others have shown that Syk kinase, Src-related tyrosine kinases (SRTKs) and phosphatidyl inositol 3-kinases (PI3K) play important roles in Fc{gamma}R phagocytosis. Herein, we demonstrate that these kinases are not required for Fc{gamma}R endocytosis. Endocytosis of heat-aggregated IgG (HA-IgG) by COS-1 cells stably transfected with Fc{gamma}RIIA or chimeric Fc{gamma}RI-{gamma}-{gamma} (EC-TM-CYT) was not significantly altered by PP2, piceatannol, or wortmannin. In contrast, phagocytosis of large opsonized particles (IgG-sensitized sheep erythrocytes, EA) was markedly reduced by these inhibitors. These results were confirmed in primary mouse bone marrow-derived macrophages and freshly isolated human monocytes. Levels of receptor phosphorylation were similar when Fc{gamma}RIIA was cross-linked using HA-IgG or EA. However, inhibition of Fc{gamma}R phosphorylation prevented only Fc{gamma}R phagocytosis. Finally, biochemical analyses of PI3K(p85)-Syk binding indicated that direct interactions between native Syk and PI3K proteins are differentially regulated during Fc{gamma}R phagocytosis and endocytosis. Overall, our results indicate that Fc{gamma}R endocytosis and phagocytosis differ dramatically in their requirement for Syk, SRTKs, and PI3K, pointing to striking differences in their signal transduction mechanisms. We propose a competitive inhibition-based model in which PI3K and c-Cbl play contrasting roles in the induction of phagocytosis or endocytosis signaling cascades.

Key Words: Fc-{gamma} • Syk kinase • Src-related tyrosine kinases • phosphatidyl inositol 3 kinase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Fc-gamma receptors (Fc{gamma}Rs) are essential for the recognition and elimination of immunoglobulin G (IgG)-opsonized pathogens and immune complexes, as well as clearance of senescent erythrocytes and platelets. They are important for immune surveillance mechanisms and have been implicated in immunohematologic and autoimmune disorders [1 2 3 4 5 ]. Fc{gamma}Rs bind to the constant region of IgG and mediate downstream events that are intimately linked to the size of the opsonized complex. Large immune complexes, such as IgG-opsonized microorganisms, are internalized by phagocytosis and culminate in pathogen death within the phagolysosomal compartment, presentation of microbial peptides in association with the MHC complex (i.e., antigen presentation), and activation of inflammatory responses, including NO production, superoxide formation, as well as chemokine, cytokine, and granule release [6 7 8 9 10 11 12 13 14 ]. Small immune complexes are internalized through endocytic pathways geared toward the efficient internalization of nutrients and other extracellular molecules, control of cell-surface receptor expression, regulation of signal transduction, modulation of plasma membrane composition, antigen presentation, and many other physiological processes [15 , 16 ].

Members of the human Fc{gamma}R family can be divided into four subclasses (Fc{gamma}RI, Fc{gamma}RII, Fc{gamma}RIII, and Fc{gamma}RIV) based on size, affinity for IgG, biochemical structure, reactivity with mAbs, and cellular and tissue distribution [1 , 2 , 17 ]. Among the activating Fc{gamma}Rs, Fc{gamma}RI and Fc{gamma}RIIA illustrate different structural requirements for induction of signaling events. Fc{gamma}RI functions as a multichain heterocomplex composed of a ligand binding {alpha} chain and a signal-transducing {gamma} subunit [1 ]. Central to the initiation of {gamma}-chain signaling is a classic immunoreceptor tyrosine-based activation motif (ITAM) consisting of two YxxL sequences separated by seven amino acids [18 ]. In contrast, Fc{gamma}RIIA functions as a single-chain transmembrane receptor containing both the ligand-binding extracellular domain and a signal-transducing cytoplasmic domain. The latter contains an ITAM-like domain where the two YxxL motifs are interspaced by 12 amino acids [19 ]. These and other structural differences provide a basis for heterogeneity in signal transduction and may be responsible for quantitative and qualitative variations in functional programs downstream of these Fc{gamma} receptors. For example, we have recently shown that Fc{gamma}RIIA and Fc{gamma}RI/{gamma} differ in their interaction with components of the phagocytic signaling cascade such as Syk- and Src-related tyrosine kinases [20 ].

The molecular processes underlying Fc{gamma}R-mediated phagocytosis and endocytosis differ dramatically, but it is unclear how the same receptors (Fc{gamma}R) binding to identical ligands (IgG) can elicit such distinct responses. Early observations suggested that distinct structural determinants on Fc{gamma}R are required for activation of endocytic and phagocytic events [21 ]. Subsequently, it was found that Fc{gamma}R-mediated endocytosis requires the assembly of clathrin at the site of receptor clustering [16 ]. Yet, Fc{gamma}R-mediated endocytosis is not dependent on the cytoskeleton nor does it require class I phosphatidyl inositol 3-kinase activity. In contrast, phagocytosis involves assembly of F-actin and is blocked by inhibitors of class I phosphatidyl inositol 3-kinase [22 , 23 ]. Most recently, we have found that Fc{gamma}R-mediated endocytosis requires clathrin, dynamin, and receptor-induced ubiquitination but is insensitive to cytochalasin, an inhibitor of actin polymerization [24 , 25 ]. In contrast, Fc{gamma}R-mediated phagocytosis is unaffected by inhibition of dynamin, reduced clathrin expression, or impairment of ubiquitination, but is blocked by cytochalasin.

In this study, we further dissect the molecular requirements that mediate Fc{gamma}R endocytosis and phagocytosis. We compared the requirement for three kinases generally associated with efficient Fc{gamma}R signaling: Syk kinase, Src-related tyrosine kinases (SRTKs) and phosphatidyl inositol 3 kinases (PI3K). First, we compared the requirement for these kinases in COS-1 cells stably-expressing Fc{gamma}RIIA or the chimeric receptor Fc{gamma}RI-{gamma}-{gamma} (EC-TM-CYT). Fc{gamma}RI requires association with the signal-transducing {gamma}-subunit for most signaling events. In the chimeric receptor Fc{gamma}RI-{gamma}-{gamma}, the transmembrane and cytoplasmic domains of the {gamma}-chain have been fused to the Fc{gamma}RI extracellular domain, thus furnishing a functional cytoplasmic ITAM for Fc{gamma}RI signaling within a single molecule. Our results in transfected COS-1 cells were confirmed in studies using freshly isolated human monocytes and primary mouse bone marrow-derived macrophages (BMM). Moreover, we determined that signaling pathways mediating Fc{gamma}R phagocytosis and endocytosis branch off early after receptor activation, as only phagocytosis requires Fc{gamma}R tyrosine phosphorylation. Interestingly, branching of these signaling pathways appears to be coupled to competitive inhibition of PI3K and c-Cbl as they bind to Syk. These competitive events may provide the basis for Fc{gamma}R signaling bias under different immune conditions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Construction of recombinant plasmids
The chimeric receptor Fc{gamma}RI–{gamma}–{gamma} [extracellular (EC)-transmembrane (TM)-cytoplasmic (CT) domains] was constructed by two-step overlap extension polymerase chain reaction as described [20 ]. Retroviral infection was used to prepare COS-1 cells permanently transfected with Fc{gamma}RIIA or Fc{gamma}RI-{gamma}-{gamma}. Fc{gamma}RIIA, or Fc{gamma}RI-{gamma}-{gamma} was inserted into the HindIII site of the retroviral vector pLNCX under the control of the CMV promoter. The resulting construct was transfected into the packaging cell line pA317. G418-resistant colonies were isolated and assessed for virus production, and supernatants from high viral producers were used to infect COS-1 cells.

Cell culture and transfection
COS-1 cells were cultured and maintained in DMEM containing glucose (4.5 mg/ml), glutamine (2 mM), streptomycin (100 U/ml), penicillin (100 µg/ml), and 10% heat-inactivated fetal bovine serum. COS-1 cell lines stably expressing the Fc{gamma} receptor Fc{gamma}RIIA or the chimeric receptor Fc{gamma}RI–{gamma}–{gamma} were constructed for neomycin resistance and selected using the neomycin analog G418 (500 µg/ml). G418-resistant cells were sorted for Fc{gamma}R expression by flow cytometry, and individual clones were obtained from anti-Fc{gamma}R antibody-positive cells by the limiting dilution method. We previously showed that only cells transfected with Fc{gamma}R-expressing constructs and not parental (untransfected) or mock-transfected (vector alone) cells mediate binding and internalization of immune complexes (EA or HA-IgG) [e.g., 26 27 28 ].

Murine bone marrow-derived macrophages (BMM) were cultured as described previously [29 ]. Femurs dissected from sacrificed C57/BL6 mice were flushed with 5 ml cold, sterile PBS (Life Technologies, Gaithersburg, MD). Bone marrow cells were washed and resuspended in DMEM (Life Technologies) containing glucose, glutamine (2 mM), and supplemented with 15% FBS (v/v), 10% L929 cell-conditioned medium (v/v, as a source of M-CSF), 100 µg/ml streptomycin, 100 U/ml penicillin, and 50 µg gentamicin. Mature BMM were harvested using ice-cold buffer, resuspended in DMEM containing glucose, glutamine (2 mM), and supplemented with 15% FBS (v/v) and 10% L929 cell-conditioned medium, and incubated at 37°C for 24 h before their use in our experiments.

Peripheral blood mononuclear cells from healthy individuals were isolated as described previously [30 ]. Briefly, the heparinized blood was centrifuged on Ficoll-Hypaque (Lymphocyte Separation Medium; Organon Teknika, Durham, NC), and interface cells were washed twice in PBS. Mononuclear cells were resuspended and maintained in RPMI 1640 medium (Life Technologies) containing 10% heat-inactivated fetal bovine serum, glutamine (2 mM), streptomycin (100 U/ml), and penicillin (100 µg/ml).

Binding and phagocytosis of IgG-sensitized erythrocytes (EA)
Antibody-coated sheep red blood cells (RBCs; Rockland, Gilbertsville, PA) were prepared in magnesium- and calcium-free PBS by incubating 109 sheep RBCs per ml with an equal volume of the highest subagglutinating concentration of IgG rabbit anti-sheep RBC antibody (Cappel Laboratories, West Chester, PA), as described previously [27 ]. The cells were overlaid with EA (108 cells/ml for a target: effector cell ratio of 10:1) and incubated at 37°C for 30 min. Unbound EA were removed by washing with PBS. Externally bound EA were removed by a short (40 s) hypotonic wash. The cells were stained with Wright's-Giemsa, and the number of cells with one or more internalized EA was determined in a blinded fashion by light microscopy. Over 300 cells were counted for each determination. For experiments with the SRTK-specific inhibitor PP2 (Calbiochem, San Diego, CA), the Syk kinase inhibitor piceatannol (Sigma, St. Louis, MO), or the PI3-kinase inhibitor wortmannin (Calbiochem, San Diego, CA), effector cells were preincubated for 30 min at 37°C with 1 ml PBS containing the specific inhibitor at the indicated concentrations or PBS alone. Following incubation, the cells were washed twice with PBS and EA added as described above to assay for phagocytosis. Phagocytosis is expressed as the phagocytic index (PI), the number of EA internalized per 100 cells. The phagocytic index was corrected for variations in cell surface receptor expression, as determined by flow cytometry.

Endocytosis assay
Human IgG (MP Biomedicals, Solon, OH) at 10 mg/ml in PBS (without calcium and magnesium) was aggregated by heating at 62°C for 20 min, followed by centrifugation at 13,000 g for 10 min to remove insoluble aggregates. The supernatant was used at a 1:100 dilution (100 µg/ml stock) to induce receptor cross-linking and endocytosis. Resolution of the immune complexes by sucrose density centrifugation indicated that the complexes contained 2 to 6 IgG molecules per complex. The protocol used for quantitation of endocytosis was modified from Odin et al. [31 ]. Briefly, paired sets of cells were incubated with heat-aggregated IgG-containing immune complexes (IC) on ice for 45 min and washed with ice-cold PBS. One set was maintained at 0°C for an additional 30 min and used as a binding control. The other set was rapidly warmed to 37°C and incubated for 30 min unless otherwise specified. Cells were then washed with ice-cold PBS, incubated for 30 min on ice with 3.5 µg/ml phycoerythrin (PE) conjugated goat anti-human IgG F(ab’)2 fragment (Jackson ImmunoResearch, West Grove, PA), and washed again with ice-cold PBS. Cells were detached from six-well culture plates in the presence of PBS (without calcium and magnesium) and fixed with 2% paraformaldehyde. The mean fluorescence of cells maintained on ice throughout the procedure was used as the binding reference (no endocytosis). Flow cytometric analysis of PE fluorescence illustrates the kinetics of IC internalization (Fig. 1 ; BD-FACScan; Becton Dickinson, San Jose, CA).


Figure 1
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  		Figure 1. FACS analysis of Fc{gamma}R-mediated endocytosis. Heat-aggregated IgG (HA-IgG) was added to COS-1 cells stably expressing human Fc{gamma}RIIA (RIIA) on ice, washed, and incubated at 37°C for different times. IgG complexes remaining on cell surface were then labeled with phycoerythrin (PE) conjugated goat anti-human IgG. Fc{gamma}R-mediated endocytosis was determined as outlined in Materials and Methods. COSIIA cells kept on ice during incubation with HA-IgG retained the greatest amount of IgG on the surface and thus appear brightest (shaded curve). 10-min (thick line), 20-min (solid line), and 30-min (dashed line) incubations at 37°C stimulated time-dependent internalization of HA-IgG, thus resulting in lower fluorescence intensities. In this representative experiment, COSIIA cells endocytosed 73% of HA-IgG after 30 min [mean fluorescence = 388 (37°C), 1431 (ice)]. Control experiments confirmed that the decrease in mean fluorescence intensity was a direct result of internalization of HA-IgG and not its dissociation from the cell surface (see Materials and Methods). We chose the 30-min time-point for subsequent experiments, as this provided optimal conditions for analysis of HA-IgG endocytosis.

 
We have previously demonstrated that cell surface expression of Fc{gamma}R is necessary for IgG binding [26 27 28 ]. IgG binding to Fc{gamma}RIIA-expressing COS-1 cells was absent when cells were preincubated with mAb IV.3 (which blocks binding sites on Fc{gamma}RIIA for IgG), verifying the specificity of IgG binding to Fc{gamma}RIIa in our system (not shown). Blocking antibodies specific for Fc{gamma}RI are not available. Instead, we used monomeric IgG, which binds Fc{gamma}RI with high affinity, to block binding sites for IgG on Fc{gamma}RI. Fc{gamma}RI transfected COS-1 cells pretreated with monomeric IgG did not bind heat-aggregated IgG.

We also have demonstrated that the decrease in surface-bound IgG after incubation of cells at 37°C is due to internalization of the small immune complexes and not their dissociation from the cell surface. In these experiments, FITC-labeled IgG was bound to the cells at 0°C. After incubation at 37°C, cells were incubated with a PE-labeled goat anti-human F(ab’)2 fragment, as described above. Our data indicated that PE fluorescence, representing surface-bound IgG, decreased dramatically in the cells exposed to 37°C, whereras FITC fluorescence, representing both surface-bound and intracellular IgG, did not significantly change (data not shown). Further, we found that binding of HA-IgG requires expression of Fc{gamma}Rs. Finally, no significant internalization of HA-IgG occurs at 0°C.

Immunoprecipitation and Western blot analysis
COS-1 cell transfectants were stimulated with human heat-aggregated IgG or EA at 37°C for 20 min and washed twice in ice-cold PBS. Cells were lysed by incubation on ice for 30 min with 1.0 ml 1% Triton X-100 lysis buffer. The cell lysis solutions contained the following protease inhibitors: 1 mM NaVO4, 1 mM PMSF, 10 µg/ml aprotinin, 50 µg/ml leupeptin and 100 µg/ml soybean trypsin inhibitor. Cleared lysates from stimulated cells were obtained by centrifugation at 13,000 g at 4°C. Cleared lysates were immunoprecipitated with 2 µg/ml anti-Myc mAb 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoprecipitates were separated by SDS-PAGE. Proteins transferred to nitrocellulose were immunoblotted with 1 µg/ml anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid, NY) or 1 µg/ml anti-Myc mAb 9E10. Immunoblots were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (BioRad, Richmond, VA) and visualized by enhanced chemiluminescence reagent (ECL) (Amersham Pharmacia Biotech, Piscataway, NJ).

For comparison of PI3K(p85)-Syk interactions during Fc{gamma}R phagocytosis and endocytosis, freshly isolated human monocytes were stimulated with heat aggregated human IgG or EA at 37°C for 20 min. Cells were lysed by 1% BRIJ lysis buffer, which contains 1 mM NaVO4, 1 mM PMSF, 10 µg/ml aprotinin, 50 µg/ml leupeptin, and 1 mM EGTA. Cleared cell lysates were obtained by centrifugation at 12,000 rpm at 4°C. The lysates were immunoprecipitated with 4 µg/ml anti-Syk antibody (Upstate Biotechnology). Immunoprecipitates were separated by SDS-PAGE. Proteins transferred to nitrocellulose were immunoblotted with 1 µg/ml anti-PI3 kinase p85 antibody (Upstate Biotechnology), or 0.3 µg/ml anti-Syk antibody 4D10 (Santa Cruz Biotechnology). Immunoblots were developed with horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) and were visualized by enhanced chemiluminescence reagent (Amersham Pharmacia Biotech).

To examine overall changes in tyrosine phosphorylation between Fc{gamma}R phagocytosis and endocytosis, as well as to confirm the specificity and efficacy of the chemical inhibitors used, freshly isolated human monocytes were stimulated with heat-aggregated human IgG or EA at 37°C for 20 min in the presence or absence of inhibitors (PP2: 10 µM; piceatannol: 25 µg/ml; wortmannin: 100 nM; genistein: 10 µg/ml). Cells were lysed by 1% Triton X-100 lysis buffer, which contains 1 mM NaVO4, 1 mM PMSF, 10 µg/ml aprotinin, 50 µg/ml leupeptin, and 1 mM EGTA. Cleared cell lysates were obtained by centrifugation at 12,000 rpm at 4°C. The proteins in the lysates were separated by SDS-PAGE. Proteins transferred to nitrocellulose were immunoblotted with 1 µg/ml antiphosphotyrosine mAb 4G10 (Upstate Biotechnology), or anti-actin antibody (Santa Cruz Biotechnology). Immunoblots were developed with horseradish peroxidase-conjugated goat anti-mouse IgG, or horseradish peroxidase-conjugated donkey anti-goat IgG, respectively (Santa Cruz Biotechnology), and were visualized by enhanced chemiluminescence reagent (Amersham Pharmacia Biotech).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
General inhibition of tyrosine phosphorylation inhibits Fc{gamma}R-mediated phagocytosis and endocytosis
Protein tyrosine kinases are known mediators of Fc{gamma}R-signaling events. Inhibitors of these kinases, such as genistein, have been shown to block phosphorylation of several proteins involved in Fc{gamma}R signaling in monocytes and macrophages ([32 ] and references therein). Genistein specifically inhibits tyrosine-dependent protein kinases with almost no inhibition observed for serine- and threonine-dependent protein kinases. To examine differences in signaling requirements by Fc{gamma}Rs, we first compared phagocytosis of IgG-opsonized erythrocytes (EA) and endocytosis of HA-IgG in COS-1 cells stably transfected with human Fc{gamma}RIIA. Pretreatment of these cells with genistein resulted in a dose-dependent inhibition of Fc{gamma}R-mediated phagocytosis (Fig. 2 ). Similarly, we observed a dose-dependent suppression of endocytosis of small immune complexes (heat-aggregated IgG, HA-IgG), suggesting that protein tyrosine kinases are required for effective signaling in both Fc{gamma}R-mediated phagocytosis and endocytosis. Interestingly, experiments using a broad concentration range of genistein suggested that Fc{gamma}RIIA-mediated phagocytosis is more sensitive to reduction in tyrosine phosphorylation than is endocytosis (Fig. 2) . As little as 5 µg/ml of genestein caused a significant decrease in Fc{gamma}RIIA-mediated phagocytosis, whereas 10 µg/ml were needed in order to obtain comparable levels of endocytic inhibition.


Figure 2
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  		Figure 2. The general tyrosine kinase inhibitor genistein inhibits Fc{gamma}RIIA-mediated endocytosis and phagocytosis. Genistein was added to COS-1 cells stably expressing human Fc{gamma}RIIA (RIIA). Phagocytosis of IgG-coated RBC (EA) and endocytosis of heat-aggregated IgG (HA-IgG) were determined as outlined in the Materials and Methods. Each bar represents the mean ± SEM of 3 independent experiments. Significant differences between genistein-treated cells and corresponding untreated controls are marked by an asterisk (*, P<0.01). The average value for phagocytosis (PI) in the absence of tyrosine kinase inhibitors was 118, and the average value for endocytosis in the absence of tyrosine kinase inhibitors in the absence of tyrosine kinase inhibitors was 73%.

 
Inhibition of Src-related tyrosine kinases (SRTKs), Syk kinase, and phosphatidyl inositol 3-kinase (PI3K) inhibits Fc{gamma}R-mediated phagocytosis but not endocytosis
Among the human-activating Fc{gamma}Rs, Fc{gamma}RI and Fc{gamma}RIIA illustrate contrasting structural requirements for induction of signaling events. We previously observed that COS-1 cells stably expressing Fc{gamma}RIIA and {gamma}-chain-dependent Fc{gamma}Rs significantly differed in their efficiency for EA phagocytosis [33 ]. These differences in phagocytic ability were at least partially mediated through differences in the association between Fc{gamma}Rs and downstream molecules required for efficient signaling, such as Syk [20 ]. To compare Fc{gamma}RI- and Fc{gamma}RIIA-mediated phagocytosis and endocytosis, we used COS-1 cells stably expressing human Fc{gamma}RIIA, as well as the chimeric receptor Fc{gamma}RI-{gamma}-{gamma} (EC-TM-CYT). The use of the Fc{gamma}RI-{gamma}-{gamma} chimeric receptor precluded the intrinsic requirement of the native Fc{gamma}RI receptor for association with a separate {gamma} chain, thus allowing us to focus on the molecular events that occur after receptor activation.

Current models indicate that cross-linking of the Fc{gamma}R ligand-binding extracellular domain results in tyrosine phosphorylation of the cytoplasmic ITAM domain by members of the Src kinase family (SRTKs) [2 , 4 , 5 , 34 35 36 37 38 39 ]. Phosphorylated ITAMs then serve as docking sites for the SH2-containing signaling molecules, most notably Syk tyrosine kinase. Syk activation subsequently leads to activation of signaling cascades that involve a variety of molecules including Ca2+, protein kinase C (PKC), phospholipase A2 (PLA2), phospholipase C{gamma} (PLC{gamma}), phospholipase D (PLD), phosphatidyl inositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and GTPases of the Rho family. To further dissect the molecular requirements that mediate Fc{gamma}R endocytosis and phagocytosis, we used three well-known inhibitors of protein kinases. We investigated the effect of the SRTK inhibitor PP2, the Syk kinase-specific inhibitor piceatannol, and the PI3Kinase inhibitor wortmannin in internalization mediated by the human Fc{gamma}Rs, Fc{gamma}RIIA, and the chimeric receptor Fc{gamma}RI-{gamma}-{gamma}. Pretreatment of COS-1 cells stably expressing Fc{gamma}RIIA or the chimeric receptor Fc{gamma}RI-{gamma}-{gamma} with PP2 resulted in a dose-dependent inhibition of Fc{gamma}R-mediated phagocytosis (Fig. 3 ). In contrast, we observed little suppression of endocytosis of HA-IgG, suggesting that SRTKs are required for effective Fc{gamma}R-mediated phagocytosis but not for endocytosis (Fig. 3) . Because endocytosis by Fc{gamma}RIIA appeared to be modestly inhibited by 1 µM of PP2, we also examined the effects of this inhibitor at lower concentrations. Further analyses at sub-1 µM concentrations of PP2 also indicated that it does not significantly affect Fc{gamma}RIIA-mediated endocytosis (data not shown). We then compared the levels of Fc{gamma}R phosphorylation following Fc{gamma}R cross-linking with large (EA) or small (HA-IgG) immune complexes, and assessed the effects of SRTK inhibition following PP2 treatment. Analysis of Fc{gamma}RIIA phosphorylation after stimulation with HA-IgG or EA indicated that both ligands induced receptor phosphorylation (Fig. 4 ). Fc{gamma}RIIA phosphorylation levels were greater after receptor cross-linking with EA compared with HA-IgG (note band intensity in parallel anti-Myc control blots). PP2-mediated inhibition of Fc{gamma}R phosphorylation prevented only Fc{gamma}R-mediated phagocytosis, whereas no significant effect on endocytosis was observed. Similar results were obtained when we analyzed Fc{gamma}RI-{gamma}-{gamma} phosphorylation after cross-linking with HA-IgG or EA (data not shown). This suggested that preferential inhibition of Fc{gamma}R phagocytosis by PP2 was the result of a differential requirement for Fc{gamma}R phosphorylation of phagocytosis and endocytosis, rather than differences in the intrinsic ability of large or small immune complexes to induce Fc{gamma}R phosphorylation upon receptor cross-linking. Conversely, we previously found that elimination of all five cytoplasmic lysine residues (sites of ubiquitination) within the cytoplasmic domain of Fc{gamma}RIIA markedly impaired Fc{gamma}R endocytosis but did not affect phagocytosis nor phagosome maturation [25 ]. Together, these observations support the thesis that distinct Fc{gamma}R cytoplasmic domains mediate induction of phagocytosis and endocytosis signaling cascades. Fc{gamma}R phagocytosis appears to require phosphorylation of Fc{gamma}R ITAM Tyr residues by SRTKs [32 ]. In contrast, Fc{gamma}R endocytosis requires Lys residues [25 ] and Tyr-282 (unpublished data) within the Fc{gamma}R cytoplasmic domain for induction of endocytosis. Therefore, signaling events mediating Fc{gamma}R phagocytosis and endocytosis appear to branch off early after phosphorylation of the Fc{gamma}R cytoplasmic domain, the earliest detectable event following Fc{gamma}R receptor activation.


Figure 3
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  		Figure 3. The SRTK inhibitor PP2 inhibits Fc{gamma}RIIA and Fc{gamma}RI-{gamma}-{gamma}-mediated phagocytosis but not endocytosis. PP2 was added to COS-1 cells stably expressing human Fc{gamma}RIIA (IIA) or Fc{gamma}RI-{gamma}-{gamma} (RI-{gamma}-{gamma}). Phagocytosis of IgG-coated RBC (EA) and endocytosis of heat-aggregated IgG (HA-IgG) were determined as outlined in the Materials and Methods. Each bar represents the mean ± SEM of 3 independent experiments. Significant differences between PP2-treated cells and corresponding untreated controls are marked by an asterisk (*, P<0.01). A dose-dependent inhibition of phagocytosis was observed in both IIA and I-{gamma}-{gamma} stable transfectants.

 

Figure 4
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  		Figure 4. The SRTK inhibitor PP2 prevents Fc{gamma}RIIA phosphorylation after receptor cross-linking with heat-aggregated human IgG (HA-IgG) or IgG-opsonized erythrocytes (EA). COS-1 cells stably expressing human Fc{gamma}RIIA tagged with Myc/His were stimulated with small (HA-IgG; A) or large (EA; B) immune complexes in the presence or absence of 10 µM PP2. Lysates were immunoprecipitated using anti-Myc antibody and immunoblotted with antiphosphotyrosine (anti-PT) or anti-Myc antibody. pFc{gamma}RIIA-Myc/His denotes phosphorylated Fc{gamma}RIIA tagged with Myc/His.

 
Syk kinase is required for phagocytosis in human and murine macrophages and neutrophils [34 , 36 , 39 , 40 ]. In accordance with these reports, our experiments indicated that pretreatment of COS-1 cells stably expressing Fc{gamma}RIIA or the chimeric receptor Fc{gamma}RI-{gamma}-{gamma} with the Syk kinase inhibitor piceatannol results in a dramatic decrease in Fc{gamma}R-mediated phagocytosis (Fig. 5 ). In contrast, Fc{gamma}RIIA and Fc{gamma}RI-{gamma}-{gamma} endocytosis were unaffected by piceatannol-mediated inhibition of Syk kinase. Thus, our experiments indicate that Syk kinase is required for Fc{gamma}R phagocytosis, but not endocytosis, and are consistent with previous studies. For example, overexpression of Syk kinase significantly enhanced phagocytosis in cells expressing Fc{gamma}R, and inhibition of Syk expression using Syk antisense oligodeoxynucleotides or Syk–/– mice abrogated macrophage Fc{gamma}R phagocytosis [34 , 39 ]. In contrast, Syk overexpression had no significant effect on Fc{gamma}R-mediated endocytosis (unpublished observations).


Figure 5
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  		Figure 5. The Syk kinase inhibitor piceatannol inhibits Fc{gamma}RIIA and Fc{gamma}RI-{gamma}-{gamma}-mediated phagocytosis but not endocytosis. Piceatannol (25 µg/ml) was added to COS-1 cells stably expressing human Fc{gamma}RIIA (IIA) or Fc{gamma}RI-{gamma}-{gamma} (RI-{gamma}-{gamma}). Phagocytosis of IgG-coated RBC (EA) and endocytosis of heat-aggregated IgG (HA-IgG) were determined as outlined in the Materials and Methods. Each bar represents the mean ± SEM of 3 independent experiments. Significant differences between piceatannol-treated cells and corresponding untreated controls are marked by an asterisk (*, P<0.01).

 
Recent characterization of the molecular associations of Syk protein tyrosine kinase with downstream partners showed a direct interaction between Syk and PI3K [41 ]. PI3K interaction with Syk occurred at Syk phosphotyrosine 317 (pTyr-317), a site phosphorylated in trans by SRTKs. Our results show that pretreatment of COS-1 cells stably expressing Fc{gamma}RIIA or Fc{gamma}RI-{gamma}-{gamma} with the PI3K kinase inhibitor wortmannin resulted in dramatic inhibition of Fc{gamma}R-mediated phagocytosis (Fig. 6 ). In contrast, we observed no significant change in Fc{gamma}RIIA or Fc{gamma}RI-{gamma}-{gamma} endocytosis upon pretreatment with wortmannin. Thus, these experiments indicate that Fc{gamma}R-mediated internalization of large immune complexes (phagocytosis) requires PI3K, while Fc{gamma}R-mediated internalization of small immune complexes (endocytosis) does not. Interestingly, we found that induction of Fc{gamma}R phagocytosis greatly enhanced the association between PI3K(p85) and Syk, and this was most pronounced early in the Fc{gamma}R phagocytic response (5 min) (Fig. 7 ). In marked contrast, our results indicated that induction of Fc{gamma}R endocytosis triggered a significant decrease in PI3K(p85)-Syk association at the earliest time point observed (5 min) compared with the untreated control group, and this continued to decrease during the rest of the observation period (15 min). These results strongly support a model where PI3K(p85)-Syk association increases after Fc{gamma}R cross-linking with large immune complexes (e.g., EA) and promotes phagocytosis, whereas induction of Fc{gamma}R endocytosis leads to a decrease in PI3K(p85)-Syk binding, thus negatively regulating Fc{gamma}R phagocytic signaling.


Figure 6
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  		Figure 6. The PI3 kinase inhibitor wortmannin inhibits Fc{gamma}RIIA and Fc{gamma}RI-{gamma}-{gamma} phagocytosis but not endocytosis. Wortmannin (100 nM) was added to COS-1 cells stably expressing human Fc{gamma}RIIA (IIA) or Fc{gamma}RI-{gamma}-{gamma} (RI-{gamma}-{gamma}). Phagocytosis of IgG-coated RBC (EA) and endocytosis of heat-aggregated IgG (HA-IgG) was determined as outlined in Materials and Methods. Each bar represents the means ± SEM of 3 independent experiments. Significant differences between wortmannin-treated cells and corresponding untreated controls are marked by an asterisk (*, P<0.01).

 

Figure 7
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  		Figure 7. Effect of kinase inhibitors on Fc{gamma}R-mediated phagocytosis and endocytosis in human monocytes. (A) Freshly isolated human monocytes were treated with PP2 (10 µM), piceatannol (25 µg/ml), or wortmannin (100 nM). Phagocytosis of IgG-coated RBC (EA) and endocytosis of heat-aggregated IgG (HA-IgG) were determined as outlined in Materials and Methods. Effective phagocytosis of EA was inhibited by PP2, piceatannol, and wortmannin. In contrast, significant inhibition of endocytosis was not observed in the presence of these kinase inhibitors. Each bar represents the mean ± SEM of 3 independent experiments. Significant differences between treated cells and corresponding untreated controls are marked by an asterisk (*, P<0.01). (B) In control experiments, HA-IgG or EA were added to freshly isolated human monocytes in order to assess their effects on protein tyrosine phosphorylation following Fc{gamma}R stimulation. In addition, PP2, piceatannol, wortmannin, and genistein were included to assess their specificity and efficacy during inhibition of Fc{gamma}R signaling. Whole cell lysates were prepared as outlined in Materials and Methods and immunoblotted with the anti-phosphotyrosine mAb 4G10 or an anti-actin antibody. Inset shows a shorter film development for the EA group in order to facilitate comparisons between samples. (C) Fc{gamma}R-mediated phagocytosis and endocytosis differentially regulate PI3K(p85)-Syk binding. Freshly isolated human monocytes were stimulated with HA-IgG or EA, cell lysates were immunoprecipitated with an anti-Syk antibody, and immunoblotted with an anti-PI3K(p85) antibody or a anti-Syk antibody (loading control). Immunoblot with anti-PI3K(p85) antibody shows the dramatic increase observed in PI3K(p85)-Syk associations following EA stimulation; in contrast, HA-IgG stimulation resulted in decreased PI3K(p85)-Syk binding compared with unstimulated monocytes.

 
In addition to PI3K(p85), the only other protein reported to bind to pTyr-317 is c-Cbl [42 and references therein]. This proto-oncoprotein is a ubiquitin ligase (E3) and is a major target of tyrosine phosphorylation after Fc{gamma}R stimulation. Importantly, it is a negative regulator of Syk kinase [42 43 44 45 46 47 ]. Competition experiments indicated that the C-terminal SH2 domain of PI3K(p85) bound more tightly than the Cbl tyrosine kinase binding domain to Syk pTyr-317 [41 ]. Thus, on the basis of these published observations and our results presented above, it is possible that c-Cbl and PI3K play contrasting roles in the induction of Fc{gamma}R-mediated cellular responses, because of competition for the unique pTyr-317 site on Syk. PI3K binding to Syk would promote activation of phagocytosis signaling cascades downstream of PI3K. In contrast, increased access of Cbl to Syk pTyr-317 would inhibit Fc{gamma}R phagocytic cascades by eliminating molecular events downstream of PI3K and by promoting Cbl-mediated Syk poly-ubiquitination and subsequent Syk degradation. Unfortunately, in contrast to our PI3K-Syk binding experiments, we were unable to compare the direct interaction between Cbl and Syk in primary mononuclear cells during Fc{gamma}R phagocytosis and endocytosis due to the relatively small amounts of Cbl found (data not shown). However, further support for this thesis comes from experiments demonstrating that a mutated form of Cbl (70Z-Cbl), which lacks E3 ubiquitin ligase activity, enhanced Fc{gamma}R-mediated phagocytosis in COS-1 cells and murine P388D1 macrophages [42 ]. In contrast, this dominant/negative 70Z-Cbl mutant inhibited Fc{gamma}RIIA endocytosis [unpublished data]. Moreover, Fc receptor stimulation with small immune complexes has been shown to result in Cbl-mediated Syk polyubiquitination with subsequent targeting for proteasomal degradation [48 , 49 ].

Heterologous expression of Fc{gamma}RIIA and Fc{gamma}RI-{gamma}-{gamma} in COS-1 cells allowed us to examine the role that protein kinases play in endocytosis and phagocytosis events mediated by individual Fc{gamma}R. We confirmed these results in cells naturally expressing Fc{gamma}Rs by examining the requirement for SRTKs, Syk, and PI3 kinases in mononuclear cells ex vivo. As in COS-1 Fc{gamma}R stable transfectants, freshly isolated human monocytes (Fig. 7) and murine bone marrow-derived macrophages (BMM) (Fig. 8 ) showed a requirement for SRTKs, Syk, and PI3 kinases during native Fc{gamma}R phagocytosis of EA. In contrast, Fc{gamma}R endocytosis of HA-IgG in human monocyte or murine BMM was not significantly altered by pretreatment of cells with PP2, piceatannol, or wortmannin, further supporting our studies with COS-1 stable Fc{gamma}R transfectants. As expected, Fc{gamma}R cross-linking with large immune complexes (EA) led to higher levels of downstream tyrosine phosphorylation when compared with cross-linking with small immune complexes (HA-IgG). This observation suggested greater overall levels of receptor activation during Fc{gamma}R phagocytosis (Fig. 7) . Addition of PP2, piceatannol, wortmannin, or genistein led to marked decreases in overall protein tyrosine phosphorylation whether EA or HA-IgG was used to stimulate Fc{gamma}Rs, confirming the specificity and efficacy of these chemical inhibitors for inhibition of Fc{gamma}R signaling (Fig. 7) . Furthermore, biochemical analyses of PI3K(p85)-Syk binding in primary human monocytes (Fig. 7) indicated that direct interactions between native Syk and PI3K(p85) proteins are differentially regulated during Fc{gamma}R-mediated phagocytosis and endocytosis.


Figure 8
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  		Figure 8. Effect of kinase inhibitors on Fc{gamma}R-mediated phagocytosis and endocytosis in murine bone marrow-derived macrophages (BMM). The SRTK inhibitor, PP2 (10 µM), the Syk kinase inhibitor, piceatannol (25 µg/ml), and the PI3 kinase inhibitor, wortmannin (100 nM), were added to mouse BMM. Phagocytosis of IgG-coated RBC (EA) and endocytosis of heat-aggregated IgG (HA-IgG) were determined as outlined in Materials and Methods. Effective phagocytosis of EA was only observed in untreated controls. The decrease in fluorescence intensity in the FACS-based endocytosis assay upon 37°C incubation (green line) compared with controls incubated on ice (black line) is indicative of endocytosis. No significant inhibition of Fc{gamma}R-mediated endocytosis was observed with PP2, piceatannol, or wortmannin compared with untreated controls. Cytochalasin D (25 µg/ml) and sucrose (17%) represent known inhibitors of phagocytosis and endocytosis, respectively, and are included as controls [26 , 54 , 55 ].

 
Overall, our results further illustrate the marked differences that exist in signaling requirements between Fc{gamma}R-mediated phagocytosis and endocytosis. Together, with reports of other researchers, these results suggest the presence of mechanisms that negatively cross-modulate Fc{gamma}R phagocytosis and endocytosis (Fig. 9 ). These mechanisms appear to operate at various levels within Fc{gamma}R signaling cascades and may play important roles in the modulation of immune responses that are central to clearance of invading pathogens and/or the pathogenesis of autoimmune disorders. For example, evolutionary pressures often lead to the development of mechanisms in microbes that can exploit immune cellular processes to gain survival advantage. The gram-positive bacterium Listeria monocytogenes, which is 20 times the maximum accepted size for an endocytic particle, was reported to hijack the clathrin-mediated endocytic machinery in order to invade mammalian cells [50 ]. Cbl plays a crucial role in the bacterial invasion process by monoubiquitinating the hijacked Met receptor. Endocytosis of several other receptor tyrosine kinases is also dependent on Cbl-mediated monoubiquitination [51 52 53 ]. It is possible that additional pathogens may use similar strategies to increase Cbl activity in the cell, thus promoting bacterial uptake through endocytosis and down-regulating PI3K-mediated activation of Fc{gamma}R phagocytic killing responses. Conversely, activation of cellular processes that increase local PI3K activity may confer an advantage to immune defense mechanisms, as this would lead to increased killing through Fc{gamma}R phagocytosis and decreased uptake of L. monocytogenes through nonmicrobicidal endocytosis mechanisms.


Figure 9
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  		Figure 9. Models of Fc{gamma}R-mediated endocytosis and phagocytosis. Fc{gamma}R-mediated phagocytosis and endocytosis signaling cascades branch off early after receptor-ligand association. SRTKs mediate the phosphorylation of Fc{gamma}R cytoplasmic domains upon binding of large and small immune complexes with the subsequent association of kinases of the Syk/ZAP-70 family, but tyrosine phosphorylation is only required for Fc{gamma}R phagocytosis. Because phagocytic cup formation requires greater levels of receptor cross-linking, Fc{gamma}R phagocytosis may require the greater efficiency of receptor clustering that occurs following Fc{gamma}R phosphorylation by SRTKs [56 ]. After Fc{gamma}R activation, phosphorylation of Syk kinase provides a unique binding site (Tyr-317) for both PI3K and Cbl. Binding of the p85 C-terminal SH2 domain of PI3K with Syk Tyr-317 may be responsible for the sharp and transient accumulation of PI(3,4,5)P3 observed on the phagosomal cup during Fc{gamma}R phagocytosis [57 ] and is essential for the internalization of large immune complexes. Because Syk Tyr-317 binds preferentially to the PI3K p85 regulatory subunit, Cbl binding to Tyr-317 through its single phosphotyrosine binding domain may be dependent on relative PI3K activity. Cbl binding to Syk during endocytosis results in Syk poly-ubiquitination and its targeting for proteasomal degradation. This process requires activated Syk [48 , 49 ] and, thus, may be dependent on Fc{gamma}R phosphorylation, observed following receptor cross-linking with HA-IgG. Overall, the PI3K:Cbl competition for Syk binding appears to be important for 1) the induction of Fc{gamma}R phagocytosis, 2) Cbl-mediated inhibition of Syk activity and Fc{gamma}R phagocytic cascades, and perhaps 3) the modulation of Fc{gamma}R endocytosis. The negative cross-modulation between Fc{gamma}R endocytosis and phagocytosis observed at the level of Syk binding would complement competition events on the Fc{gamma}R cytoplasmic domain. In this case, competition between Cbl and Syk for binding with Fc{gamma}R ITAM tyrosines results in induction of endocytic or phagocytic events, respectively. Cbl-mediated mono-ubiquitination of Fc{gamma}R, which requires Fc{gamma}R ITAM’s Y-282, promotes endocytic uptake of Fc{gamma}R [unpublished data]. In contrast, phagocytosis of large immune complexes does not depend on Fc{gamma}R ubiquitination, but requires Syk association with Fc{gamma}R ITAM phosphorylated tyrosines. This, in turn, activates downstream events, including phosphorylation of the p85 regulatory subunit of PI3K [25 , 39 ].

 


   ACKNOWLEDGEMENTS
 
This study was supported by grants from the National Institutes of Health and a Natural Sciences and Engineering Council of Canada (NSERC) postdoctoral fellowship to D.R.B. R.G.W. is an Arthritis Foundation postdoctoral fellow.


   FOOTNOTES
 
1 These authors contributed equally to this work. Back

2 Current address: Medical Microbiology and Immunology, Medical University of Ohio, Toledo, OH 43614, USA. Back

Received January 11, 2006; revised June 21, 2006; accepted July 14, 2006.


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 RESULTS AND DISCUSSION
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