Fcγ receptor (FcγR)-mediated phagocytosis is known to require tyrosine kinases (TKs). We identified c-Cbl and Cbl-b as proteins that undergo tyrosine phosphorylation during phagocytosis. Cbl-deficient macrophages displayed enhanced FcγR-mediated signaling and phagocytosis. Surprisingly, binding of IgG-coated targets (EIgG) was also enhanced. c-Cbl-deficient macrophages expressed less FcγRIIb, the inhibitory Fcγ receptor; however, this did not account for enhanced target binding. We isolated the function of one Fc receptor isoform, FcγRI, using IgG2a-coated targets (EIgG2a). Cbl-deficient macrophages demonstrated a disproportionate increase in binding EIgG2a, suggesting that signal strength regulates binding efficiency toward opsonized targets. In resting cells, FcγRI colocalized with the Src family TK Hck in F-actin-rich structures, which was enhanced in Cbl-deficient macrophages. Target binding was sensitive to TK inhibitors, profoundly inhibited following depletion of cholesterol, and ablated at 4°C or in the presence of inhibitors of actin polymerization. Sensitivity of EIgG binding to cytoskeletal disruption was inversely proportional to opsonin density. These findings challenge the view that FcγR-mediated binding is a passive event. They suggest that dynamic engagement of TKs and the cytoskeleton enables macrophages to serve as cellular “Venus fly traps”, with the capacity to capture phagocytic targets under conditions of limiting opsonin density.

Fcγ receptor-mediated phagocytosis is essential for the clearance of IgG-coated pathogens. The phagocytic process is initiated when opsonized targets engage Fcγ receptors on the surface of phagocytes. The bulk of evidence suggests that this is a passive event. Subsequent clustering of Fcγ receptors triggers tyrosine kinase (TK)4-dependent cytoskeletal assembly, pseudopod extension, and engulfment of the phagocytic targets (reviewed in Refs. 1, 2, 3). To identify TK substrates relevant to phagocytosis, we purified a large number of phosphotyrosyl-containing proteins following Fcγ receptor stimulation. One such protein that we purified was c-Cbl, an E3 ubiquitin ligase. Cbl has previously been show to negatively regulate ITAM motif-dependent Ag receptor signaling and proliferation in T cells (4, 5) and B cells (6, 7), as well as IgE-mediated degranulation in mast cells (8, 9). Cbl is also known to undergo enhanced tyrosine phosphorylation following FcγR ligation (10, 11) and to associate with the SH3 domains of various Src family TKs in macrophages before and after engagement of Fcγ receptors (12). c-Cbl is a substrate of Src family kinases and Syk (13, 14), two families of kinases that are required for many ITAM-dependent events, including phagocytosis (15, 16). c-Cbl and Cbl-b are negative regulators of Syk kinase activity (7, 17). Although phagocytosis in cells lacking either c-Cbl or Cbl-b has not been reported, expression of a transforming mutant of c-Cbl enhances phagocytosis in COS cells transfected with Fcγ receptors and in the murine macrophage-like cell line P388D1 (18). Together with analogous data in other cells, this suggests that Cbl has the capacity to regulate Fcγ receptor-mediated phagocytosis.

In this study, we show that macrophages from c-Cbl−/− and Cbl-b−/− mice demonstrate enhanced Fcγ receptor-mediated tyrosine phosphorylation, phagocytosis, and, unexpectedly, target binding. The capacity of Cbl proteins to dampen Fcγ receptor-mediated signaling prompted us to test the role of signaling and cytoskeletal alterations in Fcγ receptor-binding activity. Our results challenge the widely held view that Fcγ receptor binding is a passive event and suggest that dynamic engagement of TKs and the cytoskeleton endow macrophages with the capacity to “capture” phagocytic targets, which may be particularly important in the early phases of the primary immune response, when opsonic activity might be limited.

c-Cbl−/− line 145 and littermate control mice were obtained from the National Institute of Allergy and Infectious Diseases repository (Taconic). Cbl-b−/− and littermate control mice were a generous gift from Dr. Hua Gu (Columbia University). All mice had been backcrossed to the C57BL/6 background for at least eight generations. Mice were kept in a specific pathogen-free facility (Columbia University). All experiments were approved by the Columbia University Institutional Animal Care and Use Committee.

Bone marrow-derived macrophages (BMDMs) were generated by harvesting stem cells from bone marrow. Cells were grown in complete medium (RPMI 1640, 10% (v/v) FCS, 20% (v/v) L929 conditioned media, 100 U/ml penicillin G, and 100 μg/ml streptomycin) in a tissue culture incubator maintained at 5% CO2 and 37°C.

The following primary Abs were used: actin (I-19), c-Cbl (C-15), Cbl-b (C-20), and FcγRI (N-19), Hck (N-30), c-Src (SRC2), Syk (LR), c-Cbl (G-1), and phosphotyrosine (PY99) from Santa Cruz Biotechnology; phospho-Src (Y418) (Invitrogen) and phospho-Syk (Y525/Y526) (Cell Signaling Technology); mAb X54-3/4 against FcγRI (from Dr. Mark Hogarth, Burnet Institute, Heidelberg, Australia); mAb K9.361 against FcγRIIb (from Dr. Ulrich Hammerling, Cornell Medical Center, New York, NY); mAb 2.4G2 against FcγRII/III/IV (BD Biosciences); mAb 9E9 against FcγRIV (from Dr. Jeffery Ravetch, Rockefeller University); mAb TIB191 (IgG1 isotype) against dinitrophenyl and mAb S-S.1 (IgG2a isotype) against sheep erythrocytes (American Type Tissue Collection); mAb against flotillin (clone 18; BD Transduction Laboratories); rabbit IgG against γ-chain subunit (Upstate Biotechnology); and rabbit IgG against sheep erythrocytes (MP Biomedicals). Peroxidase-anti-peroxidase immune complexes and secondary Abs were from Jackson ImmunoResearch Laboratories.

We batch-purified phosphotyrosyl-containing proteins that accrued during phagocytosis in RAW 264.7 cells (manuscript in preparation). In brief, 2 × 109 RAW 264.7 cells were incubated with IgG-coated erythrocytes (EIgG) ghosts and lysed in a Triton X-100-containing buffer; cleared lysates were applied to a mAb 4G10 affinity column, and phosphoproteins were eluted with 10 mM phenylphosphate. Following SDS-PAGE and silver staining, gel-resolved proteins were digested with trypsin, purified, and sequenced (see Extended Methods).5

Total RNA was purified, primed with random hexamers, reverse transcribed into cDNA, and subjected to quantitative PCR (see Extended Methods). FcγRI was stained with 2 μg/ml mAb x54-3/4 or with 10 μg/ml biotin-labeled mouse IgG2a in the presence of unlabeled 50 μg/ml mouse IgG2b. FcγRII was stained with 3 μg/ml biotinylated mAb K9.361 in the presence of 50 μg/ml unlabeled mouse IgG2a and IgG2b (to block binding of K9.361, which is an IgG2a isotype, to FcγRI and FcγRIV). FcγRIII was stained with 1 μg/ml biotinylated 2.4G2 mAb in the presence of 25 μg/ml unlabeled mAb K9.361 and 50 μg/ml mouse IgG2b. FcγRIV was stained with 10 μg/ml 9E9 mAb. Following addition of fluorochrome-conjugated secondary Abs or streptavidin, cells were fixed and subjected to flow cytometry.

Immunofluorescence was performed as described (19). Quantitation of γ-chain and phosphotyrosine content associated with attached particles and/or phagosomes was performed by microspectrofluorometry (19) using MetaMorph software (Universal Imaging). Fields were selected randomly and targets were identified using Cy5 fluorescence to detect the IgG opsonin. Rhodamine and fluorescein pixel intensities of circular regions encompassing targets and associated regions of macrophages were recorded. All fluorescence intensities were corrected for auto-, bleed-through, and background fluorescence intensities. For fluorescence microscopic images depicted in this study, raw images were subjected to a one-dimensional deconvolution processing algorithm (MetaMorph). All comparison images were imaged for the same exposure times and subjected to the same deconvolution processing parameters. Quantification of the extent of fluorescence overlap was performed using ImageJ software (rsb.info.nih.gov/ij).

EIgG was obtained by incubating 2 × 109 sheep erythrocytes with a subagglutinating titer of rabbit IgG against sheep erythrocytes. EIgG2a was obtained by incubating 2 × 109 sheep erythrocytes with a subagglutinating titer of mouse IgG2a against anti-erythrocytes. EIgG1 was obtained by derivatizing 2 × 109 sheep erythrocytes with 0.3% picrylsulfonic acid followed by incubation of E-DNP with mouse IgG1 against DNP. Following addition of opsonized erythrocytes, attached, but uningested targets, cells were detected by incubation with FITC-conjugated F(ab′)2 fragments directed against the particle opsonin for 20 min at 4°C. Cells were washed, fixed, permeabilized, and stained with rhodamine-conjugated F(ab′)2 fragments directed against the opsonin to detect total (ingested and uningested) targets. Association and phagocytosis indices were calculated as follows: 5–10 microscope fields (×600) were selected randomly and scored for total attached (rhodamine-labeled) and attached, but uningested (FITC-labeled), targets. Phagocytosis was calculated by subtracting the number of attached, but uningested, targets from the total number of targets associated with each macrophage. Phagocytosis efficiency was defined as the percentage of targets ingested as a fraction of all targets associated with the macrophages at the indicated time intervals. All cells within each field were scored for target association, and at least 100 macrophages were scored per coverslip. For experiments using inhibitors, cells were preincubated with the indicated concentrations of inhibitor for 30 min at 37°C before addition of targets, which were added for 20 min at 37°C. Cells were gently washed in PBS three times to removed unattached targets. In some experiments, cells were preincubated for 5 h in 10 mM methyl-β-cyclodextrin before addition of targets. This method of cholesterol depletion results in a 60% reduction of the total cellular cholesterol content in mouse BMDMs (20). Receptor-mediated endocytosis of FcγRI was performed according to a published method (21) (see Extended Methods for details).

Heat-aggregated mouse IgG2a was made by incubating mouse IgG2a (5 mg/ml in PBS) at 63°C for 20 min followed by removal of insoluble complexes by centrifugation (13,000 × g for 5 min). Following stimulation, cells were centrifuged and lysed in RIPA buffer, followed by sonication preclearing with protein A/G agarose for 45 min followed by incubation with protein A/G agarose conjugated to IgG against the γ-subunit or nonimmune rabbit IgG control for 90 min. Beads were washed extensively and boiled in Laemmli buffer for 5 min. Proteins were resolved by SDS-PAGE, transferred onto nitrocellulose, immunoblotted using the indicated Abs, and developed with ECL. Where indicated, band intensities were quantitated by densitometry using ImageJ software (v.1.38; rsb.info.nih.gov/ij).

Data analysis was performed using either repeated measures ANOVA for matched observations followed by Bonferroni post hoc analysis or, for data that failed the test for normality, Kruskall-Wallis test followed by Dunn’s post hoc analysis, using GraphPad Prism software v.4.0c (GraphPad Software).

To identify substrates of TKs in macrophages engaged in phagocytosis, we used affinity chromatography to purify phosphotyrosyl-containing proteins that accumulate following stimulation of a RAW 264.7 cell line with IgG-coated targets (manuscript in preparation). Among the most heavily phosphorylated proteins we detected was c-Cbl, an E3 ubiquitin ligase that has been previously shown to undergo enhanced tyrosine phosphorylation following Fcγ receptor stimulation in macrophages (10, 11, 12). Phagocytosis of EIgG in murine BMDMs (Fig. 1,A) and mouse pulmonary alveolar macrophages (supplemental Fig. S1)6 was accompanied by recruitment of Cbl to phagocytic cups. Although we were unable to identify an Ab useful for immunofluorescence that specifically recognized Cbl-b, we detected Cbl-b in phagocytic cups in macrophages derived from c-Cbl−/− mice using an Ab that recognized both Cbl isoforms (Fig. 1,B). To confirm that Cbl undergoes enhanced tyrosine phosphorylation in response to Fcγ receptor stimulation, we incubated BMDMs with immune complexes and immunoprecipated c-Cbl and Cbl-b. As expected, stimulation of Fcγ receptors resulted in enhanced tyrosine phosphorylation of both Cbl family members (Fig. 2 A).

FIGURE 1.

Recruitment of Cbl to phagocytic cups during Fcγ receptor-mediated phagocytosis in BMDMs. Adherent BMDMs were challenged with EIgG for 5 min at 37°C, fixed, and processed for immunofluorescence using Abs against the indicated proteins. A, WT macrophages stained for c-Cbl. B, c-Cbl−/− macrophages stained for Cbl-b. Note colocalization of c-Cbl (green panel in A) and Cbl-b (green panel in B) with F-actin (red). Similar results were seen in four additional experiments. Bar, 10 μm.

FIGURE 1.

Recruitment of Cbl to phagocytic cups during Fcγ receptor-mediated phagocytosis in BMDMs. Adherent BMDMs were challenged with EIgG for 5 min at 37°C, fixed, and processed for immunofluorescence using Abs against the indicated proteins. A, WT macrophages stained for c-Cbl. B, c-Cbl−/− macrophages stained for Cbl-b. Note colocalization of c-Cbl (green panel in A) and Cbl-b (green panel in B) with F-actin (red). Similar results were seen in four additional experiments. Bar, 10 μm.

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FIGURE 2.

Enhanced tyrosine phosphorylation of Cbl following Fcγ receptor stimulation and enhanced association and phagocytosis of IgG-opsonized targets in Cbl−/− macrophages. A, BMDMs (107) from WT, c-Cbl−/−, and Cbl-b−/− mice were incubated with 100 μg/ml peroxidase-anti-peroxidase immune complexes for the indicated times. Cells were lysed in RIPA buffer, subjected to immunoprecipitation with c-Cbl- or Cbl-b-specific Abs, followed by immunoblotting with Abs against phosphotyrosine (PY; top) or Cbl proteins (bottom). Similar results were seen in two additional experiments. B, Adherent BMDMs were challenged with 107 EIgG for 20 min at 37°C and scored for association and phagocytosis as described in Materials and Methods. Attachment and phagocytic indices were calculated by scoring the number of EIgG targets that were associated with, or ingested by, 100 macrophages. Data represent mean ± SEM (n = 5). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

FIGURE 2.

Enhanced tyrosine phosphorylation of Cbl following Fcγ receptor stimulation and enhanced association and phagocytosis of IgG-opsonized targets in Cbl−/− macrophages. A, BMDMs (107) from WT, c-Cbl−/−, and Cbl-b−/− mice were incubated with 100 μg/ml peroxidase-anti-peroxidase immune complexes for the indicated times. Cells were lysed in RIPA buffer, subjected to immunoprecipitation with c-Cbl- or Cbl-b-specific Abs, followed by immunoblotting with Abs against phosphotyrosine (PY; top) or Cbl proteins (bottom). Similar results were seen in two additional experiments. B, Adherent BMDMs were challenged with 107 EIgG for 20 min at 37°C and scored for association and phagocytosis as described in Materials and Methods. Attachment and phagocytic indices were calculated by scoring the number of EIgG targets that were associated with, or ingested by, 100 macrophages. Data represent mean ± SEM (n = 5). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

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To test for a functional role for Cbl in Fcγ receptor-mediated phagocytosis, we challenged BMDMs from wild-type (WT), c-Cbl−/−, and Cbl-b−/− mice with EIgG for 20 min. c-Cbl−/− and particularly Cbl-b−/− BMDMs demonstrated enhanced phagocytosis compared with WT BMDMs (Fig. 2,B). This was not due to greater phagocytic efficiency, as BMDMs from all three genotypes ingested >78% of all bound particles after 20 min; instead, BMDMs from Cbl−/− macrophages exhibited an enhanced ability to bind IgG-opsonized targets. Interestingly, differences in binding could not be explained solely by receptor expression (Table I). Although there were slight increases in mRNA and surface expression of FcγRI in macrophages from c-Cbl−/− mice, expression of FcγR mRNA and protein in macrophages derived from Cbl-b−/− mice were either not different or slightly reduced, as compared with controls (Table I). Thus, we were unable to detect differences in Fcγ receptor expression in Cbl-deficient macrophages that could account for the extent of enhanced binding of EIgG that we observed (22).

Table I.

Relative expression of FcγR isoforms and the γ-subunit in BMDMs from WT, c-Cbl−/−, and Cbl-b−/− mice

FcγR/SubunitmRNA (fold WT)a95% CInp
c-Cbl−/−Cbl-b−/−c-Cbl−/−Cbl-b−/−c-Cbl−/−Cbl-b−/−c-Cbl−/−Cbl-b−/−
FcγRI 1.10 1.07 1.02–1.18 0.98–1.16 <0.05 NS 
FcγRIIb 0.67 1.09 0.55–0.79 0.96–1.22 <0.01 NS 
FcγRIII 0.71 1.19 0.51–0.91 0.89–1.49 <0.01 NS 
FcγRIV 1.04 1.23 0.83–1.25 0.91–1.55 NS NS 
γ-Subunit 0.98 1.21 0.70–1.26 0.85–1.57 NS NS 
 Protein (fold WT)b 95% CI n p 
FcγRIc 1.22 0.87 0.87–1.57 0.72–1.02 NS NS 
FcγRId 1.22 0.91 1.04–1.40 0.84–0.98 <0.05 <0.05 
FcγRIIb 0.54 0.81 0.35–0.73 0.61–1.01 <0.01 NS 
FcγRIII 0.67 1.11 0.61–0.73 0.95–1.27 <0.01 NS 
γ-Subunite 0.80 0.89 0.57–1.03 0.82–0.96 NS <0.05 
FcγR/SubunitmRNA (fold WT)a95% CInp
c-Cbl−/−Cbl-b−/−c-Cbl−/−Cbl-b−/−c-Cbl−/−Cbl-b−/−c-Cbl−/−Cbl-b−/−
FcγRI 1.10 1.07 1.02–1.18 0.98–1.16 <0.05 NS 
FcγRIIb 0.67 1.09 0.55–0.79 0.96–1.22 <0.01 NS 
FcγRIII 0.71 1.19 0.51–0.91 0.89–1.49 <0.01 NS 
FcγRIV 1.04 1.23 0.83–1.25 0.91–1.55 NS NS 
γ-Subunit 0.98 1.21 0.70–1.26 0.85–1.57 NS NS 
 Protein (fold WT)b 95% CI n p 
FcγRIc 1.22 0.87 0.87–1.57 0.72–1.02 NS NS 
FcγRId 1.22 0.91 1.04–1.40 0.84–0.98 <0.05 <0.05 
FcγRIIb 0.54 0.81 0.35–0.73 0.61–1.01 <0.01 NS 
FcγRIII 0.67 1.11 0.61–0.73 0.95–1.27 <0.01 NS 
γ-Subunite 0.80 0.89 0.57–1.03 0.82–0.96 NS <0.05 
a

Data represent mean relative mRNA expression as compared to macrophages derived from WT mice; mRNA values for indicated proteins are corrected for expression of peptidyl-prolyl-isomerase A mRNA.

b

For FcγRI, IIb, and III, cell surface expression was performed using flow cytometry as described in Materials and Methods. Staining for FcγRIV using mAb 9E9 gave negligible staining for all genotypes.

c

Measured by flow cytometry using mAb X54-3/4.

d

Measured by flow cytometry using FITC-IgG2a.

e

Measured by densitometry of γ-subunit immunoblots using tubulin as a loading control.

Rabbit IgG-opsonized targets cannot be easily used as probes for specific Fcγ receptors, as they interact with multiple Fcγ receptor isoforms. Therefore, we took advantage of the fact that IgG2a-coated targets interact almost exclusively with FcγRI in BMDMs (23), and IgG1-coated targets would be predicted to preferentially interact with FcγRIIb and, to some extent, FcγRIII, but not with FcγRI or FcγRIV (24, 25, 26). Although we were unable to detect appreciable surface expression of FcγRIV in these cells, we blocked any potential interactions with this Fc receptor isoform with monomeric mIgG2b (24). Similar to experiments using EIgG, EIgG2a was ingested with high efficiency (>77%) in all three genotypes, although c-Cbl−/− and Cbl-b−/− macrophages bound and ingested more of these targets (Fig. 3,A). The enhanced binding was not due to increased receptor number (Table I) and was not due to a reduction in endocytosis of FcγRI at the cell surface (supplemental Fig. S2). Furthermore, binding and ingestion of EigG2a in c-Cbl−/− and Cbl-b−/− macrophages was not due to an increase in functional cell surface FcγRIV, as addition of 2 μg/ml mAb 9E9 against FcγRIV did not reduce these parameters (data not shown). In the case of EIgG1, fewer targets bound to macrophages from c-Cbl−/− mice, consistent with reduced levels of mRNA and protein for both FcγRIIb and III (Table I). However, c-Cbl−/− macrophages ingested EIgG1 with a far greater efficiency (47%) than did WT (25%) and Cbl-b−/− (23%) macrophages (Fig. 3,B). This was likely due to a relatively greater reduction in levels of the inhibitory, nonphagocytic FcγRIIb as compared with the activating FcγR, FcγRIII, in macrophages lacking c-Cbl (Table I). Therefore, Cbl proteins regulate phagocytosis by multiple mechanisms in BMDMs: c-Cbl regulates the ratio of activating and inhibitory FcγRs, favoring the latter, whereas both c-Cbl and Cbl-b inhibit maximal binding and phagocytosis mediated by FcγRI.

FIGURE 3.

Lack of Cbl proteins affect association and phagocytosis of EIgG2a, as well as enhanced phagocytic efficiency of EIgG-1. Adherent BMDMs were challenged with the 2 × 107 of the indicated phagocytic targets for 20 min at 37°C and scored for association and phagocytosis as described in Materials and Methods. Attachment and phagocytic indices were calculated by scoring the number of EIgG1 or EIgG2a targets that were associated with, or ingested by, 100 macrophages. A, Data represent mean ± SEM (n = 5). ∗, p < 0.05. B, Data represent mean ± SEM (n = 8). ∗, p < 0.05; ∗∗, p < 0.01.

FIGURE 3.

Lack of Cbl proteins affect association and phagocytosis of EIgG2a, as well as enhanced phagocytic efficiency of EIgG-1. Adherent BMDMs were challenged with the 2 × 107 of the indicated phagocytic targets for 20 min at 37°C and scored for association and phagocytosis as described in Materials and Methods. Attachment and phagocytic indices were calculated by scoring the number of EIgG1 or EIgG2a targets that were associated with, or ingested by, 100 macrophages. A, Data represent mean ± SEM (n = 5). ∗, p < 0.05. B, Data represent mean ± SEM (n = 8). ∗, p < 0.05; ∗∗, p < 0.01.

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In many cell types, Cbl has been shown to act as an endogenous negative regulator of transmembrane signaling, particularly in the case of ITAM-containing receptors (reviewed in Refs. 27 , 28). Pilot experiments demonstrated that c-Cbl−/− and Cbl-b−/− BMDMs exhibited markedly enhanced levels of tyrosine phosphorylation in response to immune complexes containing rabbit IgG (data not shown). These results could be partly explained, at least in the case of c-Cbl−/− BMDMs, by the reduced expression of FcγRIIb. To exclude the contribution of this inhibitory receptor, we stimulated BMDMs with aggregates of IgG2a, an isoform that specifically binds to FcγRI (23). This resulted in enhanced and sustained tyrosine phosphorylation of multiple proteins (e.g., arrowhead 2 in Fig. 4,A) in Cbl-deficient macrophages, particularly at the 3 min time point. In fact, the only protein that was phosphorylated at consistently lower levels in c-Cbl−/− macrophages appeared to be 120 kDa (arrowhead 1 in Fig. 4,A), which probably corresponded to c-Cbl itself. The enhanced accumulation of phosphotyrosyl-containing proteins included the γ-subunit, an ITAM-containing subunit of FcγRI, and other activating receptors (Fig. 4,B). Furthermore, the levels of phosphotyrosyl-containing proteins that accumulated in the periphagosomal region, expressed as a fraction of recruited γ-subunit, were greater in macrophages from c-Cbl−/− and Cbl-b−/− mice (Fig. 4,C). We also detected enhanced activation of Src family TKs and Syk in these cells (Fig. 4 D). Collectively, these data indicate that Cbl proteins negatively regulate FcγR-mediated signaling at a very early stage following receptor ligation.

FIGURE 4.

Enhanced early signaling events following FcγRI stimulation in Cbl−/− macrophages. A, BMDMs (2 × 106) from WT, c-Cbl−/−, and Cbl-b−/− macrophages were incubated with 200 μg/ml heat-aggregated mouse IgG2a for the indicated times at 37°C. Cells were lysed in RIPA buffer and lysates were subjected to immunoblotting for phosphotyrosine as described in Materials and Methods. Molecular mass markers (kDa) appear at the left. Arrowhead denoted by 1 corresponds in molecular mass to c-Cbl; arrowhead denoted by 2 corresponds in molecular mass to Syk. Similar results were seen in six additional experiments. B, BMDMs (107) from WT, c-Cbl−/−, and Cbl-b−/− macrophages were incubated with 200 μg/ml heat-aggregated IgG2a for the indicated times at 37°C. Cells were lysed in RIPA buffer, subjected to immunoprecipitation with γ-subunit-specific Abs, followed by immunoblotting with Abs against either phosphotyrosine (PY; top) or the γ-subunit (bottom). Lane 1, Immunoprecipitation with nonimmune rabbit IgG control at 3 min time point; lanes 2, 5, and 8, WT BMDMs; lanes 3, 6, and 9, c-Cbl−/− BMDMs; lanes 4, 7, and 10, Cbl-b−/− BMDMs. Graph depicts densitometry (pYγ/γ), in relative units, of the above data. C, Adherent macrophages were challenged with EIgG2a for 30 min at 30°C, fixed, and processed for immunofluorescence microscopy using Abs against the γ-subunit, phosphotyrosine, and IgG2a. Quantitation of relative fluorescence staining intensities for the γ-subunit and phosphotyrosine was performed using microspectrofluorometry as described in Materials and Methods. Data for 75 phagosomes are depicted. Bars represent mean values. ∗, p < 0.05. D, BMDMs (2 × 106) from WT, c-Cbl−/−, and Cbl-b−/− macrophages were incubated with 200 μg/ml heat-aggregated mIgG2a for the indicated times at 37°C. Cells were lysed in RIPA buffer and equal amounts of protein were subjected to immunoblotting using Abs against the indicated proteins. Note presence of higher molecular mass forms (large arrow) and breakdown products (small arrow) of phospho-Src. The Ab used recognizes phosphorylated species of multiple Src family members, in addition to c-Src. Graphs depict densitometry (in relative units) of data above. Similar results were seen in two additional experiments.

FIGURE 4.

Enhanced early signaling events following FcγRI stimulation in Cbl−/− macrophages. A, BMDMs (2 × 106) from WT, c-Cbl−/−, and Cbl-b−/− macrophages were incubated with 200 μg/ml heat-aggregated mouse IgG2a for the indicated times at 37°C. Cells were lysed in RIPA buffer and lysates were subjected to immunoblotting for phosphotyrosine as described in Materials and Methods. Molecular mass markers (kDa) appear at the left. Arrowhead denoted by 1 corresponds in molecular mass to c-Cbl; arrowhead denoted by 2 corresponds in molecular mass to Syk. Similar results were seen in six additional experiments. B, BMDMs (107) from WT, c-Cbl−/−, and Cbl-b−/− macrophages were incubated with 200 μg/ml heat-aggregated IgG2a for the indicated times at 37°C. Cells were lysed in RIPA buffer, subjected to immunoprecipitation with γ-subunit-specific Abs, followed by immunoblotting with Abs against either phosphotyrosine (PY; top) or the γ-subunit (bottom). Lane 1, Immunoprecipitation with nonimmune rabbit IgG control at 3 min time point; lanes 2, 5, and 8, WT BMDMs; lanes 3, 6, and 9, c-Cbl−/− BMDMs; lanes 4, 7, and 10, Cbl-b−/− BMDMs. Graph depicts densitometry (pYγ/γ), in relative units, of the above data. C, Adherent macrophages were challenged with EIgG2a for 30 min at 30°C, fixed, and processed for immunofluorescence microscopy using Abs against the γ-subunit, phosphotyrosine, and IgG2a. Quantitation of relative fluorescence staining intensities for the γ-subunit and phosphotyrosine was performed using microspectrofluorometry as described in Materials and Methods. Data for 75 phagosomes are depicted. Bars represent mean values. ∗, p < 0.05. D, BMDMs (2 × 106) from WT, c-Cbl−/−, and Cbl-b−/− macrophages were incubated with 200 μg/ml heat-aggregated mIgG2a for the indicated times at 37°C. Cells were lysed in RIPA buffer and equal amounts of protein were subjected to immunoblotting using Abs against the indicated proteins. Note presence of higher molecular mass forms (large arrow) and breakdown products (small arrow) of phospho-Src. The Ab used recognizes phosphorylated species of multiple Src family members, in addition to c-Src. Graphs depict densitometry (in relative units) of data above. Similar results were seen in two additional experiments.

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Ligand engagement by a variety of receptors is thought to induce redistribution of the receptor into preexisting lipid raft microdomains within the plasma membrane (reviewed in Refs. 29, 30, 31). In unstimulated BMDMs, flotillin, a marker of lipid rafts in macrophages (32), was not organized into discrete clusters. Addition of heat-aggregated IgG2a to BMDMs at 4°C was sufficient to induce clustering of flotillin (Fig. 5). Rather than ligand inducing redistribution of receptor in lipid rafts, these data support an alternative, if not opposite interpretation; that is, ligand-induced receptor clustering induces reorganization of lipid rafts into receptor-associated macromolecular complexes. Similar results were seen with other markers of lipid rafts, such as Gαi2 and CD14 (not shown). The enhanced signaling by FcγRI that we observed in macrophages lacking Cbl raised the possibility that FcγRI engages TKs in a distinct manner. Indeed, we found that, under basal conditions, the extent of colocalization of FcγRI and Hck was greater in macrophages from Cbl-deficient mice compared with WT mice (Figs. 6 and 7). There was some basal colocalization of FcγRI with another Src family member, Lyn, although the pattern of Lyn immunofluorescence indicated that Lyn was more diffusely distributed throughout the cell, as compared with Hck (not shown). Additionally, regions of high receptor density also tended to codistribute with F-actin in Cbl-deficient macrophages (Fig. 6). The extent of colocalization of FcγRI and F-actin was greater for macrophages from c-Cbl−/− mice, with a similar trend for enhanced association between FcγRI and F-actin in macrophages from Cbl−/− mice (Fig. 7). Addition of aggregated ligand at 4°C, which was sufficient to induce receptor clustering, led to coclustering of FcγRI with Hck and F-actin (Figs. 6 and 7), Lyn, and Src (not shown), even in WT cells. Thus, clustering of FcγRI with ligand induced coclustering of Src family TKs and F-actin, although this was true even in resting conditions, particularly for Cbl-deficient macrophages. As expected, addition of aggregated IgG2a at 37°C resulted in a more exaggerated cytoskeletal response in macrophages lacking Cbl (supplemental Fig. S3).

FIGURE 5.

Addition of aggregated IgG2a is sufficient to induce redistribution of flotillin into ligand-associated microclusters. Adherent WT BMDMs were incubated in the presence or absence of 200 μg/ml heat-aggregated mIgG2a for 10 min at 4°C, fixed, and processed for immunofluorescence microscopy using the indicated Abs. Note that flotillin is in a dispersed configuration in unstimulated macrophages (right panel). Similar results were seen in seven additional experiments.

FIGURE 5.

Addition of aggregated IgG2a is sufficient to induce redistribution of flotillin into ligand-associated microclusters. Adherent WT BMDMs were incubated in the presence or absence of 200 μg/ml heat-aggregated mIgG2a for 10 min at 4°C, fixed, and processed for immunofluorescence microscopy using the indicated Abs. Note that flotillin is in a dispersed configuration in unstimulated macrophages (right panel). Similar results were seen in seven additional experiments.

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FIGURE 6.

Increased basal association of FcγRI with the Src family TK, Hck, in macrophages lacking Cbl. Adherent BMDMs from WT, c-Cbl−/−, and Cbl-b−/− macrophages were incubated in the presence or absence of 200 μg/ml heat-aggregated mIgG2a for 10 min at 4°C, fixed, and processed for fluorescence microscopy using IgG against FcγRI (red) and Hck (green), and for F-actin using Alexa 488-phalloidin (blue). Only the merged images are shown for clarity. Note that, in the absence of aggregated IgG2a ligand, there is unimpressive colocalization of FcγRI and Hck, as well as FcγRI and F-actin, in WT macrophages, whereas there is appreciable colocalization of these proteins in c-Cbl−/− and Cbl-b−/− macrophages. Following addition of ligand, the extent of overlap between FcγRI and Hck or F-actin was indistinguishable in all three genotypes. Similar results were seen in three additional experiments. Bar, 10 μm.

FIGURE 6.

Increased basal association of FcγRI with the Src family TK, Hck, in macrophages lacking Cbl. Adherent BMDMs from WT, c-Cbl−/−, and Cbl-b−/− macrophages were incubated in the presence or absence of 200 μg/ml heat-aggregated mIgG2a for 10 min at 4°C, fixed, and processed for fluorescence microscopy using IgG against FcγRI (red) and Hck (green), and for F-actin using Alexa 488-phalloidin (blue). Only the merged images are shown for clarity. Note that, in the absence of aggregated IgG2a ligand, there is unimpressive colocalization of FcγRI and Hck, as well as FcγRI and F-actin, in WT macrophages, whereas there is appreciable colocalization of these proteins in c-Cbl−/− and Cbl-b−/− macrophages. Following addition of ligand, the extent of overlap between FcγRI and Hck or F-actin was indistinguishable in all three genotypes. Similar results were seen in three additional experiments. Bar, 10 μm.

Close modal
FIGURE 7.

Dynamic association of Hck and F-actin with FcγRI, and of Hck and Syk with Cbl, in response to addition of clustered ligand. Adherent BMDMs from WT, c-Cbl−/−, and Cbl-b−/− macrophages were incubated in the absence (black bars) or presence (gray bars) of 200 μg/ml heat-aggregated mIgG2a for 10 min at 4°C and fixed and processed for fluorescence microscopy as described in the legend to Fig. 6. Fields were selected randomly, cortical regions of cells were imaged, and the extent of colocalization of FcγRI and Hck (A) and of FcγRI and F-actin (B) was quantified using ImageJ software. Data represent mean ± SEM (n = 20–30 cells). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. C, Unstimulated macrophages (2 × 107) of the indicated genotype were subjected to detergent lysis, and immunoprecipitation of the γ-subunit followed by immunoblotting for the indicated proteins was performed as described in Materials and Methods. Graph below depicts densitometry of above data. Similar results were seen in an additional experiment.

FIGURE 7.

Dynamic association of Hck and F-actin with FcγRI, and of Hck and Syk with Cbl, in response to addition of clustered ligand. Adherent BMDMs from WT, c-Cbl−/−, and Cbl-b−/− macrophages were incubated in the absence (black bars) or presence (gray bars) of 200 μg/ml heat-aggregated mIgG2a for 10 min at 4°C and fixed and processed for fluorescence microscopy as described in the legend to Fig. 6. Fields were selected randomly, cortical regions of cells were imaged, and the extent of colocalization of FcγRI and Hck (A) and of FcγRI and F-actin (B) was quantified using ImageJ software. Data represent mean ± SEM (n = 20–30 cells). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. C, Unstimulated macrophages (2 × 107) of the indicated genotype were subjected to detergent lysis, and immunoprecipitation of the γ-subunit followed by immunoblotting for the indicated proteins was performed as described in Materials and Methods. Graph below depicts densitometry of above data. Similar results were seen in an additional experiment.

Close modal

Because plasma membrane-associated lipid rafts require cholesterol for their integrity, we tested a role for cholesterol in ligand binding. Incubation of WT BMDMs for 5 h with either vehicle control (RPMI 1640) or methyl-β-cyclodextrin, which results in a gradual time-dependent decrease in macrophage cholesterol content to 40% of control (20), resulted in a >85% inhibition in both EIgG2a binding and phagocytosis (Fig. 8 A). These data are consistent with a requirement for the integrity of cholesterol-based signaling structures, which include members of the Src family, in FcγRI ligand binding activity.

FIGURE 8.

EIgG2a binding is sensitive to depletion of cellular cholesterol, inhibition of TKs and actin polymerization, and opsonin concentration. Adherent WT BMDMs were preincubated with (A) 20 mM methyl-β-cyclodextrin (MβCD) in RPMI 1640 or RPMI 1640 control for 5 h at 37°C and incubated with 2 × 107 EIgG2a for 20 min before fixation and determination of association (filled bars) and ingestion (open bars) of EIgG2a. B, Adherent WT BMDMs were preincubated with 10 μm of 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine (PP2), 100 μM genistein, or vehicle control and incubated with 2 × 107 EIgG2a for 20 min before fixation and determination of association (filled bars) and ingestion (open bars) of EIgG2a. C, Adherent WT BMDMs were preincubated with 1 μM cytochalasin D (Cyto D), 1 μM latrunculin A (Latrunc A), or vehicle control for 20 min and incubated with 2 × 107 EIgG2a for 20 min before fixation and determination of association (filled bars) and ingestion (open bars) of EIgG2a. D, Adherent WT BMDMs were challenged with 107 EIgG opsonized with the indicated dilutions of rabbit Ab for 20 min before fixation and determination of association of EIgG. Numbers above bars indicate percentage of binding in the presence of cytochalasin D, compared with vehicle control. Average phagocytosis indices in vehicle controls were 965 ± 58, 396 ± 87, and 40 ± 38, for EIgG opsonized with 1/1200 (titer used in Fig. 2 B), 1/2400, and 1/4800 dilutions of opsonizing Ab, respectively. Phagocytosis in cytochalasin D-treated cells was essentially nil. Data represent mean ± SEM (n = 5). ∗, p < 0.05; ∗∗, p < 0.01.

FIGURE 8.

EIgG2a binding is sensitive to depletion of cellular cholesterol, inhibition of TKs and actin polymerization, and opsonin concentration. Adherent WT BMDMs were preincubated with (A) 20 mM methyl-β-cyclodextrin (MβCD) in RPMI 1640 or RPMI 1640 control for 5 h at 37°C and incubated with 2 × 107 EIgG2a for 20 min before fixation and determination of association (filled bars) and ingestion (open bars) of EIgG2a. B, Adherent WT BMDMs were preincubated with 10 μm of 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine (PP2), 100 μM genistein, or vehicle control and incubated with 2 × 107 EIgG2a for 20 min before fixation and determination of association (filled bars) and ingestion (open bars) of EIgG2a. C, Adherent WT BMDMs were preincubated with 1 μM cytochalasin D (Cyto D), 1 μM latrunculin A (Latrunc A), or vehicle control for 20 min and incubated with 2 × 107 EIgG2a for 20 min before fixation and determination of association (filled bars) and ingestion (open bars) of EIgG2a. D, Adherent WT BMDMs were challenged with 107 EIgG opsonized with the indicated dilutions of rabbit Ab for 20 min before fixation and determination of association of EIgG. Numbers above bars indicate percentage of binding in the presence of cytochalasin D, compared with vehicle control. Average phagocytosis indices in vehicle controls were 965 ± 58, 396 ± 87, and 40 ± 38, for EIgG opsonized with 1/1200 (titer used in Fig. 2 B), 1/2400, and 1/4800 dilutions of opsonizing Ab, respectively. Phagocytosis in cytochalasin D-treated cells was essentially nil. Data represent mean ± SEM (n = 5). ∗, p < 0.05; ∗∗, p < 0.01.

Close modal

The above findings in Cbl-deficient macrophages prompted us to reexamine the widely held view that TK activity and actin assembly are irrelevant for FcγRI ligand binding function. We found that both 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine (PP2), a selective Src family TK inhibitor, and genistein, a nonspecific TK inhibitor, blocked binding and ingestion of EIgG2a (Fig. 8,B). These results are reminiscent those found in a recent study of TK inhibitors and IgG-opsonized zymosan (33). Additionally, cytochalasin D markedly inhibited, and latrunculin A ablated, binding of EIgG2a to macrophages (Fig. 8,C). Interestingly, the sensitivity of binding activity to cytoskeletal-disrupting agents when EIgG was used as a test particle was less than that of EIgG2a, and it was inversely proportional to opsonin density of the test particles (Fig. 8,D). These results help explain why binding of EIgG to macrophages was previously thought to be a passive event, independent of cytoskeletal integrity, as phagocytic targets are typically generated using a high titer of opsonizing Ab. Furthermore, binding of EIgG2a to WT BMDMs was abolished at 4°C (supplemental Fig. S4), whereas binding of heat-aggregated IgG2a was not only uninhibited at this temperature, but was sufficient to induce the reorganization of flotillin and Src family TKs (Figs. 5–7). These data indicate that both ingestion and binding of phagocytic targets to FcγRI is an active process, dependent on TKs, including those of the Src family, and an intact cytoskeleton.

Our results show that BMDMs from Cbl−/− mice demonstrate enhanced particulate ligand binding capacity and phagocytosis in comparison to macrophages from WT controls. These results could not be explained solely by receptor expression or phagocytic efficiency, especially in the case of macrophages from Cbl-b−/− mice. Rather, they correlated with enhanced signaling and cytoskeletal assembly in Cbl-deficient mice. We were initially surprised by these results, as a number of laboratories, including our own, have shown that phagocytic ligand binding can occur in the presence of inhibitors of TKs and actin assembly. Additionally, binding, but not phagocytosis, of IgG-coated targets can occur in cells lacking Rac proteins or function (34, 35), and many studies have demonstrated that binding of EIgG at 4°C is readily apparent. However, the test particles used in these studies were typically generated under conditions of high opsonin concentration and target multiple FcγR isoforms. In the present study, differences in sensitivity of phagocytic target binding to TK and cytoskeletal inhibitors were readily apparent when the ligand density was titrated downward or when only one FcγR isoform, FcγRI, was engaged. There are several explanations for this phenomenon, including the possibility of limiting receptor number, limiting opsonin density, limiting signal strength, or a combination of these. In addition to its quantitative expression, FcγRI function is regulated at the level of receptor affinity and interaction with other proteins, such as filamin (36) and periplakin (37, 38). However, murine FcγRI does not bind periplakin (Jeanette Leusen, unpublished observation), and receptor affinity alone is unlikely to explain our results as macrophages from c-Cbl−/− and Cbl-b−/− mice bound no more heat-aggregated IgG2a than did WT mice (our unpublished observations).

Mono- or multiubiquitylation catalyzed by E3 ubiquitin ligases promotes endocytosis of a subset of cell surface receptors, including receptor TKs, G protein-coupled receptors, transporters, and ion channels (reviewed in Ref. 39). Although we have shown that Cbl is recruited to Fcγ receptor-containing phagosomes and undergoes enhanced tyrosine phosphorylation during endocytosis of immune complexes, endocytosis of FcγRI takes place with normal kinetics in macrophages that lack either Cbl protein. There are several explanations for these results, including functional redundancy between c-Cbl and Cbl-b, or with other E3 ubiquitin ligases. Thus, we cannot conclude that endocytosis via FcγRI is independent of ubiquitylation. Interestingly, endocytosis of small immune complexes by FcγRIIA is inhibited in ts20 cells, which bear a temperature-sensitive mutation in the E1 ubiquitin-activating enzyme (40, 41). Polyubiquitylation may have distinct functions during phagocytosis, such as promoting fission of FcγRIIA into multivesicular bodies during phagosome maturation (42). Consistent with these results, we have found that the γ-subunit is more persistent in late phagosomes from Cbl-deficient cells, as compared with WT cells, by immunofluorescence (unpublished results). Multiple monoubiquitin or polyubiquitin residues are required for optimal lysosome targeting (43), probably by serving as docking sites for Hrs/STAM (44).

How do Cbl proteins regulate phagocytic signaling? The PTB domain of Cbl binds phosphorylated Tyr416 in the activation loop of Src and inhibits its kinase activity (45). This domain also interacts with Syk pY323, a negative regulatory site, and suppresses its kinase activity (46). The findings of the present study, in which both Cbl proteins associate with Hck and Syk during FcγRI clustering, and both TKs demonstrated enhanced activation following activation of FcγRI in Cbl-deficient cells, are consistent with these possibilities. Notably, macrophages from both c-Cbl−/− and Cbl-b−/− mice displayed an “activated” phenotype, exhibiting constitutive colocalization of FcγRI with the Src family TK, Hck, and F-actin. It is conceivable that FcγRI in macrophages lacking Cbl proteins has a lower threshold for activation, by virtue of its “preassociation” with microdomains of the plasma membrane containing Hck, and possibly other Src family members. Cbl may also regulate phagocytosis by its known adaptor function. c-Cbl is found in multiprotein complexes that include the inositol 5′-phosphatase SHIP and the tyrosine phosphatase SHP-1 (47), both of which negatively regulate phagocytosis (48, 49).

In c-Cbl−/− BMDMs, we saw modest increases in surface expression of FcγRI. We saw no such increase in Cbl-b−/− macrophages. We also found no quantitative differences in the expression of Fcγ receptors IIb, and III, as well as the γ-subunit, Lyn, Syk, PLC-γ, and Crk-L by immunoblotting in these cells (Table I and our unpublished observations). These findings are similar to studies of Cbl-b in other cell types, in which alterations in signaling in Cbl-b−/− cells are not a consequence of differences in expression of key signaling proteins (50, 51). We also considered the possibility that Cbl-deficient macrophages contained increased latent intracellular pools of FcγRI. However, despite multiple attempts, we could not demonstrate the existence of these pools, consistent with results in human macrophages, which demonstrate that intracellular pools of FcγRI are negligible (52).

In contrast to FcγRI, we saw a substantial reduction in the expression of cell surface FcγRIIb in c-Cbl−/− macrophages, which probably explains why these cells demonstrated an increase in the efficiency of phagocytosis of EIgG1, which binds both FcγRIIb and FcγRIII. Regulation of the expression of c-Cbl by cytokines or innate immune stimuli might therefore be expected to influence the balance of inhibitory FcγRIIb and activating Fcγ receptors, thus affecting macrophage responsiveness to IgG. It is tempting to speculate that an imbalance in expression of FcγRIIb and activating Fcγ receptors may contribute to the autoimmune phenotype observed in Cbl-deficient mice (53, 54), although this possibility has yet to be explored.

In summary, we have uncovered an unsuspected role for signaling and cytoskeletal assembly in binding of opsonized targets in mouse macrophages. Our results challenge the widely held view that Fcγ receptors are regulated exclusively at the postreceptor binding stage. We suggest that dynamic engagement of TKs and the cytoskeleton enable macrophages to “capture” phagocytic targets. In other words, engagement of receptors leads to TK-based actin polymerization, promoting pseudopod extension and increasing the probability that unligated receptors engage distant ligand. According to this view, transmembrane signaling and pseudopod extension effectively increase the dynamic avidity of receptor-ligand interactions. This is analogous to the Venus fly trap, which binds tightly to its prey and engulfs it only after triggering a motile event. We suggest that this mechanism of phagocytosis may be particularly important during the early phases of the primary immune response, when opsonic activity is limited.

We thank Tomohiro Kurosaki for his generous donation of anti-phosphotyrosine mAb 4G10-coupled agarose, Hua Gu for his donation of Cbl-b-deficient mice, and Charles Mainhart for help in procuring c-Cbl-deficient mice.

The authors have no financial conflicts of interest.

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

1

This work was supported by National Institutes of Health Grants HL054164 and AI067502 to S.G.

4

Abbreviations used in this paper: TK, tyrosine kinase; BMDM, bone marrow-derived macrophages; EIgG, IgG-coated erythrocytes; WT, wild type.

5

The online version of the article contains Extended Methods.

6

The online version of this article contains supplemental material.

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