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(Journal of Leukocyte Biology. 2001;70:801-811.)
© 2001 by Society for Leukocyte Biology

Role of Src kinases and Syk in Fc{gamma} receptor-mediated phagocytosis and phagosome-lysosome fusion

Meytham Majeed*, Elena Caveggion*, Clifford A. Lowell{dagger} and Giorgio Berton*

* Department of Pathology, Section of General Pathology, University of Verona, Verona, Italy, and
{dagger} Department of Laboratory Medicine, University of California San Francisco, San Francisco

Correspondence: Meytham Majeed, Ph.D., Department of Health and Environment, Division of Medical Microbiology, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden. E-mail: meyma{at}ihm.liu.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phagocytosis is increased by Fc{gamma} receptors (Fc{gamma}Rs), and studies with syk-/- macrophages demonstrated that Syk kinase is required for Fc{gamma}R phagocytosis. Similar studies with macrophages lacking the Src family kinases Hck, Fgr, and Lyn showed that these kinases are not required for phagocytosis but that they enhance the rate of particle engulfment. In this report we show that both wild-type and hck-/-fgr-/- macrophages expressed Fyn, Src, and Yes and that these kinases were activated on ingestion of immunoglobulin G (IgG)-coated particles and redistributed, together with Syk, to actin-rich phagocytic cups and the phagosomal membrane. At doses blocking IgG-dependent phagocytosis, the tyrosine kinase inhibitors PP1 and piceatannol inhibited both Src family kinase and Syk activities, as well as their redistribution to actin-rich phagocytic cups. Hck, Fgr, and Lyn were dispensable for lysosome-phagosome fusion (PLF) induced by IgG-coated particles. However, PP1 or piceatannol hampered unopsonized yeast-induced PLF despite the fact that they did not block yeast internalization.

Key Words: protein tyrosine phosphorylation • immune receptors • signal transduction • Fyn • Yes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phagocytosis plays an essential role in host defenses mediated by macrophages and neutrophils [1 ]. Immunoglobulins act as opsonins, increasing the ability of phagocytes to effectively internalize and kill any immunoglobulin G (IgG)-coated pathogens. IgG-coated particles are internalized via Fc{gamma} receptors (Fc{gamma}Rs) that recognize the Fc portion of the antibody molecule [2 ]. Fc{gamma}Rs belong to the family of so-called "immune receptors," also including the antigen receptors present on B and T lymphocytes and Fc{varepsilon} receptor I. According to a widely accepted model, the first event in signal transduction by immune receptors is the phosphorylation of tyrosine residues contained in conserved sequences, which are referred to as immunoreceptor tyrosine activation motifs (ITAMs) [3 4 5 ]. These motifs are found either within the cytoplasmic tail of dimeric transmembrane molecules (the 16-kDa {gamma} chain) that are associated with these receptors or, in the case of the human Fc{gamma}RIIA, in the cytoplasmic tail of the receptor itself. ITAM phosphorylation creates a specific binding site for cytoplasmic tyrosine kinases of the ZAP70/Syk family, and, once bound to ITAMs, these kinases phosphorylate downstream targets, eventually leading to cell activation.

Several findings suggest that the tyrosine kinase Syk plays an essential role in IgG-dependent phagocytosis. First, a chimeric transmembrane molecule containing the ligand-binding portion of Fc{gamma}RIIIB extracellularly and Syk intracellularly is capable of mediating phagocytosis of IgG-coated particles when transfected in COS cells [6 ]. Second, Syk expression is essential for IgG-dependent phagocytosis by both monocytes and nonphagocytic cells transfected with Fc{gamma}Rs [7 8 9 10 ]. Finally, macrophages from Syk-deficient embryos or from mouse radiation chimeras reconstituted with fetal liver cells from Syk-deficient embryos are defective in the capability to internalize IgG-coated particles [11 , 12 ].

Although Syk is clearly essential in signaling for phagocytosis through Fc{gamma}Rs, the mechanism of Syk activation in this signaling pathway is still elusive. In particular, whereas in B and T cells, recruitment of Syk and ZAP-70, respectively, to phosphorylated ITAMs and their eventual activation are believed to be secondary to Src family member activation [3 4 5 ], this relationship is less clear in the Fc{gamma}R-signaling pathway. Early studies suggested that, in the case of Fc{gamma}Rs associated with the ITAM-containing {gamma} chain dimers, i.e., Fc{gamma}RI and Fc{gamma}RIIA, Syk might endogenously associate with the {gamma} chain and be directly activated, independently of Src family kinases, by receptor cross-linking [7 ]. Indeed, {gamma} chain phosphorylation does not occur in Syk-deficient macrophages after ligation of Fc{gamma}Rs [11 ]. These observations suggest that Syk might phosphorylate the {gamma} chain directly, leading to autoactivation, independently of Src family kinases. Moreover, macrophages derived from mice deficient for the three Src kinases Hck, Fgr, and Lyn can phagocytose IgG-coated particles, although at a significantly reduced rate compared with wild-type cells [11 , 13 ].

The finding that hck-/- fgr-/- lyn-/- macrophages are not completely defective in their capability to ingest IgG-coated particles prompted us to reinvestigate the role of Src kinases in regulating Fc{gamma}R-dependent phagocytosis. In this report, we show that murine macrophages also expressed the Src family kinases Fyn, Yes, and Src and that these were activated during internalization of IgG-coated particles. We also found that Src family kinases, as well as Syk, were redistributed to the phagocytic cup and the phagosomal membrane of phagocytosing macrophages where they colocalized with filamentous actin (F-actin). The tyrosine kinase inhibitors PP1 and piceatannol inhibited IgG-dependent phagocytosis, as well as tyrosine kinase redistribution and F-actin-rich phagocytic-cup formation. Redistribution of the lysosome-associated membrane protein 1 (LAMP-1), a marker of late endosomes/lysosomes, to the phagosomal membrane was also inhibited by doses of PP1 or piceatannol that were able to block both Src family kinase and Syk activities. These findings suggest that Src family kinases and Syk play a coordinated role in both IgG-dependent phagocytosis and phagosome maturation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
Bone marrow-derived macrophages (BMDMs) were isolated from wild-type and double (hck-/- fgr-/-)- or triple (hck-/- fgr-/-; f46lyn-/-)-knockout mice as described previously [14 , 15 ]. Briefly, marrow cells from tibia and femur bones were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 mg/mL of glucose and 2 mM L-glutamine (Boehringer Ingelheim, Verviers, Belgium), 15% heat-inactivated fetal calf serum (FCS; Biochrom KG, Berlin, Germany), 10% L cell-conditioned medium, 100 U/mL of penicillin, and 100 µg/mL of streptomycin. The cells were cultivated in multiwell tissue culture plates (Greiner Labortechnik, Frickenhausen, Germany) containing round coverslips (13 mm in diameter; BDH Lab Supplies, Dorset, United Kingdom) and incubated for 5–7 days at 37°C with 5% CO2. Fresh warm DMEM was added after 2 days of incubation. For examination of the effect of inhibitory drugs on BMDM responses, cells were preincubated with (4-amino-5-(4-methylphenyl)-7-(t-butyl) pyrazolo[3,4d] pyrimidine) (PP1) (Calbiochem–Behring, Darmstadt, Germany) or piceatannol (Boehringer Mannheim, Mannheim, Germany) for 30 and 60 min at 37°C, respectively, before assay and maintained in the presence of the drugs for the time of the experiment. This treatment did not affect cell viability as determined by trypan blue exclusion (Sigma Chemical Co., St. Louis, MO).

Fluorescein-isothiocyanate (FITC)-labeling of particles
FITC labeling was performed as described previously [16 ]. Briefly, heat-killed yeast particles or polystyrene latex beads (Sigma) were washed twice in Hank’s balanced salt solution without Ca2+ and Mg2+, supplemented with 10 mM HEPES (pH 7.4) (Sigma; washing buffer), and then resuspended, at a final concentration of 109 particles/mL in 0.5% carbonate buffer (pH 9.5) supplemented with 0.5 mg/mL of FITC (Sigma). After incubation for 30 min at 37°C, the particle suspension was spun down at 2,000 rpm for 5 min at room temperature and washed five times in the washing buffer before resuspension in DMEM.

Phagocytosis assays
FITC-labeled yeast particles were used for phagocytosis assays, either without any further treatment or, in some experiments, after opsonization. For opsonization, FITC-labeled yeasts were incubated for 30 min at 37°C with 50 µg/mL of polyclonal anti-yeast antibody (IgG-yeast) as described previously [17 ]. Yeast cells (107/mL; 250 µL) were added to BMDMs, and the plates were centrifuged at 2,000 rpm for 30 s at room temperature. BMDM monolayers were washed twice in washing buffer to remove nonadherent yeast particles and, after addition of warm DMEM, were incubated for 2–15 min at 37°C in 5% CO2. At the end of the incubation, the cells were washed twice in ice-cold washing buffer and overlaid with 0.2% trypan blue (Sigma) for 2–5 min to quench fluorescence of extracellular yeasts and discriminate between extracellular and intracellular FITC-labeled yeast particles [16 ]. Internalized yeast particles were counted using a fluorescence microscope (Olympus BX59 WI; Tokyo, Japan) equipped with 440- to 480-nm excitation and 520-nm emission filters and with a 40x oil immersion objective. For phagocytosis of latex beads, 6.0-µm-diameter latex beads (Sigma) were coated with 1% BSA in phosphate-buffered saline (PBS), pH 7.4, for 30 min at 37°C and, after washing, either incubated or not with 50 µg/mL of rabbit anti-BSA antibody (Sigma) diluted in PBS. Phagocytosis of latex beads was assayed by two different methods: (1) uptake of FITC-labeled beads coated with BSA was performed exactly as described above for yeast particles, exploiting the use of trypan blue to quench the fluorescence of extracellular beads, and (2) uptake of IgG-coated latex beads was also measured by staining for the presence of the rabbit antibody used to coat the beads. After incubation at 37°C with IgG-coated latex beads, cells were washed extensively and further incubated for 30 min at 4°C with rhodamine isothiocyanate (RITC)-conjugated goat anti-rabbit antibody diluted 1:100 in PBS. After cells were washed five times with PBS to remove all the secondary antibodies, the cells were fixed in 4% paraformaldehyde in PBS to avoid particle internalization during the time required for microscopic analysis. The number of beads stained by the RITC-conjugated goat anti-rabbit antibody (extracellular beads) was subtracted from the total number of cell-associated beads counted by phase-contrast microscopic analysis. The resulting number was taken as the number of intracellular beads. When BMDMs were incubated with IgG-coated beads at 4°C, i.e., in conditions preventing particle internalization, and then were paraformaldehyde fixed and stained with RITC-conjugated secondary antibody, the count of fluorescent particles and total, cell-associated particles gave an equal number, thus validating this method of assay of phagocytosis. Independently of the particle used, the phagocytic index was calculated as previously described [18 ].

Cell lysate preparation and immunoblotting
Before lysis, BMDM monolayers were washed twice with PBS and then incubated for 30 min at 4°C in ice-cold Nonidet P-40 (NP-40) lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 5 mM EDTA, 100 µM Na3VO4), 10 µM phenylarsine oxide, 1 mM dithiothreitol, and one tablet of CompleteTM miniprotease inhibitor cocktail (Boehringer Mannheim) for each 10 mL of lysis buffer. Lysates were collected with a cell scraper and cleared by centrifugation for 10 min at 4°C. Protein concentration was determined by Bio-Rad (Richmond, CA) protein assay. Samples (30–40 µg) were subjected to electrophoresis in a sodium dodecyl sulfate-7.5% polyacrylamide gel (SDS-PAGE) under reducing conditions and electrotransferred to Hybond-C membranes (Amersham, Cardiff, UK) for 1 h at a constant voltage of 70 V. The membranes were blocked overnight at 4°C in a solution of Tris-buffered saline [TBS (170 mM NaCl, 50 mM Tris, pH 7.5)] containing 5% BSA and, after washing with TBS, incubated with rabbit antibodies against Src, Fyn, Lyn, Yes, or Syk (Santa Cruz Biotechnology, Santa Cruz, CA) diluted in TBS supplemented with 1% BSA and 0.05% Tween 20. After incubation for 2 h at room temperature, blots were washed five times in TBS-1% BSA-0.05% Tween 20 and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma) diluted in the same buffer. An enhanced chemiluminescence detection system (Amersham) was used to develop the filters.

Immunoprecipitation and in vitro kinase assays
BMDM monolayers, either left untreated or challenged with IgG-coated latex beads as described for phagocytosis assays, were washed with ice-cold TBS containing 100 µM Na3VO4 and 10 µM phenylarsine oxide and, after washing, lysed as described above. Src family kinases and Syk were immunoprecipitated from 150 µg of cell lysate using antibodies bound to protein A immobilized on Trysacryl. In vitro kinase assays for Lyn and Syk were performed essentially as described previously [19 ]. Immunoprecipitates were washed twice in lysis buffer, once in TBS, and once in kinase buffer (20 mM HEPES, pH 7.4, 10 mM MnCl2, and 1 mM dithiothreitol). The kinase assay was started by adding 20 µL of the kinase buffer containing 5 µCi of [32P]ATP (6,000 Ci/mmol; Amersham) to the samples. The reaction was terminated after 10 min at room temperature by adding 2x SDS-PAGE sample buffer. Kinase assays for Yes, Src, and Fyn were performed as described above for Lyn, with the exception that 1 µM cold ATP (Sigma) was included in the kinase buffer, and the reaction was stopped after 15 min. Increasing the ATP concentration by adding cold ATP was found to be essential to detect the activities of these kinases. After in vitro kinase assays, samples were subjected to SDS-PAGE as mentioned above, and the gels were dried and exposed to X-Omat AR films. The amount of 32P incorporated into the kinases was quantified with an InstantImager (Packard Instruments, Meriden, CT).

Immunofluorescence staining and confocal microscopy
Intracellular distribution of protein kinases, LAMP-1, and F-actin in BMDMs was investigated by fluorescence microscopy. For analysis of tyrosine kinase localization, phagocytic and nonphagocytic cells were fixed for 30 min at room temperature in 4% paraformaldehyde in PBS (pH 7.3). After fixation, cells were washed three times in PBS containing 0.1% BSA, incubated for 3 min in PBS containing 1% Triton X-100 (Sigma), and, after further washing as above, overlaid with 4% normal goat serum. The incubation was then prolonged for 30 min at room temperature in a moist chamber, and, after aspiration of the goat serum, rabbit polyclonal antibodies diluted 1:40 in PBS containing 0.1% BSA were added. After further incubation in a moist chamber for 30 min at 37°C, BMDMs were washed as above and then overlaid with RITC-conjugated goat anti-rabbit IgG (Sigma) diluted 1:150 in PBS plus 0.1% BSA. After incubation for 30 min at 37°C and washing as above, the cells were mounted in PBS plus 30% glycerol. To stain the LAMP-1, rat monoclonal antibody (1D4B) against the mouse LAMP-1 (Developmental Studies Hybridoma Bank, University of Iowa) and Texas Red-conjugated goat anti-rat secondary antibody with minimal cross-reaction to human, bovine, horse, and rabbit serum proteins (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) were used. All fluorescent specimens were labeled for F-actin using BODIPY-labeled FL phallacidin (Molecular Probes, Eugene, OR) diluted 1:40, and the cells were handled as above. Fluorescent specimens were observed with a confocal fluorescence imaging system, the Carl Zeiss LSM 510 microscope (Carl Zeiss, Oberkochen, Germany) with a 63x/1.2 C-Apochromat objective. This technique allows observation of cell structures within a narrow section (~0.2 µm thick) of the cell [20 , 21 ]. The 543-nm line of the model LSM 510 microscope was used to excite both fluorescein and rhodamine. The dicronic beam splitter (model LP 560; Carl Zeiss) and barrier filters in front of detectors 1 (rhodamine) and 2 (fluorescein) allowed separation of the fluorescent markers. The cells were serially scanned in horizontal sections 0.2 µm apart. To quantify the fluorescence intensity around the phagocytic-cup region we measured the total fluorescence intensity within a square surrounding the phagocytic cup in the images. A threshold was set to remove background fluorescence variations. The size of the square and the threshold level were the same for all measured images within each series of measurements.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Murine BMDM expression of the Src-family members Src, Fyn, and Yes and their implication in signaling by Fc{gamma}Rs
Signaling by immune receptors implicated both Src family kinases and Syk. However, whereas Syk-deficient macrophages were completely defective in Fc{gamma}R-mediated phagocytosis, macrophages obtained from triple-knockout hck-/- fgr-/- lyn-/- mice can internalize IgG-coated erythrocytes with substantially reduced kinetics [11 , 13 ]. One possible reason that hck-/- fgr-/- lyn-/- macrophages can internalize IgG-coated particles is that they express other Src kinases that can compensate for the lack of Hck, Fgr, and Lyn. To address this possibility, we examined Src family member expression and detected the presence of the Fyn, Yes, and Src proteins in BMDMs (Fig. 1 ). This finding was not surprising in light of previous studies with myelomonocytic cells and mature macrophages [22 23 24 25 26 27 ]. It is interesting that expression of Fyn, Lyn, Yes, and Syk was increased in hck-/- fgr-/- macrophages, albeit this increase was not as clear as that reported in Figure 1 for two other experiments. In addition, Src family kinase activity was comparable in hck-/- fgr-/- and wild-type BMDMs (see below). We concluded that other Src family members and perhaps Syk itself could compensate for the lack of Fgr, Hck, and Lyn in the double or the triple knockouts.



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  		Figure 1. Src family kinase expression in murine macrophages. BMDM monolayers were lysed with 1% NP-40 as described in Materials and Methods. The same amount of protein lysate (30 µg) was solubilized and subjected to SDS-PAGE in 7.5% polyacrylamide gels. Proteins were transferred to nitrocellulose, and Src family members were identified by Western blot analysis as described in Materials and Methods.

 
Phagocytosis of either IgG-coated latex beads or IgG-coated yeast particles resulted in at least a 10-fold increase in enzymatic activity of Fyn, Lyn, Src, Yes, and Syk immunoprecipitated from hck-/- fgr-/- BMDMs (Fig. 2 ) or wild-type cells (Fig. 2 legend; data not shown). In contrast, the kinase activity of these Src family members was only weakly increased in cells treated with BSA-coated latex beads, which are poorly internalized by BMDMs (see below and Fig. 5 ). Treatment of cells with plain yeasts did not enhance activity of Src consistently but stimulated Fyn, Lyn, and Yes activities at least fivefold. The results reported in Figure 2 were not dependent on genetic inactivation of fgr and hck. In fact, we obtained equal results examining activities of Src kinases and Syk in wild-type BMDMs (Fig. 2 legend; data not shown).



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  		Figure 2. IgG-coated particles enhanced kinase activity of Src family kinases and Syk in hck-/- fgr-/- BMDMs. BMDMs were either left untreated or incubated for 5 min with IgG-opsonized or -nonopsonized particles as indicated. Proteins extracted with 1% NP-40 were immunoprecipitated with antibodies of the indicated specificity, and in vitro kinase assays were performed as described in Materials and Methods. Autophosphorylated kinases were resolved by SDS-7.5% PAGE followed by autoradiography and quantitation by phosphoimager analysis. Counts per minute [cpm (mean ± SD of three independent experiments)] incorporated into the immunoprecipitated kinase are reported over each lane. Thirty micrograms of each lysate were resolved on an SDS-7.5% PAGE, transferred to nitrocellulose, and immunoblotted with antibodies of the indicated specificity to confirm equal kinase protein levels in each sample. Kinase activities assayed in lysates from wild-type BMDMs gave results comparable with those reported in the figure for hck-/- fgr-/-. In fact, radioactivity incorporated in the immunoprecipitated kinase after in vitro assays was (in cpm ± SD of two independent experiments) as follows for unstimulated cells: Fyn, 15,000 ± 500; Lyn, 27,000 ± 220; Src, 25,000 ± 805; Yes, 29,000 ± 950; and Syk, 21,000 ± 950. For cells stimulated with IgG-coated beads, radioactivity incorporated in the immunoprecipitated kinase was (in cpm ± SD of two independent experiments) as follows: Fyn, 295,000 ± 750; Lyn, 850,000 ± 220; Src, 380,000 ± 620; Yes, 325,000 ± 105; and Syk, 400,000 ± 590.

 


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  		Figure 5. PP1 and piceatannol inhibit phagocytosis of IgG-coated beads but not unopsonized yeasts. Wild-type and hck-/- fgr-/- BMDMs were left untreated or incubated at 37°C for 30 or 60 min, respectively, with 30 µM PP1 or 50 µM piceatannol and then challenged with phagocytosable particles as indicated. Phagocytosis of IgG-coated latex beads and unopsonized yeasts was performed as described in Materials and Methods. Mean results ± SD of four independent experiments are reported.

 
Redistibution of Src kinases and Syk to actin-rich phagocytic cups and to the phagosomal membrane in macrophages internalizing IgG-coated beads
To obtain further insight into the role of Src kinases in signaling for IgG-dependent phagocytosis, we addressed whether these kinases were redistributed to actin-rich phagocytic cups that form underneath the plasma membrane at the site of particle ingestion. Figure 3 illustrates the results of these studies reporting redistribution of Yes to the phagocytic cup and the phagosomal membrane. In resting, wild-type (Fig. 3A) and double-knockout hck-/- fgr-/- (Fig. 3B) BMDMs, Yes was mainly localized in discrete regions consisting of the edges of lamellopodia-like structures, thin evagination of the plasma membrane, and punctate focal-adhesion-like structures. It is important that these sites were also enriched in F-actin (Fig. 3C) , suggesting that Yes localizes to sites of actin-based cytoskeleton in spread macrophages. Similar results were obtained by analyzing distribution of other Src family members (data not shown; see Fig. 8 for distribution of Fyn). On challenge with IgG-coated latex beads, Yes was highly enriched in phagocytic cups, as well as in the phagosomal membrane, and colocalized with F-actin in both wild-type (Fig. 3G) and hck-/- fgr-/- BMDMs (Fig. 3F and 3H) . Among the members of the Src family, enrichment in the phagocytic cup and the phagosomal membrane was not restricted to Yes because we also detected Fyn, Src, and Lyn in close association with F-actin in phagocytosing wild-type and hck-/- fgr-/- BMDMs (data not shown; see Fig. 8 for distribution of Fyn).



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  		Figure 3. Confocal series showing the distributions of Yes and F-actin in wild-type and hck-/- fgr-/- BMDMs. After fixation and permeabilization, the cells were stained with polyclonal rabbit anti-Yes antibodies and with BODIPY-labeled FL phallacidin (see Materials and Methods). A, D, and G, wild-type BMDMs. B, C, E, F, and H, hck-/- fgr-/- BMDMs. A–C, distribution of Yes (A and B) and F-actin (C) in unstimulated BMDMs. D–F, distribution of Yes (D, E) and F-actin (F) in BMDMs after the cells were allowed to ingest IgG-coated beads for 2 min at 37°C. G and H, Combination of anti-Yes and F-actin staining giving simultaneous visualization of Yes and F-actin; yellow areas indicate colocalization of the proteins. G, wild-type BMDM. H, hck-/- fgr-/- BMDM. Arrows, localization of Yes and F-actin in a phagocytic-cup region; arrowheads, localization of these proteins around a phagosome. Scale bars, 10 µm.

 


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  		Figure 8. Confocal series showing the effect of PP1 and piceatannol (PCT) on the distributions of Fyn and F-actin in wild-type and hck-/- fgr-/- BMDMs. The cells were handled as described in the Figure 3 legend, except that polyclonal rabbit anti-Fyn antibodies were used. A, D, G, and J, wild-type BMDMs. B, C, E, F, H, I, K, and L, hck-/- fgr-/- BMDMs. A–C, distribution of Fyn (A and B) and F-actin (C) in unstimulated BMDMs. D–F, distribution of Fyn (D and E) and F-actin (F) in BMDMs after the cells were allowed to ingest IgG-coated beads for 2 min at 37°C. G–I, as described for panels D–F, except that the cells were preincubated with 30 µM PP1 for 30 min at 37°C and during phagocytosis. J–L, as described for panels D–F, except the cells were pretreated with 50 µM piceatannol for 60 min and during phagocytosis. Arrows, localization of Fyn and F-actin in phagocytic-cup regions. Scale bars, 10 µm.

 
Examination of Syk localization in wild type and hck-/- fgr-/- macrophages (Fig. 4 ) gave results comparable with those obtained with Src kinases. In fact, Syk was enriched in F-actin-containing phagocytic cups in both wild-type (Fig. 4D and 4G) and hck-/- fgr-/- (Fig. 4E 4F 4H) BMDMs, as well as in the phagosomal membrane (Fig. 4I) .



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  		Figure 4. Confocal series showing the distributions of Syk and F-actin in wild-type and hck-/- fgr-/- BMDMs. The cells were handled as described in the legend to Figure 3 , except that polyclonal rabbit anti-Syk antibodies were used. A, D, and G, wild-type BMDMs. B, C, E, F, H, and I, hck-/- fgr-/- BMDMs. A–C, distribution of Syk (A and B) and F-actin (C) in unstimulated BMDMs. D–F,distribution of Syk (D and E) and F-actin (F) in BMDMs after the cells were allowed to ingest IgG-coated beads for 2 min at 37°C. G–I, Combination of anti-Syk and F-actin staining giving simultaneous visualization of Syk and F-actin; yellow areas indicate colocalization of the proteins. G, wild-type BMDMs. H and I, hck-/- fgr-/- BMDMs. I, example of colocalization of Syk and F-actin around a phagosome. Arrows, localization of Syk and F-actin in a phagocytic-cup region; arrowhead, colocalization of these proteins around a phagosome. Scale bars, 10 µm.

 
The tyrosine kinase inhibitors PP1 and piceatannol blocked IgG-dependent phagocytosis and Src family kinase and Syk activities in vivo
To address whether the residual phagocytic activity of hck-/- fgr-/- or hck-/- fgr-/- lyn-/- macrophages might be explained by the compensation of other Src family kinases, we tested the effect of the tyrosine kinase inhibitors PP1 and piceatannol [28 , 29 ] on Fc{gamma}R-dependent phagocytosis. As shown in Figure 5 , at concentrations of 30 and 50 µM, respectively, both PP1 and piceatannol totally inhibited internalization of IgG-coated beads by both wild-type and hck-/- fgr-/- BMDMs (Fig. 5A) . On the other hand, PP1 had only a limited effect (~50% inhibition), and piceatannol had no effect at all on phagocytosis of unopsonized yeasts by wild-type or mutant cells (Fig. 5B) . At 30 µM, PP1 totally blocked the Fc{gamma}R-induced activation of Lyn, Src, Fyn, and Yes in hck-/- fgr-/- BMDMs, reducing kinase activities to levels well below those detected in unstimulated cells (Fig. 6 ). This concentration of PP1 also blocked the enhancement of Syk activity induced by IgG-coated beads. Results comparable with those reported in Figure 6 were obtained with wild-type BMDMs (data not shown). Inhibition of Syk by the Src family kinase blocker PP1 was understandable in light of the current model, which places Src family kinases upstream of Syk in signaling by immune receptors (see Discussion). Conversely, 50 µM piceatannol reduced activity of Syk in BMDMs internalizing IgG-coated beads to almost undetectable levels but also blocked Fc{gamma}R-dependent activation of Src family kinases. The inhibitory effect of piceatannol on Src family kinase activities is less easily understood. Recent findings cast doubts on the real selectivity of piceatannol as a Syk inhibitor (30). To find a dose of the inhibitors that would selectively block Src family kinase or Syk activities, we examined kinase activities in cells treated with different doses of PP1 or piceatannol. PP1 at doses from 1 to 30 µM inhibited Src family kinase activities dose dependently, but, in line with the model placing Src family kinases upstream of Syk, it had a comparable inhibitory effect on Syk activity. As shown in Figure 7 , piceatannol at the dose of 25 µM selectively inhibited IgG-induced Syk but not Src family kinase activity in vivo. At this concentration, it is important that IgG-dependent phagocytosis was also inhibited, albeit not more than 50%. Given the absolute requirement for Syk in Fc{gamma}R phagocytosis, these results suggest that residual Syk activity remained in cells treated with 25 µM piceatannol.



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  		Figure 6. In vivo treatment of BMDMs with PP1 and piceatannol results in inhibition of IgG-stimulated Src family kinases and Syk activities. hck-/- fgr-/- BMDMs were incubated with 30 µM PP1 or 50 µM piceatannol as described in the Figure 5 legend. Cells were lysed, and immunocomplex kinase assays were performed as described in the Figure 2 legend and Materials and Methods. Counts per minute (cpm) incorporated into the immunoprecipitated kinases are reported as means ± SD of three independent experiments.

 


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  		Figure 7. In vivo treatment of BMDMs with 25 µM piceatannol inhibits Syk but not Src family kinase activity. hck-/- fgr-/- BMDMs were incubated with 25 µM piceatannol (PCT) as described in the Figure 5 legend. Cells were lysed and immunocomplex kinase assays were performed as described in the Figure 2 legend and Materials and Methods. Counts per minute (cpm) incorporated into the immunoprecipitated kinases are reported as means ± SD of two independent experiments. Phagocytosis of IgG-coated latex beads was performed as described in Materials and Methods.

 
Inhibition of Src and Syk kinase activities also resulted in inhibition of kinase redistribution to the phagocytic cup and actin polymerization in the phagocytic cup itself
Because we found that Fyn, Yes, Lyn, and Syk become enriched in phagocytic cups underneath IgG-coated beads (Fig. 3 and 4) , we asked whether inhibitors of these kinases affected this process. As shown in Figure 8 , PP1 (Fig. 8G 8H 8I) and piceatannol (Fig. 8J 8K 8L) inhibited, albeit not completely, both Fyn redistribution to the phagocytic cup (compare Fig. 8D and 8E with 8G H J and K ) and F-actin polymerization in the cup itself (compare Fig. 8F with Fig. 8I and 8L ). An approximate quantitation of Fyn and F-actin redistribution to the phagocytic cup confirmed that both PP1 and piceatannol inhibited redistribution of Fyn and F-actin to a comparable extent (Table 1). Similar results were obtained by analyzing association of Src, Yes, and Syk with actin-rich phagocytic cups (data not shown). Because formation of F-actin-rich phagocytic cups is not affected in syk-/- macrophages [11 ], these findings suggest that Src kinases contributed to their formation and only the block of both Src family kinases and Syk resulted in inhibition of phagocytic-cup formation and in an incomplete closure of the cup itself in a phagosome.


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  		Table 1. Effect of PP1 and Piceatannol on Phagocytic-Cup Formation

 
Regulation of Src kinases and Syk PLF
An important consequence of phagocytosis is the fusion of lysosomes with the phagosomal membrane to allow the discharge of hydrolytic enzymes and bactericidal proteins into the phagosomal space. To address whether Hck, Fgr, or Lyn is implicated in the regulation of PLF, we examined this process in hck-/-fgr-/- and hck-/- fgr-/- lyn-/- mutant BMDMs. Immunofluorescence staining of BMDMs with antibodies against the marker of late endosomes/lysosomes, LAMP-1, showed distribution of LAMP-1 in a perinuclear region and in granular structures scattered throughout the cytoplasm in wild-type (Fig. 9A ) as well as hck-/- fgr-/- or hck-/- fgr-/- lyn-/- knockout macrophages (data not shown). This staining pattern changed dramatically in wild-type (Fig. 9C) , double-knockout hck-/- fgr-/- (Fig. 9E) , and triple-knockout hck-/- fgr-/- lyn-/- (Fig. 9G) BMDMs after challenge with IgG-coated beads. In fact, we detected a clear redistribution of LAMP-1 to the vicinity of IgG bead-containing phagosomes, which were clearly identified by staining of F-actin (Fig. 9D 9F and 9H) in all three cell types. The redistribution of LAMP-1 in knockout BMDMs suggested that the deficiency of Hck, Fgr, or Lyn did not affect PLF. Because this finding could result from compensation by other Src kinases, exactly as it occurred with phagocytosis (Fig. 5) , the use of inhibitory drugs seemed to be the only possible approach to address the role of this kinase family in mediating PLF. It must be noted, however, that at doses inhibiting Src kinase and Syk activities, the two drugs inhibited internalization of IgG-coated particles as well (Fig. 5) , thus hampering the possibility of investigating PLF occurrence as a consequence of Fc{gamma}R-dependent phagocytosis. We therefore exploited the evidence that internalization of yeast particles occurred also in BMDMs treated with PP1 and piceatannol (Fig. 5) , using this as a tool to address the possibility that Src kinases and Syk play a role in regulating PLF independently of their involvement in phagocytosis. Treatment of cells with both drugs prevented redistribution of LAMP-1 to yeast-containing phagosomes in both wild-type (compare Fig. 10A with 10D and G ) and hck-/- fgr-/- (compare Fig. 10B with 10E and H) BMDMs. We concluded that at concentrations inhibiting both Src kinase and Syk activities (see Fig. 6 ), PP1 and piceatannol can hamper PLF independently of their effect on phagocytosis.



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  		Figure 9. Confocal series showing the distribution of LAMP-1 and F-actin in BMDMs isolated from wild-type and double (hck-/- fgr-/-)- or triple (hck-/- fgr-/- lyn-/-)-knockout mice. A–D, Distribution of LAMP-1 (A and C) and F-actin (B and D) in wild-type BMDMs which were left untreated (A and B) or allowed to ingest IgG-coated beads for 5 min at 37°C (C and D). E and F, Distribution of LAMP-1 (E) and F-actin (F) in hck-/- fgr-/- BMDMs which were allowed to ingest IgG-coated beads for 5 min at 37°C. G and H, Distribution of LAMP-1 (G) and F-actin (H) in hck-/- fgr-/- lyn-/- BMDMs which were allowed to ingest IgG-coated beads for 5 min at 37°C. Arrows, localization of LAMP-1 and F-actin in the phagosomal regions. Scale bars, 10 µm.

 


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  		Figure 10. Confocal series showing the effect of PP1 and piceatannol (PCT) on the distributions of LAMP-1 and F-actin in wild-type and hck-/- fgr-/- BMDMs. A, D, G, and J, wild-type BMDMs. B, C, E, F, H, I, K, and L, hck-/- fgr-/- BMDMs. A–C, distribution of LAMP-1 (A, B) and F-actin (C) in BMDMs that were allowed to ingest unopsonized yeast particles for 5 min at 37°C. D–F, as described for panels A–C, except that the cells were preincubated with 30 µM PP1 for 30 min at 37°C and during phagocytosis. G–I, as described for panels A–C, except that the cells were pretreated with 50 µM piceatannol for 60 min and during phagocytosis. J–L, distribution of LAMP-1 (J, K) and F-actin (L) in BMDMs which had been treated as above described with 25 µM piceatannol and then allowed to ingest IgG-coated beads for 5 min at 37°C. Arrows, localization of LAMP-1 and F-actin in the phagosomal region. Scale bars, 10 µm.

 
To distinguish the role of Src family kinases from that of Syk in regulating PLF occurring in macrophages that internalize IgG-coated particles, we exploited the evidence illustrated in Figure 7 that at 25 µM, piceatannol selectively inhibited Syk kinase activity stimulated by IgG-coated particles but only partially blocked particle internalization. It is interesting that at this dose of piceatannol, PLF occurred normally in both wild-type (Fig. 10J) and hck-/- fgr-/- (Fig. 10K) BMDMs. These findings suggest that Src family kinases are more relevant in regulating PLF and that this depends on their enzymatic activity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Initiation of Fc{gamma}R-signaling events depends on tyrosine phosphorylation of ITAM-containing sequences, presumably by Src family kinases, within the receptors (or their associated chains), leading to recruitment and activation of Syk. Although the major Src family kinases in phagocytes, Hck, Fgr and Lyn, have been found to be physically and functionally associated with Fc{gamma}Rs [31 32 33 34 35 ], macrophages from hck-/- fgr-/- lyn-/- mutant mice have only kinetic delay and not a complete block in their ability to phagocytose IgG-opsonized red blood cells [11 , 13 ]. The capability of hck-/- fgr-/- lyn-/- macrophages to internalize IgG-coated particles could be explained by a functional association between Fc{gamma}Rs and other Src family kinases. We found that both wild-type and hck-/- fgr-/- macrophages express Fyn, Src, and Yes and that these kinases are rapidly activated on interaction of the cell with IgG-coated particles. In addition, we showed that these kinases are redistributed, together with Syk, to the membranes of both forming and closed phagosomes, in close contact with F-actin. The mechanisms responsible for this redistribution remain to be determined. Given that the periphagosomal cytoplasm is the site of assembly of multimolecular complexes containing cytoskeletal and tyrosine-phosphorylated proteins [36 ], it is possible that Src homology 2 (SH2) or 3 (SH3) protein interactions might mediate recruitment of Src family kinases to the phagocytic cup [37 38 39 ]. This kinase recruitment can be independent of Fc{gamma}R signaling per se, because we found that Fyn, Src, and Yes were concentrated in actin-rich phagocytic cups formed during internalization of unopsonized yeast particles (data not shown), and similar results have been reported for Lyn in monocytes [40 ]. It is also important to note that other immune receptors, such as the Fc{gamma}RI and antigen receptors, redistribute to specialized sphingolipid- and cholesterol-enriched membrane domains that represent sites of attachment of a variety of lipid-modified proteins, including Src family kinases [41 ]. Whether Fc{gamma}R clustering also induces increased association of Src family kinases, as well as other membrane or signaling proteins, with similar specialized membrane compartments is not known. However, it is worth noting that formation of Triton X-100-insoluble complexes containing ß2 integrins, the urokinase plasminogen activator-receptor, and Src family kinases has been described in monocytes [42 ].

That Fc{gamma}R engagement activates up to six distinct Src family members [31–34 and this paper] represents a major obstacle for the understanding of their role in phagocytosis. We addressed this issue by exploiting the availability of drugs that have been determined to selectively inhibit the activity of Src family kinases or Syk, i.e., PP1 and piceatannol, respectively [28 , 29 ]. Testing the activity of these drugs in vivo, we found that at optimal concentrations to inhibit IgG-dependent phagocytosis, both drugs were capable of inhibiting Fc{gamma}R-induced Src family kinase, as well as Syk activities. It must be noted that inhibition of Src family kinase and Syk activities resulted not only in a total block of IgG-dependent phagocytosis but also in a marked inhibition of actin-rich phagocytic-cup formation and Src-kinase redistribution to phagocytic cups. It was interesting that syk-/- macrophages were shown to assemble F-actin underneath the plasma membrane adherent to IgG-coated particles normally [12 ]. Hence, our findings indicate that Src family kinase activities were critical for maturation of actin-rich phagocytic cups into phagosomes, suggesting that, independently of their role in ITAM phosphorylation and Syk activation, these activities might play the major role of regulating actin polymerization and closure of the membrane in a phagosome during Fc{gamma}R signaling. This conclusion is consistent with recent findings showing that Lyn and Hck can drive signaling for Fc{gamma}R-mediated phagocytosis in a macrophage cell line [43 ].

An important consequence of phagocytosis is the sequestration of pathogens within the phagosome and the subsequent discharge of hydrolytic enzymes and bactericidal proteins contained in the lysosomal compartment and the phagosomal space. Despite the well-established notion that one of the mechanisms of pathogen escape from host defenses relies on the inhibition of phagosome maturation, little is known about pathways of regulation of PLF [1 , 44 , 45 ]. For this report we investigated the role of Src family kinases and Syk in regulating PLF by using hck-/- fgr-/- lyn-/- macrophages and inhibitory drugs. We found that phagosome maturation as assayed by examining redistribution of the late endosome/lysosome marker LAMP-1 was normal in hck-/- fgr-/-and hck-/- fgr-/- lyn-/-macrophages, demonstrating that these kinases are not absolutely required for PLF. Likewise, treatment of cells with 25 µM piceatannol, which results in inhibition of detectable Syk activity, also did not produce a total block of LAMP-1 redistribution to the phagosomal membrane during Fc{gamma}R-mediated particle internalization. Because study of PLF in PP1- and piceatannol-treated cells is hampered by the fact that Fc{gamma}R-mediated particle uptake is blocked, we took advantage of the fact that ingestion of unopsonized yeast particles still occurred under these conditions. We found that concentrations of both PP1 and piceatannol able to effectively inhibit Src family kinases totally blocked PLF after internalization of unopsonized yeast particles (Fig. 9) . Together with the evidence that PLF is normal in hck-/- fgr-/- lyn-/- macrophages, these findings suggested that Fyn and Yes should be implicated in regulation of PLF. Studies with Fyn and Yes knockout macrophages are needed to validate these conclusions. The evidence that Src family kinases might be implicated in regulation of PLF is of great interest in the context of the recent findings that lipoarabinomannan, a mycobacterial cell wall component possibly representing a virulence factor of Mycobacterium tubercolosis, decreased tyrosine phosphorylation signals through activation of the SH2-containing tyrosine phosphatase 1 (SHP-1) in mononuclear phagocytes [47 ]. In fact, at least as far as adhesion receptors are concerned, an inverse relationship between Src family kinases and SHP-1 in myelomonocytic-cell signaling has been demonstrated [47 , 48 ]. Hence, it is tempting to speculate that inhibition of phagosome maturation by Mycobacterium species is achieved through interference of Src family kinases. Indeed, mycobacteria hamper activation of Hck and its translocation to the phagosomal membrane in human neutrophils [49 ].

In conclusion, in this report we have demonstrated that Src family kinases are redundantly expressed in macrophages and are implicated in signaling by Fc{gamma}Rs, participating in actin polymerization in phagocytic cups and particle internalization. Additionally, we have shown that Src family kinases, as well as Syk, redistribute to the phagosomal membrane, suggesting that they play a role in postphagocytic events. The evidence that inhibition of Src family kinase activities blocked PLF adds new clues to the mechanism of regulation of phagosome maturation.


   ACKNOWLEDGEMENTS
 
This study was supported by grants from The Minister of University and Scientific Research of Italy (MURST)/University of Verona (Research Program: Inflammation: Cellular and Molecular Pathophysiology) and Cariverona (Progetto Sanità) to G. B.; the County of Östergötland and the University Hospital, Linköping, Sweden, the Swedish Society of Medicine, the Magnus Bergvall Foundation, and the Swedish Medical Research Council to M. M.; and the National Institutes of Health (DK50267 and HL54476) to C. A. L. M. M. is a recipient of a Marie Curie Research Training Grant from the European Community. We are grateful to Prof. Guido Fumagalli for his help in providing access to the confocal microscopy facility of the Institute of Pharmacology of Verona. Monoclonal antibody 1D4B developed by Dr. Thomas August was obtained from the Developmental Studies Hybridoma Bank under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City.

Received April 9, 2001; revised August 6, 2001; accepted August 8, 2001.


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