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R1
Department of Cell Biology, Nencki Institute of Experimental Biology, Warsaw, Poland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Rs is
controlled by the Src and Syk families of protein tyrosine kinases. In
this study, we demonstrate a sequential involvement of Lyn and Syk in
the phagocytosis of IgG-coated particles. The particles isolated at the
stage of their binding to FcRs (4°C) were accompanied by high
amounts of Lyn, in addition to the signaling -chain of FcRs.
Simultaneously, the particle binding induced rapid tyrosine
phosphorylation of numerous proteins. During synchronized
internalization of the particles induced by shifting the cell to
37°C, Syk kinase and Src homology 2-containing tyrosine phosphatase-1
(SHP-1) were associated with the formed phagosomes. At this
step, most of the proteins were dephosphorylated, although some
underwent further tyrosine phosphorylation. Quantitative immunoelectron
microscopy studies confirmed that Lyn accumulated under the plasma
membrane beneath the bound particles. High amounts of the -chain and
tyrosine-phosphorylated proteins were also observed under the bound
particles. When the particles were internalized, the -chain was
still detected in the region of the phagosomes, while amounts of Lyn
were markedly reduced. In contrast, the vicinity of the phagosomes was
heavily decorated with anti-Syk and anti-SHP-1 Abs. The local
level of protein tyrosine phosphorylation was reduced. The data
indicate that the accumulation of Lyn during the binding of IgG-coated
particles to FcRs correlated with strong tyrosine phosphorylation of
numerous proteins, suggesting an initiating role for Lyn in protein
phosphorylation at the onset of the phagocytosis. Syk kinase and
SHP-1 phosphatase are mainly engaged at the stage of particle
internalization. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rs are well-characterized
components engaged in phagocytosis in macrophages, monocytes, and
neutrophils (1, 2, 3). During phagocytosis, the specific
interaction of FcR with IgG-opsonized particle induces local
clustering of the receptors. As a result, the tyrosine residues in the
immunoreceptor tyrosine-based activation motifs
(ITAMs)3 of FcRIIA
and the accessory -chain of FcRI and FcRIIIA are
phosphorylated, and subsequently, phosphorylation of several downstream
proteins occurs (2, 4, 5). Biochemical data demonstrate
that Src, as well as Syk, family kinases are the enzymes activated by
clustered FcRs and that they play a crucial role in FcR-mediated
phagocytosis (6, 7, 8, 9, 10). The role of Src kinases was
confirmed by experiments on Src-deficient cells in which the
phosphorylation of FcRIIA and phagocytosis were diminished
(11). However, some data indicate that Src kinases are not
absolutely required for uptake of particles. In
hck-/-fgr-/-lyn-/-
macrophages, FcR-mediated phagocytosis did occur in a delayed
fashion, and treatment of these cells with inhibitors of Src family
kinases did not completely block phagocytosis (12). In
contrast, in monocytes in which the expression of Syk was blocked by
antisense oligodeoxynucleotides, tyrosine phosphorylation of FcRIIA
occurred but phagocytosis was completely inhibited (13).
Similarly, studies of Syk-deficient macrophages derived from transgenic
mice proved that the attendance of Syk is indispensable for
phagocytosis (14). Cotransfection of Syk with the
subunit of FcRs conferred phagocytic abilities on fibroblasts
(9). Moreover, a chimeric protein, constructed from the
extracellular and transmembrane domains of FcRIII and Syk kinase as
the cytoplasmic part, was able to induce reorganization of the actin
cytoskeleton and trigger phagocytosis (15). Comparison of
the phenotypes of the
hck-/-fgr-/-lyn-/-
and syk-/- macrophages reveals that, in
the absence of Syk kinase, the formation of actin cups was normal but
internalization of particles did not occur. In contrast, the lack of
kinases of the Src family led to a delay in actin cup formation but
successful phagocytosis was achieved. These data suggest that Src and
Syk family kinases have distinct functions in the propagation of a
phagocytic signal generated by the FcR (12) and
indicate a sequential activation of Src and Syk family kinases by the
FcR. Studies on the cellular redistribution of Src and Syk family
kinases during phagocytosis could verify the sequence of involvement of
the kinases in the process. The aim of our study was to examine the
cellular distribution of Lyn and Syk kinases at different steps of
phagocytosis in J774 macrophage-like cells. We wanted to determine
whether the kinases are sequentially translocated to the ligated
receptors and the dynamics of their association with FcRs when
phagocytosis occurs. This is the first high-resolution immunogold
observation of the sequential engagement of Lyn and Syk kinases in
phagocytosis mediated by FcRs. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse J774A.1 macrophage-like cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM (Seromed, Munich, Germany) supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Before experiments, the cells were starved for 2 h in serum-free medium at 37°C in 5% CO2. The starved cells were detached mechanically by a rubber policeman and resuspended in serum-free medium at a concentration of 5 x 106 cells/ml.
For phagocytosis, polystyrene latex beads (LB; 3 µm in diameter; Sigma-Aldrich, St. Louis, MO), zymosan A (ZA; 3–5 µm in diameter; Sigma-Aldrich), or magnetic particles (MP; 1 µm in diameter; Boehringer Mannheim, Indianapolis, IN) were used. Particles were suspended in PBS to 10 mg/ml and used for opsonization. The opsonization was performed by incubation of the particles in PBS containing 20 mg/ml rat IgG or BSA for 2 h at 37°C with constant shaking. The opsonized particles were collected by centrifugation (3000 x g, 5 min) and free protein-binding sites were blocked in 5% BSA in PBS for 30 min at 37°C. The particles were then washed and suspended again at 2 x 107 particles/ml in phosphated Dulbecco’s (PD) medium (125 mM NaCl, 5 mM KCl, 10 mM NaHCO3, 1 mM KH2PO4, 1 mM CaCl2, 1 mM MgCl2, and 20 mM HEPES; pH 7.4) and directly used for phagocytosis studies.
Before experiments, the cells were washed and preincubated for 10 min in ice-cold PD. To promote binding of the particles to the cell surface, the cells were exposed to the opsonized particles at 4°C (20 particles/cell) and incubated with a delicate shaking. Unbound particles were removed by washing with ice-cold PD. The internalization of the bound particles was performed by shifting the cells to 37°C, and the cells were incubated with a delicate shaking. At various time points, the cells were pelleted and either fixed and used for electron microscopy studies or lysed and processed for immunoblotting analysis.
Cell lysis
The cells undergoing phagocytosis of opsonized LB were collected, washed in ice-cold PD medium supplemented with 1 mM Na3VO4 10 mM NaF, and 10 mM Na4P2O7, and lysed in 75 µl of ice-cold Triton/SDS lysis buffer (1% Triton X-100 (TX-100), 0.1% SDS, 150 mM NaCl, 2 mM EDTA, and 50 mM Tris-HCl; pH 7.5) supplemented with 1 mM Na3VO4, 10 mM NaF, 50 µM PAO, 2 mM PMSF, 20 mM Na4P2O7, 10 µg/ml aprotinin, 10 µg/ml leupeptine, and 10 µg/ml pepstatin A (4). Twenty minutes later, 3.5 µl of 20% SDS was added, and nonlysed material was pelleted (3000 x g, 5 min). The supernatant was complemented with 26 µl of 4x SDS-sample buffer, boiled for 5 min, and processed for immunoblotting analysis.
Isolation of phagocytic particles
The cells undergoing phagocytosis of opsonized MP were collected, washed and solubilized in ice-cold Triton lysis buffer (0.1% TX-100, 150 mM NaCl, 5 mM EDTA, and 50 mM Tris-HCl; pH 7.5), and supplemented with the protease and phosphatase inhibitors mentioned above. After lysis (20 min, 4°C), MP were separated in a magnetic particle separator (Boehringer Mannheim). The isolated particles were resuspended in 60 µl of ice-cold Triton lysis buffer, and the suspension was supplemented with 16.2 µl of 4x SDS-sample buffer. The samples were boiled for 5 min and processed for immunoblotting analysis.
Immunoblotting analysis
The proteins (30 µg) were electrophoretically separated on
10% SDS-PAGE (16) and transferred to nitrocellulose
sheets (17). After overnight blocking in 5% nonfat milk
in TBS/0.05% Tween 20, the blots were washed in TBS/0.05% Tween 20
and probed for 1.5 h at room temperature with the following Abs:
mouse anti-phosphotyrosine PY-20 (BD Transduction Laboratories,
Lexington, KY), rabbit anti--chain 934 (generously provided by
Dr. M.-H. Jouvin (Beth Israel Deaconess Medical Center, Boston, MA)),
rabbit anti-Lyn, rabbit anti-Syk (N-19 and C-20, directed
against the N and C termini of Syk, respectively) and rabbit
anti-Src homology 2-containing tyrosine phosphatase-1 (SHP-1)
(C-19) (Santa Cruz Biotechnology, Santa Cruz, CA). In some experiments,
rabbit anti-SHP-1 (generously provided by Dr. J. C. Cambier
(University of Colorado, Denver, CO)) were also applied, yielding
results corresponding to those obtained with the Santa Cruz Ab. After
washing (six changes of TBS/0.05% Tween 20, 5 min each), the blots
were incubated for 1 h at room temperature with appropriate
secondary Abs: sheep anti-mouse IgG or sheep anti-rabbit IgG
conjugated with HRP (Amersham, Arlington Heights, IL) or with biotin
followed by streptavidin HRP (Sigma-Aldrich). All Abs were prepared in
2% nonfat milk in TBS/0.05% Tween 20. After washing, the
immunoreactive bands were visualized using SuperSignal Chemiluminescent
Substrate (Pierce, Rockford, IL). Luminescent images were analyzed in a
Fluo-S MultiImager System using QuantityOne software (Bio-Rad,
Hercules, CA). Molecular masses of polypeptides were determined
based on their relative electrophoretic mobility using prestained
molecular mass standards (Bio-Rad).
Electron microscopy
At various time points of phagocytosis of opsonized ZA, the
cells were collected, washed in PD at 4°C, and fixed with 3%
paraformaldehyde/1% glutaraldehyde in 100 mM sodium phosphate buffer
(pH 7.2) for 1 h at room temperature. The washed samples were
dehydrated in an ethanol series at room temperature. The pelleted
samples (3000 x g, 30 s) were suspended in 100%
propylene oxide for 20 min at room temperature followed by incubation
in mixtures of LR White resin/propylene oxide (Polysciences,Warrington,
PA) at ratios of 1:2 and 1:1, each for 30 min. Finally, the samples
were infiltrated twice with 100% LR White resin for 1 h at room
temperature. The probes were polymerized at 55°C for 48 h.
Ultrathin sections were placed on formvar-covered nickel grids and
blocked in 5% FCS in PBS for 1 h at room temperature. After
washing in PBS, the samples were incubated with different primary
rabbit Abs (anti--chain, anti-Lyn, anti-Syk (N-19 and
C-20), anti-SHP-1 (all as described above), and
anti-phosphotyrosine) for 3 h at room temperature in a humid
atmosphere. After washing (six times in PBS/0.5% BSA/0.05% Tween 20,
5 min each), sections were treated with goat anti-rabbit IgG
conjugated with 10-nm gold particles (Sigma-Aldrich) for 4 h at
room temperature. In control samples, either incubation with the
primary Abs was omitted or the samples were treated with rabbit IgG
(Jackson ImmunoResearch Laboratories, West Grove, PA) followed by goat
anti-rabbit IgG-10-nm gold. All Abs were prepared in PBS/0.5%
FCS/0.05% Tween 20. After washing, the samples were counterstained
with 2.5% uranyl acetate in ethanol for 15 min at room temperature in
dark, washed in 50% ethanol and three times in distilled water, and
stained with lead citrate for 2 min at room temperature. Finally, the
sections were washed three times in distilled water and examined under
a JEOL (Peabody, MA) JEM-1200EX electron microscope. The number of gold
particles in cellular compartments (in cytoplasm, at the plasma
membrane nonoccupied by ZA, at the membrane with bound ZA, and in
phagosome region) was counted from five experiments and evaluated both
per square micrometer of the compartment area and according to the
number of gold particles per cluster.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R-mediated phagocytosis
Earlier studies by Greenberg’s group (4, 6)
demonstrated that the engulfment of particles mediated by the FcR is
accompanied by enhanced tyrosine phosphorylation of numerous proteins.
We expanded Greenberg’s studies and examined the level of tyrosine
phosphorylation of proteins at two distinct steps of phagocytosis:
during particle binding and during internalization. The binding of
IgG-coated LB to the surface of J774 cells induced rapid and transient
tyrosine phosphorylation of polypeptides of 210–190, 150, 134–115,
110, 101, 97, 85–69, 64–50, 40, and 30 kDa (Fig. 1
, lane 2). The
phosphorylation was most intensive during the first 5–10 min of
particle binding. When internalization of IgG-coated LB started, most
of the proteins were dephosphorylated, although some underwent further
tyrosine phosphorylation (Fig. 1, lane 4). The proteins of
64–50 kDa were likely to belong to the Src family kinases. The
polypeptides of 75–69 kDa had been suggested to be Syk kinase and
paxilin, respectively (4). In control samples, when LB
were coated with BSA, the basic level of tyrosine phosphorylation was
seen (Fig. 1, lanes 1 and 3).
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A, lane 2,
arrows). When particles were isolated after 5–10 min of
internalization, the proteins of 40–30 kDa were found to be further
phosphorylated, whereas the phosphorylation of the others was reduced
(Fig. 2A, lanes 3 and 4). In control
experiments, when MP were opsonized with BSA, some level of nonspecific
phosphorylation was detected, although the pattern of phosphorylated
proteins was different from the pattern characteristic for IgG
stimulation (Fig. 2A, lanes 5–8). The isolated
proteins associated with MP were also analyzed for the presence of the
-chain, Lyn, Syk kinases, and SHP-1 tyrosine phosphatase. The
-chain, the accessory signaling subunit of FcRI and FcRIII
expressed in J774 cells, was found to be associated with IgG-coated MP
at the binding and internalization steps of phagocytosis (Fig. 2B, lanes 1–4). High amounts of Lyn kinase were
isolated with the IgG-coated MP at the binding step, while during
internalization the Lyn content was reduced (Fig. 2C,
lanes 1–4). Analysis of the level of Syk kinase in pellets
of IgG-coated MP with the use of Abs against both N and C termini of
Syk showed that, at the binding step, only minute amounts of the enzyme
were associated with the particles. During internalization, the level
of Syk kinase that copelleted with the particles substantially
increased, peaking at 10 min for particle engulfment (Fig. 2, D and E, lanes 1–4). As the
internalization of particles was accompanied by distinct tyrosine
dephosphorylation of numerous proteins, distribution of SHP-1 was next
examined. Recently, SHP-1 has been implicated in modulating tyrosine
phosphorylation-dependent signaling during FcR-mediated phagocytosis
(18). As shown in Fig. 2F, lanes
1–4, SHP-1 was associated with isolated IgG-coated MP mainly
during internalization of the particles. The binding and
internalization of control BSA-coated MP engaged smaller amounts of Syk
and SHP-1 (Fig. 2, D–F, lanes 5–8). Similarly,
less -chain and Lyn accompanied phagocytosis of the control
particles compared with that of the IgG-coated MP (Fig. 2, B
and C, lanes 5–8).
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R-mediated phagocytosis: immunoelectron microscopy study
Relative amounts of the -chain and downstream signaling
molecules of FcRs were estimated in the cellular compartments at
different steps of phagocytosis. In resting cells, the -chain and
Lyn kinase decorated by 10-nm gold particles were observed in
comparable amounts under the plasma membrane and in the cytoplasm (Fig. 3, A and D). The
total number of gold particles in these compartments was 23–32 gold
particles/µm2 (Fig. 4, A and B). Under
the plasma membrane and in the cytoplasm, the gold particles attributed
to the -chain existed as a mixture of individuals and aggregates of
various sizes (Table I). In these cells, Lyn located in the vicinity of
the plasma membrane was decorated mainly by single gold particles. In
the cytoplasm, Lyn was marked with gold particles more heterogeneously;
a mixture of individuals (34% of total), small aggregates of 2–4 and
5–7 particles (
40% of total), and some numbers of large aggregates
containing over 12 gold particles (26% of total) were seen (Table I). Under these conditions, the majority of total Syk kinase
decorated by both N- and C-terminal-specific Abs was located in the
cytoplasm as demonstrated in Fig. 3G for Ab N-19. Syk in the
cytoplasm was revealed by 29 ± 4 and 39 ± 5 gold
particles/µm2 occurring as individuals and also
as small and large aggregates (Fig. 4, C and D;
Table I). Under the plasma membrane, Syk
was displayed predominantly as individuals (76.5% of total for N-19
and 52% for C-20 Abs) (Table I). However, only 6–8 gold
particles/µm2 were found there (Fig. 4, C and D). The distribution of SHP-1 resembled the
cellular distribution of Syk with the majority of the phosphatase
located in the cytoplasm (26 ± 3 gold
particles/µm2) (Figs. 3J and
4E; Table I). The quantification of tyrosine-phosphorylated
proteins showed that they were mainly located in the cytoplasm (Fig. 3M). The total number of gold particles attributed to the
phosphoproteins in this compartment was 42 ± 8 gold
particles/µm2 (Fig. 4F), and they
were distributed mainly as individuals and as medium aggregates of
8–11 gold particles (both 37.5% of total) (Table I). Under the plasma
membrane, phosphotyrosine decoration was observed at a density of
22 ± 2 gold particles/µm2 (Fig. 4F) and was displayed as individuals (56% of total) and as
small aggregates of 2–4 and 5–7 gold particles (44% of total)
(Table I).
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-chain and Lyn kinase. High amounts
of these proteins were visualized under the plasma membrane beneath the
bound particles (Fig. 3
, B and E), reaching up to
51 gold particles/µm2 (Fig. 4, A and
B). Among them, 50% of the gold particles were organized
in large aggregates containing over 8 gold particles per cluster (Table I). The region of the plasma membrane outside the bound IgG-coated ZA
was poorer in gold particles, 10 ± 1 and 15 ± 1 gold
particles/µm2 for the -chain and Lyn,
respectively; also, small amounts of gold particles
per square micrometer were found in cytoplasm (Fig. 4, A and
B). In contrast to Lyn, no significant amounts of Syk were
observed beneath bound IgG-coated ZA (8–12 gold
particles/µm2) (Figs. 3H and 4,
C and D). Similarly, under the plasma membrane
not occupied by ZA, only 5–8 gold
particles/µm2 were counted (Fig. 4, C and D). The main pool of Syk was still present
in the cytoplasm (25 ± 4 and 27 ± 3 gold
particles/µm2 for N-19 and C-30, respectively)
where gold particles occurred mainly as singlets and as small
aggregates containing up to 4 gold particles (Fig. 4, C and
D; Table I). The pattern of distribution of SHP-1 at this
stage of phagocytosis reflected the localization of Syk kinase, and the
phosphatase remained dispersed in the cytoplasm, avoiding regions of
bound particles (Figs. 3K and 4E; Table I). The
results of counting gold particles specific for tyrosine-phosphorylated
proteins indicated a significant number of the label under the bound
IgG-coated ZA (Fig. 3N), reaching up to 87 ± 17 gold
particles/µm2 (Fig. 4F). The
phosphotyrosine decoration diminished under the plasma membrane not
occupied by ZA to 17 ± 2 gold
particles/µm2, and to 33 ± 3 gold
particles/µm2 in the cytoplasm (Fig. 4N). The appearance of large clusters containing over 15
gold particles attributed to phosphoproteins under bound IgG-opsonized
ZA correlated with high amounts of aggregated -chain and Lyn kinase
found in this cellular compartment (Table I).
Internalization of IgG-coated ZA induced a new distribution of the
kinases and SHP-1 in the cell. In the phagosomal region, the amount of
Lyn was dramatically decreased and only 13 ± 2 gold
particles/µm2 attributed to the kinase were
found (Figs. 3C and 4B). The large aggregates of
Lyn observed at bound ZA dispersed during internalization; only
singlets (49.5% of total) and small aggregates up to 7 gold particles
(50.5% of total) were detected around phagosomes (Table I). In
parallel, the number of gold particles attributed to Lyn in the
cytoplasm and under the plasma membrane was elevated to 29 ± 3
and 20 ± 3 gold particles/µm2,
respectively (Fig. 4B). The majority of the particles were
individuals, 46 and 41% of total, respectively (Table I). During
internalization of IgG-opsonized ZA, the -chain still accumulated
around phagosomes, forming small clusters (Figs. 3C and
4A; Table I). In contrast to the scarce labeling of Lyn, the
phagosomal region was heavily decorated by anti-Syk Abs (Figs. 3I and 4, C and D). The particles
occurred mostly as aggregates composed of over 8 particles per cluster
(50% of total gold particles) when probed with the N-19 Ab. The
C-20 Ab revealed small clusters of Syk (Table I). At this stage, the
total number of gold labels in the cytoplasm and under the plasma
membrane resembled the Syk decoration detected earlier in these
compartments (Fig. 4, C and D). The level of
tyrosine-phosphorylated proteins around phagosomes was reduced 2-fold
to 50 ± 3 gold particles/µm2 in
comparison to the amounts of phosphoproteins under the plasma membrane
beneath the bound ZA, and large aggregates of gold particles were no
longer visible (Figs. 3O and 4F; Table I). The
decrease of protein phosphorylation in phagosomal regions correlated
with significant local enrichment of SHP-1 (Figs. 3L and
4E; Table I). In control samples incubated with irrelevant
Abs, or in which incubation with the primary Abs was omitted, only a
few singular and regularly distributed gold particles were seen,
independent of the stage of phagocytosis (Figs. 3, P–S, and
4G; Table I).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rs is
questionable. In our studies, we focused on the question of the
involvement of Lyn and Syk kinases at different stages of
FcR-mediated phagocytosis distinguished by processing experiments
consecutively at 4°C and 37°C. The data presented here indicate
that there is a sequential participation of Lyn and Syk kinases in the
phagocytosis of IgG-coated particles. Our immunoelectron microscopy
data demonstrated that only Lyn accumulated under the bound particle
(4°C), whereas Syk and SHP-1 phosphatase were found in the
periphagosomal region where particle internalization took place
(37°C). Tyrosine phosphorylation of numerous proteins occurred when
particles interacted with FcRs on the cell surface, and later,
during particle internalization, the level of the protein
phosphorylation decreased (Figs. 3 and 4; Table I). The isolation of
IgG-coated MP at different steps of phagocytosis confirmed that, during
binding of particles, Lyn and the major fraction of phosphorylated
proteins were associated with MP. The engulfed particular material was
mainly occupied by Syk and SHP-1 and smaller amounts of phosphorylated
proteins (Figs. 1 and 2). Analysis of the engagement of Src and Syk
family kinases and the appearance of phosphoproteins at different steps
of phagocytosis indicate that the accumulation of Lyn kinase at the
binding sites correlates with abundant phosphorylation of numerous
proteins, thus suggesting the initiating role of Lyn in the
phagocytosis.
The role of Src family kinases in phosphorylation of the -chain was
clearly demonstrated in
hck-/-fgr-/-lyn-/-
macrophages in which no induction of the -chain phosphorylation was
detectable after FcR cross-linking (12). These data are
consistent with a report showing a role of Src family kinases in the
phosphorylation of ITAM of FcRIIA in THP-1 cells by a
pharmacological approach (19). Furthermore, the authors
revealed strong tyrosine phosphorylation of FcRIIA in COS-7 cells
cotransfected with Lyn, but not with Syk. Engagement of Src family
kinases in the phosphorylation of ITAM of other immunoreceptors,
including TCR, BCR and Fc
RI, is generally accepted
(20, 21, 22). Using a two-hybrid system, immunoprecipitation,
and chemical cross-linking, it was demonstrated that a small fraction
of Src kinases (e.g., Lyn for FcRI) was constitutively associated
with immunoreceptors. This fraction of the enzymes can be responsible
for the initial phosphorylation of the activated receptors.
Phosphorylated ITAMs serve as the docking site for additional molecules
of Src kinases recruited to the activated receptors (Refs.
23, 24, 25 and 26 and references therein).
Subsequent dissociation of Lyn from highly phosphorylated FcRI is
required for the docking of Src homology 2 domains of Syk and
signal progression (27). Sequential engagement of Src and
Syk kinases was also considered for FcRIIA signaling and
FcR-mediated phagocytosis (10, 19, 26, 28, 29, 30). In
contrast, in
hck-/-fgr-/-lyn-/-
macrophages, FcR-mediated phagocytosis accompanied by Syk activation
and actin assembly still occurred but in a delayed and diminished
fashion. These cells treated with PP1 still maintained the ability to
engulf the particles (12). Thus, Src family kinases seem
not to be absolutely required for initiation of the phagocytic signal.
Instead, a line of evidence points to Syk as the initiating kinase,
suggesting that a small number of Syk/ITAM complexes can constitutively
exist in resting cells (31). In accordance with this,
activation of FcRs in macrophages derived from Syk-knockout mice did
not induce phosphorylation of the -chain (32), and
inhibition of Syk activity by treatment of platelets with 100 µM
piceatannol resulted in significant inhibition of FcRIIA
phosphorylation (33). However, there is a possibility
that, in the absence of Syk, tyrosine residues of ITAM phosphorylated
by Src kinases were not protected against the action of tyrosine
phosphatases, and, as a result, no ITAM phosphorylation was detected
(32). Furthermore, careful investigation of Majeed et al.
(34) cast doubt on piceatannol specificity, indicating
that prolonged incubation of cells with the drug at concentrations
exceeding 25 µM results in inhibition of both Syk and Src family
kinases. Nevertheless, several findings showed that recruitment of Syk
to activated immunoreceptors, including BCR, FcRI, and FcRs, was
rapid (e.g., Refs. 4, 21, 35 , and 36). Syk
was also found in phagocytic cups during FcR-mediated phagocytosis
(33). However, it should be noted that, in these studies,
the receptors were activated at 37°C either by cross-linking or by
binding with simultaneous internalization of IgG-coated particles. This
temperature mimicked physiological conditions but may hamper
elucidation of the assembly of multimolecular signaling complex due to
the fast processing of this event. In contrast, our experimental
approach in which binding of particles at 4°C blocked particle
internalization allowed the distinction of some of the signaling events
attributed to distinct stages of phagocytosis.
In our studies, minute amounts of Syk were found to be recruited during
the binding of IgG-coated particles. For detection of Syk, Abs specific
against the N and C termini of the kinase were applied to overcome
sterical hindrances of accessibility of the Abs to their epitopes.
Similar results were obtained with the two different Abs indicating
that the plausible epitope masking caused by the interaction of
N-terminal Src homology 2 domains of Syk with phosphorylated
ITAM was not observed. However, we cannot exclude that the low
temperature during particle binding could reduce the diffusion of Syk,
as well as SHP-1, in the cytoplasm and diminish the solubility of
cytosolic proteins affecting the amounts of Syk and SHP-1 found at the
membrane under bound particles. In addition, the low temperature could
favor the enzymatic activity of Csk/Lyn over that of Syk and tyrosine
phosphatases, thus magnifying the accumulation of Lyn and
tyrosine-phosphorylated proteins under the bound particles. Despite
these perspectives, we believe that our experimental approach revealed
that Src family kinases can fulfill the initiating role in protein
phosphorylation during FcR-mediated phagocytosis. This may be
facilitated by docking of the Src kinases in the FcR-bearing plasma
membrane and compartmentalization of Syk in the cytoplasm.
Recently, it has been suggested that activated Src family kinases can
promote actin polymerization and formation of phagocytic cups either
through Syk or without Syk engagement, as demonstrated in
hck-/-fgr-/-lyn-/-
and syk-/- macrophages (12, 14) and in J774 cells (37). Syk is indispensable
for phagocytosis, because down-regulation of its expression in
monocytes (13) and in macrophages (14, 32)
abrogate internalization of IgG-coated particles. A chimeric protein,
containing the extracellular and transmembrane domains of FcRIII and
catalytic domain of Syk kinase as the cytoplasmic part, was efficient
in triggering actin filament polymerization and internalization of
particles (15). However, in Syk-deficient macrophages, the
formation of actin-rich phagocytic caps was not disturbed
(14). Thus, Syk kinase may play a critical role in closing
phagosomes and internalization of particles. Under conditions in which
expression of Src family kinases is experimentally diminished or their
activation is circumvented, Syk may solely trigger actin rearrangement
and phagocytosis.
Recently, the engagement of Src family kinases in the tyrosine
phosphorylation of immunoreceptors was strongly supported by
elucidation of a crucial role of cholesterol/sphingolipid-rich membrane
domains (rafts) in this process (38, 39, 40, 41). The rafts are
places of concentration of Src kinases (38), but not Syk
family kinases (42). Cross-linked FcRII associates with
rafts containing Lyn and undergoes phosphorylation by Src family
kinases (41, 43). Later, Syk is recruited to clusters of
the cross-linked FcRIIA (our unpublished data). The engagement of
the Src kinases residing in rafts in generation of phagocytic signals
by FcRs was recently suggested (10). From this point of
view, it is of interest that large clusters of the -chain, and
particularly prominent clusters of Lyn and tyrosine-phosphorylated
proteins, were revealed during the binding of IgG-coated particles
(Table I). These observations support the assumption that clustering of
FcRs triggers signal transduction at the onset of phagocytosis
(2, 44) and may point to the involvement of membrane rafts
in this process.
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Acknowledgments |
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-chain 934 and rabbit anti-SHP-1 Abs.
We also thank Dr. Patrick Groves for his critical reading of the
manuscript. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Andrzej Sobota, Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Street, PL 02-093 Warsaw, Poland. E-mail address: asobota{at}nencki.gov.pl
3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; SHP-1, Src homology 2-containing tyrosine phosphatase-1; LB, latex bead; MP, magnetic particle; TX-100, Triton X-100; ZA, zymosan A; PD, phosphated Dulbecco’s.
Received for publication December 12, 2001. Accepted for publication October 4, 2002.
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