By
From the G.W. Hooper Foundation, * Department of Microbiology and Immunology, and Department of Laboratory Medicine, University of California, San Francisco, California
94143-0552; and the § National Institute for Medical Research, London NW7 1AA, United Kingdom
Receptors on macrophages for the Fc region of IgG (FcR) mediate a number of responses
important for host immunity. Signaling events necessary for these responses are likely initiated by the activation of Src-family and Syk-family tyrosine kinases after Fc
R cross-linking.
Macrophages derived from Syk-deficient (Syk
) mice were defective in phagocytosis of particles bound by Fc
Rs, as well as in many Fc
R-induced signaling events, including tyrosine phosphorylation of a number of cellular substrates and activation of MAP kinases. In contrast,
Syk
macrophages exhibited normal responses to another potent macrophage stimulus, lipopolysaccharide. Phagocytosis of latex beads and Escherichia coli bacteria was also not affected.
Syk
macrophages exhibited formation of polymerized actin structures opposing particles
bound to the cells by Fc
Rs (actin cups), but failed to proceed to internalization. Interestingly,
inhibitors of phosphatidylinositol 3-kinase also blocked Fc
R-mediated phagocytosis at this
stage. Thus, PI 3-kinase may participate in a Syk-dependent signaling pathway critical for
Fc
R-mediated phagocytosis. Macrophages derived from mice deficient for the three members
of the Src-family of kinases expressed in these cells, Hck, Fgr, and Lyn, exhibited poor Syk activation upon Fc
R engagement, accompanied by a delay in Fc
R-mediated phagocytosis.
These observations demonstrate that Syk is critical for Fc
R-mediated phagocytosis, as well as for signal transduction in macrophages. Additionally, our findings provide evidence to support
a model of sequential tyrosine kinase activation by Fc
R's analogous to models of signaling by
the B and T cell antigen receptors.
Cross-linking of receptors for Fc regions of IgG
(Fc Signaling events triggered by Fc An important function of Fc Antibodies.
The hybridomas producing the 2.4G2 monoclonal rat antibody, the MAR18.5 mouse anti-rat Ig Reagents.
Wortmannin was obtained from Calbiochem Corp.
(San Diego, CA). Fluorescent phalloidins were from Molecular
Probes Inc. (Eugene, OR). Myelin Basic Protein was from Sigma
Chemical Co. The recombinant GST fused to the COOH-terminal of Jun (GST-c-Jun) fusion protein was purified from Escherichia coli lysate as described (16). Purified LPS from Salmonella
minnesota was obtained from List Biological Laboratories (Campbell, CA).
Cells.
Mice heterozygous for a disruption of the syk gene
(17) were bred, and the resulting embryos were dissected at day
17 of gestation to obtain the fetal liver. Genotyping of offspring
was performed by Southern blotting as described previously (17). Single-cell suspensions were resuspended at 106 cells per ml in
RPMI-1640 media supplemented with 10% FCS, 2 mM sodium
pyruvate, 1 mM glutamine, and 50 µM 2-mercaptoethanol, nonessential amino acids, and 10% L cell conditioned media (LCM) as a source of M-CSF and GM-CSF. The adherent monolayer
cultures generated by this protocol contained primarily cells of
the macrophage lineage as determined by adherent morphology,
expression of CD11b, CD16, and CD32, but not Gr-1, or B or T
lymphocyte markers.
Rs)1 triggers cellular events that are crucial for a
variety of immune responses. These include phagocytosis,
production of cytokines and chemokines, release of agents
that damage microorganisms or infected cells, and changes
in expression of cell surface proteins involved in cell-cell
adhesion and antigen presentation (1, 2). The important
roles for these receptors in antibody-mediated allergic and
inflammatory responses have been demonstrated in mice
made deficient for Fc
Rs by targeted gene disruption (3, 4). Thus, the Fc
Rs allow the humoral and cellular aspects of immunity to communicate and cooperate in expanding,
sustaining, and regulating immune responses.
R cross-linking are believed to be largely analogous to the events induced by engagement of B cell and T cell antigen receptors. Tyrosine
kinases of the Src and Syk families become activated and
associate with specific recognition sequences known as immunoreceptor tyrosine-based activation motifs (ITAMs),
contained within the intracellular domains of some of the
Fc
R subunits. Targets of these activated tyrosine kinases
include the Fc
R itself, enzymes that generate second messengers (e.g., phospholipase C-
1 and phosphatidylinositol 3-kinase [PI 3-kinase]), and regulators of Ras and other
Ras-like G proteins (e.g., Shc, Vav) (5).
Rs on macrophages and
monocytes is their ability to promote phagocytosis. Ingestion
of IgG-coated cells serves to remove and destroy invading
microorganisms or infected cells. In addition, phagocytosis
provides a means for internalizing antigen for processing
and presentation to T cells (6). The molecular mechanisms
by which Fc
Rs trigger the phagocytic process are poorly
understood. A role for Fc
R-mediated protein tyrosine
phosphorylation in inducing phagocytosis is suggested by
the finding that protein tyrosine kinase inhibitors block
phagocytosis of IgG-coated particles (7). Moreover, the
intracellular tyrosine kinase Syk associates with Fc
RII (10)
and with the tyrosine phosphorylated
chain of Fc
RI
(11) and Fc
RIII (12), and has been implicated in Fc
R-mediated phagocytosis. For example, COS-1 cells transfected with human Fc
Rs exhibit enhanced phagocytosis upon cotransfection of human Syk (8). Similarly, cells expressing Fc
RIII-Syk (CD16-Syk) chimeras can phagocytose particles that cross-link the CD16 portion of the molecule (13); chimeras containing kinase-inactive Syk do not
mediate internalization. How Syk promotes Fc
R-mediated phagocytosis is unclear, but inositol phospholipid metabolism is likely to be an important downstream signaling
event since wortmannin, a potent inhibitor of PI 3-kinase,
prevents Fc
R-mediated phagocytosis (8). To test directly
the importance of Syk for Fc
R-induced signaling and
phagocytosis, we have examined these events in cultured
macrophages derived from mice genetically deficient for
Syk. The role of Syk in signal transduction in response to
Fc
R engagement and stimulation with the bacterial endotoxin LPS were also examined. The results reported here
demonstrate that Syk is required for Fc
R-induced phagocytosis, but not for phagocytosis of latex beads or microorganisms. In addition, Syk was found to play an important
role for many Fc
R-induced signaling events, but not for
various LPS-induced signaling events or biological responses.
chain
monoclonal antibody, and the anti-sheep red blood cell monoclonal antibodies TIB 111, TIB 114, and TIB 109, were obtained
from the American Type Culture Collection (Rockville, MD).
Monoclonal antiphosphotyrosine-agarose was purchased from
Sigma Chemical Co. (St. Louis, MO). The 4G10 antiphosphotyrosine antibody was prepared and used as described (14). Horseradish peroxidase-conjugated sheep anti-mouse IgG antibody
was obtained from Amersham Corp. (Arlington Heights, IL).
Horseradish peroxidase-conjugated sheep anti-rabbit IgG antibody was obtained from Boehringer Mannheim Biochemicals
(Indianapolis, IL). Protein G-Sepharose was obtained from Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Anti-human
Vav protein antibody was obtained from Upstate Biotechnology
Inc. (Lake Placid, NY). Antibodies to p38, Erk1/2, and Jun
NH2-terminal (JNK) kinases were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit anti-Syk antibody has
been described (15). Anti-Shc antisera was generated by immunizing with a glutathione S-transferase (GST) fusion protein containing amino acids 359-473 of the human Shc protein. Specific antibodies were obtained by affinity column purification of antibodies reactive with the Shc-GST fusion protein, but not with GST
alone.
/fgr
/lyn
strain of mice is described
elsewhere (18). Macrophages from normal and hck
/fgr
/lyn
mice were isolated from bone marrow. Marrow from tibia and
femur bones was eluted in DMEM. Clumps were removed by
rigorous pipetting and passage through 70-µm nylon mesh.
Erythrocytes were lysed by adding 2 vol of 1.44% NH4Cl. The
cells in these suspensions were recovered by centrifugation. Cells
were cultured in media described for fetal liver cultures. Nonadherent cells were removed 1-2 d later and transferred to a fresh
150-mm plate. Nonadherent cells were removed from this secondary culture 4 d later and discarded. The adherent cultures
were used 5-7 d after initial harvest from the mice, when confluency was achieved. Cultures generated via this protocol were almost purely macrophages, as indicated by the criteria listed above,
and contained very few, if any, fibroblasts.
Binding and Phagocytosis of Antibody-coated Erythrocytes (EA). EA were prepared in calcium- and magnesium-free PBS by incubating sheep erythrocytes (Accurate Chemical and Scientific Corporation, Westbury, NY) (109/ml) with a subagglutinating concentration of rabbit anti-sheep red blood cell antibodies (Nordic Immunology, Tilburg, Netherlands). Cultured macrophages were grown on 12-mm glass coverslips and incubated at 37°C with EA (107/ml) for 30 min. Unbound EA was washed away, and some coverslips were fixed in 2% glutaraldehyde in PBS for 30 min, and were mounted for phase microscopy of rosettes as described below. To observe internalization, some coverslips were incubated in hypotonic buffer (1.4% NH4Cl) at room temperature for 5 min to lyse uninternalized EA before fixation. For studies on the rate of phagocytosis, EA particles were first bound to macrophages on ice for 30 min, followed by a 5-min centrifugation at 150 g. Phagocytosis was initiated by addition of warm medium, and cultures were placed in a 37°C incubator. Uninternalized EA were lysed by addition of H2O for 30 s, and cells were fixed as above.
Fluorescence Microscopy. To visualize actin cups, cells on coverslips were incubated in the presence or absence of cytochalasin D (1 µM) for 15 min. Cells were incubated 2-4 min with EA, and were then fixed in 3.7% formaldehyde for 10 min. Alternatively, to achieve a high number of stable actin cups, cells were incubated on ice with EA (107/ml) for 30 min. Unbound EA were washed away, and slips were processed for fluorescence microscopy. Cells on 12-mm glass coverslips were fixed in 3.7% formaldehyde for 10 min, and were then permeabilized in .01% Triton X-100 in PBS for 5 min. Cells were then stained with BODIPY-phalloidin or Oregon green-phalloidin (5 U/ml in PBS) for 15 min. Cells were observed by phase contrast and fluorescence microscopy (Axiophot microscope; Carl Zeiss, Inc., Thornwood, NY). To visualize LPS-induced actin-rich structures, cells on coverslips were incubated at 37°C with or without LPS (100 ng/ml) for 1 h.
Cell Stimulation.
Before stimulation for signaling experiments, cells were starved for 2 h in culture media lacking LCM.
For FcR stimulation, adherent cultures were incubated on ice
for 30 min with 2.4G2 hybridoma supernatant. Cells were then
warmed to 37°C and treated with cross-linking antibody (10 µg/
ml MAR18.5) for indicated times. Unstimulated cultures were
incubated on ice, and at 37°C in culture media without LCM.
For LPS activation, cells were starved as above. LPS (1 µg/ml)
was then added directly to culture vessels for the indicated times.
Plates of Fc
R- and LPS-stimulated cells were washed with cold
PBS containing 1 mM sodium vanadate. Ice-cold lysis buffer
consisting of 1% Triton-X-100, 50 mM Hepes buffer, pH 7.4, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA,
100 mM NaF, 1 mM PMSF, and 1 mM NaVO4 was added directly to the flasks. Cultures were lysed on ice for 10 min.
Immunoprecipitations and Immunoblotting. Anti-Shc immunoprecipitations were carried out as described previously (19). For antiphosphotyrosine immunoprecipitations, samples were adjusted to RIPA (150 mM NaCl, 1.0% NP-40, 0.5% deoxycholate, 0.1% SDS, and 50 mM Tris buffer, pH 8.0). Immunoprecipitations were carried out for 8-10 h at 4°C. SDS-PAGE and immunoblotting were carried out as described previously (19), with the exception that to detect the electrophoretic mobility shift of p42 Erk2, soluble lysate proteins were separated on 12.5% SDS-PAGE containing an acrylamide/bis-acrylamide ratio of 120:1, and were transferred to nitrocellulose.
Immunoprecipitation and In Vitro Protein Kinase Assays.
Cell lysates prepared as described above were precleared with 25 µl of
protein A-Sepharose, and were then incubated with 10 µl of
anti-Syk, anti-JNK, or anti-Erk2 antibody for 1 h on ice. Immune
complexes were collected on 25 µl Protein A-Sepharose, and
were washed twice with lysis buffer and twice with kinase assay
buffer (25 mM Hepes, pH 7.6, 20 mM MgCl2, 20 mM -glycerol phosphate, 1 mM DTT, or 20 mM MOPS, pH 7.6, 20 mM
MgCl2, 30 mM
-glycerol phosphate, 5 mM EGTA, and 1 mM
DTT for JNK or Erk kinase assays, respectively). Reactions were
performed at 30°C for 15 min in the presence of 10 µCi of
-[33P]ATP using 20-25 µg GST-c-Jun or Myelin Basic Protein
as substrate for JNK or Erk2, respectively. Anti-Syk immunoprecipitates were washed twice with lysis buffer and twice with kinase assay buffer (20 mM Tris HCl, pH 8.0, 10 mM MgCl2, 0.1%
NP-40). The beads were incubated in 50 µl of kinase assay buffer
plus 10 µCi of
-[33P]ATP for 10 min at room temperature.
Erk1/2, JNK, and Syk kinase reactions were stopped by adding
SDS-PAGE sample buffer, separated by SDS-PAGE and transferred to nitrocellulose, and were analyzed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Cytokine ELISA Assays.
Macrophages were incubated in culture media containing LCM, in 24-well plates at a density of 5 × 104/well in 1.5 ml of media. Cultures received LPS at the indicated doses, and supernatants were collected from cultures at the
indicated times after initial exposure. Duplicate supernatants were
collected from two separate wells at each time point. Supernatants
were stored at 80° until assay. Cytokine concentrations in the
supernatants were measured using murine cytokine ELISA detection kits from Biosource International (Camarillo, CA).
The engulfment of cells and particulate matter is
an important function of the tissue macrophage. Among
the receptors known to promote phagocytosis are the
FcRs and the complement receptor. To examine the relative importance of Syk and members of the Src-family of
tyrosine kinases for Fc
R-mediated phagocytosis, we established primary cultures of fetal liver-derived wild-type and
Syk-deficient macrophages, or of bone marrow-derived
wild-type and Src-family kinase-deficient (Hck
/Fgr
/
Lyn
) macrophages in CSF-1-containing medium. None
of the other known Src-family tyrosine kinases were detected in the latter macrophages (18).
The capacity of these cultured fetal liver-derived or bone
marrow-derived macrophages to mediate particle uptake
via their FcRs was tested (Fig. 1). Macrophages were incubated with IgG-coated sheep erythrocytes (EA) either on
ice or at 37°C. Wild-type, Syk-deficient, and Hck
/Fgr
/
Lyn
cells bound EA equally well under either condition.
At 37°C, wild-type and Hck
/Fgr
/Lyn
cells internalized
the bound EA particles, although the Src family-deficient macrophages internalized the bound particles more slowly.
To examine this difference more readily, the macrophages
were allowed to bind the EA particles at 0°C, and were
then warmed to 37°C to permit internalization to proceed.
Under these conditions, wild-type macrophages internalized the EA particles rapidly, with a substantial percentage
internalized by 5 min at 37°C, and near maximal internalization by 10 min. In contrast, the Hck
/Fgr
/Lyn
macrophages had internalized very few particles by 5 min, but had internalized a substantial percentage by 10 min. At this
time, there was less internalization than that seen with wild-type macrophages, but after 30 min the difference was minimal (data not shown). In contrast, Syk-deficient macrophages began engulfment, but were unable to complete
phagocytosis of the bound EA particles. This effect was
readily apparent when the macrophages were subjected to
hypotonic conditions that lysed the erythrocytes attached to the surface of the macrophage, but not the fully engulfed EA
(Fig. 1, b and c). Additionally, wild-type macrophages bound
and internalized erythrocytes coated with IgG3, IgG2a, or
IgG2b antibodies, whereas Syk
macrophages bound but
failed to internalize these particles (data not shown). Antibody-coated erythrocytes remained bound to Fc
Rs on
the surface of Syk
macrophages for several days in culture,
indicating that the inhibition of phagocytosis was complete,
and was not simply delayed. The phagocytosis observed in
these experiments was Fc
R dependent as uncoated erythrocytes were not phagocytosed or bound by normal or
Syk
macrophages. These findings demonstrate an absolute
requirement for the presence of Syk to accomplish Fc
R-mediated phagocytosis in macrophages.
The difference in phagocytic ability of Syk macrophages
compared to Hck
/Fgr
/Lyn
macrophages was unexpected, as both of these types of kinases are thought to play
an important role in signaling by the family of receptors
that includes Fc
Rs and the antigen receptors of B and T
cells (5, 20). Studies in a variety of systems have suggested
that Src-family tyrosine kinases are responsible for initiating
signaling of these immunoreceptors by phosphorylating the
two tyrosines of the receptor ITAM, and thereby providing a mechanism for recruiting and activating the tyrosine kinase Syk (or ZAP-70 in T cells) to the engaged receptor
(21). Indeed, as predicted by this general model, we
found that Syk was only poorly activated after Fc
R cross-linking in Hck
/Fgr
/Lyn
macrophages as compared to
its activation in normal cells (Fig. 2). It should be noted,
however, that there was a small amount of Fc
R-induced
activation of Syk in the Src family mutant cells, indicating
that Syk could participate in Fc
R signaling to some extent, even in the absence of these three Src-family tyrosine kinases.
The defect in FcR-mediated phagocytosis of Syk-deficient macrophages did not represent a general defect in the
phagocytic ability of these cells. There were no discernible
differences in the abilities of wild-type, Syk-deficient, or
Hck
/Fgr
/Lyn
macrophages to phagocytose latex beads
ranging in size from 0.2 to 5.5 µm, or to engulf and destroy yeast and E. coli (data not shown). Furthermore, after
pretreatment with LPS, all three macrophage populations
exhibited increased phagocytic capacity for bacteria (data
not shown). The attachment and engulfment of microorganisms most likely represents binding of complex carbohydrates on the surface of the microbe to lectin-like glycoproteins or integrins expressed by the macrophage (24).
Evidently these receptors do not require Syk for triggering
phagocytosis.
To characterize further the defect in FcR-mediated
phagocytosis of Syk
macrophages, we next examined the
position of actin structures after EA binding. Actin is likely
to play an important role in phagocytosis since it is concentrated in the macrophage cytoplasm surrounding the particle being engulfed, and since cytochalasin D treatment,
which blocks actin polymerization, abrogates phagocytosis without affecting particle binding (25, 26). We used fluorescent phalloidin to visualize the polymerized actin cytoskeleton in Syk-deficient and wild-type cells. Phagocytic,
actin-lined cups formed around antibody-coated erythrocytes bound by Syk-deficient cells, and were indistinguishable from those formed by wild-type cells (Fig. 3, a-d) or
by Hck
/Fgr
/Lyn
cells (data not shown). Actin cup formation occurred at 4°C or at 37°C, and was blocked by cytochalasin D treatment. Syk-deficient cells were presumably unable to effect the closure of the pocket generated by
the advancing membrane edges that created the phagocytic cup. How macrophages and other professional phagocytes
achieve this final stage of phagocytosis is unknown. At
37°C, but not at 4°C, the actin cup structures were dynamic, and their dissolution occurred at about the same
time as internalization was completed in wild-type macrophages. Interestingly, Syk
cells also exhibited dissolution of the actin cups around the bound EA, although these
particles were never internalized or released.
We next examined whether Syk participates in other
macrophage-signaling pathways involving cytoskeletal rearrangements. Macrophages responding to LPS develop distinct morphological changes, including the development of
actin-rich ruffles. As LPS stimulation of macrophages leads
to tyrosine phosphorylation of Syk (19), we asked whether Syk participated in LPS-induced cytoskeletal changes. When
exposed to LPS, Syk cells developed actin-rich ruffles indistinguishable from those induced on normal cells (data
not shown). Thus, LPS does not require Syk to effect these
changes in the actin-based cytoskeleton.
To examine the mechanism behind the observed phagocytic defect of Syk macrophages, we looked for defects in tyrosine
phosphorylation of cellular proteins and defects in signal
transduction pathways known to be dependent upon receptor-activated tyrosine kinases. After cross-linking of Fc
RII and Fc
RIII on the surface of Syk
and wild-type
macrophages with the antibody 2.4G2, total cellular proteins were analyzed by SDS-PAGE and antiphosphotyrosine immunoblotting. We observed a number of proteins
that were inducibly tyrosine phosphorylated in normal
cells. The most obvious of these had apparent molecular
weights of 180, 150, 145, 120, 100, 85, 70, 60, 56, 52, 48,
and 42 kD, and are indicated by arrows in Fig. 4 a. Syk
macrophages were defective in most aspects of this response. Some of the induced tyrosine phosphoproteins, for
example, the 42-kD band corresponding to Erk2 (Fig. 4 c),
were conspicuously absent upon Fc
R stimulation in Syk
cells, and many other proteins became only weakly phosphorylated after receptor cross-linking. In contrast, a few
proteins, including two at 56 and 52 kD which may correspond to members of the Src family of tyrosine kinases, did
exhibit induced tyrosine phosphorylation comparable to
that seen in wild-type cells.
We then examined the induced tyrosine phosphorylation
of the important signaling components Shc and p145Ship (Fig.
4 b). Within 5 min of FcR cross-linking in normal cells, we observed increased tyrosine phosphorylation of Shc
and p145Ship as well as the induced association of Shc with
p145Ship, in agreement with previous results (19). In contrast, in Syk-deficient cells, Fc
R cross-linking induced only
weak tyrosine phosphorylation of Shc and p145Ship. These
results show that Syk plays a principal role in the rapid tyrosine phosphorylation of many cellular substrates after
Fc
R cross-linking.
The lack of FcR-induced tyrosine phosphorylation of p42MAPK (Erk2) in the Syk
macrophages (Fig. 4)
was quite striking. MAP kinases become activated upon dual
phosphorylation of threonine and tyrosine residues in a TxY
motif. In the case of Erk1 and Erk2, this dual phosphorylation results in a mobility shift upon SDS-PAGE. Therefore,
we examined the effect of Fc
R stimulation on Erk1 and
Erk2 electrophoretic mobility. As expected, p42MAPK (Erk2)
and p44MAPK (Erk1) exhibited a clear shift in mobility in
normal macrophages stimulated through the Fc
Rs. This
response was absent in Syk
macrophages (Fig. 5 a and data
not shown). Moreover, in vitro kinase assays revealed that
Fc
R stimulation led to a large increase in Erk2 MAP kinase activity in wild-type macrophages, but to a small or no
increase in Syk
macrophages (Fig. 5 b).
These results do not reflect a generalized defect in
MAPK activition in the Syk-deficient macrophages, since
the ability of LPS to induce a mobility shift of p44 Erk1
and the activation of Erk2 were normal (Fig. 5, c and d).
LPS stimulation of macrophages strongly activates not only
the Erk1/2 MAP kinases, but also the other two known
types of mammalian MAP kinases, p38 and JNK1/JNK2. Enzymatic activation of JNK occurred normally in LPS-stimulated Syk macrophages (Fig. 6 a). Similarly, LPS-
induced tyrosine phosphorylation of p38 occurred normally in Syk
macrophages (Fig. 6 b). Fc
R cross-linking
also induced a modest increase in p38 immunoprecipitated
with antiphosphotyrosine antibodies from wild-type macrophages, although this response was less robust than was
the response seen with LPS (Fig. 6 c). As was seen with Erk1 and Erk2, the p38 response to Fc
R engagement was
largely absent in Syk-deficient macrophages. We did not
find JNK kinase activity increased in response to Fc
R
cross-linking in the fetal liver-derived macrophages, or in
murine macrophage cell lines such as P388D1 and RAW
264.7 (data not shown).
As the activation of MAP kinases in response to LPS was
not affected by the absence of Syk, we next examined LPS-induced tyrosine phosphorylation of cellular proteins. We
did not observe any defects in LPS-induced tyrosine phosphorylation of the examined cellular substrates, including
Vav, Shc, and p145 Ship (Fig. 7). Thus, Syk-deficient macrophages were not defective in any of the examined signaling responses to LPS.
Fc
We next examined the effect of loss of Syk
on FcR-induced tyrosine phosphorylation of Vav and of
the p85 subunit of PI 3-kinase. In normal macrophages,
p85 and Vav became immunoprecipitable with antiphosphotyrosine antibodies after Fc
R cross-linking (Fig. 8).
It is possible that the immunoprecipitated proteins were
themselves phosphorylated on tyrosine, or alternatively, that
they were precipitated by virtue of an association with another protein that was tyrosine phosphorylated upon cell
stimulation. Relatively harsh conditions were used for the
antiphosphotyrosine immunoprecipitations, so weak protein-
protein interactions were likely disrupted. In Syk
macrophages, immunoprecipitation of Vav by antiphosphotyrosine antibodies was still induced by Fc
R stimulation,
but immunoprecipitation of p85 was abolished (Fig. 8).
Vav or its associated proteins may be substrates for members of the Src family of kinases that are associated with the
Fc
Rs (27). The absence of p85 in antiphosphotyrosine immunoprecipitates, however, suggested that signaling reactions involving PI 3-kinase were defective in Syk-deficient macrophages.
The perturbation of PI 3-kinase signaling in Syk-deficient
macrophages was particularly interesting as PI 3-kinase has
been implicated in FcR-mediated phagocytosis in the human monocyte cell line U937 (28), and in murine bone
marrow-derived macrophages (29) by virtue of the blocking effect of a potent and selective inhibitor of PI 3-kinase,
wortmannin. We found that wortmannin also prevented
phagocytosis of EA by fetal liver-derived cultured macrophages. This inhibitor did not affect Fc
R-induced polymerized actin accumulation around bound particles (Fig.
9), and thus blocked phagocytosis at a stage similar to the
block seen in Syk-deficient macrophages. In contrast to its
effects on Fc
R-mediated phagocytosis, wortmannin did
not affect macrophage phagocytosis of yeast, latex beads, or
E. coli (data not shown).
Cytokine Production By Syk
Cross-linking of
FcR receptors on some macrophage populations can trigger the production and release of a variety of proinflammatory cytokines, bioactive lipids, and cytotoxic oxidants
(30). The nature of the responses are dependent upon the
tissue origin of the macrophage, since different macrophage
types have distinctive properties in this regard. Unfortunately, cross-linking of Fc
Rs of fetal liver-derived cultured
macrophages did not generate a respiratory burst in either
wild-type or Syk-deficient cells. Fc
R cross-linking also
failed to trigger detectable production of TNF
, IL-1
, or
IL-6 (data not shown). In contrast, LPS caused these macrophages to release a number of proinflammatory mediators, including TNF
, IL-1
, IL-6, and IL-12 (Fig. 10).
LPS induction of TNF
release from Syk-deficient macrophages was indistinguishable from that induced from wild-type macrophages (Fig. 10 a). TNF
production during the
4 h after cell stimulation was no different in cells that were
or were not starved of the growth factors in L-Cell-conditioned media before and during such short-term assays (data
not shown). Cell viability beyond 4 h could not be maintained without conditioned medium, however, so later times
were not tested in its absence. In addition, LPS-induced release of IL-1
, IL-6, and IL-12 (Fig. 10, b and c) were not
impaired in Syk-deficient macrophages. Thus, the signaling
pathways that are involved in production of proinflammatory cytokines by macrophages responding to LPS can
function independently of the Syk tyrosine kinase.
In prior experiments, we and others observed that stimulation of macrophages with LPS or FcR cross-linking resulted in increased tyrosine phosphorylation and enzymatic
activation of Syk (11, 19, 31). In this study, we have addressed the role of Syk in LPS- and Fc
R-mediated signaling events and in downstream biological events by examining these responses in vitro in cultured macrophages from
mice in which the gene for Syk had been deleted by gene
targeting. In each case examined, the responses of Syk
macrophages to LPS were not different from the responses
of normal macrophages. These responses included early signaling events such as activation of the Erk1/2 and JNK
MAP kinases, and downstream events such as production
of the cytokines TNF-
, IL-1
, IL-6, and IL-12. It is still
possible that Syk participates in one or more responses to
LPS that were not examined. Nonetheless, it is clear that
Syk does not play a central role in mediating many of the
LPS-triggered signaling events in macrophages. As tyrosine kinase inhibitors block many LPS responses of macrophages
(32), it seems likely that tyrosine kinases other than Syk
play critical roles in mediating LPS responses.
In contrast to the normal LPS responses of Syk-deficient
macrophages, a number of FcR-mediated signaling events
were severely compromised, and Fc
R-mediated phagocytosis was completely blocked in these mutant macrophages.
Fc
R-triggered tyrosine phosphorylation of an array of cellular proteins, including Shc and p145Ship, was impaired in
Syk
macrophages. Fc
R-triggered activation of Erk2 and
p38 MAP kinases was likewise impaired. Since the tyrosine
phosphorylation of Shc triggers its association with Grb-2
and SOS (35), which may lead to the activation of the
Ras-Raf pathway, and subsequently to Erk1/2 MAP kinase activation, the defect in Erk2 activation in Syk
cells
may be attributable to deficient Shc-related signaling events.
Additionally, Syk appeared to be important for Fc
R-
induced signaling events involving the p85 subunit of PI
3-kinase. In wild-type macrophages, stimulation through
the Fc
R resulted in increased quantities of this protein
and Vav in antiphosphotyrosine immunoprecipitates. In
Fc
R-stimulated Syk
cells, increased amounts of Vav, but
not p85, were observed in antiphosphotyrosine immunoprecipitates. These results revealed an important role for Syk in
some, but not all Fc
R-induced signaling events.
Since FcRs share subunits and ITAM signaling sequences with B and T cell antigen receptors and Fc
RI, the
importance of Syk for Fc
R signaling observed here is consistent with studies indicating a requirement for Syk or the
related tyrosine kinase in T cells, ZAP-70, for signaling by
these receptors (36). Antigen or Fc receptor cross-linking triggers phosphorylation of receptor subunit ITAMs,
probably by different members of the Src family of kinases (41), and subsequent recruitment of Syk or ZAP-70 tyrosine kinases to the phosphorylated ITAMs. It is currently
thought that Syk and ZAP-70 are then primarily responsible
for phosphorylation of important signaling targets. Given
the similarities of the lymphocyte antigen receptors and
Fc
Rs, it is likely that initiation of Fc
R signaling similarly
requires the sequential activity of these two families of tyrosine kinases. With this in mind, we examined Fc
R-mediated signaling events in macrophages genetically deficient for the three Src family members ordinarily expressed in macrophages Hck, Fgr, and Lyn. We found that Fc
R-mediated Syk activation was impaired in Hck
/Fgr
/Lyn
macrophages. This observation provides additional strong
evidence for a model of sequential activation of Src family
and Syk tyrosine kinases by the Fc
Rs.
In addition to its role in early signal transduction events,
Syk was also found to be absolutely required for FcR-mediated phagocytosis in macrophages. Syk
macrophages
bound antibody-coated particles by their Fc
Rs, and initiated engulfment of the particles with actin-based cytoplasmic extensions. In the absence of Syk, however, the cells were
unable to internalize fully these antibody-coated particles.
Evidently, Syk was important for the Fc
R-triggered cellular events necessary to extend further and fuse the leading
edge of the macrophage membrane that surrounded the particle. These results are in agreement with findings demonstrating a role for Syk in FcR-mediated phagocytosis in various artificial systems. For example, coexpression of Syk in COS-1 transfectants expressing Fc
Rs promoted efficient Fc
R-mediated phagocytosis (8), and chimeric transmembrane proteins containing Syk kinase domains were
capable of triggering phagocytosis by COS-1 cells (13). In
the latter case, the kinase activity of Syk was sufficient and
necessary to trigger phagocytosis in COS cells. Interestingly, we found that Fc
R-mediated phagocytosis was
only delayed in Hck
/Fgr
/Lyn
macrophages, despite severely diminished Syk activation. Evidently, the reduced
number of activated Syk molecules clustered beneath cross-linked Fc
Rs in Hck
/Fgr
/Lyn
macrophages were still
sufficient to allow phagocytosis to proceed with only a
moderate delay.
The phagocytic defect in Syk macrophages may reflect
the lack of Syk kinase action needed to activate signaling
pathways necessary for Fc
R-mediated phagocytosis, as suggested by experiments in Cos-1 cells (13). Alternatively,
the physical association of Syk with phosphorylated receptor tails may allow it to serve an adaptor-type function at
cross-linked receptors to allow recruitment of other important proteins to the site of particle binding. Chacko et. al
have described such a role for Syk in Fc
RII signaling in
platelets (42). In any case, the experiments reported here demonstrate that Syk is a crucial element in Fc
R-mediated phagocytosis by cells in which it is a naturally occurring and biologically important response after cross-linking
of these receptors. Importantly, the phagocytic defect in Syk
macrophages was limited to phagocytosis via activated Fc
Rs. Phagocytosis of microorganisms and synthetic particles was
unaffected by the absence of Syk. Interesting in this regard
are observations indicating that there are distinct cytoskeletal structures involved in phagocytosis of particles bound to
different receptors (43).
Several FcR-induced signaling events have been implicated as participating in phagocytosis. Fc
R cross-linking
triggers phospholipase A2 activation and subsequent arachidonic acid (AA) release, and it was found that the AA was a
critical component in phagocytosis (44). In those studies, a
block in Fc
R-mediated phagocytosis was overcome by
providing exogenous AA in the culture media. The phagocytic defect in Syk
macrophages was not remedied by addition of exogenous AA (data not shown). It is still possible
that local production of AA plays an important role in
Fc
R-mediated phagocytosis, but that this is only one of
several required Syk-dependent signaling events.
Another signal transduction protein implicated in FcR-mediated phagocytosis is PI 3-kinase (28). Fc
R engagement
induced an increase in p85 protein immunoprecipitable by
antiphosphotyrosine antibodies in normal macrophages, but
not in Syk
macrophages. Moreover, we noted a striking
similarity between the phagocytic defect in Syk
cells, and
the stage at which phagocytosis was blocked by treatment of normal cells with the PI 3-kinase inhibitor wortmannin.
In both cases, actin cups formed, but could not close off to
internalize the bound particle (Figs. 3 and 9). In agreement
with our results, another group has recently reported that
wortmannin blocks phagocytosis at this stage (29). Thus, it
is attractive to propose that the defect in Syk
macrophages
results from a failure to activate a signaling pathway involving PI 3-kinase. Syk may be required to activate and/or to
properly localize PI 3-kinase activity during phagocytosis. Interesting in this regard is the observation that Fc
RII
cross-linking on platelets induced the association of the
Fc
RII with PI 3-kinase (42).
It should be noted that FcR signaling in Syk
macrophages also showed a defect in a second signaling component that acts on inositol-containing phospholipids,
p145Ship. This molecule is an inositol phosphatase that hydrolyzes the 5-phosphate of phosphatidylinositol (3,4,5)P3,
the product of PI 3-kinase, to generate phosphatidylinositol
(3,4)P2 (45, 46). It is not known how the activity of
p145Ship is regulated, but Fc
R engagement leads to tyrosine phosphorylation of p145Ship and its association with
both Syk and Shc (19). These signaling events were also
defective in the Syk-deficient macrophages, and thus,
p145Ship function is also likely to be impaired in Syk
cells
stimulated via Fc
R cross-linking.
How PI 3-kinase and/or p145Ship may be acting in the phagocytic process is of course not known. One possibility is suggested by the connection between PI 3-kinase and small G proteins involved in cytoskeletal rearrangements. The Rho family GTPases Rho, Rac, and CDC42 play important roles in the regulation of the actin cytoskeleton to produce membrane ruffling, filopodia, and lamellipodia (47), and likely play an important role in phagocytosis. PI 3-kinase is thought to play a role in coordinating these responses. For example, activation of Rac via the PDGF receptor requires synthesis of PI 3-kinase phosphoinositide products and association of PI 3-kinase with receptor cytoplasmic domains, indicating that PI 3-kinase can act upstream of Rac (48). Similarly, inhibitors of PI 3-kinase prevent agonist-stimulated guanine nucleotide exchange on Rac (49).
The phagocytic process may be analogous to other processes that require the combined activities of Rho-family
proteins. Nobes et al. concluded that stimulation of CDC42
induced the formation of filopodia (47). CDC42 also can
stimulate Rac, which in turn initiates a process whereby the
filopodia are filled in by advancing lamella. This process
may be related to the events in FcR-mediated phagocytosis. That is, the phagocytic cup may be a scaffold of filopodia-like structures. The formation of actin cups may be followed by filling in over the cup by advancing lamellipodia. According to this hypothesis, a key issue is how the activities of Rho-family members are orchestrated to allow phagocytosis to proceed successfully. The final events in phagocytosis may be defective in Syk-deficient macrophages
because signaling events, perhaps involving Rho family
proteins, PI 3-kinase, and p145Ship, are not activated or localized properly in the absence of Syk.
Address correspondence to Dr. Mary T. Crowley, Scripps Clinic and Research Foundation, 10550 N. Torrey Pines Rd., IMM 25, La Jolla, CA 92037. Phone: 619-784-2195; FAX: 619-784-9180; E-mail: mtcrow{at}scripps.edu
Received for publication 23 January 1997 and in revised form 29 July 1997.
1 Abbreviations used in this paper: AA, arachidonic acid; EA, erythrocytes coated with antibody; FcWe thank Drs. Vivien Chan, Julie Hambleton, Stacey Harmer, James Richards, and Steven Weinstein, for many helpful discussions and for critical reading of the manuscript.
This work was supported by grants AI20038 and AI33442 from the U.S. National Institutes of Health. M.T. Crowley was supported by National Institutes of Health training grant T32AI07334. P.S. Costello and V. Tybulewicz were supported in part by the Medical Research Council and the Imperial Cancer Research Fund, and by grants from the Oiver Bird Fund (V. Tybulewicz) and the Leukaemia Research Fund (V. Tybulewicz).
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