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INTRODUCTION |
Phagocytosis via Fc
receptors in macrophages is
accompanied by actin assembly, pseudopod extension, and phagosomal
closure (1). Fc
R-directed actin assembly is blocked by
tyrosine kinase inhibitors (2) and requires the participation of Rac1 and Cdc42 (3), two members of the Rho family of GTPases. However, it is
not known precisely how enhanced protein tyrosine phosphorylation leads
to changes in either the cytoskeleton or the membrane. Signaling by
Fc
receptors shares many elements in common with that of
growth factor receptors. For example, both classes of receptors signal
directly or indirectly through tyrosine kinases, and ligation of
multiple growth factor receptors and
Fc
Rs1
culminates in net actin assembly and plasma membrane-based protrusions (1, 4, 5). Studies of the PDGF receptor indicate a prominent role for
PI 3-kinase in the generation of F-actin-rich membrane ruffles.
Phosphotyrosine residues within the kinase insert region of the
cytosolic domain of the PDGF receptor bind the p85/p110 isoform of PI
3-kinase, and mutation of these residues abolishes membrane ruffling
induced by this receptor (6-8). Addition of wortmannin, a fungal
metabolite that inhibits PI 3-kinases in the nanomolar range, blocks
PDGF receptor-induced membrane ruffling and actin assembly (8, 9).
Similarly, addition of PI 3-kinase inhibitors abrogated membrane
ruffling and actin polymerization in response to insulin (10-12).
Precisely how PI 3-kinases participate in actin assembly is not known,
but pharmacological inhibition of PI 3-kinase inhibits GTP loading of
Rac1 stimulated by PDGF, and addition of constitutively active forms of
Rac1 induces membrane ruffling despite the presence of PI 3-kinase
inhibitors (10, 13). These data suggest that PI 3-kinase lies upstream
of Rac1. In contrast, recent studies of epithelial cells spreading on
collagen suggest that PI 3-kinase, which is required for motility, may lie downstream of Rho family GTPases (14).
The role of PI 3-kinase in actin assembly mediated by other types of
receptors is less clear. For G protein-linked receptors, such as the
thrombin receptor (15) and the chemotactic peptide receptor (16, 17),
inhibition of PI 3-kinase has been reported to have no effect on
stimulus-induced actin polymerization. For immunoreceptor tyrosine
activation motif-containing receptors, such as Fc
RI,
addition of wortmannin does not inhibit IgE-induced actin
polymerization but does block the appearance of well-formed membrane
ruffles in response to antigen (18). Several studies have demonstrated
a role for PI 3-kinase in Fc
R-mediated phagocytosis
(19-21). In one, a role for this enzyme was suggested for the closure
of phagosomes (21). Although quantitation of PI 3-kinase activity or
F-actin was not performed, addition of wortmannin did not appear to
inhibit the formation of "phagocytic cups," as determined by
fluorescence micrographs of phalloidin-stained cells interacting with
phagocytic targets (21).
A study of Fc
RI in mast cells suggests that stimulation
of actin polymerization may not necessarily lead to membrane ruffles
(18). Similarly, in DT40 lymphocytes expressing chimeric receptors
encoding CD16 and the
subunit of Fc receptors, addition of
IgG-coated targets resulted in localized actin assembly and rudimentary
plasma membrane protrusions, but phagocytosis did not occur (22). This
suggests that actin polymerization at the plasma membrane is not always
coupled to pseudopod extension; distinct signals may be required for
this function. Interestingly, a recent study suggested that pseudopod
extension by Fc
RI expressed in COS cells occurred in the
absence of net actin assembly (23). Collectively, these studies suggest
that actin assembly and pseudopod extension, two cellular events that
normally coincide spatially and temporally, may be regulated by
distinct signal transduction cascades.
PI 3 kinases have been implicated in multiple aspects of membrane
trafficking, including endocytosis, exocytosis, and membrane recycling
(for review, see Ref. 24). During phagocytosis, significant amounts of
plasma membrane are internalized in the form of phagocytic vacuoles.
However, this is accompanied by no apparent decrease in membrane
surface area (25), suggesting that surface membrane is replenished from
an intracellular source. To define the role of PI 3-kinase in
phagocytosis, we used a variety of approaches to identify the stage in
phagocytosis that was blocked during PI 3-kinase inhibition. These
studies indicate that the block occurs during pseudopod extension, not
during the very early phases (i.e. F-actin accumulation) or
late phases (i.e. phagosomal closure) of ingestion and could
be bypassed when requirements for pseudopod extension were minimized.
The block in pseudopod extension coincided with a decrease in exocytic
insertion of membrane, suggesting that PI 3-kinases are required for
coordinating membrane insertion events and pseudopod extension.
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EXPERIMENTAL PROCEDURES |
Cells and Reagents--
RAW LacR/FMLP.2 cells were derived from
RAW 264.7 cells as described previously (3). Primary macrophages were
isolated from the peritoneal cavities of C57Bl6 mice 3-5 days after
the intraperitoneal injection of thioglycollate (thio-macrophages) as
described previously (26). Cells were maintained in RPMI 1640 medium
containing 10% fetal calf serum, 100 units/ml penicillin G, and 100 µg/ml streptomycin. Wortmannin and sulfhydral-modified BSA were from
Calbiochem (La Jolla CA). LY294002 was from Biomol (Plymouth Meeting,
PA). IgG against sheep erythrocytes was from Diamedix (Miami, FL). A
monoclonal antibody directed against the p85 subunit of
phosphatidylinositol 3-kinase was from Transduction Laboratories
(Lexington, KY). [32P]Orthophosphate and
[
-32P]ATP were from Dupont NEN. Aluminum-backed Silica
Gel 60 thin layer chromatography plates were from EM Separations
(Gibbstown, NJ). Rabbit serum against BSA was from Dako Corp.
(Carpinteria, CA). Fluorescein isothiocyanate- and rhodamine-conjugated
F(ab')2 fragments of anti-rabbit IgG were from Jackson
ImmunoResearch (West Grove, PA). Rhodamine-phalloidin, YO-PRO 1 iodide,
and FM1-43 were from Molecular Probes (Eugene, OR).
Carboxylate-modified latex beads of various sizes were from Bangs
Laboratories (Fishers, IN).
Phagocytosis Assays--
5 × 106 sheep
erythrocytes opsonized with rabbit IgG (EIgG) were added to adherent
cells for 30 min as described previously (27). For opsonization of
latex beads, carboxylate-modified latex beads ranging in size from 1 to
6 µm were incubated with 4 mg/ml sulhydryl-modified BSA dissolved in
0.05 M 2-[N-morpholino]ethanesulfonic acid, pH
5.5, and 1 mg/ml 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, followed by washing and incubation in various dilutions of rabbit antiserum against BSA for 1 h at 25 °C. After washing three
times in phosphate-buffered saline, aliquots of beads were removed for immunoblotting to determine the quantity of IgG deposited on the beads.
The density of IgG/µm2 was determined based on the
calculated surface areas of the different bead size. Batches were
discarded if the density of IgG deposited varied >15% of the mean,
which was 5000 molecules/µm2. For phagocytosis assays,
cells were incubated with beads for 45 min at 37 °C. Excess beads
were washed away and the extent of binding and ingestion of the beads
was determined. To detect attached, but uningested beads, cells were
incubated with fluorescein isothiocyanate-conjugated anti-rabbit IgG
for 45 min at 4 °C, followed by fixation in 3.7% formaldehyde.
Cells were subsequently permeabilized with 0.2% Triton X-100, and
total cell-associated beads were stained with rhodamine-conjugated
anti-rabbit IgG. Phagocytosis was quantified as the total number of
beads per cell (i.e. rhodamine-stained) minus the number of
bound but uningested beads (i.e. those that were accessible
to staining with fluorescein isothiocyanate-conjugated anti-rabbit
IgG). 5-10 high power fields were scored for attached and ingested
particles, and phagocytosis assays were performed in duplicate.
Determination of PIP3 Content of RAW 264.7 Cells--
Adherent RAW LacR/FMLP.2 cells were grown to 90%
confluence in 6-cm dishes and were washed and incubated in minimal
essential medium minus phosphates (Life Technologies, Inc.) containing
1% BSA and 37 MBq/ml [32P]orthophosphate acid for 2 h at 37 °C. Cells were washed and incubated further with 8 × 107 EIgG for varying times followed by phospholipid
extraction and thin layer chromatography as described in (17). Samples
were run with a PIP3 standard obtained by incubation of
purified phosphatidylinositol-4,5-bisphosphate with
[
-32P]ATP and anti-phosphatidylinositol 3-kinase
immunoprecipitates derived from RAW LacR/FMLP.2 cells.
Fluorescence Microscopy--
Adherent RAW LacR/FMLP.2 cells on
coverslips were incubated with 3 × 106 EIgG on ice
for 30 min to allow for particle binding, and after washing with
ice-cold buffer, cells were incubated for a further 5 min at 37 °C
before fixation in 3.7% formaldehyde and staining with 0.33 µM rhodamine-phalloidin. Cells were imaged using a
confocal scanning system (Bio-Rad MRC 600) as described previously
(22).
Quantitation of F-actin--
Total cellular F-actin was
quantitated as described (22) with the following modifications: RAW
LacR/FMLP.2 cells were plated at 2 × 104 cells/well
in 96 well tissue culture plates and treated with vehicle (dimethyl
sulfoxide), 100 nM wortmannin, or 100 µM
LY294002 at 37 °C for 30 min. 2 × 106 EIgG were
added at 37 °C for 5 min, and cells were stained for F-actin with
rhodamine-phalloidin as described above. Rhodamine fluorescence
(excitation, 540 nm; emission, 590 nm) was measured using a
fluorescence plate reader (CytoFluor II; Millipore) and normalized
to cell number by dividing by a subsequent measurement of fluorescence
(excitation, 485 nm; emission, 530 nm) after addition of 5 µM YO-PRO. Experiments were performed in triplicate.
Spreading of Macrophages on Human IgG--
Thio-macrophages were
pretreated with vehicle (dimethyl sulfoxide), 100 nM
wortmannin, or 100 µM LY294002 for 30 min at 37 °C and
then applied to 13-mm2 round coverslips previously coated
with 1 mg/ml human IgG. Cells were allowed to spread for varying
intervals at 37 °C followed by fixation and staining for F-actin (to
facilitate delineation of cell margins). The cell diameter in close
contact with the coverslip was measured, and the apparent adherent
surface area was calculated. The adherent surface areas of ~30
cells/coverslip were measured, and experiments were performed in duplicate.
Quantitation of Exocytosis--
Thio-macrophages were
preincubated with various concentrations of FM1-43 for 3 min at
25 °C and added to 96-well plates that had been previously coated
with 1 mg/ml human IgG. Using a fluorescence plate reader (CytoFluor
II), fluorescence (excitation, 485 nm; emission, 530 nm) was monitored
in the continual presence of dye for varying time intervals at
25 °C. Cells and dye alone gave negligible fluorescence readings.
FM1-43 fluorescence values were normalized to cell number after a
subsequent fluorescence measurement (excitation, 360; emission, 460)
after addition of 2 µM 4,6diamidino-2-phenylindole.
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RESULTS |
Wortmannin and LY294002 Inhibit Fc
-mediated
Phagocytosis--
PI 3 kinase has been shown to be activated by most
receptors capable of mediating actin polymerization. To test the role
of this family of enzymes in phagocytosis, we determined the effects of
two structurally unrelated PI 3-kinase inhibitors, wortmannin and
LY294002, on Fc
R-mediated phagocytosis. Addition of
either wortmannin or LY294002 produced a
concentration-dependent inhibition of phagocytosis with an
IC50 of ~4.5 nM and 3 µM,
respectively (Fig. 1). These results are
similar to those reported for phagocytosis in guinea pig neutrophils
(using wortmannin) and bone marrow-derived macrophages (19, 21). The
IC50 values in this study were similar to those reported
for inhibition of PI 3-kinases, but not for a PI 4-kinase (28) and
myosin light chain kinase (29). Because mTOR is another target of
wortmannin (30), we tested the effects of rapamycin on phagocytosis;
however, 10 µM rapamycin had no effect on phagocytosis
(not shown). These results suggest that the inhibitory effects of
wortmannin and LY294002 on Fc
R-mediated phagocytosis
were attributable to inhibition of one or more members of the PI
3-kinase family of enzymes.

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Fig. 1.
Wortmannin or LY294002 inhibits phagocytosis
by macrophages in a concentration-dependent manner.
Phagocytosis assays were performed for RAW 264.7 cells incubated with
EIgG in the presence of wortmannin (A) or LY294002
(B) as described under "Experimental Procedures." Data,
expressed as the mean ± S.E. (n = 3), are
reported as percent of control phagocytosis in the presence of vehicle
alone.
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Inhibition of Phagocytosis by Wortmannin Is Not via Inhibition of a
PLA2--
A previous study demonstrated that wortmannin
inhibits PLA2 in vivo but not in
vitro (31), suggesting that this class of phospholipases may lie
downstream of a PI 3-kinase. In addition, Fc
R-mediated
phagocytosis is accompanied by enhanced activation of one or more
isoforms of PLA2, and inhibition of PLA2
activity leads to decreased phagocytosis (32). To determine whether the inhibition of phagocytosis by wortmannin is mediated via inhibition of
PLA2, we preincubated cells with either 100 nM
wortmannin or 10 µM bromophenacyl bromide (an
irreversible inhibitor of PLA2) followed by the absence or
presence of 2 µM exogenous arachidonate to attempt to
bypass PLA2 blockade (32). Both wortmannin and bromophenacyl bromide inhibited phagocytosis of EIgG. Addition of
exogenous arachidonate restored phagocytosis by 38 ± 3% in cells
incubated with bromophenacyl bromide but did not do so in cells treated
with wortmannin. This indicates that the decrease in phagocytosis in
cells treated with wortmannin was not attributable to inhibition of
PLA2 activity.
Fc
-mediated Phagocytosis Is Accompanied by Enhanced
Production of PIP3--
To test whether ligation of
Fc
receptors results in the activation of PI 3 kinase(s)
in vivo, [32P]orthophosphate-labeled
macrophages were challenged with EIgG, phospholipids were extracted,
and the phosphoinositide content was analyzed by thin layer
chromatography. There was a rapid increase in the accumulation of
PIP3 after Fc
R ligation, which peaked at
15 s and declined by 1-2 min (Fig.
2). Immunoprecipitation of the 85-kDa
subunit of PI 3-kinase did not reveal enhanced phosphotyrosine content
of this subunit (not shown). The increase in the accumulation of
PIP3 that occurred during phagocytosis is consistent with
an increase in the activity of one or more isoforms of PI 3-kinase.

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Fig. 2.
Fc R-mediated phagocytosis is
accompanied by enhanced production of PIP3. EIgG were
added to adherent [32P]orthophosphate-labeled RAW 264.7 cells for the indicated times, and lipids were extracted, separated by
thin layer chromatography, and processed for autoradiography as
described under "Experimental Procedures." The arrow
denotes position of PIP3 standard.
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Actin Assembly in Response to Fc
Ligation Is Not
Inhibited by Wortmannin or LY294002--
PI 3 kinase activity has been
previously shown to be necessary for actin assembly mediated by PDGF or
insulin (12, 33). Like receptor tyrosine kinases, ligation of
Fc
receptors results in activation of several tyrosine
kinases, such as Syk, and tyrosine kinase activity is required for
actin assembly mediated by this class of immunoreceptor tyrosine
activation motif-bearing receptors (2). To determine whether the
inhibition of phagocytosis by wortmannin or LY294002 correlated with an
inhibition of Fc
R-directed actin polymerization, we
stained RAW 264.7 cells undergoing phagocytosis for F-actin using
rhodamine-phalloidin. F-actin-rich phagocytic cups were visible in
control cells treated with vehicle alone (Fig.
3, A and B),
similar to those seen previously (27). In contrast to results obtained
using growth factors, addition of wortmannin (Fig. 3, C and
D) and LY294002 (not shown) had no obvious effect on the
accumulation of F-actin beneath attached particles. However, careful
observation of the cortical cytoplasm beneath attached cells revealed
that, in nearly all cases, F-actin-rich pseudopods did not extend
beyond ~50-70% of the circumferences of the particles (Fig. 3,
C and D). This was apparent only for particles
attached to the sides of the macrophages, because the degree of
pseudopod extension around particles that were attached to the dorsal
surfaces of macrophages was difficult to appreciate.

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Fig. 3.
Phagocytic cup formation and actin assembly
induced by EIgG in macrophages is not inhibited by wortmannin or
LY294002. RAW 264.7 cells were preincubated with vehicle
(A and B) or 100 nM wortmannin
(C and D) followed by a further incubation with
EIgG for 5 min at 37 °C before fixation and staining for F-actin
with rhodamine phalloidin as described under "Experimental
Procedures." A and C, phase-contrast;
B and D, rhodamine-phalloidin fluorescence.
Scale bar, 10 µm. E, F-actin content of RAW
264.7 cells pretreated with vehicle, 100 nM wortmannin, or
100 µM LY294002 was determined after addition of EIgG for
5 min at 37 °C. Data, expressed as the mean ± S.E.
(n = 7), are reported as percent increase compared with
cells not challenged with EIgG.
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To determine whether the inhibition of PI 3 kinase in RAW 264.7 cells
resulted in a failure to polymerize a sufficient quantity of actin
necessary for complete pseudopod extension, we quantitated F-actin
content in cells challenged with EIgG in the presence or absence of
wortmannin or LY294002. In vehicle-treated cells there was an increase
in the total amount of F-actin present after the addition of EIgG (Fig.
3E), which was blocked by the presence of 2 µM
cytochalasin D (data not shown). The presence of either wortmannin or
LY294002 did not inhibit increases in F-actin content after addition of
EIgG and, in fact, led to slightly enhanced accumulations of F-actin
(Fig. 3E; p = 0.08 and 0.04, respectively). These data indicate that inhibition of PI
3-kinase did not impair actin assembly in response to
Fc
R ligation.

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Fig. 4.
Sensitivity of phagocytosis to PI 3-kinase
inhibition is independent of the number of particles added but is
dependent on particle size in RAW LacR/FMLPR.2 cells. A,
model for potential sources of recruited membrane necessary for
pseudopod extension and successful completion of phagocytosis. In
Model 1, the source of membrane is directly from the plasma
membrane itself (i.e. from areas adjacent to those directly
participating in particle ingestion). In Model 2, the source
of membrane is from a latent intracellular vesicular pool.
B, phagocytosis of EIgG as a function of increasing total
number of EIgG added in the absence ( ) or presence ( ) of 100 nM wortmannin. Data are expressed as the mean ± S.E.
(n = 3). C, phagocytosis of IgG-coated beads
of the indicated diameters in the presence of 100 nM
wortmannin ( ) or 100 µM LY294002 ( ). Data,
expressed as the mean ± S.E. (n = 3), are
reported as the percent ingestion of beads in the presence of
vehicle.
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Efficacy of PI 3-Kinase Inhibition Is a Function of Particle Size,
Not Number--
The apparent block in maximal pseudopod extension in
the presence of wortmannin (Fig. 3, C and D),
with a concomitant preservation of Fc
R-directed actin
polymerization (Fig. 3E), suggested that pathways leading to
cytoskeletal assembly were not the target of PI 3-kinase inhibition.
Because PI 3 kinase(s) have been implicated in the regulation of
various membrane-trafficking events (for review, see Ref. 24), we
considered the possibility that inhibition of this pathway produced a
limitation in the membrane available for participation in pseudopod
extension. In principle, the source of this membrane can be from the
plasma membrane itself (e.g. from areas adjacent to those
directly participating in particle ingestion) or from an intracellular
membrane source (Fig. 4A). To distinguish these
possibilities, we first assessed the effect of reducing the total
number of particles added to the macrophages on sensitivity to PI
3-kinase inhibition. However, reduction in the number of particles did
not lead to a restoration of phagocytosis in the presence of wortmannin
(Fig. 4B), indicating that availability of plasma membrane
was not a limiting factor for phagocytosis during PI 3-kinase inhibition.
To determine whether inadequate pseudopod extension in the presence of
PI 3-kinase inhibitors was attributable to a failure of the recruitment
of membrane derived from an intracellular source, we challenged
macrophages with phagocytic particles of varying sizes. We determined
whether the phagocytic blockade by PI 3-kinase inhibitors was relieved
by reducing particle size and, hence, the magnitude of pseudopod
extension required for complete particle engulfment. By screening a
variety of latex particles derivatized by several proteins, we found
that covalent modification of carboxylated polystyrene beads with
sulfhydryl-modified BSA afforded the lowest extent of nonspecific
binding to macrophages. We chose a subclone of RAW 264.7 cells to
study, RAW LacR/FMLPR.2, because this clone among several others
demonstrated the least tendency to bind latex particles
nonspecifically. Finally, we chose to opsonize the BSA-derivatized particles with an equivalent surface density of IgG, because this would
allow for changes in particle size without altering ligand density and,
hence, efficiency of Fc
R engagement. Quantitative immunoblotting was performed to determine IgG opsonin density, and
batches of beads of varying sizes were prepared that varied by no more
than 15% in opsonin density.
IgG-opsonized latex beads ranging from 1 to 6 µm in diameter were
ingested by RAW LacR/FMLP.2 cells. Ingestion of these beads resembled
phagocytosis of EIgG, because cytochalasin D inhibited phagocytosis by
>90% regardless of the bead size. However, inhibition by cytochalasin
D was slightly more effective in larger beads (e.g. 100 ± 0 versus 91 ± 6% for 6- and 1-µm beads,
respectively). The greater sensitivity to cytochalasin for the larger
beads may be attributable to a requirement for a greater magnitude of
actin assembly needed to completely surround the 4.5- and 6-µm
particles. Similar to results using EIgG (2), phagocytosis of all sizes of latex beads was completely blocked by genistein, a tyrosine kinase
inhibitor (data not shown).
The efficacy of PI 3-kinase blockade on phagocytosis varied with bead
size. Wortmannin or LY294002 potently inhibited the ingestion of beads
of 4.5 and 6 µm in diameter but had progressively less efficacy of
inhibiting ingestion of smaller beads (Fig. 4C). Because the
magnitude of pseudopod extension required for complete internalization
varies with particle diameter, these results suggest that intact
signaling through PI 3-kinase(s) is critical when pseudopods are
required to extend from the margins of the cells by more than the
circumference of 2-3-µm beads. These results are consistent with a
requirement for surface recruitment of a latent intracellular pool of
membrane for optimal pseudopod extension.
Spreading of Thio-macrophages on Human IgG and Concomitant Exocytic
Insertion of Membrane Are Inhibited by Wortmannin or LY294002--
The
above results using beads of 1-2 µm in diameter suggested that the
PI 3-kinase blockade impaired pseudopod extension rather than
phagosomal closure (21). To confirm this, we used an assay of
Fc
R-based motility that does not require phagosomal
closure. We coated coverslips with human IgG and observed the extent of spreading as a function of time and PI 3-kinase activity. We chose thio-macrophages because RAW 264.7 cells and similarly derived lines do
not spread readily on this substrate (data not shown). Thio-macrophages, like RAW LacR/FMLP.2 cells, ingested small beads in
the presence of wortmannin of LY294002 (data not shown).
Thio-macrophages spread rapidly on IgG-coated substrates, demonstrating
an ~4-fold increase in their apparent adherent surface areas.
Spreading of macrophages on IgG was inhibited by either wortmannin or
LY294002, whereas the peripheral appearance of F-actin was not,
consistent with a lack of inhibition of cytoskeletal assembly by PI
3-kinase inhibitors (Fig. 5). The extent
of spreading in controls was appreciable and suggested that newly
recruited membrane might be required for efficient spreading. We
measured the recruitment of membrane to the cell surfaces using FM1-43, a styryl dye with fluorescence that increases after binding the outer
leaflet of plasma membranes (34, 35). As new membrane is recruited to
the surface during exocytosis, fluorescence increases in cells
continuously maintained in the presence of the dye. Thus, the dye
reports cumulative exocytic membrane insertion. We measured FM1-43
fluorescence using saturating concentrations of dye (Fig. 6A) and found that macrophages
displayed a time-dependent increase in FM1-43 fluorescence
during spreading on human IgG. Similar to results of cell spreading
(Fig. 5), increases in FM1-43 fluorescence were inhibited by wortmannin
and LY294002 (Fig. 6B). Collectively, these data indicate
that spreading of macrophages on human IgG is accompanied by an
apparent increase in the plasma membrane surface area concomitant with
an increase in exocytic insertion at the plasma membrane from an
intracellular source. Both processes are regulated in a PI
3-kinase-dependent fashion.

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Fig. 5.
Spreading but not F-actin accumulation of
thio-macrophages on human IgG is inhibited by wortmannin or LY294002.
A, cells, preincubated with vehicle, 100 nM
wortmannin, or 100 µM LY294002, were allowed to spread on
IgG-coated coverslips at 37 °C for the indicated times before
fixation. Data, expressed as the mean ± S.E. (n = 3), are reported as mean adherent surface areas.
B-G, thio-macrophages spreading on IgG-coated
coverslips in the presence of vehicle (B and C),
100 nM wortmannin (D and E), or 100 µM LY294002 (F and G) were fixed
and stained for F-actin with rhodamine-phalloidin. Phase-contrast
(B, D, and F) and fluorescence
micrographs (C, E, and G) depict
presence of F-actin-rich areas at the peripheries of the cells despite
the presence of wortmannin or LY294002.
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Fig. 6.
Exocytic insertion of plasma membrane is
inhibited by wortmannin or LY294002. A, FM1-43 fluorescence
saturates with increasing dye concentration. 5 × 104
cells were incubated with the indicated concentrations of FM1-43. Data
are expressed as the mean ± S.E. of FM1-43 fluorescence
normalized to cell number (n = 3). B,
fluorescence of cells spreading on adherent human IgG in the continuous
presence of 8 µM FM1-43 and vehicle, 100 nM
wortmannin, or 100 µM LY294002. Data, expressed as the
mean ± S.E. (n = 5), are reported as percent
increase in baseline fluorescence (t = 0). The
difference between control- and wortmannin- or LY294002-treated cells
is statistically significant (p < 0.01).
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 |
DISCUSSION |
The data presented here demonstrate a requirement for one or more
isoforms of PI 3-kinase in Fc
R-mediated phagocytosis. Although these results are not surprising in light of earlier reports
of the requirement for this family of enzymes in phagocytosis, the role
for PI 3-kinase in phagocytosis that we are proposing is somewhat
unexpected. Studies of the PDGF receptor signaling cascade indicate
that PI 3-kinase activity is required for membrane ruffling and actin
polymerization (8, 33), both of which occur in a
Rac1-dependent manner. Similar to growth factor receptor signaling, Fc
R-dependent signaling pathways
that culminate in actin assembly are tyrosine kinase- and
Rac1-dependent (2, 3). However, our results clearly
indicate that PI 3-kinase activity is not required for
Fc
R-mediated actin assembly. Rather, blockade of PI
3-kinase(s) appears to result in the functional dissociation between
cytoskeletal assembly and pseudopod extension, events that are normally
coupled. These data are somewhat different than those of Araki et
al. (21), who described a block in phagosomal closure and
macropinocytosis in the presence of PI 3-kinase inhibitors. Our results
support of role for PI 3-kinase in an earlier step in phagocytosis.
Because the cellular components required for the terminal fusion of
vesicles, an event akin to phagosomal closure, are likely to be
different than those governing process extension, this distinction has
mechanistic consequences. Indeed, given the results described in Fig. 6
and studies that show a requirement for PI 3-kinase in membrane
trafficking (36-40), we propose that up-regulation of one or more
intracellular membrane compartments is required for optimal pseudopod
extension, and that this event is dependent on one or more isoforms of
PI 3-kinase.
The identity of PI 3-kinase-sensitive membrane compartments is under
extensive study by many groups (for review, see Ref. 24). The bulk of
evidence shows a requirement for PI 3-kinase in trafficking from one or
more recycling compartments that contain transferrin receptors and/or
glucose transporters to the plasma membrane (10, 36, 41-44). In
addition, several studies demonstrate a requirement for PI 3-kinase in
the secretory pathway (45-47). Conceivably, one or more of these
pathways are up-regulated during, and are required for, pseudopod
extension. Interestingly, a recent ultrastructural study of
phagocytosis in monocytes demonstrated the accumulation of plasma
membrane-derived, electron-lucent vesicles beneath nascent phagosomes,
particularly in the presence of PLA2 inhibitors (48).
However, the lack of restoration of phagocytosis by arachidonate in the
presence of PI 3-kinase blockade and the results of Fig. 4B
indicate that this membrane compartment is not likely to be directly
regulated by PI 3-kinase.
This study did not address the specific isoforms of PI 3-kinase
involved in phagocytosis and pseudopod extension. The list of PI
3-kinase family members is long (for review, see Ref. 49), and many
have been only partially characterized. The isoform most often
implicated in cellular signaling, p85/p110, is activated by many growth
factor receptors; during receptor clustering, the p85 regulatory
subunit is recruited to the receptor itself (50-52) or interacts with
adaptor proteins such as Grb2 (53), c-Cbl (54, 55), and cytosolic
tyrosine kinases (56, 57). p85/p110 has been shown to associate with
Fc
receptors and with Syk, although it is not clear
whether such an association is direct (58, 59). We could not
definitively determine whether this isoform was required for
phagocytosis. Although transfection of RAW LacR/FMLPR.2 cells with
p85, a p110 binding-defective allele of p85, did not result in
impaired phagocytosis despite apparent expression (data not shown), it
also produced no detectable phenotypic changes in the cells; therefore,
we could not verify that it functioned in a dominant-negative fashion.
Other members of the PI 3-kinase family, in addition to p85
binding isoforms, may be activated after Fc
receptor
activation (60).
Several molecular targets of the lipid products of PI 3-kinase have
been identified. One of these, ARNO (cytohesin-2), contains a PH domain
that is capable of interacting with acidic phospholipids in
vitro (61). Recent studies indicate that cytohesin-2 has guanine
nucleotide exchange activity for ARF6 (62), and intact ARF6 has been
shown to be required in phagocytosis (63). Other likely targets include
proteins that interact with or stimulate guanine nucleotide exchange
activity of members of the Rab family of GTPases. Wortmannin inhibits
Rab5-mediated stimulation of endocytosis (64) and blocks
insulin-stimulated binding of 35S-GTP
S to Rab4 (65). It
is possible that lipid products of PI 3-kinase bind to PH domains on
guanine nucleotide exchange factors for ARF and Rab family members,
thereby increasing their exchange activity. For example, a role for
phosphoinositide-stimulated guanine nucleotide exchange factor activity
has been described for Vav (66). Another product of PI 3-kinase,
phosphatidylinositol-3-phosphate, may be required for
Rab-dependent membrane fusion.
Phosphatidylinositol-3-phosphate binds the FYVE finger domain of EEA1,
a protein that is required for Rab5-dependent endosomal
fusion in vitro. This phospholipid is required for membrane
localization of EEA1 (for review, see Ref. 67). Thus, multiple lipid
products of PI 3-kinase may be required for promoting membrane fusion
events in vivo, including those that accompany phagocytosis.
Although it could be argued that ingestion of beads of 1-2 µm in
diameter differ mechanistically from ingestion of larger beads, our
results suggest that they share certain common elements, including
cytochalasin sensitivity and a requirement for one or more tyrosine
kinases. The slightly greater sensitivity of large bead phagocytosis to
cytochalasin may reflect a requirement for sustained actin
polymerization necessary to support the formation of large pseudopods.
Phagocytosis of small (i.e. 1-2 µm) beads would be
expected to require less sustained actin assembly to achieve complete
particle engulfment. Thus, the typically incomplete inhibition of
barbed end actin filament growth by cytochalasin may result in complete
failure in the engulfment of large beads and only partial inhibition in
the engulfment of small beads. These data are similar to recent
findings by Koval et al. (68), in which complete inhibition
of the ingestion of latex particles by macrophages required high (2.5 µM) concentrations of cytochalasin D. Although we did not
test the phagocytosis of beads smaller than 1 µm, it is anticipated
that the cellular machinery involved in the ingestion of progressively
smaller test particles may not require the active participation of the
actin-based cytoskeleton, as suggested by Koval et al. (68),
or the membrane recruitment of tyrosine kinases. It is difficult to
draw general conclusions regarding the requirement of PI 3-kinase in
the phagocytosis of other phagocytic targets, such as bacteria, because
they are geometrically distinctive and may not necessarily be opsonized
uniformly, as is the case here. However, a demonstration of a
requirement for PI 3-kinase was reported for the ingestion of
Listeria monocytogenes by epithelial cells (69); it is
possible that the role of this enzyme in phagocytosis of
Listeria is similar to that described in the current study.
Several studies that attempt to elucidate how a cell extends surface
protrusions focus on the role of actin as a protrusive force driving
pseudopod extension and view the membrane as a passive component in
this process (for review, see Ref. 70). Other models for membrane
protrusion have proposed a role for a polarized endocytic cycle that
involves exocytosis of vesicles at the leading edge (71). In support of
this model, recycled transferrin receptors are concentrated at the
leading lamella in migrating fibroblasts (72), and protrusion of the
cell surface in plasmodia has been linked with exocytic events (73).
Studies of macrophage phagosomes indicate the presence of multiple
syntaxins, and addition of tetanus toxin light chain, a v-SNARE
protease, inhibited Fc
R-mediated phagocytosis,
underscoring the close association of the endocytic and exocytic
compartments (74, 75). Our data support a model of pseudopod extension
that requires active participation of tyrosine kinases, actin
polymerization, and PI 3-kinase. All are necessary for the coordinated
extension of pseudopods that culminate in the formation and
eventual closure of the phagosome.