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*
Center for Cell Biology and Cancer Research,
Department of Biochemistry,
Center for Cardiovascular Sciences, and
§
Center for Neuropharmacology and Neurosciences, Albany Medical College, Albany, NY 12208; and
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Department of Biology, Kobe University, Kobe, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Rs on
macrophages results in phagocytosis of the particles and generation of
a respiratory burst. Both IgG-stimulated phagocytosis and respiratory
burst involve activation of protein kinase C (PKC). However, the
specific PKC isoforms required for these responses have yet to be
identified. We have studied the involvement of PKC isoforms in
IgG-mediated phagocytosis and respiratory burst in the mouse
macrophage-like cell line, RAW 264.7. Like primary
monocyte/macrophages, their IgG-mediated phagocytosis was calcium
independent and diacylglycerol sensitive, consistent with novel PKC
activation. Respiratory burst in these cells was Ca2+
dependent and inhibited by staurosporine and calphostin C as well as by
the classic PKC-selective inhibitors Gö 6976 and CGP 41251,
suggesting that classic PKC is required. In contrast, phagocytosis was
blocked by general PKC inhibitors but not by the classic PKC-specific
drugs. RAW 264.7 cells expressed PKCs 
, ßI, 
, 
, and 
.
Subcellular fractionation demonstrated that PKCs , , and
translocate to membranes during phagocytosis. In
Ca2+-depleted cells, only novel PKCs and increased
in membranes, and the time course of their translocation was consistent
with phagosome formation. Confocal microscopy of cells transfected with
green fluorescent protein-conjugated PKC or confirmed that
these isoforms translocated to the forming phagosome in Ca-replete
cells, but only PKC colocalized with phagosomes in
Ca2+-depleted cells. Taken together, these results suggest
that the classic PKC mediates IgG-stimulated respiratory burst in
macrophages, whereas the novel PKCs and/or are necessary for
phagocytosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rs on the surface of
monocyte/macrophages and neutrophils. Phagocytosis requires the cells
to coordinate changes in cytoskeleton and membrane structure during
pseudopod extension and particle internalization. In addition to
phagocytosis, FcR ligation activates other signaling pathways,
including those regulating intracellular Ca2+
flux, mitogen-activated protein kinase cascades, and the respiratory
burst (1, 2). Current research has focused on identifying
signaling pathways activated by FcR. Intracellular events resulting
from FcR clustering include tyrosine phosphorylation, actin
rearrangement, arachidonic acid release, and activation of signaling
enzymes phosphoinositol 3-kinase, protein kinase C
(PKC),3 phospholipase
C, phospholipase D, and phospholipase A2
(1, 2, 3, 4). However, the specific functions of these
components in FcR-mediated signaling remain unknown.
Our previous work has confirmed a role for PKC activation during
IgG-mediated phagocytosis in human monocytes and the human monocytic
cell line, Mono Mac 6 (5, 6). PKC is a family of related
enzymes, which is divided into three groups on the basis of structure
and cofactor requirements (7). The classic PKC (cPKC)
isoforms , ßI, ßII, and require Ca2+,
diacylglycerol (DAG), and phosphatidylserine (PS) for optimal activity.
The novel PKC (nPKC) isoforms , , 
, and 
lack the
Ca2+ requirement but are activated by DAG and PS.
The atypical PKC isoforms and 
/
bind PS, but are insensitive
to Ca2+ and DAG. Despite the differing cofactor
requirements, there is little difference between the in vitro substrate
specificities of the isoforms. Because cells usually express several
PKC isoforms, in vivo function is thought to be regulated by
intracellular location and binding to specific targeting proteins
(7).
Although PKC activation is necessary for phagocytosis in
monocyte/macrophages, it is not known which PKC isoform(s) function in
FcR-mediated signaling (5, 6, 8). Both cPKC ( and
ß) and nPKC () isoforms have been reported to translocate to
membranes during FcR cross-linking or phagocytosis (9, 10), but there is no direct evidence that these isoforms are
required for FcR-mediated signaling. Phagocytosis proceeds normally
in the absence of a Ca2+ signal (11, 12) and can be increased by the treatment of cells with the PKC
activators PMA and DAG (5, 6). These characteristics are
consistent with the cofactor requirements for nPKC isoforms, i.e.,
Ca2+ independent and DAG sensitive. Therefore, we
tested the hypothesis that one or more of the nPKC isoforms is required
for phagocytosis.
IgG-mediated phagocytosis is accompanied by the generation of a
respiratory burst; however, the role of specific PKC isoforms in
respiratory burst is not certain. Respiratory burst can be activated by
PMA in both neutrophils and monocyte/macrophages (13, 14, 15).
In neutrophils, IgG-stimulated respiratory burst is decreased by
pharmacological inhibition of PKC (16) or by selective
antisense down-regulation of cPKC ß (14), indicating a
requirement for PKC. In comparison with neutrophils, studies in
monocyte/macrophages have not been as consistent. In human monocytes,
respiratory burst stimulated by opsonized zymosan was reduced to
background levels by selective down-regulation of cPKC
(17). However, in guinea pig macrophages, pharmacological
inhibition of PKC produced only a modest decrease in FcR-mediated
respiratory burst (15). We addressed the question of
whether the mouse macrophage cell line RAW 264.7 was similar to human
monocytes in requiring cPKC for FcR-mediated respiratory
burst.
We have previously demonstrated that IgG-mediated phagocytosis requires PKC and that PKC activation is upstream of mitogen-activated protein kinase and Ca2+-independent phospholipase (2, 5). Phagocytes also require PKC to produce the respiratory burst that accompanies IgG-mediated phagocytosis (1). To determine the functions of PKC in these two signaling pathways, it is necessary to identify the relevant PKC isoform(s). Activation of cPKC and nPKC is associated with their translocation to specific sites (6). Therefore, we examined the translocation of cPKC and nPKC isoforms during phagocytosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-PMA were obtained from LC Laboratories (Woburn, MA). DAG was
purchased from Avanti Polar Lipids (Alabaster, AL). GF109203X was
obtained from Biomol Research (Plymouth Meeting, PA). CGP 41251 was a
gift from Novartis (Basil, Switzerland). Gö 6976 was purchased
from Alexis Biochemicals (San Diego, CA). Cell culture
The RAW 264.7 mouse macrophage cell line was maintained in RPMI 1640 media (Life Technologies, Grand Island, NY) plus sodium pyruvate, nonessential amino acids, glutamate (BioWhittaker), and 10% newborn calf serum (HyClone, Logan, UT). For phagocytosis experiments, cells were seeded in 24-well culture plates at 6 x 105 cells per well. For PKC translocation experiments, cells were grown in 100-mm culture plates and used when confluent. Unless otherwise specified, cells were incubated in above media without the 10% calf serum overnight before each experiment.
Buffers
HBSS (Life Technologies) buffer was composed of: 4 mM sodium bicarbonate, 10 mM HEPES, containing either 1.5 mM each CaCl2 and MgCl2 (HBSS2+) or 2 mM MgCl2 and 1 mM EGTA (Mg/EGTA). Lysis buffer was composed of: 25 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 2.5 mM DTT, and 2.5 mM EDTA, and contained the following inhibitors: 5 mM benzamidine, 50 µg/ml leupeptin, 50 µg/ml aprotinin, 50 µg/ml trypsin inhibitor, 5 µg/ml pepstatin, 1 mM PMSF, 20 mM NaF, 1 mM Na3VO4, 1 mM para-nitrophenylphosphate, and 5 mM imidazole (Sigma). DNA assay buffer was composed of: 2 M NaCl, 2 mM EDTA, and 50 mM Na2HPO4. TBST was composed of: 50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20, and 0.01% thimerosal.
Targets
Ig-G opsonized, radiolabeled erythrocytes were referred to as EIgG. SRBC (Crane, Syracuse, NY) were radiolabeled by incubation (45 min, 37°C) with 51Cr (New England Nuclear, Boston, MA) and opsonized with rabbit anti-SRBC IgG (Cappel, Durham, NC) (18). Glass beads (Duke Scientific, Palo Alto, CA) were opsonized by sequential incubation with poly-L-lysine, BSA, and anti-BSA IgG (Sigma) as previously described (5), and referred to as BIgG. Control beads were incubated with poly-L-lysine and BSA only (BBSA). For confocal analysis, the BSA-coating step included 5 µg of Alexa 568 (Molecular Probes, Eugene, OR)-conjugated BSA. BSA was conjugated to the Alexa dye according to manufacturer’s directions.
Phagocytosis assay
If cells were described as Ca depleted, they were incubated (45 min, 37°C) in Mg/EGTA buffer (-Ca cells), otherwise the assay was performed in HBSS2+ (+Ca cells). When used, inhibitors were added for the last 30 min of the incubation. EIgG (6 x 106) were added to each well for the designated time at 37°C. The cells were washed twice with 0.83% NH4Cl, once in PBS, and solubilized in 2 M NaOH. Phagocytosis is defined as the cell-associated radioactivity after hypotonic lysis of noninternalized targets and expressed as a percentage of phagocytosis in control cells. Nonspecific binding was determined using nonopsonized SRBC and subtracted from the phagocytosis values.
Synchronized phagocytosis of BIgG
To determine the time course of phagocytosis of BIgG, 2 x 105 cells were plated on 13-mm glass coverslips (Ernest F. Fullam, Latham, NY) in 24-well plates. The cells were incubated in buffer as described above and then chilled on ice for 20 min. The buffer was removed, and 0.5 ml of ice-cold buffer containing 1 x 106 BIgG was added. After 5 min on ice for target binding, the plates were transferred to a 37°C water bath, and, at each time point, phagocytosis was stopped by fixing the cells with 3.7% formaldehyde. The coverslips were washed three times with PBS and blocked with 10% sheep serum (30 min, 21°C). The following incubations were performed with Ab diluted in 10% sheep serum (30 min, 21°C): 1) rabbit anti-BSA, 1:250; 2) FITC-conjugated goat anti-rabbit, (Rockland, Gilbertsville, PA), 1:200; 3) permeabilize 5 min in 0.01% Triton X-100; 4) block as above; 5) rabbit anti-BSA; 6) Texas-red conjugated goat anti-rabbit, (ICN Pharmaceuticals, Aurora, OH), 1:250; and 7) Hoechst nuclear stain (Molecular Probes). Coverslips were washed three times with PBS after each incubation. After washing, the coverslips were mounted on glass slides with Prolong antifade medium (Molecular Probes) and viewed with a triple-band filter. The cell nuclei stained blue, internalized beads were red, and external beads or portions of beads were yellow/green. The number of completely internalized beads were counted in a minimum of 100 cells and expressed as the phagocytic index: (no. of red beads/number of cells counted) x 100.
H2O2 production
H2O2 production was determined as
described previously (18). Briefly, 1.2 x
106 cells were incubated for 1 h in 1 ml
HBSS containing 100 nM homovanillic acid and 1 IU HRP, with 1 x
107 BIgG (see Figs. 5
and 7) or 50 ng PMA (see Fig. 6) as
the stimulus. The homovanillic acid oxidation product was measured
fluorometrically.
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For PMA stimulation, confluent cultures of RAW 264.7 cells in 10-cm dishes were treated with 100 nM PMA or an equivalent volume of DMSO (carrier) (10 min, 37°C), the media was aspirated, and the cells were scraped and sonicated in 0.4 ml of lysis buffer. Lysates were centrifuged 45 min at 100,000 x g. The supernatant (designated cytosol) was removed, and the pellet was solubilized in lysis buffer plus 0.2% Triton X-100. The samples were centrifuged again as above. The supernatant (designated membrane) was removed from the pellet (designated insoluble fraction). For phagocytosis experiments, the protocol for synchronized phagocytosis of BIgG was followed using cells in 100-mm culture dishes and 1 x 108 targets. Phagocytosis was stopped at the various time points by scraping and sonicating the cells in lysis buffer; cell lysates were processed as described above. As no PKCs were detected in the insoluble cell fractions following phagocytosis (data not shown), PKC levels were quantified only in the membrane and cytosolic fractions.
DNA assay
DNA was measured by the method of Labarca and Paigen (19). Briefly, aliquots of whole cell lysate were diluted with assay buffer and 2% (final concentration) bisbenzamide, and the fluorescence was compared with a standard curve prepared with calf thymus DNA.
Western blots
For PMA experiments, protein in cytosolic and membrane fractions
was quantified by the Bradford protein assay. Different proportions of
the cell fractions were loaded to obtain a measurable signal; however,
the amount in each fraction was matched between treatments to allow
comparison. This corresponded to 
6% of the cytosol, 25% of the
membrane, and 25% of the insoluble fractions. For phagocytosis
experiments, equal volumes of the membrane fractions were loaded.
Proteins were transferred to nitrocellulose membranes, and standards
were located with Ponceau S stain. The membranes were blocked with 3%
BSA in TBST and probed with the appropriate primary and secondary Ab in
1% BSA in TBST. For the primary Ab, mAb against PKCs , , ,
, and , (Transduction Laboratories, San Diego, CA) and polyclonal
Ab against PKCs ßI, ßII, and (Santa Cruz Biotechnology, Santa
Cruz, CA) were used. For the secondary Ab, goat anti-rabbit HRP
(Santa Cruz Biotechnology) and rabbit anti-mouse HRP (The Jackson
Laboratory, Bar Harbor, ME) were used. Bands were detected with Ultra
Supersignal ECL reagent (Pierce, Rockford, IL) and were quantified by
densitometry. In phagocytosis experiments, the densities of the PKC
bands were normalized for cell DNA before comparison by ANOVA.
Transfections
The construction and characterization of the green fluorescent
protein (GFP) PKC and plasmids have been previously described
(20). These constructs are enzymatically active and
translocate to membranes in response to a variety of stimuli. DNA (14
µg/6 x 106 cells) was transfected into
RAW 264.7 cells using Superfect (Qiagen, Valencia, CA) as per
manufacturer’s instructions. Cells were exposed to DNA for 3 h,
then washed and incubated for an additional 12–18 h to allow maximal
expression of fluorescence.
Confocal imaging
Transfected cells were replated onto 13-mm coverslips (5 x 105 cells/coverslip) for 3 h. Media were removed and the cells were washed and incubated in either HBSS2+ or Mg/EGTA (45 min, 37°C). Cells were cooled on ice (30 min), and ice-cold Alexa 568-conjugated BIgG were added at a 5:1 BIgG/cell ratio. Following target binding (15 min on ice), cells were placed in a 37°C water bath for 0–15 min. At varying times, the cells were fixed (5 min, 3.7% formaldehyde), mounted in Prolong Antifade (Molecular Probes), and analyzed by confocal microscopy. Z series images of GFP-expressing cells were analyzed on a Noran-OZ (Noran Instruments, Middleton, WI) confocal laser scanning microscope, interfaced with a Nikon Diaphot 200 inverted microscope (Nikon, Melville, NY) equipped with a PlanApo x60, 1.4 NA oil-immersion objective lens. GFP and Alexa 568 were independently imaged using excitation/emission wavelengths of 488/500–550 nm band pass and 568/590 nm long pass, respectively. Images were analyzed using Noran InterVision software.
Statistics
Unless otherwise stated, all measurements were made in
triplicate on at least three separate cell preparations. Data are
expressed as the mean ± SEM. Comparisons were made by ANOVA.
Results with p 
0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We identified the PKC isoforms expressed in RAW 264.7 cells by
Western blot analysis with isoform-specific Abs (Fig. 1). cPKC , cPKC ßI, nPKC , nPKC
, and atypical PKC were detected in RAW 264.7 cell lysates. cPKC
, nPKC , and nPKC could be detected only in positive controls
but not in RAW 264.7 cell lysate (data not shown). PKC ßII could not
be reproducibly detected in cell fractions.
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Earlier studies demonstrated that IgG-mediated phagocytosis is
Ca2+ independent in monocytes and macrophages
(11, 12). Ca2+ depletion did not
affect the extent or rate of IgG-mediated phagocytosis by RAW 264.7
cells (Fig. 2), indicating that this is
an appropriate model for studying Ca-independent phagocytic signaling.
The cells were Ca2+ depleted by incubation in
Mg/EGTA buffer (45 min, 37°C). We have previously shown that this
treatment virtually eliminates free intracellular calcium concentration
([Ca2+]i) in monocytes as
determined by fura-2 fluorescence in cells stimulated with
IgG-opsonized particles (12). Similar results were
obtained with RAW 264.7 cells stimulated with either
platelet-activating factor or immune complexes (data not shown).
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and ßI, but not PKC or
Because Ca2+ depletion did not alter
phagocytosis, we examined its affect on PKC translocation. We tested
the hypothesis that Ca2+ depletion would inhibit
membrane localization of PKCs and ßI in response to PMA, a potent
stimulus of PKC translocation. The baseline distribution of PKC
isoforms was the same in +Ca and -Ca cells, indicating that
Ca2+ depletion alone did not alter PKC location
(data not shown). PKCs , ßI, , and are primarily cytosolic
in unstimulated cells (Fig. 3). Although
the intensity of the bands in the cytosol and membrane fractions for
ßI appear similar, the differences in loading must be taken into
account. As 25% of the membrane fraction vs 6% of the cytosol was
loaded, the majority of PKC ßI, like the other isoforms, is
cytosolic.
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(Fig. 3
, lane 1 vs 4) and a
substantial increase in the particulate (membrane + insoluble)
fractions (>12-fold; lanes 2 and 3 vs
lanes 5 and 6). In contrast,
Ca2+ depletion prevented the translocation of PKC
to membrane and insoluble fractions in response to PMA, resulting
in a PKC distribution similar to that of resting cells (Fig. 3, lanes 1–3 vs 7–9). PMA stimulated PKC ßI
translocation to the insoluble fraction in the presence, but not the
absence, of Ca2+. In the presence of
Ca2+, PKC ßI in the cytosol and membrane
fractions decreased by 9-fold (Fig. 3, lanes 1 and
2 vs 4 and 5) and the insoluble
fraction increased >40-fold (lane 3 vs
6). In the absence of Ca2+, the
distribution of PKC ßI was virtually identical in PMA-treated and
control cells (Fig. 3, lanes 1–3 vs 7–9). This
confirms that the cells were indeed Ca depleted, and that Ca depletion
inhibited the PMA-induced translocation of cPKCs and ßI.
In contrast, Ca2+ depletion did not block the
translocation of nPKC isoforms to the particulate (membrane +
insoluble) fractions. PMA treatment decreased cytosolic PKC
(2-fold) and increased its levels in the particulate fractions
(3-fold), as shown in Fig. 3. This pattern occurred in both
Ca2+-containing and
Ca2+-depleted cells (Fig. 3, lanes
4–6 are similar to lanes 7–9). In cells treated with
PMA, PKC was decreased in the cytosol (>3-fold) and increased in
the particulate fractions (3- to 4-fold). Similar translocation
occurred in Ca2+-containing and
Ca2+-depleted cells (Fig. 3, lanes
4–6 are similar to lanes 7–9). The translocation of
nPKCs and in Ca2+-depleted cells was
predicted, as these isoforms lack the functional
Ca2+ binding domain found in cPKC isoforms
(7).
Phagocytosis requires a Ca2+-independent, DAG-sensitive PKC
Phagocytosis in human monocytes is increased by PMA or DAG,
consistent with a role for PKC activation in phagocytosis (5, 8). Likewise, phagocytosis in RAW 264.7 cells was also PMA/DAG
sensitive, increasing >60% when the cells were treated with either of
these PKC activators (Fig. 4).
Conversely, treatment with the PKC inhibitors calphostin C or
staurosporine decreased phagocytosis in a dose-dependent fashion (Fig. 5, A and B). Thus,
similar to our results in primary monocytes and MonoMac 6 cells
(5, 6), phagocytosis in RAW 264.7 cells requires a PKC
that is both Ca2+ independent and PMA/DAG
sensitive, matching the characteristics of the nPKC isoforms.
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Unlike particle uptake via complement receptors or endocytosis,
IgG-mediated phagocytosis is accompanied by a respiratory burst
(1, 21), which requires translocation and assembly of
NADPH oxidase components at the membrane, and results in production of
O2
and
H2O2 (18, 22).
NADPH oxidase activation has been shown to require an increase in
intracellular Ca2+ and PKC activity (22, 23). As predicted, calphostin C and staurosporine decreased
H2O2 production by RAW
264.7 cells >75% at doses similar to those that affect phagocytosis
(Fig. 5, A and B). Additionally, depletion of
intracellular Ca2+ abolished
H2O2 production (Fig. 6). As PKCs and ßI did not
translocate to the membrane in Ca2+-depleted
cells (Fig. 3), these results are consistent with the hypothesis that
cPKC activation is required for IgG-mediated respiratory burst in RAW
264.7 cells.
This hypothesis predicts that the cPKC selective inhibitors CGP 41251
and Gö 6976 will block the IgG-stimulated burst, but not affect
phagocytosis. Fig. 7 (A and
B) demonstrates that CGP 41251 and Gö 6976 caused a
dose-dependent decrease in H2O2 production, but not
phagocytosis. Interestingly, the nonselective PKC inhibitor GF109203X
also blocked respiratory burst but not phagocytosis (Fig. 7C). These results are further evidence for a role for cPKC
and/or ßI in the respiratory burst in RAW 264.7 cells.
Phagocytosis was not affected, implying that cPKCs are not involved in
FcR-mediated ingestion and that these drugs are not otherwise
interfering with FcR signaling.
nPKCs translocate to the membrane fraction during phagosome formation
cPKC and nPKC isoforms associate with membranes by binding DAG and
PS (7). We reasoned that those PKC isoforms involved in
FcR-initiated signaling would translocate to the membrane fraction
during IgG-mediated phagocytosis, so we measured membrane-bound cPKC
, cPKC ßI, nPKC , and nPKC during synchronized phagocytosis
of IgG-opsonized glass beads. Time course studies showed that
phagocytosis took at least 7.5 min (Fig. 8A). Few completely
internalized targets were observed before 7.5 min. Internalized targets
were present in most cells at 7.5 min, and phagosomes continued to form
and close by the 10- and 15-min time points. Therefore, fractions were
prepared from cells at time points between 0 and 15 min to study
translocation of PKC isoforms during phagosome formation (0–5 min) and
formation/closure (7.5–15 min).
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B shows Western blots of PKC isoforms in the membrane
fractions from +Ca and -Ca cells. In +Ca cells, PKCs and ßI were
present in the membrane at all time points. In contrast, PKCs and
ßI were not detectable in the membrane fractions in cells depleted of
intracellular Ca2+, although they could be
readily detected in mouse brain lysate included as a positive control
on the blots. As phagocytosis proceeded normally in
Ca2+-depleted cells, these results indicate that
membrane localization of PKCs and ßI is not required for
phagocytosis. In the +Ca experiments, PKC ßI did not increase in the
membranes during phagocytosis. PKC always increased in the +Ca
membrane during phagocytosis, but the magnitude of the increase at the
specific time points was variable. To test for an effect of
phagocytosis on PKC distribution, ANOVA indicated that levels of
membrane-bound PKC increased during phagocytosis
(p < 0.05).
In contrast to PKCs and ßI, membrane-bound PKCs and were
present during phagocytosis in both +Ca and -Ca cells (Fig. 8B). ANOVA indicated that phagocytosis increased PKCs
and in the membrane in both +Ca and -Ca cells
(p < 0.05). To examine the pattern of nPKC
translocation during phagosome formation, the levels of membrane PKCs
and in Ca2+-depleted cells were
quantified (Fig. 8C). PKC increased gradually and was
maximally elevated after 7.5 min, corresponding to the appearance of
closed phagosomes. PKC was significantly increased in the membrane
fraction by 5 min, suggesting that this isoform translocates during the
formation of phagosomes.
Although the amount of membrane-bound PKC increased during phagocytosis, the cytosolic levels did not change (data not shown). Unlike PKC activation during PMA stimulation, the amount of PKC associated with the membranes during phagocytosis was small compared with that in the cytosol. This is consistent with phagocytosis being a localized membrane event, with a relatively small proportion of the PKC mass targeted to membrane sites involved in phagocytosis.
GFP PKCs and translocate to the phagosome membrane
The detection of PKCs and in membrane fractions during
phagocytosis is consistent with their localization to phagosomes. To
identify the membranes to which these isoforms move, we transfected
GPF-conjugated PKCs and into RAW 264.7 cells and examined their
location during phagocytosis. In the presence of
Ca2+, GFP PKC concentrated in membrane
regions associated with targets at 2.5 and 7.5 min, but at 15 min, when
phagosome closure was complete, little localization remains (Fig. 9, a–f). In contrast, GFP PKC
was cytosolic at all times in Ca2+-depleted
cells; no concentration of the GFP signal was detected (Fig. 9, g–l). Analysis of topographic images confirmed the lack of
GFP PKC concentration in Ca2+-depleted cells
(data not shown).
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also translocated to forming phagosomes (Fig. 9, m–r). The localization of GFP PKC was similar in the
presence (data not shown) and absence (Fig. 9, m–r) of
Ca2+. That is, GFP PKC localized to targets
at early time points (2.5 min, 7.5 min; Fig. 9, m,
n, p, and q) but this localization was
largely lost by 15 min. These images demonstrate that PKC is
associated with the phagocytic cup. The results confirm the biochemical
data (Fig. 8) demonstrating translocation of PKC to cell membranes
during phagocytosis in Ca2+-depleted cells. The
confocal data extend these findings by localizing the PKC to the
target-associated membranes, consistent with our hypothesis that nPKC
is involved in IgG-mediated phagocytosis. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R) results in particle
internalization, but unlike the phagocytosis of
complement-opsonized particles or apoptotic cells, FcR ligation
induces macrophages to produce a respiratory burst (1, 21, 24). In monocyte/macrophages, phagocytosis and respiratory burst
are increased by PMA and reduced by PKC inhibitors, suggesting a
requirement for PKC (5, 6, 8, 15, 18, 25).
Different PKC isoforms are likely to be involved in the multiple
signaling pathways from FcR, and research has focused on identifying
required isoforms to study their functions in these pathways. Increased
Ca2+-dependent PKC activity has been reported
during IgG-mediated phagocytosis in monocytes (8). When
monocytes were stimulated by cross-linking FcR,
Ca2+-dependent PKC activity increased and both
cPKC ß and nPKC translocated to the membrane fraction
(9). Importantly, PKC has been observed in nascent and
fully formed phagosomes by immunostaining (10). Although
these reports provide evidence that cPKCs are activated upon FcR
ligation, they are not consistent with repeated observations that
phagocytosis proceeds at exceedingly low
[Ca2+]i in
monocyte/macrophages and related cell lines (6, 11, 12).
The [Ca2+]i measured in
these studies (2 nM) is well below that required for cPKC isoforms to
bind to membranes (0.8–1 µM) or to be fully activated (1.5–38 µM)
(26). Thus, although cPKCs may be activated during
phagocytosis, they are apparently not required for particle
ingestion.
Phagocytosis in RAW 264.7 cells was Ca2+
independent (Fig. 2), increased by PMA and DAG (Fig. 4), and decreased
by staurosporine and calphostin C (Fig. 5), indicating that PKC is
critical for phagocytosis in these cells. As calphostin C acts as a
competitive inhibitor for the DAG binding site on cPKC and nPKC
isoforms (27, 28), inhibition of phagocytosis by
calphostin C is further evidence that a DAG-requiring PKC isoform is
involved. Thus, phagocytosis responded to conditions consistent with
activation of a DAG-dependent, Ca2+-independent
nPKC isoform.
We hypothesized that PKC activated during phagocytosis would
translocate to, and be detected in, the membrane fraction. In this
study, PKC increased in the membrane fraction during phagocytosis,
consistent with its activation and previous reports documenting PKC
and Ca2+-dependent PKC activity in phagosomes
(8, 9, 10). However, Ca2+ depletion,
which did not inhibit phagocytosis, completely prevented membrane
association of PKC and ßI (Figs. 3, 8B, and 9). Both
PKCs and increased in the membrane fraction with a time course
consistent with their participation in phagocytosis. PKC was
present at time points corresponding to the presence of fully formed
phagosomes. Importantly, membrane-associated PKC increased before
and during particle internalization. This agrees with previous reports
that PKC activity increases after FcR ligation but before phagosome
closure (8, 9) and that membrane-associated PKC is
increased by 2–5 min after FcR cross-linking in monocytes
(9). Thus participation of nPKC and/or in
phagocytic signaling is supported by their rapid translocation to
membranes upon initiation of IgG-mediated phagocytosis as well as by
their activation characteristics.
PKC activity has been implicated in regulation of respiratory burst
(14, 25, 34, 35), with cPKC isoforms identified as the
primary mediators (13, 14, 17, 23). In our studies, CGP
41251 and Gö 6976 inhibited respiratory burst and not
phagocytosis, thus separating these pathways at the level of PKC
activation (Fig. 7, A and B). Because PKC was
the only cPKC that translocated to membranes during phagocytosis, this
suggests that respiratory burst is mediated by PKC in RAW 264.7
cells, consistent with results in primary human monocytes
(17). A recent report that overexpression of a
dominant-negative PKC in RAW 264.7 cells decreased their ability to
kill the intracellular pathogen Leishmania donovani, but had
no effect on their phagocytosis (36), provides additional
evidence that PKC is activated, but not required, during
phagocytosis.
Our results that the cPKC inhibitor Gö 6976 reduced, but did not
eliminate, respiratory burst in neutrophils are in agreement with those
reported by Pongracz and Lord (13). In their studies, the
magnitude of the reduction (50–60%) was similar to that seen in our
experiments (Fig. 7) using the same cPKC inhibitors. Similarly, the
down-regulation of PKC ß in differentiated HL-60 cells using
antisense oligonucleotides produced a partial inhibition of superoxide
release (14). In contrast, nonselective PKC inhibitors
(Fig. 5 and 7C) gave a much more complete inhibition of
respiratory burst. Although the absence of a respiratory burst in the
Ca2+-depleted cells may seem to contradict the
partial inhibition of respiratory burst achieved with pharmacological
inhibition of cPKC, the lack of respiratory burst in the
Ca2+-depleted cells may also be due to the
inhibition of other Ca2+-dependent enzymes,
notably cPLA2, also required for superoxide
production (37). Taken together, these data suggest that
the respiratory burst is regulated by multiple signaling pathways, with
part of the burst mediated by cPKC and part by other PKC isoforms.
GF109203X is used as a nonisoform-selective inhibitor of PKC (38, 39). Unexpectedly, GF109203X inhibited respiratory burst but not
phagocytosis in the same manner as the cPKC-selective drugs (Fig. 7).
There are several possible explanations for this discrepancy. One is
that the concentrations of GF109203X used in our study were sufficient
to inhibit the cPKC but not the nPKC isoforms. The
IC50 values given for GF109203X vary with the
different isoforms and are lower for PKC than for PKC or
(39). However, our dose curve (1–10 µM) is at and above
the levels previously shown to effect PKC activity and function in vivo
(38, 39, 40, 41), so this interpretation seems unlikely.
Alternatively, phagocytosis may require PKC translocation but not
kinase activity. GF109203X is a competitive inhibitor of the PKC
ATP-binding site (38, 39), and as such should not inhibit
the translocation of the PKC isoforms. Calphostin C inhibits the DAG
binding site and translocation of PKC (27) and
presumably other PKC isoforms. This suggests that the translocation and
presence of the PKC enzyme, but not its kinase activity, may be
required during phagocytic signaling. Precedence for such a mechanism
comes from studies on the regulation of neurite outgrowth by PKC .
Zeidman and coworkers have shown that the catalytic domain of PKC
is not required to induce neurite outgrowth, and the effect cannot be
inhibited by GF109203X, indicating that kinase activity is not required
(42). In a similar manner, PKC has been shown to
activate phospholipase D even in the absence of its kinase activity
(43). Our results are consistent with the hypothesis that
the role of PKC in phagocytosis is independent of its kinase
activity.
In conclusion, we have separated the signaling pathways for
IgG-mediated phagocytosis and respiratory burst at the level of PKC
activation. We have demonstrated that cPKC inhibition or
Ca2+ depletion inhibited respiratory burst but
not phagocytosis. IgG-mediated phagocytosis was
Ca2+ independent and DAG sensitive, implying
activation of the nPKC isoforms. Both nPKCs and increased in
cell membranes during phagocytosis with a time course consistent with
particle ingestion. Our results suggest that PKCs and
translocate and are likely candidates for signal transduction during
IgG-mediated phagocytosis in RAW 264.7 cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michelle R. Lennartz, Center for Cell Biology and Cancer Research, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208.
3 Abbreviations used in this paper: PKC, protein kinase C; DAG, diacylglycerol; GFP, green fluorescent protein; [Ca2+]i, intracellular calcium concentration; PS, phosphatidylserine; nPKC, novel PKC; cPKC, classic PKC; Mg/EGTA, 2 mM MgCl2 and 1 mM EGTA in HBSS; BIgG, IgG opsonized glass beads.
Received for publication September 13, 1999. Accepted for publication June 13, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
receptor-mediated intracellular killing of Staphylococcus aureus by human monocytes. J. Immunol. 155:776.[Abstract]
receptor-mediated superoxide generation in macrophages. J. Immunol. 138:4353.[Abstract]
regulates human monocyte O2- production and low density lipoprotein lipid oxidation. J. Biol. Chem. 274:3764.- and - subspecies of protein kinase C. J. Cell Biol. 143:511. mediates phorbol ester inhibition of calphostin C- and tumor necrosis factor--induced apoptosis in U937 histiocytic lymphoma cells. J. Biol. Chem. 273:24115. chain of the high affinity receptor for immunoglobulin E by receptor-associated protein kinase C-. J. Biol. Chem. 269:23102., novel PKC- and PKC-, but not atypical PKC- are MARCKS kinases in intact NIH 3T3 fibroblasts. J. Biol. Chem. 272:4072.. Exp. Cell Res. 29:327.
and filamentous actin. J. Biol. Chem. 273:26790. in the control of infection by intracellular pathogens in macrophages. J. Immunol. 163:5505.-protein kinase C enhances nerve growth factor-induced phosphorylation of mitogen-activated protein kinases and neurite outgrowth. J. Biol. Chem. 270:30134. and IL-1ß production. Shock 9:256.[Medline]
, via its regulatory domain and independently of its catalytic domain, induces neurite-like processes in neuroblastoma cells. J. Cell Biol. 145:713.This article has been cited by other articles:
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|
  B. Diouf, A. Collazos, G. Labesse, F. Macari, A. Choquet, P. Clair, C. Gauthier-Rouviere, N. C. Guerineau, P. Jay, F. Hollande, et al. A 20-Amino Acid Module of Protein Kinase C{epsilon} Involved in Translocation and Selective Targeting at Cell-Cell Contacts J. Biol. Chem., July 10, 2009; 284(28): 18808 - 18815. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Dai, M. Jayapal, H. K. Tay, R. Reghunathan, G. Lin, C. T. Too, Y. T. Lim, S. H. Chan, D. M. Kemeny, R. A. Floto, et al. Differential signal transduction, membrane trafficking, and immune effector functions mediated by Fc{gamma}RI versus Fc{gamma}RIIa Blood, July 9, 2009; 114(2): 318 - 327. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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|
|
|
|
|
|
N. K. Basu, L. Kole, M. Basu, K. Chakraborty, P. S. Mitra, and I. S. Owens The Major Chemical-detoxifying System of UDP-glucuronosyltransferases Requires Regulated Phosphorylation Supported by Protein Kinase C J. Biol. Chem., August 22, 2008; 283(34): 23048 - 23061. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
D. Yadav and N. Sarvetnick B7-2 Regulates Survival, Phenotype, and Function of APCs J. Immunol., May 15, 2007; 178(10): 6236 - 6241. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
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|
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|
|
|
|
|
|
M. Yadav, L. Clark, and J. S. Schorey Macrophage's Proinflammatory Response to a Mycobacterial Infection Is Dependent on Sphingosine Kinase-Mediated Activation of Phosphatidylinositol Phospholipase C, Protein Kinase C, ERK1/2, and Phosphatidylinositol 3-Kinase J. Immunol., May 1, 2006; 176(9): 5494 - 5503. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
K. Reville, J. K. Crean, S. Vivers, I. Dransfield, and C. Godson Lipoxin A4 Redistributes Myosin IIA and Cdc42 in Macrophages: Implications for Phagocytosis of Apoptotic Leukocytes J. Immunol., February 1, 2006; 176(3): 1878 - 1888. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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|
|
|
|
|
|
A. von Knethen, A. Tautenhahn, H. Link, D. Lindemann, and B. Brune Activation-Induced Depletion of Protein Kinase C{alpha} Provokes Desensitization of Monocytes/Macrophages in Sepsis J. Immunol., April 15, 2005; 174(8): 4960 - 4965. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Swanson and A. D. Hoppe The coordination of signaling during Fc receptor-mediated phagocytosis J. Leukoc. Biol., December 1, 2004; 76(6): 1093 - 1103. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ueyama, M. R. Lennartz, Y. Noda, T. Kobayashi, Y. Shirai, K. Rikitake, T. Yamasaki, S. Hayashi, N. Sakai, H. Seguchi, et al. Superoxide Production at Phagosomal Cup/Phagosome through {beta}I Protein Kinase C during Fc{gamma}R-Mediated Phagocytosis in Microglia J. Immunol., October 1, 2004; 173(7): 4582 - 4589. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
J. Jongstra-Bilen, R. Harrison, and S. Grinstein Fc{gamma}-receptors Induce Mac-1 (CD11b/CD18) Mobilization and Accumulation in the Phagocytic Cup for Optimal Phagocytosis J. Biol. Chem., November 14, 2003; 278(46): 45720 - 45729. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Muro, R. Wiewrodt, A. Thomas, L. Koniaris, S. M. Albelda, V. R. Muzykantov, and M. Koval A novel endocytic pathway induced by clustering endothelial ICAM-1 or PECAM-1 J. Cell Sci., April 15, 2003; 116(8): 1599 - 1609. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. C. Larsen, T. Ueyama, P. M. Brannock, Y. Shirai, N. Saito, C. Larsson, D. Loegering, P. B. Weber, and M. R. Lennartz A role for PKC-{varepsilon} in Fc{gamma}R-mediated phagocytosis by RAW 264.7 cells J. Cell Biol., December 23, 2002; 159(6): 939 - 944. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Garcia-Garcia and C. Rosales Signal transduction during Fc receptor-mediated phagocytosis J. Leukoc. Biol., December 1, 2002; 72(6): 1092 - 1108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Edberg, H. Qin, A. W. Gibson, A. M. F. Yee, P. B. Redecha, Z. K. Indik, A. D. Schreiber, and R. P. Kimberly The CY Domain of the Fcgamma RIa alpha -Chain (CD64) Alters gamma -Chain Tyrosine-based Signaling and Phagocytosis J. Biol. Chem., October 18, 2002; 277(43): 41287 - 41293. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. T. Myers and J. A. Swanson Calcium spikes in activated macrophages during Fc{gamma} receptor-mediated phagocytosis J. Leukoc. Biol., October 1, 2002; 72(4): 677 - 684. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mitchell, G. Thomas, K. Harvey, D. Cottell, K. Reville, G. Berlasconi, N. A. Petasis, L. Erwig, A. J. Rees, J. Savill, et al. Lipoxins, Aspirin-Triggered Epi-Lipoxins, Lipoxin Stable Analogues, and the Resolution of Inflammation: Stimulation of Macrophage Phagocytosis of Apoptotic Neutrophils In Vivo J. Am. Soc. Nephrol., October 1, 2002; 13(10): 2497 - 2507. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Todt, B. Hu, A. Punturieri, J. Sonstein, T. Polak, and J. L. Curtis Activation of Protein Kinase C beta II by the Stereo-specific Phosphatidylserine Receptor Is Required for Phagocytosis of Apoptotic Thymocytes by Resident Murine Tissue Macrophages J. Biol. Chem., September 20, 2002; 277(39): 35906 - 35914. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Garcia-Garcia, R. Rosales, and C. Rosales Phosphatidylinositol 3-kinase and extracellular signal-regulated kinase are recruited for Fc receptor-mediated phagocytosis during monocyte-to-macrophage differentiation J. Leukoc. Biol., July 1, 2002; 72(1): 107 - 114. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Hu, A. Punturieri, J. Todt, J. Sonstein, T. Polak, and J. L. Curtis Recognition and phagocytosis of apoptotic T cells by resident murine tissue macrophages require multiple signal transduction events J. Leukoc. Biol., May 1, 2002; 71(5): 881 - 889. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Christensen, J. T. Myers, and J. A. Swanson pH-dependent regulation of lysosomal calcium in macrophages J. Cell Sci., January 2, 2002; 115(3): 599 - 607. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Castrillo, D. J. Pennington, F. Otto, P. J. Parker, M. J. Owen, and L. Bosca Protein Kinase C{epsilon} Is Required for Macrophage Activation and Defense Against Bacterial Infection J. Exp. Med., October 29, 2001; 194(9): 1231 - 1242. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Garcia-Garcia, G. Sanchez-Mejorada, and C. Rosales Phosphatidylinositol 3-kinase and ERK are required for NF-{kappa}B activation but not for phagocytosis J. Leukoc. Biol., October 1, 2001; 70(4): 649 - 658. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. May and L. Machesky Phagocytosis and the actin cytoskeleton J. Cell Sci., January 3, 2001; 114(6): 1061 - 1077. [Abstract] [PDF]
|
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