Enhanced Phagocytosis through Inhibition of de
Novo Ceramide Synthesis*
Vania
Hinkovska-Galcheva
,
Laurence
Boxer
,
Pamela J.
Mansfield
,
Alan D.
Schreiber§, and
James A.
Shayman¶
From the
Department of Pediatrics, Division
of Hematology Oncology, the ¶ Department of Internal
Medicine, Division of Nephrology, University of Michigan,
Ann Arbor, Michigan 48109 and the § Department of
Medicine, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104
Received for publication, June 21, 2002, and in revised form, October 28, 2002
 |
ABSTRACT |
Fc
receptors are important mediators of
the binding of IgG to and induction of phagocytosis in neutrophils.
COS-1 cells provide a potentially useful model for studying these
receptors because transfection with the Fc
RIIA renders these
cells phagocytic. During Fc
RIIA-mediated phagocytosis in COS-1
cells, endogenous ceramide levels increased 52% by 20 min
(p < 0.01). Phospholipase D activity
increased by 62% (p < 0.01).
Correspondingly, the phagocytic index increased by 3.7-fold
by 20 min. Two inhibitors of ceramide formation were used to assess the
consequences of reduced ceramide generation. L-Cycloserine,
an inhibitor that blocks serine palmitoyltransferase activity, lowered
both sphingosine and ceramide levels. Under these conditions, the
phagocytic index increased 100% in the presence of 2 mM
L-cycloserine. The formation of ceramide resulting from the
N-acylation of dihydrosphingosine or sphingosine by
ceramide synthase is inhibited by the fungal toxin fumonisin
B1. When cells were treated with 5-50 µM
fumonisin B1, the cellular level of ceramide decreased in a
concentration-dependent manner, while simultaneously the
phagocytic index increased by 52%. Concomitantly, three indirect
measures of Fc
RIIA activity were altered with the fall in ceramide
levels. Syk phosphorylation, phospholipase D activity, and
mitogen-activated protein (MAP) kinase phosphorylation were increased
at 30 min. When Syk phosphorylation was blocked with piceatannol and
cells were similarly challenged, phosphatidylinositol 3-kinase
activation was blocked, but no changes in either ceramide accumulation
or MAP kinase activation were observed. Ceramide formation and MAP
kinase activation are therefore not dependent on Syk kinase activity in
this system. These results indicate that COS-1 cells provide a useful
model for the recapitulation of sphingolipid signaling in the study of
phagocytosis. Ceramide formed by de novo synthesis may
represent an important mechanism in the regulation of phagocytosis.
 |
INTRODUCTION |
An important function of
PMN1 and tissue macrophages
is the phagocytosis of IgG-coated cells. Receptors for the
constant region of IgG, the Fc
receptors, enable these cells to
detect and destroy IgG-coated microorganisms during infection and
IgG-coated blood cells in autoimmune disorders. There are three major
classes of Fc
receptors, designated Fc
RI, Fc
RII, and
Fc
RIII. The various isoforms of Fc
Rs have highly conserved
extracellular portions, but their cytoplasmic domains are
heterogeneous. This heterogeneity suggests that all Fc
Rs may not be
involved in phagocytosis. One of the main problems for understanding
the Fc
R requirements for phagocytosis has been that multiple
isoforms are expressed on each type of phagocytic cell. The expression
of Fc
R in a cell line that does not have endogenous Fc
Rs provides
a model for defining the molecular signals associated with
phagocytosis. Fc
receptor-transfected COS-1 established that a
single class of human Fc
receptor can induce phagocytosis of
IgG-sensitized red blood cells (1).
Fc
RIIA, which is restricted to cells of megakaryocytic and myeloid
lines, mediates several functions. Stimulation of Fc
RIIA leads to
antibody-dependent cellular cytotoxicity, superoxide production, phagocytosis in monocytes (2, 3), and platelet activation
(4, 5). Fc
RIIA expression is increased by interferon-
and
granulocyte-macrophage colony-stimulating factor and is decreased by
glucocorticoids (6, 7). Thus COS-1 cells transfected with Fc
RIIA
provide a potentially useful model for the study of phagocytosis.
The tyrosine kinase Syk plays a critical role in the phagocytic pathway
mediated by Fc
receptors. A cytoplasmic amino acid motif, known as
ITAM, is present on Fc
RIIA, Fc
RI/
, and Fc
RIIIA/
and is
essential for phagocytosis. Cross-linking of the Fc receptor induces
binding of Src family protein tyrosine kinase to the ITAM, leading to
an activation of the Src family protein tyrosine kinases and ITAM
tyrosine phosphorylation (8, 9). This serves to recruit Syk, an
SH2-domain-containing tyrosine kinase, which when activated
phosphorylates multiple substrates, including neighboring ITAMs. Syk is
recruited from a cytosolic pool. It is also possible that a small
number of preformed Syk-ITAM complexes exist in resting cells (10).
The induction of Syk phosphorylation by cross-linking of Fc
RII
therefore suggests that Syk plays an important role in phagocytic signaling through Fc
RIIA (8, 11, 12). The essential role for Syk in
phagocytosis signal transduction is supported by the observation that
Syk is a necessary component in ITAM-dependent activation
of actin assembly (13). Down-modulation of Syk expression in monocytes
using Syk antisense oligonucleotides also results in the decreased
ability of these cells to ingest IgG-coated particles (14). When
expressed in COS-1 cells, Syk kinase enhances the phagocytic signal
induced by Fc
RIIA and increases the number of cells able to mediate
phagocytosis (12).
The agonist-stimulated metabolism of membrane lipids produces potent
second messengers that regulate phagocytosis. These not only include
glycerolipids but sphingolipids as well. Sphingolipids are composed of
all lipids carrying a long chain sphingoid amine. Although the role of
sphingolipids in membrane structure and organization has been
appreciated for some time, members of this diverse group of lipids have
recently emerged as a novel class of signaling molecules that also
affect phagocytosis.
We observed previously (15-17) that ceramide is formed in PMN
following ingestion of EIgG and that ceramide plays a negative modulatory role in both phagolysosome formation and oxidant release. Sphingosine is known to inhibit phagocytosis by blocking the action of
protein kinase C
or protein kinase C
as well as inhibiting the
formation of diacylglycerol by phosphatidic phosphohydrolase (18, 19).
Sphingolipids are interconvertible molecules. Thus when using
exogenously added sphingolipids, it is often difficult to ascertain
which metabolite is responsible for a specific biological response (20,
21). Inhibitors of sphingolipid metabolism provide an alternative
method for altering the intracellular concentration of sphingolipids.
In contrast to short chain sphingolipid analogs, these inhibitors alter
the levels of endogenous sphingolipids (22, 23). The first step in
sphingolipid synthesis is the condensation of a fatty acyl-CoA, usually
palmitoyl-CoA, with serine in a reaction catalyzed by serine
palmitoyltransferase (3-ketosphingosine synthase).
L-Cycloserine and
-chloro-L-alanine block
sphingosine and ceramide biosynthesis by inhibiting serine palmitoyltransferase activity both in vitro and in
vivo (24, 25). The next step, the formation of ceramides resulting
from the N-acylation of dihydrosphingosine or sphingosine by
ceramide synthase, is inhibited by the fungal toxin fumonisin
B1. These inhibitors will provide the first step in
determining if de novo synthesis of either sphingosine or
ceramide is required for phagocytosis.
In the present study we report that COS-1 cells transfected with
Fc
IIA receptor replicate many of the events associated with phagocytosis in PMNs. We also observe that phagocytosis is associated with the formation of ceramide through de novo synthesis and
that inhibition of ceramide synthesis increases phagocytosis.
 |
EXPERIMENTAL PROCEDURES |
Materials--
All phospholipids, imidazole, fumonisin
B1, L-cycloserine,
diethylenetriaminepentaacetic acid,
n-octyl-
-D-glucopyranoside, and proteinase
inhibitors pepstatin and leupeptin were obtained from Sigma.
sn-1,2-Diacylglycerol kinase from Escherichia
coli and dithiothreitol were purchased from Calbiochem. Silica Gel 60 thin layer chromatography plates were purchased from VWR (Chicago, IL); [
-32P]ATP was obtained from ICN Pharmaceuticals,
Inc. (Irvine, CA), 1-O-[3H]Octadecyl-sn-glycero-3-phosphocholine,
Western blotting detection reagents, and horseradish
peroxidase-conjugated sheep anti-mouse antibody were from Amersham
Biosciences, and [3H]acetic anhydride was purchased from
American Radiolabeled Chemicals (St. Louis, MO). Polyclonal and
monoclonal antibodies against Syk were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Monoclonal anti-phosphotyrosine
antibody 4G10 and antibody against phosphatidylinositol 3-kinase 85 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY).
Polyclonal antibodies against ERK1 and ERK2, recognizing the
phosphorylated and nonphosphorylated forms of p44 and p42, were
obtained from New England Biolabs (Beverly, MA). Dulbecco's modified
Eagle's medium, trypsin EDTA, L-glutamine, penicillin/streptomycin, and geneticin (G118 sulfate) were from Invitrogen. Sheep erythrocytes were purchased from BioWhittaker (Walkersville, MD) and were opsonized with anti-sheep erythrocyte IgG
(Chapel Organon Teknika, Durham, NC). Polyvinylidene difluoride membranes were from Schleicher & Schuell. Piceatannol
(3,4,3',5'-tetra-hydroxytrans-stilbene) was obtained from Alexis
(San Diego, CA).
Cell Cultures--
COS-1 cells stably transfected with Fc
RIIA
receptor cDNA were maintained in Dulbecco's modified Eagle's
medium containing glucose 4.5 mg/ml, glutamine 25 mg/ml, streptomycin
100 units/ml, penicillin (100 mg/ml), and 10% heat-inactivated fetal
calf serum. After 24 h the medium was replaced with fresh medium
(for control cells) or fresh medium containing inhibitors or agonists.
Sheep Erythrocytes--
Sheep red blood cells were sensitized
with rabbit IgG anti-sheep erythrocyte antibody as described previously
(16). Erythrocytes (109/ml) were incubated for 30 min at
37 °C with anti-sheep erythrocyte IgG (1:350), followed by
incubation on ice for 30 min. Antibody-treated erythrocytes (EIgG) were
washed three times and suspended in the same buffer at 2-3 × 109.
Phagocytosis Assays--
Phagocytosis assays were conducted as
described previously (26). For studies with fumonisin B1 or
cycloserine, cells were incubated 24 or 6 h, respectively, prior
to the experiment. Fumonisin B1 was applied to cultured
cells dissolved in Me2SO, and L-cycloserine was
dissolved in water.
Assay for Ceramide Formation--
Lipids were extracted by the
method of Van Veldhoven and Bell (27). Total cellular ceramide was
assayed by the method of Preiss et al. (28).
Phosphatidic Acid and Phosphatidylethanol Formation--
COS-1
cells were cultured at 1.7 × 106/ml in 10 ml of
Dulbecco's modified Eagle's media and incubated with fumonisin
B1 (24 h) or L-cycloserine (6 h) in the
concentrations shown in the figure legends. Cells were labeled with
1-O-[3H]octadecyl-sn-glycero-3-phosphocholine
(10
8 mol/liter) for 30 min at 37 °C. The labeled cells
were washed with phosphate-buffered saline and incubated in 5 ml of
phosphate-buffered saline containing 1 mmol/liter Ca2+ and
1 mmol/liter Mg2+. Ethanol (200 mmol/liter) was added for 5 min at 37 °C. COS-1 cell phagocytosis was initiated as outlined
above. Thirty minutes after the addition of opsonized targets, the
cells were harvested with trypsin-EDTA; the EIgG not internalized were
lysed, and COS-1 cells resuspended in phosphate-buffered saline. The
lipids were extracted, and 3H-labeled phosphatidylethanol
and phosphatidic acid were assayed as described previously (29).
Assay for Sphingosine Formation--
Sphingosine was quantitated
by acetylation with [3H]acetic anhydride to form
C2-[3H]ceramide as described by Yatomi
et al. (30). Samples were spotted on high performance thin
layer chromatography plates and developed in a
chloroform/methanol/acetic acid (92:2:8, v/v) solvent. Sphingosine
levels were calculated by interpolation from sphingosine standards that
were run through the same procedure.
Immunoprecipitation--
For the Syk phosphorylation studies,
COS-1 cells were lysed in buffer containing 1% Triton X-100 along with
50 mM Tris (pH 8.0), 100 mM NaCl, 1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml soybean trypsin inhibitor,
and 1 µg/ml each of leupeptin and aprotinin. Lysates were precleared
with protein A-Sepharose for 30 min and incubated overnight
at 4 °C with anti-Syk antibody. Protein A-Sepharose was added to
each sample and incubated for 2 h with rotation at 4 °C. The
beads were washed briefly three times with lysis buffer and twice with
buffer containing 20 mM Tris (pH 7.5), 150 mM
NaCl, and 1 mM Na3VO4. Absorbed
proteins were solubilized in sample buffer and separated on 10%
SDS-PAGE minigels. Proteins were transferred to PVDF for 2 h at
100 V and immunoblotted with 4G10 antiphosphotyrosine antibody. The
membrane was washed three times with 0.2% Tween 20 in 50 mM Tris (pH 8.0) and 100 mM NaCl and then
incubated with a secondary antibody (horseradish peroxidase-conjugated)
in wash buffer containing 5% nonfat dry milk. Phosphorylated bands
were visualized by using ECL. The PVDF membranes were stripped with 100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris
(pH 6.5) at 50 °C and reprobed with the appropriate antibody to
demonstrate equivalent amounts of immunoprecipitated protein.
Immunoprecipitations for the measurement of phosphatidylinositol 3-kinase activity were performed as follows. Lysates were precleared with protein A-Sepharose and incubated with anti-phosphatidylinositol 3-kinase p85 subunit antibody overnight. The kinase activity was measured using phosphatidylinositol and 5 µCi per sample of
[
-32P]ATP as substrates.
MAP kinase phosphorylation experiments were performed as follows. The
COS-1 cells were cultured in medium containing 0.25% fetal bovine
serum to reduce the basal levels of phosphorylation. One hour prior to
phagocytosis, the media were aspirated, and the cells were maintained
in phosphate-buffered saline containing 1 mM calcium
chloride and 1 mM magnesium chloride followed by the
addition of EIgG. COS-1 cells were lysed in 50 µl of lysis buffer.
The cleared lysates were combined with sample buffer, boiled for 5 min,
and run on 10% SDS-PAGE minigels. The proteins were transferred to
PVDF membranes. The membranes were probed with antibody against
phosphorylated p44/42 in blocking buffer, washed three times, and then
incubated with a secondary antibody consisting of horseradish
peroxidase-conjugated goat anti-rabbit antibody in wash buffer
containing 5% nonfat dry milk.
 |
RESULTS |
In the present study the role of ceramide in the phagocytic
response of COS-1 cells transfected with Fc
RIIA was investigated. It
was first determined whether the COS-1 cell model recapitulates the
changes in ceramide content observed in PMNs. In the PMN ceramide acts
as a negative regulator of phagocytosis (16). COS-1 cells transfected
with Fc
RIIA were challenged with EIgG. The ceramide mass and
phagocytic index were measured (Fig. 1).
In the absence of phagocytic targets ceramide formation did not occur,
but following phagocytosis, ceramide levels increased significantly in
a time-dependent manner. When COS-1 cells were challenged
with EIgG (1:200) endogenous ceramide levels increased 52% over
control levels by 20 min (p < 0.01) and remained
elevated at 30 min. Correspondingly, the phagocytic index increased by
3.7-fold by 20 min.

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Fig. 1.
Ceramide formation and phagocytic index
during COS-1 cell phagocytosis. COS-1 cells (1.7 × 106/ml) transfected with the Fc IIA receptor were
incubated with EIgG (108/ml) at 37 °C for the indicated
times. EIgG not internalized were lysed, and COS-1 cells were
resuspended in phosphate-buffered saline. Ceramide was extracted and
measured as described under "Experimental Procedures." Values
represent the mean ± S.D., n = 5. **,
p < 0.01; ***, p < 0.001 compared with the
zero time point.
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Phospholipase D activation is a principal signal in PMN activation and
has been linked to the uptake of complement and EIgG-opsonized particles. Furthermore, phospholipase D has been identified as an
intracellular target of ceramide action (15). Phospholipase D activity
was measured as the formation of phosphatidylethanol. A
time-dependent increase in phosphatidylethanol was observed in EIgG-stimulated COS-1 cells (62% at 30 min, Fig.
2). A corresponding increase in the
phagocytic index was also observed under these conditions suggesting
that the presence of ethanol did not impair phagocytic activity (Fig.
2).

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Fig. 2.
Phosphatidylethanol formation and phagocytic
index during EIgG-induced phagocytosis. COS-1 cells were labeled
with
1-O-[3H]octadecyl-sn-glycero-3-phosphocholine.
At the indicated time points cells were removed with trypsin; EIgG not
internalized were removed by lysis, and the lipids were extracted and
analyzed as described under "Experimental Procedures." Values
represent the mean ± S.D. for n = 6 experiments.
*, p < 0.05; **, p < 0.01; ***,
p < 0.001, significantly different from zero time
point.
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|
The formation of ceramide resulting from the
N-acylation of dihydrosphingosine or sphingosine by ceramide
synthase is inhibited by the fungal toxin fumonisin B1. The
cellular level of ceramide decreased in a
concentration-dependent manner to 28% of control levels in
cells treated with 5-50 µM fumonisin B1
for 24 h (Fig. 3A). A parallel decrement in
ceramide levels was observed in fumonisin B1-treated COS-1
cells with EIgG exposure. Simultaneously the phagocytic index increased
by 52% at 30 min (Fig. 3B). The dose dependence of ceramide
depletion during treatment of cells with fumonisin B1
closely paralleled the phagocytic index. These results, therefore,
suggest an inverse relationship between ceramide formation and
phagocytosis.

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Fig. 3.
A and B, effect of fumonisin
B1 on ceramide depletion and phagocytic index in COS-1
cells. COS-1 cells were treated with different concentrations of
fumonisin B1. At the indicated time points, COS-1 cells
were removed with trypsin; EIgG not internalized were removed by lysis,
and the lipids were extracted and analyzed as described under
"Experimental Procedures." Values represent the mean ± S.D.
of 6 experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 significantly different from
zero time point.
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Phospholipase D activity was measured during EIgG-stimulated
phagocytosis to assess the consequences of reduced ceramide generation by fumonisin B1. During fumonisin B1 treatment
of COS-1 cells, phospholipase D activity increased further by 62%
(p < 0.02) (Fig. 4A). EIgG red blood cell
stimulated ceramide levels were significantly decreased with fumonisin
B1 treatment (Fig. 4B). Both the decrease in
ceramide content and increase in phospholipase D activity correlated with increased phagocytosis as measured by the phagocytic index. The
inclusion of ethanol in these experiments had no significant effect on
the phagocytic index (Fig. 4C).

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Fig. 4.
A-C, effect of fumonisin on
phosphatidylethanol and ceramide formation and phagocytic index during
EIgG-induced phagocytosis. COS-1 cells were labeled with
1-O-[3H]octadecyl-sn-glycero-3-phosphorylcholine
for 1 h followed by 20 min of incubation with EIgG
(108/ml) at 37 °C. The cells were collected; EIgG not
internalized were removed by lysis, and the lipids were extracted and
analyzed as described under "Experimental Procedures." Values
represent the mean ± S.D., n = 4. *,
p < 0.05; ***, p < 0.001;
****, p < 0.0001.
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The activation of MAP kinase has been demonstrated previously (31) to
be an early event preceding phagolysosome formation in PMNs. Ceramide
also inhibits MAP kinase activation and phosphorylation in the PMN.
When COS-1 cells were exposed to EIgG, a time-dependent increase in the phosphorylation of both p42 and p44 MAP kinase was
observed (Fig. 5). Preincubation with
fumonisin B1 resulted in an increase in MAP kinase
phosphorylation in concert with an increase in the phagocytic index.
The enhancement of MAP kinase activity by fumonisin B1 was
concentration-dependent with a maximal change observed at
35 µM.

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Fig. 5.
Effect of fumonisin B1 on MAP
kinase activation in COS-1 cells during EIgG-stimulated
phagocytosis. COS-1 cells (3.2 × 106/dish) were
cultured in medium containing 0.25% fetal bovine serum. One hour
before phagocytosis, the medium was aspirated, and the cells were
maintained in phosphate-buffered saline followed by the addition of
EIgG (1 × 108/ml) for 30 min at 37 °C.
Unstimulated control cells were incubated in parallel for equal times
with no addition or in the presence of 10 or 25 µM
fumonisin B1. In the upper panel the membranes
were probed with anti-MAP kinase antibody recognizing the
phosphorylated forms of ERK1 and ERK2. In the lower panel,
membranes were stripped and reprobed with an antibody that recognizes
the nonphosphorylated forms of ERK1 and ERK2.
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The tyrosine phosphorylation of Syk kinase is activated by the ligation
of Fc
R and has also been implicated in the regulation of
phagocytosis. To evaluate whether enhanced Syk phosphorylation occurs
during ingestion of EIgG in COS-1 cells after fumonisin B1
treatment, anti-phosphotyrosine immunoblotting was performed on lysates
of EigG-stimulated COS-1 cells. Syk phosphorylation increased 4.9-fold
with increased concentrations of fumonisin B1 and increased
phagocytosis (Fig. 6). There was no
tyrosine phosphorylation of Syk when cells were treated with fumonisin B1 alone and not challenged with EIgG.

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Fig. 6.
Effect of fumonisin B1 on Syk
phosphorylation in COS-1 cells transfected with
Fc IIA receptor. The COS-1 cells (3.2 × 106/dish) were treated with different concentrations of
fumonisin B1 for 24 h and then were activated with
EIgG, and at 30 min phagocytosis was terminated. The samples were
immunoprecipitated with anti-Syk antibody and were run on 10% SDS-PAGE
followed by protein transfer to PVDF membranes. Tyrosine
phosphorylation was detected by Western blotting. The blot was
re-probed with anti-Syk antibody to compare protein loading.
1st to 4th lanes indicate cells
treated without EIgG stimulation; 5th to 8th
lanes indicate cells with EIgG.
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The first step in sphingolipid synthesis is the condensation of
palmitoyl-CoA and serine in a reaction catalyzed by serine palmitoyltransferase. L-Cycloserine blocks sphingosine and
ceramide biosynthesis by inhibiting serine
palmitoyltransferase activity. Cells treated for 6 h with 0.25 to
2 mM L-cycloserine showed a decline in
sphingosine and ceramide levels (Fig. 7).
In the presence of L-cycloserine, cellular levels of
sphingosine decreased by almost 70%. The depletion of ceramide levels
by 50% was also observed. Simultaneously, the phagocytic index
increased more than 100%. The peak effect on phagocytosis was observed
when ceramide and not sphingosine was maximally lowered.

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Fig. 7.
A and B, effect of
L-cycloserine on ceramide and sphingosine formation and
phagocytic index during EIgG-induced phagocytosis. COS-1 cells were
treated for 6 h with L-cycloserine and then EIgG were
added for 30 min at 37 °C. At the indicated time points cells were
collected, and EIgG that were not internalized were removed by lysis.
Ceramide (squares) and sphingosine (triangles)
were determined as described under "Experimental Procedures."
Values represent the mean ± S.D., n = 3. *,
p < 0.05; **, p < 0.01; ***,
p < 0.001; ****, p < 0.0001, significantly
different from zero time point.
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When cells were treated with fumonisin B1, a significant
decrease in ceramide was observed, but no significant change in
sphingosine levels was seen. The effects of exogenously added
sphingosine on the reconstitution of ceramide and the impact on
phagocytosis and tyrosine phosphorylation of Syk were evaluated. In
COS-1 cells treated with fumonisin B1 alone (25-50
µM) ceramide levels declined and the phagocytic index
increased as observed previously. Treatment with sphingosine (0.5, 1, and 5 µM) resulted in increased ceramide levels and a
decrease in the phagocytic index. However, the effect of sphingosine
was blocked by the concomitant addition of fumonisin B1
(Fig. 8A). In samples that
were immunoprecipitated with anti-Syk antibody and run on 10% SDS-PAGE
followed by protein transfer to PVDF membranes, the protein
phosphorylation detected by Western blot showed decreased Syk
phosphorylation with increased ceramide levels (Fig. 8B).
These data are consistent with the interpretation that ceramide and not
sphingosine is primarily responsible for the inhibition of
phagocytosis.

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Fig. 8.
A, effect of fumonisin and sphingosine
treatment on ceramide content and phagocytic index of COS-1 cells
stably transfected with Fc RIIA. Cells were treated with fumonisin
B1 (25 or 50 µM) and then sphingosine (0.5, 1, and 5 µM) for 30 min. COS-1 cells were removed with
trypsin; EIgG not internalized were removed by lysis. Samples were
extracted, and ceramide recovery was analyzed as described under
"Experimental Procedures." Immunoprecipitation was then performed
(see Fig. 9). Values represent the mean ± S.D. for 3 experiments.
**, p < 0.01; ***, p < 0.001. B, effect of fumonisin B1 and sphingosine on Syk
phosphorylation in FC IIA receptor-transfected COS-1 cells. The COS-1
cells (3.2 × 106/dish) were treated with different
concentrations of fumonisin B1 for 24 h. Cells were
then incubated with sphingosine at the indicated concentrations for 30 min. Cells were then activated with EIgG for 30 min. The samples were
immunoprecipitated with anti-Syk antibody and were run on 10% SDS-PAGE
followed by protein transfer to PVDF membranes. Tyrosine
phosphorylation was detected by Western blot. The blot was reprobed
with anti-Syk antibody to show equal loading.
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COS-1 cells were incubated with N-acetylsphingosine
(C2-ceramide), N-acetyldihydrosphingosine, and
sphingosine to evaluate further the role of ceramide in Syk
phosphorylation. Syk phosphorylation decreased with increased
C2-ceramide and sphingosine concentrations, whereas
C2-dihydroceramide had no impact on tyrosine
phosphorylation (Fig. 9).

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Fig. 9.
Effect of C2-ceramide,
C2-dihydroceramide, and sphingosine on Syk phosphorylation
in COS-1 cells transfected with FC IIA.
The COS-1 cells (3.2 × 106/dish) were treated with
N-acetylsphingosine (10 and 20 µM),
N-acetyldihydrosphingosine (10 µM), and
sphingosine (10 and 20 µM) for 30 min, and then
phagocytosis proceeded for 30 min. The samples were immunoprecipitated
with anti-Syk antibody and were run on 10% SDS-PAGE followed by
protein transfer to PVDF membranes. Tyrosine phosphorylation was
detected by Western blot. The blot was reprobed with anti-Syk antibody
to show equal loading.
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In order to ascertain the potential role of Syk kinase in phagocytosis,
ceramide formation, and MAP kinase activation, a selective inhibitor of
Syk kinase was employed. Preincubation of cells with piceatannol (100 µM) for 30 min resulted in a marked inhibition of both
Syk phosphorylation (Fig.
10A) and phagocytosis (Fig.
10B) in response to EIgG. Cells were next incubated with or
without piceatannol (100 µM) for 30 min and then with
EIgG for different times. In the absence of piceatannol,
phosphatidylinositol 3-kinase activity increased over time as measured
by the appearance of phosphatidylinositol 3-phosphate. Inhibition of
Syk kinase, however, blocked the phosphatidylinositol 3-kinase activity
(Fig. 11A). By contrast
piceatannol had no effect on MAP kinase activation (Fig.
11B) or ceramide formation (Fig. 11C).

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Fig. 10.
Effect of piceatannol on Syk phosphorylation
and phagocytic index in COS-1 cells transfected with
Fc IIA receptor during EIgG-stimulated
phagocytosis. The COS-1 cells (3.2 × 106/dish)
were cultured in medium containing 0.25% fetal bovine serum. One hour
before phagocytosis media were aspirated, cells were kept in
phosphate-buffered saline with 1 mM Ca2+ and 1 mM Mg2+, followed by the addition of
piceatannol (100 µM) for 30 min. Control cells were
incubated in parallel for equal times with no addition of piceatannol.
Following the incubation, COS-1 cells underwent phagocytosis with EIgG
(1 × 108/ml) for different times at 37 °C.
A, the samples were immunoprecipitated with anti-Syk
antibody and were run on 10% SDS-PAGE followed by protein transfer to
PVDF membranes. Tyrosine phosphorylation was detected by Western
blotting. The blot was reprobed with anti-Syk antibody to compare
protein loading. B, phagocytic index was determined as
described under "Experimental Procedures." Values represent
the mean ± S.D. of n = 6. **, p < 0.01; ***, p < 0.001 compared with the zero time
point.
|
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Fig. 11.
Effect of piceatannol on
phosphatidylinositol 3-kinase, MAP kinase, and ceramide formation and
phagocytic index during EIgG-stimulated phagocytosis. The COS-1
cells (3.2 × 106/dish) were cultured in medium
containing 0.25% fetal bovine serum. One hour before phagocytosis the
media were aspirated, and the cells were kept in phosphate-buffered
saline with 1 mM Ca2+ and 1 mM
Mg2+, followed by the addition of piceatannol (100 µM) for 30 min. Control cells were incubated in parallel
for equal times with no addition of piceatannol. Following the
incubation COS-1 cells underwent phagocytosis with EIgG (1 × 108/ml) for different times at 37 °C. EIgG not
internalized were lysed, and COS-1 cells were resuspended in
phosphate-buffered saline. Phosphatidylinositol 3-kinase activity
(A), MAP kinase activity (B), and ceramide levels
(C) were measured as described under "Experimental
Procedures." Values represent the mean ± S.D. of
n = 3 (ceramide). The MAP kinase and
phosphatidylinositol 3-kinase data are representative of 3 or more
experiments.
|
|
 |
DISCUSSION |
In the present study the effects of de novo synthesis
of ceramides and sphingoid bases on Fc
RIIA-mediated phagocytosis in COS-1 cells were studied. Four significant findings were observed. First, Fc
RIIA-transfected COS-1 cells demonstrated phagocytosis and
associated increases in ceramide content. Phagocytosis was accompanied
by phospholipase D and MAP kinase activation. Therefore these changes
recapitulate those previously observed in PMNs. Second, the increase in
COS-1 cell ceramide levels was the result of de novo
synthesis, a result not previously observed in PMNs. The rise in
ceramide was blocked by inhibitors of ceramide synthase, fumonisin
B1, and of 3-ketodihydrosphingosine synthase,
L-cycloserine. Third, inhibition of ceramide formation
resulted in an increase in phagocytosis as measured by the phagocytotic
index. This inhibition was associated with corresponding changes in
Syk, MAP kinase, phosphatidylinositol 3-kinase, and phospholipase D
activities as well. Fourth, inhibition of Syk kinase impaired
phosphatidylinositol 3-kinase activation and phagocytosis but had no
effect on ceramide generation or MAP kinase activation. These data
suggest that de novo ceramide formation is not dependent on
Syk kinase but that ceramide inhibits Syk kinase activity.
Sphingolipids have been studied previously in signaling events in PMNs
where they have been implicated as endogenous modulators of leukocyte
function. Ceramide has been observed to inhibit both the respiratory
burst in freshly isolated PMNs (15) and the phagocytosis of opsonized
erythrocytes (16). Ceramide is formed as a late response to formyl
peptide in adherent PMNs. The time course of ceramide generation is
consistent with the down-regulation of oxidant formation, attaining
significance 90 min following fMet-Leu-Phe exposure. Ceramide levels
rise during phagocytosis in PMNs exposed to EIgG. Significant changes
in ceramide are observed 30 min following EIgG exposure. Ceramide
accumulation correlates with the termination of both oxidant formation
and the phagocytic response. The inhibitory effects of endogenous
ceramide are reproduced by the addition of short chain ceramides,
specifically N-acetylsphingosine. By contrast, tumor
necrosis factor-
, a well characterized agonist known to stimulate
ceramide formation, has been reported to stimulate sphingomyelin
hydrolysis in human PMNs within minutes. In this model tumor necrosis
factor-
primes PMNs at low concentrations and inhibits oxidant
formation at higher concentrations. In each system ceramide changes
have been correlated with the activation of a neutral sphingomyelinase.
Cell fractionation studies indicate that both the sphingomyelinase and
ceramide formed are localized to the plasma membrane (17).
Although freshly isolated human PMNs provide an important model for the
study of phagocytosis and oxidant formation, the use of these cells for
the study of sphingolipid metabolism is problematic. PMNs cannot be
cultured for prolonged periods without losing much of their functional
responsiveness over the course of hours following isolation. As a
result, PMNs are not amenable to study with the use of many of the
inhibitors of sphingolipid synthesis commonly employed, most of which
require a prolonged period of preincubation. Short chain sphingolipid
analogs have been used extensively for evaluating phagocytotic
signaling. The use of these analogs is predicated on the assumption
that they are metabolically inert. However, short chain ceramides are
metabolized to their free base, and sphingosine is converted to
ceramide. Inhibitors of sphingolipid metabolism provide an alternative
approach to using sphingolipid analogs for ascertaining the role of
ceramides and sphingolipid bases in phagocytosing COS-1 cells. In
contrast to short chain sphingolipid analogs, these inhibitors alter
the levels of endogenous sphingolipids.
During cell activation, ceramide is frequently derived from the
hydrolysis of sphingomyelin by the action of a neutral or acidic
sphingomyelinase, resulting in the generation of ceramide and
phosphorylcholine. Ceramide can also be formed as a result of the
N-acylation of sphingosine or through de novo
biosynthesis. The de novo biosynthesis of ceramide is
initiated by the condensation of serine and palmitoyl-CoA, which
results in the formation of 3-ketosphinganine, which is subsequently
reduced to dihydrosphingosine. Dihydroceramide is formed by the
addition of a fatty acid in amide linkage. Ceramides formed through
this pathway usually serve as precursors for complex sphingolipids such
as galactosylceramide and glucosylceramide. The accumulation of
ceramide resulting from de novo synthesis was first reported
by Kolesnick and colleagues (32) and resulted from anthracycline exposure.
Phospholipase D activation is a principal signal in PMN activation and
has been associated with the uptake of both complement and
IgG-opsonized particles (33-35). Furthermore, phospholipase D has been
identified as an intracellular target of ceramide during phagocytosis
in PMNs (15, 16) and in fibroblasts (36). We reported previously (16)
that N-acetylsphingosine blocked both phagocytosis and
activation of phospholipase D in PMNs. In the present study we observed
a fumonisin B1-dependent depletion of ceramide
with an associated increase in phospholipase D activity, consistent
with the regulation of phospholipase D by ceramide (Fig. 4). During
L-cycloserine treatment of cells, we were unable to detect
changes in phospholipase D activity, but the levels of sphingosine and
ceramide decreased after 6 h of treatment of the cells, and the
phagocytic index increased even more compared with cells treated with
fumonisin B1. Sphingosine and ceramide are known to have
different effects on cellular processes. Sphingosine is known to
inhibit phosphatidic acid phosphohydrolase and protein kinase C
activation and activation of phospholipase D. Sphingosine also alters
levels of Ca2+, inositol 1,4,5-trisphosphate, and cAMP. An
independent role for sphingosine in cell signaling remains to be proven
because it is actively metabolized to other products. The mitogenic
effect of sphingosine on Swiss 3T3 fibroblast is clearly independent of
protein kinase C (37, 38), and inhibition of the respiratory burst of
human neutrophils is due to a combination of inhibition of phosphatidic
acid phosphohydrolase and protein kinase C (39). At the same time that
ceramide is activating protein phosphatases and kinases and protein
kinase C
, ceramide inhibits phospholipase D activity (40).
In our previous work we evaluated the effects of
C2-ceramides and sphingoid bases on PEt and PA formation
during phagocytosis. N-Acetylsphingosine inhibited both PEt
and PA formation, but N-acetyldihydrosphingosine did not
have a significant effect on either PEt or PA formation. In contrast,
the sphingoid bases markedly stimulated both PEt and PA formation (16).
Thus, even though both C2-ceramides and sphingoid bases
inhibit phagocytosis they act on different signaling pathways. Even
though C2-ceramide completely inhibited the phagocytosis of
EIgG and phospholipase D activation, treatment of these cells with 1%
ethanol only partially (50%) inhibited phagocytosis. These findings
suggest that other components of the signaling pathways, in addition to
phospholipase D, are susceptible to ceramide-mediated inhibition.
Fc
RIIA mediates several functions. Stimulation of monocyte Fc
RII
leads to antibody cellular cytotoxicity, superoxide production, and
phagocytosis (2, 3). Transfecting COS-1 cells with Fc
RIIA in the
absence of other Fc
R renders them capable of phagocytosis of
IgG-coated erythrocytes (41). Tyrosine phosphorylation is one of the
earliest responses in PMN activation and is required for
Fc
R-mediated phagocytosis by macrophages (11, 42). Two classes of
protein tyrosine kinases, Src and Syk families, have been found to play
a role in Fc
R signaling. Syk belongs to the ZAP-70 kinase family.
Fc
R-transfected COS-1 cells, although phagocytic, presented lower
activity levels of Syk than macrophages (43), suggesting that there was
another element present in leukocytes that was important for
phagocytosis. Because Syk is exclusively present in leukocytes, it was
a good candidate to be this component. COS-1 cells transfected with
Fc
RIIA and treated with fumonisin showed 4.9 times increased Syk
phosphorylation and increased phagocytosis. There was no
phosphorylation of Syk in control cells pretreated with fumonisin
B1 in the absence of EIgG challenge. Treating the cells
with fumonisin B1 led to a depletion of ceramide content, which was restored by incubation with sphingosine for 30 min (Fig. 8B). Sphingosine treatment led to a decrease in the
phagocytic index. Protein phosphorylation detected by Western blot
showed decreased Syk phosphorylation most directly correlated with
increased ceramide levels.
To evaluate the role of Syk kinase activation on downstream signaling,
the inhibitor piceatannol was used. Inhibition of Syk kinase completely
blocked EigG-mediated phagocytosis and phosphatidylinositol 3-kinase
activity, but no effects on ceramide formation or MAP kinase activation
were observed. Presumably a domain on the Fc
RIIA receptor not
associated with ITAM mediates these signals. Because most systems in
which de novo ceramide formation occurs are not associated
with agonist receptor interactions, this COS-1 cell system may provide
a useful model for delineating the downstream signals associated with
ceramide formation.
Our studies have shown that cross-linking Fc
receptors with EIgG
results in increased tyrosine phosphorylation and activation of Syk in
COS-1 cells. High levels of ceramide and sphingosine inhibited
phagocytosis and Syk activation, thus blocking activation of
phagocytosis. Inhibitors of sphingolipid metabolism provide an
alternative approach for using sphingolipid analogs for assessing the
role of ceramides and sphingoid bases in phagocytosis. They demonstrate
that most of the ceramide formed during Fc
RIIA activation is through
de novo ceramide synthesis and may provide a novel mechanism
in the regulation of phagocytosis in COS-1 cells. COS-1 transfection
with the Fc
RIIA renders these cells phagocytic and recapitulates the
changes in ceramide content observed in PMNs.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant AI20065 (to L. A. B).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Nephrology
Division, Dept. of Internal Medicine, University of Michigan, Box 0676, Rm. 1560 MSRB II, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0676. Tel.: 734-763-0992; Fax: 734-763-0982; E-mail:
jshayman@umich.edu.
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M206199200
 |
ABBREVIATIONS |
The abbreviations used are:
PMN, polymorphonuclear leukocytes;
EIgG, opsonized red blood cells;
ITAM, immunoreceptor tyrosine-based activation motif;
MAP kinase, mitogen-activated protein kinase;
PA, phosphatidic acid;
PEt, phosphatidylethanol;
PVDF, polyvinylidene difluoride.
 |
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