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Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 686-693
Sphingosine Blocks Human Polymorphonuclear Leukocyte Phagocytosis
Through Inhibition of Mitogen-Activated Protein Kinase Activation
By
Evelin M.B. Raeder,
Pamela J. Mansfield,
Vania Hinkovska-Galcheva,
Lars Kjeldsen,
James A. Shayman, and
Laurence A. Boxer
From the Department of Pediatrics, Division of Hematology/Oncology,
and Department of Internal Medicine, Division of Nephrology, University
of Michigan, Ann Arbor, MI.
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ABSTRACT |
In the present study, we investigated the mechanism by which
sphingosine and its analogues, dihydrosphingosine and phytosphingosine, inhibit polymorphonuclear leukocyte (PMN) phagocytosis of IgG-opsonized erythrocytes (EIgG) and inhibit ERK1 and ERK2 phosphorylation. We used
antibodies that recognized the phosphorylated forms of ERK1 (p44) and
ERK2 (p42) (extracellular signal-regulated protein kinases 1 and 2).
Sphingoid bases inhibited ERK1 and ERK2 activation and phagocytosis of
EIgG in a concentration-dependent manner. Incubation with glycine,
N,N -[1,2-ethanediylbis(oxy-2,1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]]-bis[(acetyloxy)methyl]ester (BAPTA,AM), an intracellular chelator of calcium, failed to block either phagocytosis or ERK1 and ERK2 phosphorylation, consistent with
the absence of a role for a calcium-dependent protein kinase C (PKC) in
ERK1 and ERK2 phosphorylations. Western blotting demonstrated that
sphingosine inhibited the translocation of Raf-1 and PKC from PMN
cytosol to the plasma membrane during phagocytosis. These data are
consistent with the interpretation that sphingosine regulates ERK1 and
ERK2 phosphorylation through inhibition of PKC, and this in turn
leads to inhibition of Raf-1 translocation to the plasma membrane.
Consistent with this interpretation, the sphingosine-mediated inhibition of phagocytosis, ERK2 activation, and PKC translocation to the plasma membrane could be abrogated with a cell-permeable diacylglycerol analog. The increase in the diacylglycerol mass correlated with the translocation of PKC and Raf-1 to the plasma membrane by 3 minutes after the initiation of phagocytosis.
Additionally, the diacylglycerol analog enhanced phagocytosis by
initiating activation of PKC and its translocation to the plasma
membrane. Because PMN generate sufficient levels of sphingosine by 30 minutes during phagocytosis of EIgG to inhibit phagocytosis, it appears that sphingosine can serve as an endogenous regulator of EIgG-mediated phagocytosis by downregulating ERK activation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
A WIDE RANGE OF surface molecules,
including those of the Fc receptor family, are expressed on
hematopoietic cells.1,2 These receptors are important in
host defense because they represent a major mechanism for the detection
and phagocytosis of IgG-opsonized particles.3 After
receptor engagement, the functional responses involve many intermediate
steps. These include a complex cascade of biochemical events that link
receptor engagement to the microbicidal response. One of the early
events in polymorphonuclear leukocyte (PMN) stimulation is the rapid
induction of protein phosphorylation of several proteins by activation
of multiple kinases, including tyrosine kinases, protein kinase C
(PKC), and MAP kinases.4,5 Cross-linking of the Fc
receptor induces tyrosine phosphorylation of FcRIIA itself and has
been implicated in the regulation of phagocytosis.6-8 The
phosphorylated tyrosines then serve as an interaction domain for
protein tyrosine kinases of the Src and syk/ZAP 70 families.6,9 Once stimulated, these kinases catalyze the
phosphorylation of several cellular substrates, including MAP
kinases.10
The MAP kinases, extracellular signal-regulated protein kinases 1 and 2 (ERK1 and ERK2), a pair of closely related enzymes, are common
intermediates in intracellular signaling cascades and are involved in
diverse cellular functions. We previously correlated FcRII
engagement during N-formyl-methionyl-leucyl-phenyl-alanine (fMLP)-primed phagocytosis of EIgG in PMN with activation of the MAP
kinases, ERK1 and ERK2.5 Activation of ERK1 and ERK2
requires the dual phosphorylation of Thr and Tyr residues after
activation of MAP kinase kinases (MEK1 and MEK2). In turn, MEK
activation is also regulated by phosphorylation. The signaling pathway
activating the MAP kinase cascade has been studied intensively and has
been shown to be dependent on c-Raf activation via
Ras.11,12 It has been proposed that these kinases are
arranged in a linear cascade (Ras  Raf MEK
ERK).13 MEK1 and MEK2 are directly phosphorylated and activated by c-Raf. Recently, a Ras-independent pathway has been described in which PKC has been implicated in activation of the Raf MEK ERK
cascade.14 Because of the diversity of signals, there are
still open questions concerning the mechanism of upstream activation of
ERK1 and ERK2 in this cascade as it relates to phagocytosis.
The metabolism of sphingolipids gives rise to second messengers that
regulate cell activation, including free sphingosine. Sphingosine has
been shown to inhibit PMN by inhibiting PKC and cellular functions (eg,
the respiratory burst and protein secretion) dependent on this
enzyme.15 Besides inhibiting PKC, other cell signaling
pathways may be influenced by sphingosine. In particular, sphingosine
can inhibit the enzyme phosphatidic acid phosphohydrolase, which is
involved in generating diacylglycerol (DAG) from phosphatidic acid
generated by phospholipase D (PLD).16,17
The purpose of the present study was to examine the mechanism by which
sphingosine inhibits phagocytosis of antibody-coated red blood cells
(EIgG). We assessed the effect of sphingoid bases on ERK1 and ERK2
activation and phagocytosis of EIgG, because we have shown the former
to be crucial in mediating phagocytosis. In turn, we examined some of
the enzymes upstream of ERK1 and ERK2. Thus, we determined whether
sphingosine blocked translocation of PKC and Raf-1 to the plasma
membrane during PMN phagocytosis. Finally, we examined whether we could
abrogate the effect of sphingosine by adding a cell permeable analog of
DAG.
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MATERIALS AND METHODS |
Reagents.
Sphingosine, DL-erythro-dihydrosphingosine, phytosphingosine
hydrochloride, fatty acid free bovine albumin (BSA), fMLP,
diethylenetriaminepenta-acetic acid (DETAPAC), ceramide type III,
n-octyl  -D-glucopyranoside, 1,2-dioleoyl-sn-glycerol, and
diisopropylfluorophosphate (DFP) were purchased from Sigma Chemical Co
(St Louis, MO). N-acetyldihydrosphingosine was synthesized from
DL-erythro-dihydrosphingosine as previously described.16
sn-1,2-Diacylglycerol kinase (Escherichia coli) and
dithiothreitol were purchased from Calbiochem (San Diego, CA). The MEK
inhibitor, PD098059, was a generous gift of Alan R. Saltiel
(Parke-Davis Pharmaceutical Research Division, Ann Arbor,
MI).18 Polyclonal antibodies (Abs) against
ERK1 and ERK2 (p44/42) recognizing the phosphorylated form of both p42
and p44 were obtained from New England BioLabs (Beverly, MA).
Polyclonal Ab against ERK2 was obtained from Santa Cruz Biotechnology
(Santa Cruz, CA) and monoclonal Ab against PKC, PKC,
and Raf-1 from Transduction Laboratories Inc (Lexington, KY).
Monoclonal antiphosphotyrosine Ab 4G10 was purchased from Upstate
Biotechnology (Lake Placid, NY). Horseradish peroxidase
(HRP)-conjugated sheep antimouse Abs were from Amersham (Arlington
Heights, IL), and HRP-conjugated antirabbit Ab was obtained from Santa
Cruz Biotechnology. [3H]-Acetic anhydride was purchased
from American Radiolabeled Chemicals Inc (St Louis, MO) and
sn-1,2-didecanoylglycerol (DiC10) from Avanti Polar Lipids (Alabaster,
AL).
Glycine,N,N-[1,2-ethanediylbis(oxy-2,1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]]-,bis [(acetyloxy)methyl]ester (BAPTA,AM) was obtained from Molecular Probes (Eugene, OR) and
[-32P]-adenosine-5-triphosphate was from ICN
Pharmaceuticals, Inc (Irvine, CA).
Cells.
Human PMN were isolated from human peripheral blood as described
previously.19 Briefly, fresh whole blood was obtained by venipuncture from healthy volunteers and immediately added to acid
citrate dextrose. The PMN were purified by dextran sedimentation followed by hypotonic lysis to remove the majority of erythrocytes and
then centrifuged through Ficoll-Paque (Pharmacia LKB Biotechnology Inc,
Piscataway, NJ) to remove contaminating mononuclear cells. Before
activation of cells and subcellular fractionation, the cells were
incubated for 5 minutes on ice with 5 mmol/L diisopropylfluorophosphate (DFP), washed, and resuspended in the desired buffer.
BAPTA,AM loading of PMN.
PMN (2 × 106/mL) were incubated with the
intracellular Ca2+ chelator BAPTA,AM at 20 µmol/L in
Ca2+-free phosphate-buffered saline (PBS) for 30 minutes at
37°C. The PMN were then washed twice with PBS containing 1 mmol/L
Ca2+ and 1 mmol/L Mg2+. The phagocytosis assay
was started as outlined below.
Phagocytic targets.
Sheep erythrocytes were purchased from BioWhittaker
(Walkersville, MD) and were opsonized with antisheep erythrocyte IgG
(Cappel Organon Teknika, Durham, NC) as previously
described.5,20
Phagocytosis assay.
The phagocytosis assay was conducted essentially as outlined by Pommier
et al.21 For studies with sphingoid bases, lipid stocks as
well as DiC10 stock (50 mmol/L) were prepared to form a BSA complex as
described by Merrill et al.22 PMN, suspended at 2 × 106/mL in PBS containing 1 mmol/L Ca2+ and 1 mmol/L Mg2+, were incubated with different concentrations
of lipids, DiC10, or 50 µmol/L PD098059 for 30 minutes at 22°C.
In other experiments, PMN were preincubated with DiC10 and then treated
with sphingosine, or PMN were preincubated with sphingosine and then
treated with DiC10, or cells were treated with both lipids
simultaneously. After the incubation, PMN underwent phagocytosis with
EIgG at once or were preactivated with fMLP (10 7
mol/L) for 10 minutes at 37°C, and then EIgG (1 × 108/mL) were added to the activated PMN and the incubation
was continued for an additional 30 minutes at 37°C. The assays were
stopped and counted as described previously.5 Inhibition of
phagocytosis in the presence of lipids was expressed as the percentage
of control, with control being phagocytosis by fMLP-treated and
non-fMLP-treated PMN in the absence of lipid treatment.
Immunoblotting.
PMN lysates (1 to 2 × 106 PMN in 30 to 40 µL
buffer) were combined with sample buffer, boiled for 5 minutes, and run
on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) minigels. The proteins were transferred to polyvinylidene
difluoride (PVDF) membranes (Schleicher and Schuell, Keene, NH) for 2 hours at 100 V, and the membrane was blocked with 2% BSA in PBS
containing 1 mmol/L EDTA, 0.05% Tween-20, and 1 mmol/L
Na3VO4. The membrane was probed with antibody
against phosphorylated p44/42 in blocking buffer, washed three times
with 0.2% Tween-20 in 50 mmol/L Tris (pH 8.0) and 100 mmol/L NaCl, and
then incubated with a second antibody (HRP-conjugated goat antirabbit
Ab) in wash buffer containing 5% nonfat dry milk. Phosphorylated bands
were visualized using the enhanced chemiluminescence (ECL) system
(Amersham). Membranes were stripped with 100 mmol/L
-mercaptoethanol, 2% SDS, and 62.5 mmol/L Tris (pH 6.7) at
50°C, and reprobed with polyclonal anti-ERK. Immunoblotting was
also conducted using anti-Raf-1, anti-PKC, and anti-PKC Ab.
HRP-conjugated sheep antimouse Ab served as a second antibody for
anti-Raf-1, anti-PKC, and anti-PKC. For these experiments, PMN
were treated as described in the section cell fractionation for
immunoblotting. Tyrosine phosphorylation of ERK2 was
detected by immunoprecipitating ERK2 as previously described by Suchard
et al.5 Subsequent immunoblotting with 4G10
antiphosphotyrosine Ab was conducted and then incubated with HRP-conjugated sheep antimouse Ab.
Immunocomplex ERK activity.
This assay was conducted essentially as outlined by Suchard et
al.5 PMN (2 × 106) were lysed in 800 µL
of buffer containing 50 mmol/L HEPES (pH 7.5), 100 mmol/L NaCl, 2 mmol/L EDTA, 1% Nonidet P-40, 1 µmol/L pepstatin, 1 µg/mL
leupeptin, 0.2 mmol/L phenylmethyl sulfonyl fluoride (PMSF), 0.2 mmol/L
Na3VO4, 2 µg/mL aprotinin, and 40 mmol/L
4-nitrophenyl phosphate. Cleared lysates were incubated with 1 µg
anti-ERK2 (Santa Cruz Biotechnology) overnight with rotation at
4°C. Protein A-Sepharose was added to each sample and incubated for
30 minutes with rotation at 4°C. Beads were washed twice with cold
lysis buffer and twice with 10 mmol/L HEPES (pH 7.5), 10 mmol/L
magnesium acetate, and 1 mmol/L Na3VO4. Beads were resuspended in kinase buffer containing 10 mmol/L HEPES (pH 7.5),
10 mmol/L magnesium acetate, 50 µmol/L ATP, 5 µCi/sample [-32P]ATP, and 20 µg of myelin basic protein (Life
Technologies, Gaithersburg, MD). Samples were incubated for 5 minutes
at 30°C and the reaction was terminated by adding sample buffer.
Proteins were separated on 12% SDS-PAGE minigels. Phosphorylated
myelin basic protein was visualized by autoradiography and the bands
were excised and counted in a liquid scintillation counter (Wallac,
Gaithersburg, MD). Activity was expressed as the percentage of
unstimulated controls.
Cell fractionation for immunoblotting with PKC and Raf-1.
For fractionation studies, phagocytosis was stopped as described
previously 5 minutes after initiating the ingestion of
EIgG.5 PMN were resuspended at 2 × 108/mL
in extraction buffer (50 mmol/L Tris [pH 7.5], 2 mmol/L EGTA, 1 mmol/L PMSF, leupeptin [1 µg/mL], 10 µmol/L benzamidine, 10 µmol/L pepstatin, and aprotinin [0.2 µg/mL]). The cells were
disrupted by sonication on ice, and the resulting homogenate was
centrifuged (400g for 10 minutes at 4°C) to remove unbroken
cells and nuclei. The supernatant of each sample was applied to a 15%
to 40% discontinuous sucrose gradient and centrifuged for 30 minutes
at 150,000g at 4°C to obtain cytosolic, membrane, and
granule fractions.23 The cytosol was removed from the top
of the gradient, and the membrane fraction was collected at the 15% to
40% interface. The granule fraction was seen as the pellet. The
cytosol and the membrane fraction were combined with sample buffer and
boiled for 5 minutes. Protein was measured in the different samples by
the BCA method (Pierce, Rockford, IL) using BSA as a
standard.
Cell fractionation for assay of sphingosine formation.
Subcellular fractionation was performed as previously described by
Kjeldsen et al.24 For this assay, phagocytosis was stopped 30 minutes after initiating the ingestion of EIgG. PMN were resuspended at 1.5 to 5 × 107/mL and disrupted by nitrogen
cavitation, and the postnuclear supernatant was centrifuged over a
two-layer Percoll gradient as described.24 This resulted in
three visible bands containing azurophilic granules,
specific/gelatinase granules, and secretory vesicles and plasma
membranes, respectively, with the clear cytosol on top. All fractions
were assayed for specific marker proteins as described
previously.24,25
Assay for sphingosine, ceramide, and diacylglycerol formation.
Sphingosine was quantitated by acetylation with
[3H]-acetic anhydride to form [3H]
C2-Ceramide as described previously.26 Ceramide
and diacylglycerol were assayed by the method of Preiss et
al.27 This assay is based on the formation of
[32P]-phosphatidic acid from endogenous diacylglycerol
and [32P]-ceramide phosphate from ceramide. The
sphingosine, ceramide, and diacylglycerol content in the cell extracts
was calculated by extrapolation from standards treated similarly.
Statistical analysis.
Two-tailed Student's t-tests were used to assess statistical
significance.
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RESULTS |
The effect of DiC10 on phagocytosis.
We previously reported that sphingoid bases are potent inhibitors of
IgG-dependent phagocytosis in fMLP-stimulated PMN.20 Because sphingosine is a competitive inhibitor of DAG, we evaluated the
effect of a cell permeable DAG analog, DiC10, on EIgG-mediated phagocytosis. In the presence of 50, 100 and 200 µmol/L DiC10, phagocytosis was significantly increased by 9%, 42%, and 46%, respectively (Fig 1A).
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| Fig 1.
Concentration-dependent restoration of phagocytosis of
EIgG by sphingosine-treated nonprimed (A) and fMLP-primed (B) PMN in
the presence of DiC10. PMN (2 × 106/mL) were preincubated
with different concentrations of sphingosine for 30 minutes at 22°C
and washed twice, and then the PMN were incubated with DiC10 at
different concentrations at 22°C for 30 minutes. The PMN were then
challenged with EIgG (A) or primed with fMLP (107 mol/L)
and then EIgG (B). The control phagocytic index was 16.4 ± 2.4 (100%
control) in nonprimed cells and was 59.2 ± 8 in fMLP-primed PMN. The
values represent the mean ± SD for three experiments. Comparisons
were made between DiC10 versus cells not treated with DiC10:
***P < .0001, **P < .001, *P < .005.
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We next examined whether the addition of DiC10 could abrogate the
inhibition by sphingosine of phagocytosis of EIgG by PMN. PMN were
preincubated with sphingosine at different concentrations, and then
varying concentrations of DiC10 were added. A concentration of 1 µmol/L sphingosine inhibited EIgG-mediated phagocytosis by 50% (Fig
1A). After the addition of 50 µmol/L DiC10, phagocytosis was
normalized. At higher concentrations of DiC10 (100 and 200 µmol/L),
phagocytosis was further enhanced above controls. When 5 µmol/L
sphingosine was used, 200 µmol/L DiC10 was required to restore
phagocytosis to control value (P was not significant). When PMN
were incubated with 10 µmol/L sphingosine, the addition of 200 µmol/L DiC10 was only able to restore phagocytosis to 69% of control
value. These results indicate that the DAG analog was able to reverse
sphingosine inhibition of phagocytosis in a concentration-dependent manner and that the presence of DAG is a likely requirement for Fc-receptor-mediated phagocytosis to occur.
The effect of DiC10 on PMN, incubated with sphingosine was compared
between phagocytosis of EIgG by nonprimed PMN and fMLP-primed PMN (Fig
1B). The addition of DiC10 significantly increased phagocytosis in a
concentration-dependent manner. The addition of all concentrations of
DiC10 augmented fMLP-primed phagocytosis of EIgG in the presence of 5 µmol/L sphingosine to the same extent as observed with 10 µmol/L
sphingosine (Fig 1B). In fMLP-primed PMN, DiC10 did not restore
phagocytosis to control values in the presence of 5 or 10 µmol/L
sphingosine. In contrast, the addition of 200 µmol/L DiC10 augmented
phagocytosis to control values in PMN treated with 1 µmol/L
sphingosine.
To determine whether the lipid-mediated inhibition of phagocytosis was
[Ca2+]i-dependent, PMN were incubated with an
intracellular Ca2+ chelator, BAPTA,AM. BAPTA,AM-treated PMN
were not impaired in their ability to ingest EIgG. The
concentration-dependent inhibition of phagocytosis by 1 µmol/L
sphingosine was unaffected by Ca2+ chelation (Fig 1A and
B). Similar to Ca2+ replete cells, the BAPTA,AM-treated PMN
underwent a similar increase in EIgG-mediated phagocytosis in the
presence of 1 µmol/L sphingosine with various concentrations of
DiC10.
Effect of sphingoid bases on ERK1 and ERK2 activation during
Fc-receptor-mediated phagocytosis in fMLP-primed PMN.
Tyrosine phosphorylation is one of the earliest responses in PMN
activation and is required for FcR-mediated phagocytosis by
macrophages.28 Recently, we correlated ERK1 and ERK2
phosphorylation with the engagement of FcRII.5 We
investigated the effect of sphingoid bases on ERK1 and ERK2 activation
during fMLP-primed phagocytosis using an antibody that recognizes the
phosphorylated forms of both ERK1 and ERK2, which have molecular masses
of 44 and 42 kD, respectively. ERK1 and ERK2 phosphorylation was
suppressed by sphingosine and its analogs dihydrosphingosine and
phytosphingosine in a concentration-dependent manner (>95%
inhibition at 10 µmol/L and 55% inhibition at 5 µmol/L;
Fig 2A). At 1 µmol/L
sphingosine and 1 µmol/L dihydrosphingosine, ERK1 and ERK2 activation
was reduced by 17% and 11%, respectively; whereas phytosphingosine failed to inhibit ERK1 and ERK2 activation (data not shown). These observations were correlated with the effect of these compounds at 1 µmol/L on their ability to modulate the phagocytic response. We also
measured ERK2 activity using myelin basic protein as the substrate
(Table 1). ERK2 activity increased during
phagocytosis, which was reduced to basal levels in the presence of 10 µmol/L sphingosine. In our previous studies, we found that PD098059, a specific MEK inhibitor, blocked ERK1 and ERK2 activation by greater
than 80% which corresponded to a reduction in phagocytosis of
63%.5 In contrast, the tyrosine phosphorylation of ERK1 and ERK2 was not reduced in the presence of N-acetyldihydrosphingosine (Fig 2A). As expected, N-acetyldihydrosphingosine had no influence on
FcR-mediated phagocytosis.
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| Fig 2.
(A) Effect of sphingoid bases on ERK1 and ERK2 activation
during fMLP-primed phagocytosis of EIgG in PMN. PMN (2 × 106/mL) were incubated with different sphingoid bases (5 and 10 µmol/L), buffer (control), or 50 µmol/L PD098059 for 30 minutes at 22°C. The PMN were primed with fMLP (107
mol/L) for 10 minutes at 37°C, followed by the addition of EIgG (1 × 108/mL) for 3 minutes at 37°C. The membranes were
probed with anti-MAP kinase Ab that recognizes both phosphorylated
isoforms, ERK1 (p44) and ERK2 (p42). (B) Effect of sphingosine on ERK1
and ERK2 activation. PMN (2 × 106/mL) were preincubated
with 10 µmol/L sphingosine or buffer (control) and subsequently
activated with 107 mol/L fMLP and EIgG. Phagocytosis was
allowed to proceed for 3 minutes. The samples were run on 10% SDS-PAGE
and protein transferred to PVDF membranes. The membranes were probed
with Ab against phosphorylated ERK1 and ERK2. The figure is
representative of three experiments. (C) Kinetics of ERK1 and ERK2
phosphorylation in PMN during phagocytosis of EIgG. PMN were incubated
with EIgG. At the indicated times, phagocytosis was terminated
and the PMN were treated as noted in (B).
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In unstimulated PMN, there was no evidence of basal ERK1 and ERK2
phosphorylation (Fig 2B). We observed greater phosphorylation of ERK1
and ERK2 in PMN when primed with fMLP and then challenged with EIgG
(100%) compared with activation of ERK1 and ERK2 treated with fMLP
alone (63%) or by EIgG challenge alone (49%) (Fig 2B and Table 1).
Sphingosine (10 µmol/L) suppressed the phosphorylation of ERK1 and
ERK2 in cells either stimulated with fMLP alone or cells stimulated
with fMLP followed by addition of EIgG. In contrast, the addition of
sphingosine completely inhibited ERK1 and ERK2 activation in cells
stimulated with EIgG alone (Fig 2B).
The kinetics of ERK1 and ERK2 phosphorylation were monitored in PMN
challenged with EIgG alone (Fig 2C). At time 0, using nonstimulated
PMN, there was no activation of ERK1 and ERK2. After the addition of
EIgG, we observed increased phosphorylation of these proteins beginning
at 60 seconds, which reached maximal phosphorylation by 5 minutes and
was sustained for 10 minutes. At 20 minutes, dephosphorylation of ERK1
and ERK2 was apparent. Chelation of [Ca2+]i
with BAPTA,AM had no influence on the activation of ERK1 and ERK2,
indicating a [Ca2+]i-independent process
(data not shown).
After the subcellular fractionation of PMN into primary and secondary
granules, cytosol, and plasma membranes, an antibody to ERK2 was used
to determine its intracellular location. ERK2 was distributed in both
the cytosol and plasma membrane (data not shown). ERK2 failed to
translocate to the plasma membrane during phagocytosis.
Effect of DiC10 on sphingosine-induced inhibiton of ERK activation.
Because ERK2 activation rather than ERK1 activation is primarily
involved in mediating phagocytosis, the role of DiC10 in restoring ERK2
activation in the presence of sphingosine was studied.5 PMN
were incubated initially with 10 µmol/L sphingosine, followed by 200 µmol/L DiC10. DiC10 alone increased phosphorylation of ERK2 during
fMLP-primed phagocytosis of EIgG (condition A) 32% above the control.
The control consisted of fMLP-primed PMN challenged with EIgG alone.
After fMLP treatment alone (condition D), ERK2 phosphorylation was
increased by DiC10 14% above the control. Ingestion of EIgG alone
(condition B) by PMN led to an increase of 8% above the control. Even
without the addition of fMLP or EIgG, DiC10 led to ERK2 phosphorylation
in PMN (condition C; panel III in Fig 3 and
Table 2).
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| Fig 3.
Restoration of ERK2 phosphorylation by DiC10 in PMN
treated with sphingosine. PMN (2 × 106/mL) were
pretreated for 30 minutes with 10 µmol/L sphingosine followed by
incubation for 30 minutes with 200 µmol/L DiC10 (panel I) or treated
with 10 µmol/L sphingosine alone (panel II) or 200 µmol/L DiC10
alone (panel III). The PMN were then primed with 107
mol/L fMLP and then challenged with EIgG (column A), challenged with
EIgG alone (column B), were not treated (column C), or were stimulated
with 107 mol/L fMLP (column D). ERK2 phosphorylation was
determined by Western blotting. See Fig 2B for an example of control
values.
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Table 2.
The Quantification of ERK2 Activation on the Basis of
Cell Equivalents by Densitometry Is Shown for Data Presented in Fig
3
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When PMN were preincubated with 10 µmol/L sphingosine followed by
addition of 200 mmol/L DiC10 (panel I in Fig 3 and Table 2), an
increase in ERK2 phosphorylation was observed under all conditions (A,
B, C, and D; panel I). As demonstrated in panel II of Fig 3 and Table
2, the addition of sphingosine alone inhibited ERK2 phosphorylation
under conditions in which PMN were stimulated with various agonists (A,
B, and D). These results indicate that DiC10 was able to restore ERK2
phosphorylation and to improve phagocytosis when sphingosine was
present.
Sphingosine, ceramide, and diacylglycerol generation in PMN during
phagocytosis.
Because exogenous sphingosine inhibited phagocytosis of EIgG by PMN, we
measured endogenous sphingosine levels during phagocytosis to ascertain
whether cessation of phagocytosis correlated with a rise in sphingosine
levels. Subcellular fractions of unstimulated (control) and
EIgG-phagocytosing PMN were analyzed for sphingosine content.
Sphingosine was associated with azurophilic and specific granule
subsets and with the plasma membrane fraction
(Table 3). After 30 minutes of phagocytosis
of EIgG, the amount of sphingosine increased significantly in PMN to
275% above control in isolated azurophilic granules after
phagocytosis, to 237% above control in specific granules, and to 222%
above control in plasma membranes (Table 3). When the free sphingosine
generation was adjusted for the volume and water content in PMN, it was
about 5 µmol/L, which correlated with the concentration of
sphingosine used in several of our experiments.
Priming of PMN with FMLP did not alter the time at which maximal levels
of diacylglycerol, ceramide, and sphingosine were generated in PMN
compared with nonprimed cells. The diacylglycerol mass increased from
20.2 pmol/106 PMN to 40.2 ± 13.8 and 34.1 ± 6.1 pmol/106 PMN (n = 4) and decreased to control
levels at 10 minutes in nonprimed and primed PMN, respectively. The
ceramide level increased 2.6% by 1 minute from a basal level of 0.8 pmol/nmol Pi and achieved a maximal level of 4.8 pmol/nmol Pi by 30 minutes, which was similar in our previous study using primed
PMN.20
In nonprimed PMN challenged with EIgG, the ceramide level increased by
twofold at 30 minutes. The levels of sphingosine, which is derived from
ceramide, increased from a basal value of 14.4 ± 3.4 pmol/106 PMN to 47.3 ± 12.8 pmol/106 PMN
(n = 6) at 30 minutes in cells primed with fMLP followed by
EIgG addition. In PMN challenged with EIgG alone, the sphingosine amount increased by 1.9-fold. Between 0 and 30 minutes, sphingosine levels gradually increased and did not achieve maximal levels before
ceramide reached its maximal level.
Translocation of Raf-1 and PKC to PMN plasma membrane during
phagocytosis.
Because the generation of DAG is a prerequisite for phagocytosis, the
effect of DAG on PKC isoenzyme translocation and function in
phagocytosing PMN was evaluated. Of the DAG-stimulated PKCs, only
PKC is functional in the absence of
[Ca2+]i in PMN; therefore, the translocation
of PKC to the plasma membrane during phagocytosis was studied. Raf-1
was also studied as a possible intermediate between PKC and MEK.
Both Raf-1 and PKC were present in the cytosol
(Fig 4A and B). Raf-1 and PKC
were translocated to the plasma membrane as early as 1 minute and
reached maximal levels by 3 minutes after the initiation of
phagocytosis (data not shown), which correlated with the increase in
the diacylglycerol mass. The translocation to the plasma membrane was
quantified on the resultant autoradiographs by densitometry on the
basis of protein equivalents. The sum of the control peptides
(unstimulated PMN) in the cytosol and plasma membrane was assigned
100%. EIgG challenge of PMN resulted in the greatest loss of Raf-1 and
PKC from the cytosol about 39% ± 18% (mean ± SD, n = 4, P < .02) and 31% ± 12% (mean ± SD, n = 5, P < .005), respectively, at 3 minutes after initiation of phagocytosis.
The increase of Raf-1 and PKC was about 30% and 27%, respectively,
in the plasma membrane at 3 minutes after initiation of ingestion of
EIgG. At concentrations of 10 µmol/L sphingosine, Raf-1 and PKC
translocation to the plasma membrane was inhibited after 3 minutes of
phagocytosis by greater than 80%. As shown in Fig 4B, the role of
DiC10 restoring PKC translocation in the presence of sphingosine was
examined. DiC10 alone led to increased translocation of PKC greater
than 63% to the plasma membrane during phagocytosis of EIgG. When PMN
were preincubated with 10 µmol/L sphingosine followed by the addition
of 200 µmol/L DiC10, an increase in translocation of PKC to the
plasma membrane occurred during phagocytosis of EIgG compared with
unstimulated PMN or PMN treated with sphingosine alone (Fig
4B). These results demonstrate that DiC10 was able to restore
PKC translocation during phagocytosis of EIgG when sphingosine was
present. The ability of DiC10 to restore PKC translocation
correlated with the restoration of both phagocytosis and ERK2
activation in the presence of sphingosine. Similar to the findings of
others, less than 100% of the total PKC and Raf-1 content of PMN
was recovered from the cytosol and membrane fraction of EIgG-stimulated
cells compared with control PMN.23 These findings suggest
that these enzymes may be proteolyzed or translocated to other
subcellular fractions.
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| Fig 4.
(A) Inhibition by sphingosine of the translocation of
Raf-1 to the plasma membrane during phagocytosis of EIgG in PMN. PMN (1 × 108/mL) were incubated with EIgG (5 × 109/mL) at 37°C. The PMN were then separated into
cytosolic and membrane fractions. The fractions were analyzed for the
presence of Raf-1 by employing SDS-PAGE and immunoblotting using
specific Abs. (A) indicates the translocation of Raf-1 to the plasma
membrane within 3 minutes in column B, a decrease in translocation at
20 minutes phagocytosis in column C, respectively; columns D indicate
PMN pretreated 10 µmol/L sphingosine and activated with EIgG for 3 minutes. (B) Restoration of PKC translocation from cytosol to the
plasma membrane in PMN treated with sphingosine by DiC10. (B) indicates
translocation of PKC to the plasma membrane at 3 minutes from the
cytosol in the presence or in the absence of 10 µmol/L sphingosine
and DiC10.
|
|
These experiments were also performed in
[Ca2+]i-depleted PMN using BAPTA,AM and were
not different, indicating that Raf-1 and PKC translocation to the
plasma membrane was a [Ca2+]i-independent
process. The experiments using Ca2+ chelation were
performed to examine specially the role of Ca2+-independent
PKC isozymes in phagocytosis. To assure that sufficient BAPTA,AM was
used to inhibit translocation of the PKC isoenzyme, PKC, a
[Ca2+]i-dependent isoenzyme, the
translocation of PKC to the plasma membrane during phagocytosis was
also studied. When [Ca2+]i was chelated with
BAPTA,AM, PKC was not translocated to the plasma membrane during
phagocytosis of EIgG (data not shown).
| |
DISCUSSION |
The stimulation of human PMN with EIgG occurs via binding of
FcRII.5 It was shown that FcRII-mediated engagement
and associated signal transduction steps led to ERK1 and ERK2
phosphorylation and activation. In turn, ERK2 activation was better
correlated with phagocytosis of EIgG than ERK1 activation.5
The upstream signaling events leading to ERK1 and ERK2 phosphorylation
during EIgG-mediated phagocytosis have not been completely elucidated. In contrast, an extensively studied model for ERK1 and ERK2
phosphorylation exists for fMLP-primed activation of PMN. Ligation of
the fMLP receptor results in the activation of Ras. In turn, Ras
activates the serine/threonine kinase Raf.11 Upon
activation, Raf phosphorylates MEK1 and MEK2. MEK, in turn,
phosphorylates ERK1 and ERK2. Ueda et al14 showed that a
constitutively active mutant form of PKC activated MEK and then ERK
in a Ras-independent but Raf-dependent manner. This provides a basis
linking PKC activation to the phagocytic response.
PKC is a [Ca2+]i-independent isoenzyme of
PKC. PKC is found in PMN and is one of four PKC isoenzymes that
translocate to the plasma membrane during
phagocytosis.23,29 Unlike PKC , which is also
[Ca2+]i-independent and found in PMN, PKC
is diacylglycerol dependent. In our study, we found that EIgG is a
potent stimulus for the translocation of PKC to the plasma membrane
fraction of PMN during phagocytosis of EIgG, where we observed ERK in
part to be located. The association of ERK1 and ERK2 with membrane and
cytoskeletal structures has been suggested. Gonzalez et
al30 observed the localization of ERK2 to membrane ruffles
in COS cells. Coincident with the PKC translocation to the plasma
membrane during phagocytosis, Raf-1 was translocated. The appearance of
PKC and Raf-1 in the plasma membrane fraction by 3 minutes during
phagocytosis of EIgG was sustained over 10 minutes of phagocytosis
(data not shown). We confirmed that PKC was participating in a
[Ca2+]i-independent signaling pathway in
these cells because chelation of intracellular Ca2+ with
BAPTA,AM did not influence the translocation of PKC and Raf-1.
Because phagocytosis is
[Ca2+]i-independent,31 these
findings are consistent with the notion that PKC activation is
linked to Raf-1 activation.14
Because sphingosine inhibits phagocytosis and PKC activation, the role
of sphingosine in blocking activation of key components of the signal
transduction pathway affected by PKC activation was
studied.15,20 In contrast, C2-ceramide does not
directly inhibit PKC activity, which implies that ceramide regulates
phagocytosis by a mechanism different than sphingosine.32
We previously observed that ceramide completely suppressed
PLD activation,16,33 but it only suppressed ERK2 activation
by 70% to 80%.5 Sphingosine completely blocked ERK2
activation. Because sphingosine was more efficient in inhibiting ERK
activity than ceramide, it is likely that it is the preferred metabolic
inhibitor of phagocytosis. At 10 minutes, when the rate of phagocytosis
of EIgG was decreasing,20 the free sphingosine level
reached 3 µmol/L when adjusted for PMN water content and volume. In
contrast, ceramide levels were only minimally elevated in nonprimed PMN
ingesting EIgG,20 which again supports the critical role of
sphingosine in regulating phagocytosis. Sphingosine inhibited
translocation of PKC and Raf-1 to the plasma membrane during
phagocytosis. Because PKC and Raf-1 activation are upstream events
leading to MEK activation followed by ERK phosphorylation, these data
suggest that PKC and Raf-1 activation are key components of the
phagocytic pathway. The maximal translocation of PKC and Raf-1 to
the plasma membrane by 3 minutes after the initiation of ingestion of
EIgG correlated with the increase in the endogenous diacylglycerol
mass. In accordance with our results, Della Bianca et al34
showed that phagocytosis of EIgG in PMN was associated with an increase
in the formation of the diacylglycerol mass at 4 minutes after
challenge with EIgG, which is largely generated through the activity of
phospholipase D. Sphingosine can inhibit phosphatidic acid
phosphohydrolase activity in PMN, primed with C5a, phorbol myristate
acetate, fMLP, or primed PMN undergoing phagocytosis of EIgG, which
results in diminished diacylglycerol formation.16,20,35
Because diacylglycerol is a necessary requirement for PKC activation,
the sphingosine-induced failure to generate diacylglycerol could
explain the failure of PKC to translocate to the plasma membrane
during phagocytosis.23
Others have observed that sphingosine is a competitive inhibitor of
diacylglycerol and prevents the interaction of this activator with the
ternary complex of PKC.15 The findings that diacylglycerol can increase phagocytosis, ERK activation, and PKC translocation to
the plasma membrane in PMN treated with sphingosine would support the
notion that diacylglycerol was required to permit PKC activation. We
also observed that PKC translocation to the plasma membrane could
occur in PMN pretreated with sphingosine after the addition of DiC10,
which supports the hypothesis that diacylglycerol is a competive
agonist with sphingosine in terms of regulatory PKC activity.
Additionally, it was observed that DiC10 augmented phagocytosis by
nonprimed PMN. In contrast, DiC10 failed to enhance phagocytosis beyond
control values in PMN primed with fMLP. Because fMLP activation leads
to diacylglycerol formation, the fMLP-dependent diacylglycerol formation may contribute to the priming effect mediated by
fMLP.36-38 Finally, sphingosine formation may contribute to
events terminating phagocytosis in activated PMN. Maximal generation of
sphingosine required 30 minutes of phagocytic activation. Others have
also reported that sphingosine is formed in PMN during
activation.39 The amount of sphingosine that was generated
was sufficient to inhibit phagocytosis of EIgG. In conclusion, these
studies indicate a mechanism by which endogenous sphingosine generation
can modulate PMN activation through inhibition of PKC activation and
subsequent phosphorylation of kinases that are required for
phagocytosis to occur.
| |
FOOTNOTES |
Submitted April 3, 1998;
accepted September 22, 1998.
Supported by Deutsche Forschungsgemeinschaft Grant No. Ra 789/1-1 (to
E.M.B.R.); by National Institutes of Health Grants No. AI20065 (to
L.A.B.) and DK41487 and DK39255 (to J.A.S.); and by The Danish Medical
Research Council (L.K.). J.A.S is an Established Investigator of the
American Heart Association.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Laurence A. Boxer, MD, Department of
Pediatrics, University of Michigan, F6515 Mott Children's Hospital,
Ann Arbor, MI 48109; e-mail: laboxer{at}umich.edu.
| |
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Abstract
Introduction