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Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4761-4769
Activation of a Plasma Membrane-Associated Neutral
Sphingomyelinase and Concomitant Ceramide Accumulation During
IgG-Dependent Phagocytosis in Human Polymorphonuclear Leukocytes
By
Vania Hinkovska-Galcheva,
Lars Kjeldsen,
Pamela J. Mansfield,
Laurence A. Boxer,
James A. Shayman, and
Suzanne J. Suchard
From the Department of Pediatrics, the Division of
Hematology/Oncology and the Department of Internal Medicine, the
Division of Nephrology, University of Michigan, Ann Arbor, MI.
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ABSTRACT |
The sphingomyelin cycle, which plays an important role in regulation
of cell growth, differentiation, and apoptosis, involves the formation
of ceramide by the action of a membrane-associated, Mg2+-dependent, neutral sphingomyelinase and/or a
lysosomal acid sphingomyelinase. In human polymorphonuclear leukocytes
(PMNs), ceramide production correlates with and plays a role in the
regulation of functional responses such as oxidant release and Fc
receptor-mediated phagocytosis. To increase our understanding of the
sphingomyelin cycle in human PMNs, the cellular location of neutral and
acid sphingomyelinases was investigated in resting,
formylmethionylleucylphenylalanine (FMLP)-activated, and FMLP-activated
PMNs engaged in phagocytosis. In resting PMNs, a
Mg2+-dependent, neutral sphingomyelinase was the
predominant activity and was localized to the plasma membrane fractions
along with the majority of ceramide. Upon FMLP-activation, there was a
1.9-fold increase in this neutral, Mg2+-dependent
sphingomyelinase activity, which increased to 2.7-fold subsequent to
phagocytosis of IgG opsonized targets. This increase in
sphingomyelinase activity was restricted to the plasma membrane fractions, which were also the site of increased ceramide levels. Phospholipase D (PLD) activity, which is a target of ceramide action
and is required for phagocytosis, was also found primarily in the
plasma membrane fractions of FMLP-activated and phagocytosing PMNs. Our
findings indicate that in human PMNs engaged in phagocytosis, the
sphingomyelin cycle is restricted to the plasma membrane where intracellular targets of ceramide action, such as PLD, are localized.
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INTRODUCTION |
POLYMORPHONUCLEAR leukocytes (PMNs) serve
a crucial role in host defense by phagocytosing and killing invading
microorganisms. An array of microbicidal activities are produced in
human PMNs and include several acid hydrolases, natural antibiotics,
and reactive oxygen species generated by assembly of the multicomponent NADPH oxidase.1,2 These substances are secreted by
activated PMNs from at least three granule subsets either
extracellularly or to the phagosome through fusion of granules with the
plasma membrane or the phagosomal membrane, respectively. The phagosome contains the opsonized particle that has been engulfed through binding
to Fc and/or complement receptors expressed on the PMN surface.
The metabolism of phospholipids has long been recognized as an
important event in intracellular signal transduction. Recently, it has
become evident that sphingolipid metabolism via the sphingomyelin cycle
plays an important regulatory role in growth factor responses, such as
proliferation, differentiation and apoptosis, gene regulation, and
intracellular vesicle transport.3-15 The sphingomyelin
cycle involves activation of sphingomyelinases that hydrolyze
sphingomyelin to form ceramide, a key second messenger of this cycle.
At least three distinct sphingomyelinase activities have been described in mammalian cells: (1) a lysosomal acid sphingomyelinase; (2) a
cytosolic, Mg2+-independent, neutral sphingomyelinase; and
(3) a membrane-associated, Mg2+-dependent, neutral
sphingomyelinase.16,17 By crude subcellular fractionation
the latter activity has been localized to membrane fractions in human
brain,18 bovine adrenal medulla,19 rat liver,20 human renal proximal tubular cells,21
and in cultured neuroblastoma cells, where it was suggested to be
externally oriented.22,23 The intracellular targets of
ceramide are not yet fully elucidated, but a recent report by Wiegmann
et al11 indicated that in U937 cells stimulated with tumor
necrosis factor (TNF), the subcellular topology of ceramide formation
determined its action because the acidic, endosomal sphingomyelinase
was involved in activation of the transcription factor NF- B, whereas
the neutral, plasma membrane-associated sphingomyelinase was involved
in activation of a proline directed ser/thr protein kinase. Ceramide
has also been shown to activate a cytosolic ser/thr protein
phosphatase, and thus could potentially regulate a protein
phosphorylation-dephosphorylation cascade.24,25
Recent data from this and other laboratories have indicated that PMN
functional responses are regulated by the sphingomyelin cycle.
Specifically, (1) preincubation of adherent PMNs with cell permeable
short-chain ceramides results in diminished
formylmethionylleucylphenylalanine (FMLP)-induced
H2O2 production and inhibition of phospholipase D (PLD) activity.26 (2) Concomitantly, ceramide is formed
in parallel with cessation of H2O2 production
and specific granule release in FMLP-stimulated PMNs adherent to
fibrinogen.26 (3) Exogenously added ceramide also inhibits
FMLP-induced superoxide formation and calcium influx in PMNs held in
suspension.27 (4) Exogenously added C2-ceramide
inhibits PLD activity, MAP kinase activation, and IgG-dependent
phagocytosis.28,29 Consistent with these findings,
IgG-dependent phagocytosis is accompanied by activation of a neutral
sphingomyelinase and generation of ceramide at a time when the rate of
ingestion is declining.28 Thus, in both phagocytosing and
adherent FMLP-activated PMNs, ceramides inhibit the activity of PLD and
affect PMN functional responses.26,28 This suggests that
both ceramide formation and PLD activation are occurring at the same
intracellular sites.
The subcellular topology of sphingomyelinase activity and ceramide
formation has not previously been addressed in human PMNs. It is
generally believed from investigations in other cell types that acidic
sphingomyelinase localizes to the lysosomal compartments, whereas
neutral sphingomyelinase is associated with plasma membranes. However,
PMNs are specialized cells with several intracellular compartments
including at least three granule subtypes (of which the azurophil
granules are believed to be the lysosomal compartment), and the highly
mobilizable secretory vesicles. These granules and vesicles fuse with
the plasma membrane during cell activation and introduce functional
proteins and enzymes to the cell surface.1 To increase our
understanding of the sphingomyelin cycle in PMNs, we investigated the
subcellular localization of sphingomyelinase activity and ceramide
formation in resting, FMLP-activated, and phagocytosing human PMNs.
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EXPERIMENTAL PROCEDURES |
Cells.
Human PMNs were isolated from peripheral blood donated by healthy
volunteers, as previously described.26 In short, blood was
anticoagulated in acid citrate dextrose and red blood cells (RBCs)
sedimented by addition of dextran (Abbott Laboratories, Abbott Park,
NC). Remaining RBCs were removed by hypotonic lysis, followed by centrifugation through Ficoll-Paque (Pharmacia LKB Biotechnology Inc, Piscataway, NJ) to remove mononuclear
cells. Before activation of cells and subcellular fractionation, cells were incubated for 5 minutes on ice with diisopropylfluorophosphate (DFP, 5 mmol/L; Sigma Chemical Co, St Louis, MO), washed,
and resuspended in the desired buffer.
Opsonization of sheep RBCs.
Sheep erythrocytes were purchased from Biowhittaker (Walkersville, MD).
Five milliliters of stock erythrocytes were washed three times with
buffer containing 2.5% dextrose, 0.05% gelatin, 2.5 mmol/L sodium
barbital (pH 7.5), 75 mmol/L NaCl, 0.15 mmol/L CaCl2, and
0.5 mmol/L MgCl2. Erythrocytes (109/mL) were
incubated for 30 minutes at 37°C with anti-sheep erythrocyte IgG
(1:300 to 1:500 dilution; Cappel, Durham, NC), followed by a 30-minute
incubation on ice. The dilution of antibody used was that which caused
slight aggregation of erythrocytes as determined by titration.
Antibody-treated erythrocytes (EIgG) were washed three times and
suspended in the same buffer for incubation with PMNs in suspension.
Phagocytosis of EIgG.
Phagocytosis assays were conducted essentially as outlined by Pommier
et al.30 PMNs, suspended at 2 × 106/mL in
phosphate-buffered saline (PBS) with 1 mmol/L CaCl2, 1 mmol/L MgCl2, were warmed to room temperature for 1 hour.
PMNs were activated with 100 nmol/L FMLP for 10 minutes at 37°C,
then EIgG (at 1 × 108/mL, final concentration) were
added and cells allowed to phagocytose for 30 minutes. The supernatant
after the first spin was saved to measure the amount of marker proteins
released during activation. EIgG that were not ingested were lysed
twice in ice-cold water and tonicity restored by addition of 0.6 mol/L
KCl. Phagocytosis was quantitated microscopically and expressed as the
number of EIgG ingested per 100 PMNs (phagocytic index). Control cells
(room temperature cells), FMLP-activated cells, and FMLP-activated, phagocytosing cells were pelleted by centrifugation and resupended in
cold disruption buffer (see below) at 1.5 to 5 × 107/mL for subsequent subcellular fractionation.
Subcellular fractionation.
DFP-treated PMNs (4°C control cells, room temperature cells,
FMLP-activated cells, or FMLP-activated, phagocytosing cells) were
resuspended at 1.5 to 5 × 107/mL in disruption buffer
(100 mmol/L KCl, 3 mmol/L NaCl, 1 mmol/L Na2ATP, 3.5 mmol/L
MgCl2, 10 mmol/L piperazine
N,N -bis2{ethane-sulfonic acid}, pH 7.2, containing 0.5 mmol/L phenylmethylsulfonyl fluoride [PMSF]). Cells were disrupted by
nitrogen cavitation as previously described.31 Nuclei and
intact cells were pelleted by centrifugation at 400g for 15 minutes (P1). Ten milliliters of postnuclear supernatant (S1) was applied on top of either a three-layer Percoll
(Pharmacia LKB Biotechnology) gradient (1.05/1.09/1.12
g/mL, 9 mL of each density32) or two-layer Percoll gradient
(1.05/1.12 g/mL, 14 mL of each density31) containing 0.5 mmol/L PMSF. The gradient was centrifuged at 37,000g for 30 minutes. This resulted for the two-layer gradient in three visible
bands from the bottom designated the  -band, containing azurophil
granules, the  -band containing specific and gelatinase granules, and
a -band containing light membranes including secretory vesicles and
plasma membranes, with the clear cytosol on top. On the three-layer
gradient, the -band was separated into a lower 1-band
containing specific granules and an upper 2-band
containing gelatinase granules. The gradient was collected in fractions
by aspiration from the bottom of the tube (25 fractions for two-layer
gradient, 35 fractions for three-layer gradient). All fractions were
assayed for myeloperoxidase (azurophil granule marker), lactoferrin
(specific granule marker), gelatinase (gelatinase granule marker), HLA
class I (plasma membrane marker), and latent alkaline phosphatase
(alkaline phosphatase activity only measurable in the presence of a
detergent, a marker for secretory vesicles). Except for alkaline
phosphatase enzymatic assay,33 all marker proteins were
measured by enzyme-linked immunosorbent assay (ELISA), as previously
described.32 For ceramide and sphingomyelin analysis,
Percoll was removed from fractions by ultracentrifugation, the
fractions washed once in PBS and stored at  20°C until
extracted for lipid analysis.
Assays for ceramide formation and sphingomyelinase activity.
Sphingomyelinase assays were always performed on the same day as cell
fractionation. Acid and neutral sphingomyelinase assays were based on
the method of Gatt et al34 using liposomes containing NBD-sphingomyelin (10 µmol/L substrate; Molecular Probes, Inc, Eugene, OR) and 30 µmol/L 1,2 dioleoyl-sn-glycerol-3
phosphocholine (Avanti, Alabastar, AL). The assay medium for the
neutral enzyme contained 50 mmol/L Tris-HCl (pH 7.2), 25 mmol/L KCl, 5 mmol/L MgCl2, and 100 µL of the subcellular fraction in a
total volume of 0.25 mL. Samples were probe sonicated and incubated at
37°C for 30 minutes in a temperature-regulated bath sonicator. The fluorescent product, NBD-ceramide, was isolated by partitioning the
assay mixture with 0.45 mL 2-propanol, 1.5 mL heptane, and 0.2 mL
water. Samples were centrifuged at 2,000g and 0.9 mL of the
upper phase transferred to a clean tube containing 0.35 mL of water to
remove traces of contaminating NBD-sphingomyelin. After repeat
centrifugation, the upper layer was analyzed on a fluorimeter (460 nm
excitation and 515 nm emission). Assays were performed in duplicate or
triplicate. Sphingomyelinase activity was expressed as picomoles of
sphingomyelin hydrolyzed per minute per milliliter
fraction. The acid sphingomyelinase was assayed similarly
in 125 mmol/L sodium acetate buffer, pH 5.0. To distinguish between the
acid and neutral sphingomyelinase activity, the assays were performed
in the presence of 0.05 mmol/L HgCl2, which augments the
acid but inhibits the neutral activity, or 1 mmol/L dithiothreitol, which inhibits the acid but augments the neutral
activity.18 Spingomyelinase assays were also performed in
the presence of 0.1% Triton X-100 (Sigma Chemical Co) to rule out the
presence of latent sphingomyelinase activity; ie, activity present in
secretory vesicles or granules that is not "available" in the
absence of detergent treatment. Although the enzyme activity was
somewhat higher (50%) in the presence of Triton X-100, the overall
distribution of sphingomyelinase activity in the gradient fractions was
the same in the presence and absence of detergent. Unless indicated, the data represent experiments conducted in the absence of detergent. Some sphingomyelinase assays were performed in the presence of protease
inhibitors (1 mmol/L PMSF, 10 µg/mL soybean trypsin inhibitor, 1 µg/mL leupeptin, 1 µg/mL aprotinin) to determine whether granule proteases affected sphingomyelinase activity.
Lipids were extracted by the method of Van Veldhoven and
Bell35 as recently described.36 Specifically,
material from subcellular fractions was pelleted at 100,000g,
extracted with methanol, and each sample mixed with chloroform and
water to obtain a chloroform:methanol:water ratio of 10:20:8. Samples
were vortexed and incubated at room temperature for 1 hour, centrifuged
for 10 minutes at 2,000g, and the supernatants transferred to
clean tubes. The pellets were re-extracted and both supernatants
combined. Chloroform and water were added to achieve a final ratio of
10:10:9 (chloroform:methanol:water). After vortexing and
centrifugation, the lower phase was removed, washed with equal volume
of 1 mol/L NaCl:methanol (9:1), and centrifuged. The lower phase was
transferred to a clean tube, stored at 20°C, and analyzed
within 48 hours. Total cellular ceramide was assayed by the method of
Preiss et al37 as previously reported.26
Phosphatidic acid (PA) and phosphatidylethanol (PEt) formation.
PMNs were resuspended at 1 × 107/mL in
PBS and labeled with
1-O-[3H]-octadecyl-sn-glycero-3-phosphocholine
(108 mol/L; Amersham, Arlington Heights, IL)
for 30 minutes at 37°C. The labeled cells were washed with PBS and
resuspended at 2 × 106 cells/mL in PBS containing 1 mmol/L Ca2+ and 1 mmol/L Mg2+. EtOH (200 mmol/L) was added for 5 minutes at 37°C. PMNs were activated with
FMLP and phagocytosis initiated as outlined above. Thirty minutes after
the addition of opsonized targets, EIgG not internalized were lysed,
and PMNs resuspended in cavitation buffer. Cells were fractionated as
described above and the lipids from each fraction were extracted
according to the method of Van Veldhoven and Bell.35 Assays
for [3H]-labeled PEt and PA were performed as previously
decribed.38
Protein measurement.
Protein was measured by the BCA protein kit from Pierce (Rockford,
IL) using bovine serum albumin as a standard.
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RESULTS |
Initial subcellular fractionation experiments of resting PMNs were
performed on three-layer Percoll density gradients, which resolve all
known mobilizable subsets of granules and secretory vesicles in human
PMNs.32 These include azurophil granules, identified by
myeloperoxidase; specific granules, identified by lactoferrin;
gelatinase granules, identified by gelatinase; and secretory vesicles,
identified by latent alkaline phosphatase (Fig 1, top panel). Secretory vesicles were
present in the light membrane region together with plasma membranes,
recognized by HLA class I. The profiles of the two markers differed,
with secretory vesicles being slightly denser and extending into the
gradient, while plasma membranes were slightly lighter and remained in
the upper part of the gradient where very little latent alkaline
phosphatase was detected (Fig 1, top panel). The cytosol was present
above the light membrane fractions, beginning with fraction number 25 and extending to fraction number 35. Subcellular fractions were assayed
for acid and neutral sphingomyelinase activity as described in
Experimental Procedures. The major activity detected was the neutral,
Mg2+-dependent sphingomyelinase, which colocalized with the
HLA plasma membrane marker (Fig 1, middle panel). The two profiles were
virtually superimposable, indicating that the neutral sphingomyelinase
was confined to the plasma membrane. No activity was detected in any of
the granule subsets.
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| Fig 1.
Subcellular localization of neutral sphingomyelinase and
ceramide in resting human PMNs. Isolated, DFP-treated PMNs at 1.5 to 5 × 107 cells/mL, were disrupted by nitrogen cavitation.
Ten milliliters of postnuclear supernatant was layered on a three-layer
Percoll density gradient and centrifuged as described in Experimental Procedures. The gradient was fractionated into 35 fractions by aspiration from the bottom of the tube. Fractions were assayed for
neutral sphingomyelinase, myeloperoxidase (MPO), lactoferrin, gelatinase, HLA class I, and latent alkaline phosphatase (latent AP).
Numbers are average of four experiments, normalized to a cell number of
3 × 108 cells, and expressed in percent of the total
amount measured in the fractions 1 through 35. The subcellular
distribution of ceramide is shown in the bottom panel. Ceramide was
measured as described in Experimental Procedures after removal of
Percoll by ultracentrifugation. The absolute value for neutral
sphingomyelinase activity in the peak fraction was 65.5 pmol
sphingomyelin hydrolyzed/min/mL. The peak value for ceramide was 0.44 nmol/mL. Results of one representative experiment are shown.
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In Fig 1, no detergent was included in the sphingomyelinase assays. To
rule out the possibility that there was latent sphingomyelinase activity in either secretory vesicle or granule fractions, we repeated
the sphingomyelinase assays in the presence of 0.1% Triton X-100. No
sphingomyelinase activity was detected in the granule fractions in the
presence of Triton X-100. In the light membrane fractions, there was a
50% increase in sphingomyelinase activity across all fractions, with
no change in the overall distribution of sphingomyelinase activity
(data not shown).
When the sphingomyelinase assay was performed in the absence of
Mg2+ and at pH 5.0, a small amount of activity was detected
in the light membrane fractions (data not shown). This activity most likely represented residual neutral sphingomyelinase activity because
it was completely inhibited by ionic mercury and enhanced by
dithiothreitol (Table 1), features of the
neutral sphingomyelinase.18,20 A small amount of
sphingomyelinase activity was also detected in the azurophil granule
fractions at pH 5.0. This activity was enhanced in the presence of
ionic mercury and inhibited by dithiothreitol (Table 1), characteristic
of a "true" acid sphingomyelinase. However, this acid
sphingomyelinase activity constituted less than 1% of the total PMN
sphingomyelinase activity. Recently, a cytosolic,
Mg2+-independent, neutral sphingomyelinase was isolated
from HL60 cells. We found that cytosolic activity constituted less than 1% of the total sphingomyelinase activity, indicating that in human
PMNs the predominant sphingomyelinase activity is a plasma membrane-associated, Mg2+-dependent, neutral
sphingomyelinase.
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Table 1.
Acid and Neutral Sphingomyelinase Activity in Azurophil
and Specific Granules and in Plasma Membranes in the Presence or
Absence of Dithiothreitol or Hg2+
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The involvement of ceramide in intracellular signaling raises an
important question as to its cellular localization. Subcellular fractions of resting PMNs were also analyzed for ceramide content (Fig
1, bottom panel). Ceramide was detectable in all granule subsets, but
the majority was present in the light membrane fractions colocalizing
with HLA. These results indicated that ceramide was primarily localized
to the plasma membrane fractions of resting PMNs. Interestingly,
sphingomyelin, the substrate of sphingomyelinases, was equally
distributed in all the granule subsets and light membrane fractions
(Table 2).
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Table 2.
Subcellular Distribution of Sphingomyelin in Granules
and Light Membranes (secretory vesicles/plasma membranes)
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Previous experiments from our laboratory showed that exogenously added
cell-permeable, short-chain ceramides inhibit phagocytosis of EIgG by
FMLP-activated PMNs.28 In addition, there is activation of
an Mg2+-dependent, neutral sphingomyelinase and ceramide
formation that occur during phagocytosis. To investigate the
subcellular localization of these events, control (resting),
FMLP-activated, and FMLP-activated phagocytosing PMNs were subjected to
subcellular fractionation. The gradient fractions were assayed for
neutral sphingomyelinase activity and ceramide. Because the majority of
sphingomyelinase activity was present in the light membrane fractions
in unactivated cells, subcellular fractionation was performed on
two-layer Percoll gradients. The two-layer gradient, when compared to
the three-layer gradient, improves the resolution between secretory
vesicles and plasma membranes but fails to separate specific granules
from gelatinase granules. When measurements were performed in intact cells in this and previous studies,28 the increase in
neutral sphingomyelinase activity was only observed during
phagocytosis; ie, we did not see an increase in sphingomyelinase
activity with FMLP-treatment alone (Fig 2).
The addition of protease inhibitors to these assays did not affect
sphingomyelinase activity, suggesting that granule-associated proteases
were not inhibiting activity in whole cell assays (data not shown).
This is in contrast to our findings in subcellular fractions, where an
increase in sphingomyelinase activity was observed in the plasma
membrane fractions after stimulation with FMLP (1.9-fold above control,
Figs 3 and 4).
Upon phagocytosis of EIgG by FMLP-primed PMNs, a further increase in
sphingomyelinase activity to 2.7-fold above control was observed (Figs
3 and 4). Sphingomyelinase activity increased similarly during
EIgG-mediated phagocytosis whether PMNs were pretreated with FMLP or
not pretreated with FMLP (Fig 3). These increases in sphingomyelinase
activity could not be explained by differences in the disruption of
PMNs or differences in the total number of disrupted PMNs loaded onto the gradient, because no significant difference could be shown between
control, FMLP-activated, and FMLP-activated phagocytosing cells in
total content of the plasma membrane marker HLA, or the granule markers
myeloperoxidase, lactoferrin, and gelatinase (Fig 4). As seen in Fig 4,
there was a significant increase in sphingomyelinase activity from
control to FMLP-activated cells, and also from FMLP-activated cells to
FMLP-activated cells engaged in phagocytosis. The activation of the
neutral sphingomyelinase occurred in the plasma membrane fractions
since the distribution profile of sphingomyelinase colocalized with the
HLA profile in control (as also shown above on the three-layer gradient), FMLP-stimulated, and FMLP-stimulated phagocytosing cells
(Fig 5).
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| Fig 2.
Sphingomyelinase activity was measured in intact PMNs
after FMLP activation, and after phagocytosis of EIgG. Sphingomyelinase activity was measured in control cells, FMLP-activated cells (100 nmol/L), and in cells phagocytosing EIgG (for 30 minutes) after preincubation with FMLP. Values represent the mean ± SD for three experiments.
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| Fig 3.
Subcellular fractionation of resting, FMLP-activated, and
phagocytosing human PMNs. Distribution profiles of neutral
sphingomyelinase and ceramide. Isolated and DFP-treated PMNs were
resuspended at 2 × 106/mL in PBS with CaCl2
(1 mmol/L) and MgCl2 (1 mmol/L), and warmed to room
temperature for approximately 1 hour. Cells were left untreated
(control), stimulated with FMLP (100 nmol/L) for 10 minutes (FMLP), or
stimulated with FMLP followed by addition of EIgG and incubation for 30 minutes (phagocytosis). Cells were resupended in disruption buffer,
disrupted by nitrogen cavitation, and the postnuclear supernatant
centrifuged on a two-layer Percoll density gradient. An equal number
(ranging from 1.8 to 3.3 × 108 cells) of control cells,
FMLP-activated cells, and phagocytosing, FMLP-activated cells were
fractionated in each experiment. The gradients were fractionated into
25 fractions by aspiration from the bottom of the tubes, and fractions
assayed for neutral sphingomyelinase (SMase) activity, and every second
fraction assayed for ceramide after removal of Percoll by
ultracentrifugation. Results are the average of three experiments,
normalized to a cell number of 3 × 108.
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| Fig 4.
Increase in neutral sphingomyelinase activity in
FMLP-activated and phagocytosing PMNs. Control, FMLP-activated, and
phagocytosing FMLP-activated cells were fractionated as described in
Experimental Procedures and in the legend to Fig 3. The bars show fold
increase (+SEM) above control (set to 1) of total measured activity
of neutral sphingomyelinase (SMase) and the various marker proteins after FMLP activation and phagocytosis. The total amount is calculated as the sum of measured amount in fractions 1 through 25, in nuclei and
unbroken cells, and in exocytosed material (cell supernatant after
activation). Data are the average of five experiments. *P < .05 compared with the control by paired t-test. None of the marker proteins differed significantly between control, FMLP, or
phagocytosing cells.
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| Fig 5.
Subcellular fractionation of resting, FMLP-activated, and
phagocytosing human PMNs. Distribution profiles of neutral
sphingomyelinase, HLA, and latent alkaline phosphatase. Cells were
processed for fractionation as described in Experimental Procedures and
in the legend to Fig 3. Fractions were assayed for neutral
sphingomyelinase activity, myeloperoxidase, lactoferrin, gelatinase,
HLA class I, and latent alkaline phosphatase. Numbers are the average
of three experiments (same data as shown in Fig 3), normalized to a
cell number of 3 × 108 cells, and expressed in percent of
the total amount measured in fractions 1 through 25. Latent alkaline
phosphatase is only shown in control cells, because secretory vesicles
are almost completely mobilized (and latent AP thus disappearing) after
FMLP stimulation. The localization of the majority of azurophil
granules (AG) and specific/gelatinase granules (SG+GG) is marked.
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The sphingomyelinase data presented in Fig 5 were obtained in the
absence of detergent. To rule out the possibility that the increase in
sphingomyelinase activity seen with FMLP activation and phagocytosis
was the result of the translocation of sphingomyelinase from an
intracellular store of secretory vesicles, the sphingomyelinase measurements were also repeated in the presence of 0.1% Triton X-100.
Similar to unactivated PMNs, there was an equal increase in
sphingomyelinase activity ( 50%) in every fraction from gradients of
control, FMLP-activated, and FMLP-activated phagocytosing PMNs (data
not shown). On the other hand, there was only an increase in alkaline
phosphatase activity with Triton X-100 treatment in control cells, in
agreement with all secretory vesicles being mobilized to the plasma
membrane (ie, no latent alkaline phosphatase) in FMLP-activated and
FMLP-activated phagocytosing cells. This supports our observation that
sphingomyelinase was present in the plasma membrane and not in
secretory vesicles, and that the effect of Triton X-100 appeared to be
a direct effect on the enzyme.
The activation of sphingomyelinase was accompanied by an accumulation
of ceramide, which was also confined to the plasma membrane (Fig 3). No
changes in ceramide content were observed in granule fractions (Fig 3).
Interestingly, the relative increase in ceramide levels during
phagocytosis was greater than the relative increase in sphingomyelinase
activity. In addition, ceramide levels increased substantially during
phagocytosis in both the presence and absence of FMLP. Interestingly,
the increase in ceramide levels in FMLP-primed cells was more than
additive to those seen with FMLP and EIgG alone.
PLD activation is a principal signal in PMN activation and has been
associated with the uptake of both complement- and IgG-opsonized particles.39-41 Furthermore, PLD is one of the
intracellular targets of ceramide action during
phagocytosis.28 Thus, we thought it was likely that PLD
activity would increase in the plasma membrane fractions in parallel
with ceramide formation during PMN activation. A unique property of PLD
which provides a specific assay for this enzyme is the
transphosphatidylation reaction in which an alcohol is transferred to
the phosphatidyl group of a phospholipid substrate to form a
phosphatidylalcohol.42 Cells prelabeled with
1-O-[3H]-octadecyl-sn-glycero-3-phosphocholine
and incubated in the presence of 200 mmol/L ethanol will synthesize
radioactive PEt if PLD is active. Figure 6
shows the cellular localization of PA and PEt formation in PMN
fractions. Both PA and PEt were found in azurophil, specific, and
plasma membrane fractions in control (resting) cells. In FMLP-activated
and FMLP-activated phagocytosing cells, PLD activity, as indicated by
PEt formation, was markedly greater in the plasma membrane fractions in
comparison with the granule fractions (Fig 6). These data show
increased PLD activity in plasma membranes during PMN activation,
indicating colocalization of ceramide with its cellular target, PLD.
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| Fig 6.
Subcellular fractionation of resting, FMLP-activated, and
phagocytosing human PMNs. Distribution profiles of the PLD products, PA
and PEt. PMNs were labeled with 1-O-[3H]
octadecyl-sn-glycero-3 phosphocholine, activated and
fractionated as described in Experimental Procedures. PA and PEt were
detected as previously described.38
|
|
| |
DISCUSSION |
In this report we describe the subcellular localization of
sphingomyelinases in human PMNs. Our findings clearly indicate that a
Mg2+-dependent, neutral sphingomyelinase is the predominant
form in PMNs, with localization confined to the plasma membrane. This confirms findings in several other cell types, where crude
fractionation localized the neutral sphingomyelinase to membranes
enriched in plasma membrane markers and possibly
microsomes.18-21,23 Our fractionation scheme allowed us to
compare the distribution profiles of various well-established markers
for the PMN subsets with the profile of sphingomyelinase activity. This
showed a strict colocalization with the plasma membrane marker HLA,
rather than with the secretory vesicle marker latent alkaline
phosphatase (Figs 1 and 5).
In the presence of ionic mercury, we detected an acidic
sphingomyelinase activity in azurophil granules, but this activity was
less than 1% of the neutral activity found in plasma membranes. The
low amount of acidic sphingomyelinase activity in subcellular fractions
confirms our findings in intact cells.28 Although azurophil
granules are rich in various acid hydrolases, this is unlikely to
explain the low level of acidic sphingomyelinase obtained because this
enzyme is believed to be insensitive to the action of phosphatases and
proteases.11 This is supported by the inability of protease
inhibitors to increase acid sphingomyelinase activity in these
fractions. The localization of the acidic activity in azurophil
granules, regarded as the lysosomal compartment of the PMN, is in
agreement with the lysosomal localization of acidic sphingomyelinases
in other cell types.16 It appears that PMNs are among the
relatively few cell types investigated thus far that largely contain
neutral sphingomyelinase rather than acidic sphingomyelinase.43 In comparison, acidic sphingomyelinase
activity has been found to be several-fold higher than the neutral
activity in rat liver and in the monocytic cell line,
U937.20,44
The subcellular localization of ceramide, the product of
sphingomyelinase action, has not previously been addressed, although it
was shown to increase concomitantly with sphingomyelinase activation in
isolated, TNF--activated membranes from HL60 cells.45
Others have described compartmentalization of ceramide to
sphingomyelin-rich membrane domains with the characteristics of
caveolae in interleukin-1 (IL-1)-activated
fibroblasts.46 However, it is unclear if caveolae are
present in human PMNs. The majority of the phosphatidylinositol-linked proteins including alkaline phosphatase, known to be concentrated in
caveolae in other cells, are primarily present in an intracellular compartment in resting PMNs, namely in secretory vesicles1.
Our fractionation data showed that neutral sphingomyelinase and
ceramide were localized in the plasma membrane fractions of resting as
well as activated PMNs, but did not allow any conclusions to be drawn
regarding compartmentalization within subdomains of the membranes.
Although sphingomyelin, the substrate for sphingomyelinase, was present
in equal amounts in the three granule subsets and light membrane
fractions (Table 2), it appears that only the plasma membrane pool
contributes to the sphingomyelin cycle.
A number of activators of sphingomyelinase have been identified,
including growth factors like TNF-, interferon-, and vitamin D3 in HL60 cells, TNF- in U937 cells, and IL-1 in
fibroblasts.6-8,11,45 We have now shown that Fc
receptor-mediated phagocytosis induces a plasma membrane-associated
neutral sphingomyelinase in FMLP-activated human PMNs. In contrast to
our measurements in intact cells, we were able to demonstrate that FMLP
activation alone resulted in an increase in sphingomyelinase activity
that was detected when cells were fractionated. The discrepancy between
intact and fractionated PMNs with regard to FMLP activation of
sphingomyelinase may be explained by segregation of the plasma
membrane-associated sphingomyelinase from an unidentified inhibitor of
sphingomyelinase activity during subcellular fractionation. For
example, Liu and Hannun43 reported that neutral
sphingomyelinase is inactive in the presence of physiological concentrations of glutathione. However, cytosolic glutathione is
unlikely to be the source of this inhibition because combining cytosol
with plasma membrane fractions did not diminish sphingomyelinase activity in our assays (data not shown). Similar experiments with granule and plasma membrane fractions also failed to implicate granules
as the source of this "inhibitory" activity. The activation of
neutral sphingomyelinase by FMLP in suspended, fractionated PMNs is in
agreement with the observation that FMLP induces ceramide accumulation
in PMNs adherent to fibrinogen.26 Future studies will
determine whether other inflammatory mediators that activate PMNs
through serpentine, G-protein coupled receptors such as IL-8, C5a,
leukotriene B4, and platelet-activating factor47 also
activate the neutral sphingomyelinase.
Surprisingly, the ceramide content of plasma membranes of phagocytosing
cells was twofold to fourfold greater than that of FMLP-stimulated
cells (Fig 3). This is considerably more than predicted from the
observed 50% difference in sphingomyelinase activity between
FMLP-stimulated and phagocytosing cells. This may be a consequence of
the assay conditions, because the sphingomyelinase measurements reflect
the enzymatic activity present at a single timepoint, whereas ceramide
was detected as an endpoint measurement and measures ceramide
accumulation. Alternatively, the observed difference may be explained
by discrepancies between FMLP-activated and phagocytosing PMNs in
activation of other enzymes regulating the ceramide content of
membranes, such as ceramidase, ceramide kinase, and ceramide synthase.
The sphingomyelin cycle has hitherto been implicated in responses such
as growth, differentiation, apoptosis, gene regulation, and
intracellular vesicle transport in a variety of cells and cell
lines.3-13 Gene transcription and protein synthesis are
unlikely to be of importance for the very rapid responses of PMNs to
soluble and particulate stimuli. The lack of significant lysosomal,
acidic sphingomyelinase activity in PMNs may support this notion,
because ceramide formed in endosomes/lysosomes as a result of
TNF--induced acid sphingomyelinase activity was reported to be
responsible for activation of the NF-B pathway11 and,
thus, for gene regulation in monocytic U937 cells. In PMNs, exogenously
added short-chain ceramides inhibit important PMN responses including
FMLP-induced oxidant release in adherent and suspended cells, and
Fc-receptor-mediated phagocytosis.26-28 Therefore, it
seems that the sphingomyelin cycle regulates membrane trafficking in
PMNs, in accordance with the findings of Pagano et al,12,13
who showed that short-chain ceramides inhibited fluid phase and
receptor-mediated endocytosis and membrane traffic in Chinese hamster
ovary cells.
The mechanisms underlying the effects of ceramide in PMNs are not
completely delineated, but the localization of ceramide in the plasma
membrane of activated cells indicates that the targets are likely to be
associated with or in proximity to the plasma membrane. One of the
targets of ceramide action during IgG-mediated phagocytosis is
PLD.28 This would suggest that a plasma membrane-associated PLD is activated during FMLP treatment and phagocytosis. Consistent with this hypothesis, increased levels of PEt, indicating increased PLD
activity, were found in the plasma membrane fractions of activated PMNs. These findings do not exclude the possibility that PLD and ceramide are in different, but adjacent, membrane compartments or
microdomains that interact during phagocytosis. Overall, our present
findings indicate that the sphingomyelin cycle in PMNs is restricted to
the plasma membrane during both FMLP activation and phagocytosis, and
that the intracellular targets of ceramide action, such as PLD, are
likely to be proximal to the sites of ceramide formation. This
localization is consistent with our hypothesis that ceramide, by
accumulating at strategic sites during activation, plays a negative
feedback role in modulating PMN phagocytosis.
| |
FOOTNOTES |
Submitted June 24, 1997;
accepted February 5, 1998.
V.H.-G. and L.K. contributed equally to this work.
Supported by National Institutes of Health Grants No. HL53074 (to
S.J.S.), AI20065 (to L.A.B.), DK41487 and DK39255 (to J.A.S.); a grant
from the American Heart Association of Michigan (S.J.S.); and by The
Danish Medical Research Council (L.K.). J.A.S. is an Established
Investigator of the American Heart Association.
Address reprint requests to Suzanne J. Suchard, PhD, Zeneca
Pharmaceuticals, 1800 Concord Pike, PO Box 15437, Wilmington, DE
19850-5437.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Isolati |
Abstract
Introduction
Abstract