Activation
of human
by three SANDRA THOMAS
separable
L. REINHOLD” M. McINTYRE2
‘Departments Institute,
neutrophil
of Utah,
D
mechanisms
STEPHEN
of Pharmacology,
University
phospholipase
tMedicine,
M. PRESCOTT,tI
and tBiochemistry,
Salt Lake City,
Utah 84112,
GUY
A. ZIMMERMAN,1
Nora Eccles Harrison
AND
Cardiovascular
Research and Training
USA
D, but neither is required for receptor-mediated activation of phospholipase D activity. Wortmannin is an irreversible inhibitor of the oxidative burst, but does not
of the elements that transduce the signal from the external environment into a functional response have been defined. Thus, GTP-binding proteins, an increase in intracellular Ca2 concentration, and protein kinases all play a role in PMN activation. Stimulatable phospholipase activities are also an important element in the activation of PMN. Activation of phospholipase C produces diglyceride, which can stimulate or inhibit protein kinase C depending on the nature of the sn-i bond (1), and can generate inositol triphosphate, which can stimulate an increase in intracellular free Ca2 concentration. Activated PMN also accumulate phospholipase A2 products (2, 3), which are precursors for lipid metabolites such as platelet-activating factor and leukotriene B4 (4), that amplify the inflammatory process. Recently it has been found that a variety of cells (5-10), PMN (11), and the promeylocytic HL6O cell line (12, 13) demonstrate a phospholipase D activity that can be stimulated. Thus, binding of an appropriate agonist to its receptor, or direct stimulation of protein kinase C (5,
inhibit
7, 8, 11, 12), leads
ABSTRACT Activation of human neutrophils by receptor-mediated agonists, the Ca2 ionophore A23187, or the protein kinase C activator phorbol myristate acetate all stimulated
phospholipase
D activity.
by the increased formation the presence of ethanol, accumulation.
EGTA
This
was
demonstrated
of phosphatidic acid, and in phosphatidylethanol (PEt)
completely
inhibited
A23187-
induced PEt formation, but only one-half of the fMLP-induced PEt accumulation. Staurosporin, an inhibitor of protein kinase C, strongly inhibited PMAinduced PEt formation, but actually stimulated the formation of PEt in response to fMLP by several-fold. Thus, increased cytosolic Ca2 and activated protein kinase
nal
C can
each
NADPH
lead
oxidase
transduction.
to activation
or known
Wortmannin
of phospholipase
components
inhibited
of sig-
activation
of
phospholipase D in response to fMPL. It did not directly inhibit phospholipase D, as the response to A23187 was
unaffected.
Wortmannin
stimulated
events,
such
did not inhibit as aggregation
other
fMPL-
or adherence.
We conclude that inhibition by wortmannin defines a third pathway to activation of phospholipase D. Further, its effect on phospholipase D correlates with its effect on the respiratory burst. -REINHOLD, S. L.; PRESCOTT, S. M.; ZIMMERMAN, G. A.; MCINTYRE, T M. Activation of human neutrophil D by three separable mechanisms. 208=214;
phospholipase FASEB J.
4:
1990.
Key Words: phospholipase mannin neutrophil
D
phosphatidylethanol
wort-
STIMULATION OF NEUTROPHIL OR polymorphonuclear leukocytes (PMN)3 by receptor-mediated agonists leads to responses, such as the respiratory burst, degranulation, aggregation, and adherence, that are essential to
the
208
role
of PMN
in the
suppression
of infection.
Several
to the
activation
of phospholipase
D.
The lipid product of the phospholipase D reaction, phosphatidic acid, may modulate cell function. It stimulates intracellular C a2 release (14) and inhibits adenylate cyclase (EC 4.6.1.1) (15) in whole cells. It is mitogenic (14, 16-18), and this mitogenic effect is totally dependent on c-ms activity (17). Phosphatidic acid also stimulates NADPH oxidase (which is responsible for the respiratory burst) in PMN lysates (19). Wortmannin is a fungal metabolite with potent antiinflammatory activity (20). Wortmannin ablates the respiratory burst of activated PMN, not by inhibiting the NADPH oxidase activity, but apparently by interfering with signal transduction in the stimulated PMN. The affected step is unknown (21). As activation of PMN
has
recently
been
shown
to stimulate
a phospho-
‘This work was performed as a partial fulfillment of the requirements for a Ph.D. degree in the Department of Pharmacology, University of Utah. 2Address to whom correspondence should be sent, at: CVRTI, Bldg. 100, University of Utah, Salt Lake City, UT 84112, USA. 3Abbreviations: fMLP, N-formyl-Met-Leu-Phe; PMN, neutro-
phil or polymorphonuclear leukocytes; PMA, phorbol myristate acetate; TLC, HEPES,
PEt, phosphatidylethanol; thin-layer chromatography;
N-2-hydroxyethylpiperazine-N’-ethanesulfonic
0892-6638/90/0004-0208/$01
acid.
.50.
©
FASEB
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lipase D activity, we examined the hypothesis that activation of phospholipase D is the process inhibited by wortmannin. We found that phospholipase D activity was inhibited by wortmannin, not by direct inhibition of the enzyme but by inhibition of a process leading to its activation. Although this inhibition correlated with inhibition of the respiratory burst, aggregation and adherence to a protein matrix were insensitive to the effects of wortmannin. The mechanism affected by wortmannin could be differentiated from two other events, activation of protein kinase C and an increase in intracellular Ca2, each of which also stimulated phospholipase D activity. Thus, wortmannin identified a novel route to the activation of phospholipase D activity in agonist-stimulated PMN. EXPERIMENTAL
OF HUMAN
NEUTROPHIL
cytochalasin B, and any this 5-mm preincubation
then initiated by centrated agonist A23187, 10 ,zM; 100 nM. Half ethanol. After 5 transferring
agents period.
to be present The assay was
the addition of 100 tl of 10-fold conor buffer. Final concentrations were fMLP, PAF, or LTB4, 1 tM; PMA, of the assays also contained 0.5% mm, the reaction was terminated by
the
entire
volume
to
glass
extraction
tubes containing 3.75 ml chloroform-methanol (1:2) plus carrier phosphatidylethanol. This Bligh and Dyer (23) monophase was split with 1.25 ml CHC13 and 1.25 ml water, the lower phase containing the phospholipids was dried under nitrogen, and the lipids were resolved by TLC on silica gel 60 with chloroform-methanolacetic acid (65:15:2) as before. The amount of radioactivity comigrating with a phosphatidylethanol standard (Rf 0.6) was determined by liquid scintillation spectrometry after scraping the gel into a scintillation vial. Each experiment has been performed a minimum of twice. The respiratory burst was measured by chemiluminescence in the presence of Luminol (24): 10 cells in 1 ml HBSS were preincubated with wortmannin or buffer for 3-10 mm before the mixture was transferred to scintillation vials containing 4 ml HBSS, 50 tM luminol, plus an agonist. The counts detected from a single photomultiplier tube were monitored over time by counting the vial for 10 s every 30 s. Response of control cells in the absence of agonist served to monitor the background luminescence. Cell aggregation was assayed with a Payton 800B cell =
PROCEDURES
The [9,10-3HJoleic (8.9 Ci/mmol) and tetra(triethylammonium) [-y-32P]adenosine 5’-triphosphate were from DuPont-New England Nuclear (Boston, Mass.). Staurosporin was obtained from Kamiya Biomedical Co. (Thousand Oaks, Calif.), diglyceride kinase from Lipidex (Westfield, N.J.), and Hanks’ buffered saline solution (HBSS) from M.A. Bioproducts (Walkersville, Md.). Diglyceride (dioleoyl), phosphatidylcholine (egg), and phosphatidic acid (egg) were from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Leukotriene B4 was kindly supplied by Dr. Joshua Rokach (Merck-Frosst Canada, Ontario, Canada). Phospholipase D (cabbage type I), A23187, fMLP, sphingosine, cytochrome c, phorbol 12-myristoyl 13-acetate, and luminol were from Sigma Chemical Corp. (St. Louis, Mo.). Wortmannin was the generous gift of Dr. T. G. Payne (Sandoz Research Institute, Berne, Switzerland). Phosphatidylethanol was prepared by dissolving 200 tg of phosphatidylcholine in 1 ml diethylether and adding this to 10 units of phospholipase D in I ml 80 mM Na acetate/140 mM CaC12 that contained 170 mM ethanol. This mixture was incubated at room temperature for 30 mm with continuous mixing. The lipids were then extracted with diethylether:ethanol (4:1) and the upper phase was dried under nitrogen before being separated by thin-layer chromatography (TLC) on silica gel 60 plates in chloroform-methanol-acetic acid (65:15:2) (13). Phospholipids were localized by 12 vapor. Phosphatidylethanol was identified by its unique appearance in reactions carried out in the presence of ethanol and by comparison to published Rf values (13). Phospholipase D activity in intact cells was assayed by first labeling the cells with [3H]oleate, treating the cells with an agonist or with buffer, and then determining the amount of [3H-oleoyl]phosphatidylethanol formed (8). Neutrophils were isolated as previously described (22). Cells (107/ml) were suspended in buffer A [137 mM NaC1, 5.4 mM KC1, 0.8 mM P04, 5.4 mM glucose, 10 mM HEPES (N-2-hydroxyethylpiperazine-N’-ethanesulfonic acid), pH 7.2, with freshly added 1 mM Ca2] and rocked for 10 mm at 37#{176}C in the presence of 2.5 tCi [3H]oleate/ml. Then 0.5 ml of labeled cells were added to microfuge tubes containing 0.4 ml buffer A, ACTIVATION
5 tg during
PHOSPHOLIPASE
D
aggregometer calibrated 2.75 x 106 cells/0.5 ml,
at and
0% transmission 1.38 x 106 cells for
with 100%
transmission as previously described (22). Cells in each cuvette were stirred continuously at 37#{176}C. After 2-3 mm to allow for stabilization of baseline, 50 tl of agonist was added to the cuvette and transmission monitored for approximately 5 mm. In some experiments cells were pre-exposed to 50 nM wortmannin for 2 mm before establishment of the baseline. Adherence of PMN
to gelatinized
as previously Diglyceride
tissue
described was
culture
dishes
was assayed
(25).
quantified
by the method
of Preiss
et
al. (26). Briefly, cells were extracted by the method Bligh and Dyer (23), and the diglyceride contained the organic phase was quantitatively converted [32P]phosphatidic
acid
eride kinase in the specific radioactivity. uct
(Rf
of 0.4)
was
by Esc/zerichia
coli-derived
presence of [32P]ATP The [32P]phosphatidic separated
with CHCI3:methanol:acetic mobile phase.
from
[32P]ATP
acid
(325:75:25)
of in to
diglyc-
of known acid prodby TLC
as the
RESULTS Receptor-mediated activation is inhibited by wortmannin Receptor-mediated and the protein
of phospholipase
D
agonists, the Ca2 ionophore A23187, kinase C activator PMA all stimulated 209
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the synthesis of phosphatidylethanol (Fig. 1). Since this unique phospholipid is generated by phospholipase D activity (13, 27), we conclude that each agonist stimulated phospholipase D by two- to fivefold. We also observed an increase in phosphatidic acid, the hydrolytic product of phospholipase D, in stimulated cells (not shown). We next examined the effect of pretreating the PMN with 50 nM wortmannin, a concentration that completely inhibited the respiratory burst (Fig. 2) (21, 28). PMN that had been pretreated with wortmannin did not show an alteration in the basal rate of phosphatidylethanol formation, nor did it demonstrate an altered response to A23187 or to PMA (Fig. 1). This result demonstrated that wortmannin did not directly inhibit phospholipase D. However, the response to each receptor-mediated agonist was reduced by pretreatment with wortmannin. The degree of inhibition varied for the
different
fMLP
being
agonists,
reduced
with
the
to the level
strong
activation
of PMA
by
activation,
and the weak response to LTB4 reduced to the basal value. Thus, wortmannin affected a component between the activation of a receptor by its agonist and the
phospholipase D activity. We found that wortmannin was an irreversible inhibitor of the process leading to phospholipase D activation, since three washes in buffered saline did not reverse the inhibition. Wortmannin appears to be an alkylating agent, as its effects are sensitive to modifications of its structure that decrease its nucleophilic nature (28), and its effect on the respiratory burst is also irreversible (21). This suggests that the inhibition of phospholipase D activation by wortmannin is similar to its inhibition of the respiratory burst in that both result from an irreversible, possibly covalent, modification of cellular protein (or proteins).
A23187
Control
PMA
tMLP
PAF
LTB4
Figure 1. Effect of wortmannin on receptorand non-receptormediated stimulation of PEt formation. Human PMN were prelabeled with [3H]oleate for 10 mm, exposed to 50 nM wortmannin in DMSO or solvent control for 5 mm, and then stimulated in the
presence
of 0.5% ethanol
and the stated agonist for 5 mm. Data are
presented as percent (mean ± SEM for three experiments) of the A23187 response (control, 3922 ± 1083 dpm: wortmannin-treated, 4399
210
± 1826 dpm).
Vol. 4
Feb. 1990
uw
w
o
(I,
E EQ. -J
0
1
2
Time
3
(mm)
Figure 2. Effect of wortmannin on the respiratory burst of fMLPstimulated PMN. Cells were exposed to 50 nM wortmannin for 3 mm before quantitation of luminol-dependent chemiluminescence during stimulation with M fMLP.
io
Activation of phospholipase D by fMLP depends on Ca21-dependent and -independent pathways Stimulation of phosphatidylethanol formation was induced by the Ca2 ionophore A23187, indicating that an increase in intracellular Ca2 was sufficient to activate phospholipase D. We examined the postulate that activation of phospholipase D in response to fMLP was due solely to a receptor-mediated lar Ca2’. We incubated PMN
increase in EGTA
in intracellufor 5 mm be-
fore stimulation to deplete both extracellular and intracellular Ca2 stores (21). This treatment was effective in depleting Ca2 as activation of phospholipase D by A23187 was completely inhibited (not shown). fMLPinduced activation of phospholipase D was also inhibited by the EGTA pretreatment, but the inhibition was only about one-half of the stimulated response (2725 vs. 1175 dpm in EGTA-treated cells). This suggests that fMLP induction of phospholipase D proceeds approximately equally by Ca2-dependent and Ca2independent pathways. The weak stimulation of phospholipase D activation by PMA was not affected by Ca2 availability. This is consistent with the ability of PMA to activate other PMN responses in the presence of EGTA (21). It also showed that PMA had not activated a voltage-dependent Ca2 gate (29). Activation of phospholipase D occurs kinase C-dependent and -independent
by protein pathways
Phospholipase D activity could be activated by PMA 1), although the level of stimulation was less than that induced by fMLP. However, induction of phospholipase D activity by this protein kinase C-dependent route was not inhibited by wortmannin, whereas fMLP-induced phospholipase D activity was inhibited. This suggested either that phospholipase D could be ac(Fig.
The FASEB Journal
REINHOLD
ET AL.
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tivated by a protein kinase C-independent route, or that protein kinase C activity was not appropriately activated in wortmannin-treated cells. The latter might occur if wortmannin blocked the accumulation of diglyceride, the endogenous lipid activator of protein kinase C, in response to fMLP. This would explain why PMA could induce phospholipase D activity and the respiratory burst (21, 28) in wortmannin-treated cells, since it would be supplying the missing activator. We examined this postulate by measuring the amount of diglyceride in control and wortmannin-treated PMN. The
diglyceride
content
increased
from
55 to 220 pmol/
nmol phospholipid over 180 s in fMLP-stimulated cells. This increase was not affected by wortmannin treatment. This implies that wortmannin does not exert its effect by altering the accumulation of diglyceride in stimulated PMN. We next determined whether activation of protein kinase C was essential for activation of phospholipase D in fMLP-stimulated cells by using staurosporin to inhibit the kinase. Staurosporin has previously been reported to inhibit protein kinase C activity in vitro, and to
inhibit
events
thought
to
be
mediated
by
protein
kinase C in intact cells (30). Staurosporin proved to be an effective inhibitor of PMA-induced phospholipase D activation (Fig. 3). Staurosporin did not, however, inhibit
fMLP-induced
activation
B
A
of phospholipase
D. In
fact, it led to a marked enhancement of phosphatidylethanol formation in fMLP-stimulated cells. This enhancement was evident throughout the period of IMLP stimulation, such that after 15 mm of stimulation, staurosporin-treated cells had accumulated 4.3 times as much phosphatidylethanol as untreated control cells. Staurosporin by itself had no effect on phospholipase D activity, so this result was not simply an additive effect.
4. Effect of wortmannin on fMLP-induced PMN aggregation. Aggregation in response to fMLP was measured after pretreatment with (A) buffer or (B) 50 nM wortmannin. Figure
A similar, although less dramatic, result when sphingosine, a structurally unrelated protein kinase C (31), was substituted for These data indicate that protein kinase sential for the activation of phospholipase to fMLP, and further that it apparently hibitory
exerts
an
in-
effect.
Wortmannin or adhesion
does
not prevent
PMN
aggregation
Since wortmannin is a potent inhibitor of the respiratory burst and PEt accumulation, and at higher concentrations inhibits neutrophil degranulation (21), we examined
next
its effect
on adhesion
and
aggregation
to
determine if it is a pleotropic inhibitor of PMN function. Under conditions where fMLP-induced oxygen radical production was totally inhibited (Fig. 2), fMLP-induced aggregation was unaffected (Fig. 4), and adherence to a gelatin matrix (Fig. 5) was reduced
300
by only
30%.
Increasing
the concentration
of wortman-
nm from 100 nM to 1 M did not increase the degree of inhibition of fMLP-induced adherence. Since these two adhesive responses were relatively unaffected under conditions where phospholipase D activity was significantly reduced, it appears that activation of phospholipase D, and the accumulation of its endogenous product phosphatidic acid, are not essential components in the signaling pathways that lead to the activation of PMN-adhesive glycoproteins.
200
0.0
0.5
[staurosporinej
1.0
DISCUSSION
SM
3. Effect of staurosporine on fMLPor PMA-stimulated PEt formation. Cells were prelabeled with [3H]oleate, exposed to the stated concentration of staurosporine for 5 mm, and then stimu-
Figure
lated with IMLP or PMA in the presence of 0.5% ethanol. Data are presented as percent of the stimulated response in the absence of staurosporine (2573 and 4726 dpm, respectively). The average increase in the IMLP response (n = 6) was 2.9 ± 1.2-fold, the average decrease (n - 5) of the PMA response was 64 ± 17%.
ACTIVATION
was obtained inhibitor of staurosporin. C was not esD in response
OF HUMAN
NEUTROPHIL
PHOSPHOLIPASE
PMN and other cells demonstrate tivity that utilizes phosphatidylcholine 11,
13).
clearly further
11).
The
products
phospholipase D acas a substrate (6,
phosphatidic
acid
and
choline
arise from a phospholipase D and are not simply metabolites of a phospholipase C reaction (9, Compelling
phosphatidic
evidence
acid
can
result
that
from
the
accumulation
of
a phospholipase
D
D
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We explored the tivity in stimulated
hypothesis that phospholipase D acPMN might be the site of wortman-
nm action, and that this activated phospholipase D therefore was essential in signal transduction leading to 02 and H2O2 generation. We found that phospholipase D activity itself was not inhibited by wortmannin. However, its activation was affected by wortmannin treatment when PMN were stimulated with receptormediated hibition
1o
[fMLPI, Figure
PMN with
M
5. Effect of wortmannin on IMLP-induced adherence of to a gelatin matrix. Adherence of PMN pretreated, or not, 100 nM wortmannin
a function
of IMLP
to gelantinized
plastic
was
examined
as
concentration.
reaction is the accumulation of phosphatidylalcohols when primary alcohols, such as ethanol, are present (7, 8, 10-13). This is a well-defined reaction of plant phospholipase D where a phosphatidyl-phospholipase D intermediate can be attacked either by water or alcohol (27). These phospholipids are not normal cellular constituents, and there is no known mechanism, other than transphosphatidylation, by which mammalian cells form such phospholipids. The formation of phosphatidylethanol is therefore a sensitive and specific assay for phospholipase D activity (7). PMN phospholipase D activity undergoes a marked enhancement in cells that have been stimulated with a variety of agonists. The role for the natural product of this reaction, phosphatidic acid, has not been defined in stimulated PMN. However, it increases intracellular Ca2, apparently by inducing the release of intracellular stores of Ca2 (14), and it stimulates NADPH oxidase activity in the lysates from unstimulated cells (19). Although it is not known if such activation occurs in intact cells, our data show that the oxidative burst is much more closely associated with activation of phospholipase D than are adhesive functions of stimulated PMN. The fungal metabolite wortmannin, which inhibits the
respiratory
burst
in
PMN
without
inhibiting
Vol. 4
Feb. 1990
Thus, the site of wortmannin to stimulation of phospholipase
inD.
A scheme consistent with these observations is presented in Fig. 6. Activation of a receptor, and its functionally coupled G protein (11, 12), leads to the activation of phospholipase D by three separable, but interacting, routes. Receptor activation leads to the translocation of protein kinase C and its activation (32). Since PMA stimulated phospholipase D function, one route to stimulated phosphatidic acid formation depends on the activation of protein kinase C. This is consistent with the observations of Tettenborn and Mueller (12) that pretreatment of HL6O cells with PMA enhances the subsequent formation of phosphatidylethanol in cell lysates. These authors also observed that GTP-yS activated phospholipase D by an independent mechanism. Activation of phospholipase D by protein kinase C has also been observed in whole cells (5). Although we found that direct activation of protein kinase C stimulated phosphatidylethanol formation, activation of this kinase did not appear to be essential for the activation of phospholipase D. This was suggested
by the
effects
of staurosporin
or sphingosine,
which prevented activation by PMA, hibit receptor-induced phospholipase Similarly,
receptor
activation
by
but did not D activation. fMLP
leads
in-
to an
increase in intracellular free Ca2 concentration (21). Since directly increasing the Ca2 concentration with A23187 leads to activation of phospholipase D activity,
the
NADPH oxidase activity (21), promises to be a valuable tool to dissect the signal transduction pathways in activated PMN. Its intriguing attribute is that it does not inhibit any of the known components of the signal transduction pathway. Thus, it apparently does not inhibit receptor function, receptor-coupled G proteins, phosphoinositide metabolism, the increase in intracellular free Ca2, or the catalytic activity of protein kinase C (21). We found that it also did not interfere with receptor-mediated accumulation of diglyceride, or aggregation or adhesion to a protein matrix induced by fMLP. Thus, wortmannin must exert its effect on an unidentified Ca2-insensitive component (or components) whose function was essential for the activation of the respiratory burst. 212
agonists. is proximal
C&’2
0
#{176}--.‘..pKC
Phospholipase Figure
6. Scheme
for three
separate
tion of phospholipase D. The IMLP lipase D activity by a Ca2-dependent, and a wortmannin-inhibitable designated with a dashed
The FASEB Journal
route.
routes
that
receptor
lead
to the stimula-
activates
phospho-
protein kinase C-dependent, Modulatory interactions are
arrow.
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ET AL.
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this second Ca2-dependent. centration
route by
to phospholipase D activation is Although it increased the Ca2 con-
entry
of
external
Ca2
activated
phos-
pholipase D, this event was not essential for its activation. Cells stimulated in the presence of extracellular EGTA, to prevent Ca2 entry, retained half of their fMLP-stimulated phosphatidylethanol formation. Therefore, both Ca2-dependent and Ca2’-independent pathways lead to the activation of phospholipase D. Part of the function of the increase in intracellular Ca2 was to activate protein kinase C. This was shown by the inhibition of 50% of the A23187-induced formation of phosphatidylethanol by inhibitors of protein kinase C (not shown). Thus, although either direct activation of protein kinase C or increasing the concentration of intracellular Ca2 activated phospholipase D, neither appeared to be essential for phospholipase D activation by receptor-mediated agonists. We conclude that there is a third pathway leading to phospholipase D activation in receptor-activated cells, and it is this pathway that is sensitive to wortmannin. This conclusion is based on the finding that wortmannm inhibited fMLP-dependent activation, but not that due to Ca24 influx or activation of protein kinase C. The components of this signaling pathway are currently undefined. It is, however, clear that this pathway was not the only route to phospholipase D activation, since wortmannin
only
depressed
fMLP-induced
phosphati-
dylethanol formation to the level of activity produced by direct activation of protein kinase C. Stimulation of phospholipase D, perhaps by the wortmannin-sensitive pathway, was also inhibited by protein kinase C. This became evident when staurosporin or sphingosine was found to markedly stimulate fMLP-induced
phosphatidylethanol
formation
under
We thank study.
Dr.
We thank
T Eric
G.
Payne
Stroud
for supplying for his analysis
wortmannin
for
this
of diglyceride content for their preparation of
of PMN, Holly Nichols and Susan Cowley PMN, and Margaret Vogel for her technical assistance. This work was supported by funds from the Nora Eccles Treadwell Foundation, grants HL35828 and HL34127 from the National Institutes of Health, and Established Investigator awards (G. A. Z., S. M. P.) from
the
American
Heart
Association.
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15370-15376 5. Cabot, M. C., Welsh, C. J., Zhang, dence for a protein kinase C-directed diester-induced phospholipase D generation from phosphatidylcholine.
6. Loffelholz,
K. (1989) Receptor
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regulation
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lipid tidic
hydrolysis: a novel source of diacylglycerol and phosphaacid. Biochem. Pharmacol. 38, 1543-1549 7. Pai,J. K., Liebl, E. C., Tettenborn, C. S., Ikegwuonu, F. I., and Mueller, G. C. (1987) 12-O-Tetradecanoylphorbol-13-acetate activates the synthesis of phosphatidylethanol in animals cells exposed to ethanol. Carcinogenesis 8, 173-178 8. Liscovitch, M. (1989) Phosphatidylethanol biosynthesis in ethanol-exposed NG1O8-l5 neuroblastoma x glioma hybrid
conditions where the PMA-induced effect was sharply cells: evidence for activation of a phospholipase D phosphatidyl attenuated. This inhibitory effect of protein kinase C transferase activity by protein kinase C. J. Biol. Chem. 264, might be at least partially responsible for the inability 1450-1456 of PMA to activate phospholipase D to the same extent 9. Bocckino, S. B., Blackmore, P. F., Wilson, P. B., and Exton, as the Ca24 ionophore. This mechanism might also J. H. (1987) Phosphatidate accumulation in hormone-treated affect the amount of lipase activation by the various hepatocytes via a phospholipase D mechanism. J. Biol. Chem. 262, 15309-15315 agonists, if the different PMN agonists activate protein 10. Martin, T. W., and Michaelis, K. (1989) P2-purinergic agonists kinase C to varying extents. stimulate phosphodiesteratic cleavage of phosphatidylcholine in The observation that aggregation and adherence in endothelial cells. j Biol. Chem. 264, 8847-8856 response to LMLP was either not affected, or only 11. Agwu, D. E., McPhail, L. C., Chabot, M. C., Daniel, L. W., minimally so, under conditions where the respiratory Wylde, R. L., and McCall, C. E. (1989) Choline-linked phosphoglycerides: a source of phosphatidic acid and diglycerides in burst was completely inhibited shows that wortmannin stimulated neutrophils. j BioL Chem. 264, 1405-1413 affected a pathway that did not lead to the activation of 12. Tettenborn, C. S., and Mueller, G. C. (1988) 12-0-Tetrathe adhesive glycoprotein CD11 b/CD18. Adhesive indecanoylphorbol-13-acetate activates phosphatidylethanol and teractions in these experiments are completely depenphosphatidylglycerol synthesis by phospholipase D in cell dent on activation of this heterodimer (25). The data lysates. Biochem. Biophys. Res. Commun. 155, 249-255 13. Pai,J. K., Siegel, M. I., Egan, R. W., and Billah, M. M. (1988) also suggest that the activation of phospholipase D was Phospholipase D catalyzes phospholipid metabolism in chemonot an essential event in the activation of CD11b/CD18, tactic peptide-stimulated HL-60 granulocytes. j Biol. Chem. since phospholipase D was significantly inhibited under 263, 12472-12477 conditions where there was only a trivial effect on adhe14. Moolenaar, W. H., Kruijer, W., Tilly, B. C., Verlaan, I., Bierman, A. J., and de Laat, S. W. (1986) Growth factor-like action sive responses. Thus, although the nature of this novel of phosphatidic acid. Nature (London) 323, 171-173 pathway remains to be defined, wortmannin has clearly 15. Murayama, T., and Ui, M. (1987) Phosphatidic acid may dissociated signal transduction leading to activation of stimulate membrane receptors mediating adenylate cyclase inthe respiratory burst from that which leads to PMN adhibition and phospholipid breakdown in 3T3 fibroblasts. j Biol. herence. E!II Chem. 262, 5522-5529
ACTIVATION
OF HUMAN
NEUTROPHIL
PHOSPHOLIPASE
D
213
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16. Imagawa, W., Bandyopadhyay, G. K., Wallace, D., and Nandi, S. (1989) Phospholipids containing polyunsaturated fatty acyl
groups cells
are mitogenic in serum-free
for normal primary
mouse
cell culture.
mammary
epithelial Sci.
Proc. NatL
Acad.
USA 86, 4122-4126 17. Yu, C. L., Tsai, M. H., and Stacey, D. W. (1988) Cellular ras activity and phospholipid metabolism. Cell 52, 63-71 18. Siegmann, D. W. (1987) Stimulation of quiescent 3T3 cells by phosphatidic acid-containing liposomes. Biochem. Biophys. Res. Commun. 145, 228-233 19. Bellavite, P., Corso, F., Dusi, S., Grzeskowiak, M., DellaBianca, V., and Rossi, F. (1988) Activation of NADPHdependent superoxide production of pig neutrophils by phosphatidic 8210-8214
20. Wiesinger,
D., Gubler,
(1974) Antiinfiammatory 1l-desacetoxy-wortmannin ha 30, 135-136
21. Dewald, duction
in plasma acid.
membrane
extracts
J. Biol. Chem. 263,
H. U., Haeflinger, W., and Hauser, D. activity of the new mold metabolite and
some
of its derivatives.
Experien-
B., Thelen, M., and Baggiolini, M. (1988) Two transsequences are necessary for neutrophil activation by agonists. j Biol. Chem. 263, 16179-16184
receptor 22. Zimmerman, G. (1985) Thrombin human endothelial
A.,
McIntyre,
T
M.,
and
Prescott,
S. M.
stimulates
the adherence of neutrophils to cells in vitro. j Clin. Invest. 76, 22 35-2246 23. Bligh, E. G., and Dyer, W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. PhysioL 37, 911-917 24. Allen, R. C. (1984) Chemiluminescence study of phagocyte biochemistry and nisms. In Analytical Applications of luminescence (Kricka, L. J., Stanley, P. Whitehead, T. P., eds) pp. 279-288,
214
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as an approach humoral immune
Bioluminescence E., Thorpe, Academic,
to the mecha-
and Chemi-
25. Zimmerman, G. A., and McIntyre, T. M. (1988) Neutrophil adherence to human endothelium in vitro occurs by CDwI8 (Mol,
MAC-l/LFA-1/GP
150,95)
glycoprotein-dependent
and
-inde-
pendent mechanisms. j Clin. Invest. 81, 531-537 26. Preiss, J., Loomis, C. R., Bishop, W. R., Stein, R., Niedel, J. E., and Bell, R. M. (1986) Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and rasand sis-transformed normal rat kidney cells. j Biol. Chem. 261, 8597-8600 27. Yang, S. F., Freer, S., and Benson, A. A. (1967) Transphosphatidylation by phospholipase D. j Biol. Client. 242, 477-484 28. Baggiolini, M., Dewald, B., Schnyder, J., Ruch, W., Cooper, P. H., and Payne, T G. (1987) Inhibition of the phagocytosisinduced respiratory burst by the fungal metabolite wortmannin and some analogues. Exp. Cell Res. 169, 408-418 29. Hockberger, P., Toselli, M., Swandulla, D., and Lux, H. D. (1989) A diacylglycerol analogue reduces neuronal calcium currents independently of protein kinase C activation. Nature (London) 338, 340-342 30. Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto, M., and Tomita, F. (1986) Staurosporine, a potent inhibitor of
phospholipid/Ca2 dependent Res. Commun. 135, 397-402 31. Hannun,
Y. A.,
Loomis,
R. M. (1986) Sphingosine
protein
C. R.,
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Merrill,
inhibition
A.
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Biochem. Biophys. H., Jr.,
and
Bell,
kinase C activity
and of phorbol dibutyrate binding in vitro and in human platelets. J. BioL Chem. 261, 12604-12609 32. Nishihira, J., McPhail, L. C., and O’Flaherty, J. T. (1986) Stimulus-dependent mobilization of protein kinase C. Biochem.
Biophys. Res. Commun.
G. H. G., and New York
The FASEB Journal
134, 587-594 Received for publication August 18, 1989. Acceptedfor publication October 10, 1989.
REINHOLD
El AL.
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of human
by three SANDRA THOMAS
separable
L. REINHOLD” M. McINTYRE2
‘Departments Institute,
neutrophil
of Utah,
D
mechanisms
STEPHEN
of Pharmacology,
University
phospholipase
tMedicine,
M. PRESCOTT,tI
and tBiochemistry,
Salt Lake City,
Utah 84112,
GUY
A. ZIMMERMAN,1
Nora Eccles Harrison
AND
Cardiovascular
Research and Training
USA
D, but neither is required for receptor-mediated activation of phospholipase D activity. Wortmannin is an irreversible inhibitor of the oxidative burst, but does not
of the elements that transduce the signal from the external environment into a functional response have been defined. Thus, GTP-binding proteins, an increase in intracellular Ca2 concentration, and protein kinases all play a role in PMN activation. Stimulatable phospholipase activities are also an important element in the activation of PMN. Activation of phospholipase C produces diglyceride, which can stimulate or inhibit protein kinase C depending on the nature of the sn-i bond (1), and can generate inositol triphosphate, which can stimulate an increase in intracellular free Ca2 concentration. Activated PMN also accumulate phospholipase A2 products (2, 3), which are precursors for lipid metabolites such as platelet-activating factor and leukotriene B4 (4), that amplify the inflammatory process. Recently it has been found that a variety of cells (5-10), PMN (11), and the promeylocytic HL6O cell line (12, 13) demonstrate a phospholipase D activity that can be stimulated. Thus, binding of an appropriate agonist to its receptor, or direct stimulation of protein kinase C (5,
inhibit
7, 8, 11, 12), leads
ABSTRACT Activation of human neutrophils by receptor-mediated agonists, the Ca2 ionophore A23187, or the protein kinase C activator phorbol myristate acetate all stimulated
phospholipase
D activity.
by the increased formation the presence of ethanol, accumulation.
EGTA
This
was
demonstrated
of phosphatidic acid, and in phosphatidylethanol (PEt)
completely
inhibited
A23187-
induced PEt formation, but only one-half of the fMLP-induced PEt accumulation. Staurosporin, an inhibitor of protein kinase C, strongly inhibited PMAinduced PEt formation, but actually stimulated the formation of PEt in response to fMLP by several-fold. Thus, increased cytosolic Ca2 and activated protein kinase
nal
C can
each
NADPH
lead
oxidase
transduction.
to activation
or known
Wortmannin
of phospholipase
components
inhibited
of sig-
activation
of
phospholipase D in response to fMPL. It did not directly inhibit phospholipase D, as the response to A23187 was
unaffected.
Wortmannin
stimulated
events,
such
did not inhibit as aggregation
other
fMPL-
or adherence.
We conclude that inhibition by wortmannin defines a third pathway to activation of phospholipase D. Further, its effect on phospholipase D correlates with its effect on the respiratory burst. -REINHOLD, S. L.; PRESCOTT, S. M.; ZIMMERMAN, G. A.; MCINTYRE, T M. Activation of human neutrophil D by three separable mechanisms. 208=214;
phospholipase FASEB J.
4:
1990.
Key Words: phospholipase mannin neutrophil
D
phosphatidylethanol
wort-
STIMULATION OF NEUTROPHIL OR polymorphonuclear leukocytes (PMN)3 by receptor-mediated agonists leads to responses, such as the respiratory burst, degranulation, aggregation, and adherence, that are essential to
the
208
role
of PMN
in the
suppression
of infection.
Several
to the
activation
of phospholipase
D.
The lipid product of the phospholipase D reaction, phosphatidic acid, may modulate cell function. It stimulates intracellular C a2 release (14) and inhibits adenylate cyclase (EC 4.6.1.1) (15) in whole cells. It is mitogenic (14, 16-18), and this mitogenic effect is totally dependent on c-ms activity (17). Phosphatidic acid also stimulates NADPH oxidase (which is responsible for the respiratory burst) in PMN lysates (19). Wortmannin is a fungal metabolite with potent antiinflammatory activity (20). Wortmannin ablates the respiratory burst of activated PMN, not by inhibiting the NADPH oxidase activity, but apparently by interfering with signal transduction in the stimulated PMN. The affected step is unknown (21). As activation of PMN
has
recently
been
shown
to stimulate
a phospho-
‘This work was performed as a partial fulfillment of the requirements for a Ph.D. degree in the Department of Pharmacology, University of Utah. 2Address to whom correspondence should be sent, at: CVRTI, Bldg. 100, University of Utah, Salt Lake City, UT 84112, USA. 3Abbreviations: fMLP, N-formyl-Met-Leu-Phe; PMN, neutro-
phil or polymorphonuclear leukocytes; PMA, phorbol myristate acetate; TLC, HEPES,
PEt, phosphatidylethanol; thin-layer chromatography;
N-2-hydroxyethylpiperazine-N’-ethanesulfonic
0892-6638/90/0004-0208/$01
acid.
.50.
©
FASEB
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lipase D activity, we examined the hypothesis that activation of phospholipase D is the process inhibited by wortmannin. We found that phospholipase D activity was inhibited by wortmannin, not by direct inhibition of the enzyme but by inhibition of a process leading to its activation. Although this inhibition correlated with inhibition of the respiratory burst, aggregation and adherence to a protein matrix were insensitive to the effects of wortmannin. The mechanism affected by wortmannin could be differentiated from two other events, activation of protein kinase C and an increase in intracellular Ca2, each of which also stimulated phospholipase D activity. Thus, wortmannin identified a novel route to the activation of phospholipase D activity in agonist-stimulated PMN. EXPERIMENTAL
OF HUMAN
NEUTROPHIL
cytochalasin B, and any this 5-mm preincubation
then initiated by centrated agonist A23187, 10 ,zM; 100 nM. Half ethanol. After 5 transferring
agents period.
to be present The assay was
the addition of 100 tl of 10-fold conor buffer. Final concentrations were fMLP, PAF, or LTB4, 1 tM; PMA, of the assays also contained 0.5% mm, the reaction was terminated by
the
entire
volume
to
glass
extraction
tubes containing 3.75 ml chloroform-methanol (1:2) plus carrier phosphatidylethanol. This Bligh and Dyer (23) monophase was split with 1.25 ml CHC13 and 1.25 ml water, the lower phase containing the phospholipids was dried under nitrogen, and the lipids were resolved by TLC on silica gel 60 with chloroform-methanolacetic acid (65:15:2) as before. The amount of radioactivity comigrating with a phosphatidylethanol standard (Rf 0.6) was determined by liquid scintillation spectrometry after scraping the gel into a scintillation vial. Each experiment has been performed a minimum of twice. The respiratory burst was measured by chemiluminescence in the presence of Luminol (24): 10 cells in 1 ml HBSS were preincubated with wortmannin or buffer for 3-10 mm before the mixture was transferred to scintillation vials containing 4 ml HBSS, 50 tM luminol, plus an agonist. The counts detected from a single photomultiplier tube were monitored over time by counting the vial for 10 s every 30 s. Response of control cells in the absence of agonist served to monitor the background luminescence. Cell aggregation was assayed with a Payton 800B cell =
PROCEDURES
The [9,10-3HJoleic (8.9 Ci/mmol) and tetra(triethylammonium) [-y-32P]adenosine 5’-triphosphate were from DuPont-New England Nuclear (Boston, Mass.). Staurosporin was obtained from Kamiya Biomedical Co. (Thousand Oaks, Calif.), diglyceride kinase from Lipidex (Westfield, N.J.), and Hanks’ buffered saline solution (HBSS) from M.A. Bioproducts (Walkersville, Md.). Diglyceride (dioleoyl), phosphatidylcholine (egg), and phosphatidic acid (egg) were from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Leukotriene B4 was kindly supplied by Dr. Joshua Rokach (Merck-Frosst Canada, Ontario, Canada). Phospholipase D (cabbage type I), A23187, fMLP, sphingosine, cytochrome c, phorbol 12-myristoyl 13-acetate, and luminol were from Sigma Chemical Corp. (St. Louis, Mo.). Wortmannin was the generous gift of Dr. T. G. Payne (Sandoz Research Institute, Berne, Switzerland). Phosphatidylethanol was prepared by dissolving 200 tg of phosphatidylcholine in 1 ml diethylether and adding this to 10 units of phospholipase D in I ml 80 mM Na acetate/140 mM CaC12 that contained 170 mM ethanol. This mixture was incubated at room temperature for 30 mm with continuous mixing. The lipids were then extracted with diethylether:ethanol (4:1) and the upper phase was dried under nitrogen before being separated by thin-layer chromatography (TLC) on silica gel 60 plates in chloroform-methanol-acetic acid (65:15:2) (13). Phospholipids were localized by 12 vapor. Phosphatidylethanol was identified by its unique appearance in reactions carried out in the presence of ethanol and by comparison to published Rf values (13). Phospholipase D activity in intact cells was assayed by first labeling the cells with [3H]oleate, treating the cells with an agonist or with buffer, and then determining the amount of [3H-oleoyl]phosphatidylethanol formed (8). Neutrophils were isolated as previously described (22). Cells (107/ml) were suspended in buffer A [137 mM NaC1, 5.4 mM KC1, 0.8 mM P04, 5.4 mM glucose, 10 mM HEPES (N-2-hydroxyethylpiperazine-N’-ethanesulfonic acid), pH 7.2, with freshly added 1 mM Ca2] and rocked for 10 mm at 37#{176}C in the presence of 2.5 tCi [3H]oleate/ml. Then 0.5 ml of labeled cells were added to microfuge tubes containing 0.4 ml buffer A, ACTIVATION
5 tg during
PHOSPHOLIPASE
D
aggregometer calibrated 2.75 x 106 cells/0.5 ml,
at and
0% transmission 1.38 x 106 cells for
with 100%
transmission as previously described (22). Cells in each cuvette were stirred continuously at 37#{176}C. After 2-3 mm to allow for stabilization of baseline, 50 tl of agonist was added to the cuvette and transmission monitored for approximately 5 mm. In some experiments cells were pre-exposed to 50 nM wortmannin for 2 mm before establishment of the baseline. Adherence of PMN
to gelatinized
as previously Diglyceride
tissue
described was
culture
dishes
was assayed
(25).
quantified
by the method
of Preiss
et
al. (26). Briefly, cells were extracted by the method Bligh and Dyer (23), and the diglyceride contained the organic phase was quantitatively converted [32P]phosphatidic
acid
eride kinase in the specific radioactivity. uct
(Rf
of 0.4)
was
by Esc/zerichia
coli-derived
presence of [32P]ATP The [32P]phosphatidic separated
with CHCI3:methanol:acetic mobile phase.
from
[32P]ATP
acid
(325:75:25)
of in to
diglyc-
of known acid prodby TLC
as the
RESULTS Receptor-mediated activation is inhibited by wortmannin Receptor-mediated and the protein
of phospholipase
D
agonists, the Ca2 ionophore A23187, kinase C activator PMA all stimulated 209
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the synthesis of phosphatidylethanol (Fig. 1). Since this unique phospholipid is generated by phospholipase D activity (13, 27), we conclude that each agonist stimulated phospholipase D by two- to fivefold. We also observed an increase in phosphatidic acid, the hydrolytic product of phospholipase D, in stimulated cells (not shown). We next examined the effect of pretreating the PMN with 50 nM wortmannin, a concentration that completely inhibited the respiratory burst (Fig. 2) (21, 28). PMN that had been pretreated with wortmannin did not show an alteration in the basal rate of phosphatidylethanol formation, nor did it demonstrate an altered response to A23187 or to PMA (Fig. 1). This result demonstrated that wortmannin did not directly inhibit phospholipase D. However, the response to each receptor-mediated agonist was reduced by pretreatment with wortmannin. The degree of inhibition varied for the
different
fMLP
being
agonists,
reduced
with
the
to the level
strong
activation
of PMA
by
activation,
and the weak response to LTB4 reduced to the basal value. Thus, wortmannin affected a component between the activation of a receptor by its agonist and the
phospholipase D activity. We found that wortmannin was an irreversible inhibitor of the process leading to phospholipase D activation, since three washes in buffered saline did not reverse the inhibition. Wortmannin appears to be an alkylating agent, as its effects are sensitive to modifications of its structure that decrease its nucleophilic nature (28), and its effect on the respiratory burst is also irreversible (21). This suggests that the inhibition of phospholipase D activation by wortmannin is similar to its inhibition of the respiratory burst in that both result from an irreversible, possibly covalent, modification of cellular protein (or proteins).
A23187
Control
PMA
tMLP
PAF
LTB4
Figure 1. Effect of wortmannin on receptorand non-receptormediated stimulation of PEt formation. Human PMN were prelabeled with [3H]oleate for 10 mm, exposed to 50 nM wortmannin in DMSO or solvent control for 5 mm, and then stimulated in the
presence
of 0.5% ethanol
and the stated agonist for 5 mm. Data are
presented as percent (mean ± SEM for three experiments) of the A23187 response (control, 3922 ± 1083 dpm: wortmannin-treated, 4399
210
± 1826 dpm).
Vol. 4
Feb. 1990
uw
w
o
(I,
E EQ. -J
0
1
2
Time
3
(mm)
Figure 2. Effect of wortmannin on the respiratory burst of fMLPstimulated PMN. Cells were exposed to 50 nM wortmannin for 3 mm before quantitation of luminol-dependent chemiluminescence during stimulation with M fMLP.
io
Activation of phospholipase D by fMLP depends on Ca21-dependent and -independent pathways Stimulation of phosphatidylethanol formation was induced by the Ca2 ionophore A23187, indicating that an increase in intracellular Ca2 was sufficient to activate phospholipase D. We examined the postulate that activation of phospholipase D in response to fMLP was due solely to a receptor-mediated lar Ca2’. We incubated PMN
increase in EGTA
in intracellufor 5 mm be-
fore stimulation to deplete both extracellular and intracellular Ca2 stores (21). This treatment was effective in depleting Ca2 as activation of phospholipase D by A23187 was completely inhibited (not shown). fMLPinduced activation of phospholipase D was also inhibited by the EGTA pretreatment, but the inhibition was only about one-half of the stimulated response (2725 vs. 1175 dpm in EGTA-treated cells). This suggests that fMLP induction of phospholipase D proceeds approximately equally by Ca2-dependent and Ca2independent pathways. The weak stimulation of phospholipase D activation by PMA was not affected by Ca2 availability. This is consistent with the ability of PMA to activate other PMN responses in the presence of EGTA (21). It also showed that PMA had not activated a voltage-dependent Ca2 gate (29). Activation of phospholipase D occurs kinase C-dependent and -independent
by protein pathways
Phospholipase D activity could be activated by PMA 1), although the level of stimulation was less than that induced by fMLP. However, induction of phospholipase D activity by this protein kinase C-dependent route was not inhibited by wortmannin, whereas fMLP-induced phospholipase D activity was inhibited. This suggested either that phospholipase D could be ac(Fig.
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ET AL.
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tivated by a protein kinase C-independent route, or that protein kinase C activity was not appropriately activated in wortmannin-treated cells. The latter might occur if wortmannin blocked the accumulation of diglyceride, the endogenous lipid activator of protein kinase C, in response to fMLP. This would explain why PMA could induce phospholipase D activity and the respiratory burst (21, 28) in wortmannin-treated cells, since it would be supplying the missing activator. We examined this postulate by measuring the amount of diglyceride in control and wortmannin-treated PMN. The
diglyceride
content
increased
from
55 to 220 pmol/
nmol phospholipid over 180 s in fMLP-stimulated cells. This increase was not affected by wortmannin treatment. This implies that wortmannin does not exert its effect by altering the accumulation of diglyceride in stimulated PMN. We next determined whether activation of protein kinase C was essential for activation of phospholipase D in fMLP-stimulated cells by using staurosporin to inhibit the kinase. Staurosporin has previously been reported to inhibit protein kinase C activity in vitro, and to
inhibit
events
thought
to
be
mediated
by
protein
kinase C in intact cells (30). Staurosporin proved to be an effective inhibitor of PMA-induced phospholipase D activation (Fig. 3). Staurosporin did not, however, inhibit
fMLP-induced
activation
B
A
of phospholipase
D. In
fact, it led to a marked enhancement of phosphatidylethanol formation in fMLP-stimulated cells. This enhancement was evident throughout the period of IMLP stimulation, such that after 15 mm of stimulation, staurosporin-treated cells had accumulated 4.3 times as much phosphatidylethanol as untreated control cells. Staurosporin by itself had no effect on phospholipase D activity, so this result was not simply an additive effect.
4. Effect of wortmannin on fMLP-induced PMN aggregation. Aggregation in response to fMLP was measured after pretreatment with (A) buffer or (B) 50 nM wortmannin. Figure
A similar, although less dramatic, result when sphingosine, a structurally unrelated protein kinase C (31), was substituted for These data indicate that protein kinase sential for the activation of phospholipase to fMLP, and further that it apparently hibitory
exerts
an
in-
effect.
Wortmannin or adhesion
does
not prevent
PMN
aggregation
Since wortmannin is a potent inhibitor of the respiratory burst and PEt accumulation, and at higher concentrations inhibits neutrophil degranulation (21), we examined
next
its effect
on adhesion
and
aggregation
to
determine if it is a pleotropic inhibitor of PMN function. Under conditions where fMLP-induced oxygen radical production was totally inhibited (Fig. 2), fMLP-induced aggregation was unaffected (Fig. 4), and adherence to a gelatin matrix (Fig. 5) was reduced
300
by only
30%.
Increasing
the concentration
of wortman-
nm from 100 nM to 1 M did not increase the degree of inhibition of fMLP-induced adherence. Since these two adhesive responses were relatively unaffected under conditions where phospholipase D activity was significantly reduced, it appears that activation of phospholipase D, and the accumulation of its endogenous product phosphatidic acid, are not essential components in the signaling pathways that lead to the activation of PMN-adhesive glycoproteins.
200
0.0
0.5
[staurosporinej
1.0
DISCUSSION
SM
3. Effect of staurosporine on fMLPor PMA-stimulated PEt formation. Cells were prelabeled with [3H]oleate, exposed to the stated concentration of staurosporine for 5 mm, and then stimu-
Figure
lated with IMLP or PMA in the presence of 0.5% ethanol. Data are presented as percent of the stimulated response in the absence of staurosporine (2573 and 4726 dpm, respectively). The average increase in the IMLP response (n = 6) was 2.9 ± 1.2-fold, the average decrease (n - 5) of the PMA response was 64 ± 17%.
ACTIVATION
was obtained inhibitor of staurosporin. C was not esD in response
OF HUMAN
NEUTROPHIL
PHOSPHOLIPASE
PMN and other cells demonstrate tivity that utilizes phosphatidylcholine 11,
13).
clearly further
11).
The
products
phospholipase D acas a substrate (6,
phosphatidic
acid
and
choline
arise from a phospholipase D and are not simply metabolites of a phospholipase C reaction (9, Compelling
phosphatidic
evidence
acid
can
result
that
from
the
accumulation
of
a phospholipase
D
D
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We explored the tivity in stimulated
hypothesis that phospholipase D acPMN might be the site of wortman-
nm action, and that this activated phospholipase D therefore was essential in signal transduction leading to 02 and H2O2 generation. We found that phospholipase D activity itself was not inhibited by wortmannin. However, its activation was affected by wortmannin treatment when PMN were stimulated with receptormediated hibition
1o
[fMLPI, Figure
PMN with
M
5. Effect of wortmannin on IMLP-induced adherence of to a gelatin matrix. Adherence of PMN pretreated, or not, 100 nM wortmannin
a function
of IMLP
to gelantinized
plastic
was
examined
as
concentration.
reaction is the accumulation of phosphatidylalcohols when primary alcohols, such as ethanol, are present (7, 8, 10-13). This is a well-defined reaction of plant phospholipase D where a phosphatidyl-phospholipase D intermediate can be attacked either by water or alcohol (27). These phospholipids are not normal cellular constituents, and there is no known mechanism, other than transphosphatidylation, by which mammalian cells form such phospholipids. The formation of phosphatidylethanol is therefore a sensitive and specific assay for phospholipase D activity (7). PMN phospholipase D activity undergoes a marked enhancement in cells that have been stimulated with a variety of agonists. The role for the natural product of this reaction, phosphatidic acid, has not been defined in stimulated PMN. However, it increases intracellular Ca2, apparently by inducing the release of intracellular stores of Ca2 (14), and it stimulates NADPH oxidase activity in the lysates from unstimulated cells (19). Although it is not known if such activation occurs in intact cells, our data show that the oxidative burst is much more closely associated with activation of phospholipase D than are adhesive functions of stimulated PMN. The fungal metabolite wortmannin, which inhibits the
respiratory
burst
in
PMN
without
inhibiting
Vol. 4
Feb. 1990
Thus, the site of wortmannin to stimulation of phospholipase
inD.
A scheme consistent with these observations is presented in Fig. 6. Activation of a receptor, and its functionally coupled G protein (11, 12), leads to the activation of phospholipase D by three separable, but interacting, routes. Receptor activation leads to the translocation of protein kinase C and its activation (32). Since PMA stimulated phospholipase D function, one route to stimulated phosphatidic acid formation depends on the activation of protein kinase C. This is consistent with the observations of Tettenborn and Mueller (12) that pretreatment of HL6O cells with PMA enhances the subsequent formation of phosphatidylethanol in cell lysates. These authors also observed that GTP-yS activated phospholipase D by an independent mechanism. Activation of phospholipase D by protein kinase C has also been observed in whole cells (5). Although we found that direct activation of protein kinase C stimulated phosphatidylethanol formation, activation of this kinase did not appear to be essential for the activation of phospholipase D. This was suggested
by the
effects
of staurosporin
or sphingosine,
which prevented activation by PMA, hibit receptor-induced phospholipase Similarly,
receptor
activation
by
but did not D activation. fMLP
leads
in-
to an
increase in intracellular free Ca2 concentration (21). Since directly increasing the Ca2 concentration with A23187 leads to activation of phospholipase D activity,
the
NADPH oxidase activity (21), promises to be a valuable tool to dissect the signal transduction pathways in activated PMN. Its intriguing attribute is that it does not inhibit any of the known components of the signal transduction pathway. Thus, it apparently does not inhibit receptor function, receptor-coupled G proteins, phosphoinositide metabolism, the increase in intracellular free Ca2, or the catalytic activity of protein kinase C (21). We found that it also did not interfere with receptor-mediated accumulation of diglyceride, or aggregation or adhesion to a protein matrix induced by fMLP. Thus, wortmannin must exert its effect on an unidentified Ca2-insensitive component (or components) whose function was essential for the activation of the respiratory burst. 212
agonists. is proximal
C&’2
0
#{176}--.‘..pKC
Phospholipase Figure
6. Scheme
for three
separate
tion of phospholipase D. The IMLP lipase D activity by a Ca2-dependent, and a wortmannin-inhibitable designated with a dashed
The FASEB Journal
route.
routes
that
receptor
lead
to the stimula-
activates
phospho-
protein kinase C-dependent, Modulatory interactions are
arrow.
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ET AL.
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this second Ca2-dependent. centration
route by
to phospholipase D activation is Although it increased the Ca2 con-
entry
of
external
Ca2
activated
phos-
pholipase D, this event was not essential for its activation. Cells stimulated in the presence of extracellular EGTA, to prevent Ca2 entry, retained half of their fMLP-stimulated phosphatidylethanol formation. Therefore, both Ca2-dependent and Ca2’-independent pathways lead to the activation of phospholipase D. Part of the function of the increase in intracellular Ca2 was to activate protein kinase C. This was shown by the inhibition of 50% of the A23187-induced formation of phosphatidylethanol by inhibitors of protein kinase C (not shown). Thus, although either direct activation of protein kinase C or increasing the concentration of intracellular Ca2 activated phospholipase D, neither appeared to be essential for phospholipase D activation by receptor-mediated agonists. We conclude that there is a third pathway leading to phospholipase D activation in receptor-activated cells, and it is this pathway that is sensitive to wortmannin. This conclusion is based on the finding that wortmannm inhibited fMLP-dependent activation, but not that due to Ca24 influx or activation of protein kinase C. The components of this signaling pathway are currently undefined. It is, however, clear that this pathway was not the only route to phospholipase D activation, since wortmannin
only
depressed
fMLP-induced
phosphati-
dylethanol formation to the level of activity produced by direct activation of protein kinase C. Stimulation of phospholipase D, perhaps by the wortmannin-sensitive pathway, was also inhibited by protein kinase C. This became evident when staurosporin or sphingosine was found to markedly stimulate fMLP-induced
phosphatidylethanol
formation
under
We thank study.
Dr.
We thank
T Eric
G.
Payne
Stroud
for supplying for his analysis
wortmannin
for
this
of diglyceride content for their preparation of
of PMN, Holly Nichols and Susan Cowley PMN, and Margaret Vogel for her technical assistance. This work was supported by funds from the Nora Eccles Treadwell Foundation, grants HL35828 and HL34127 from the National Institutes of Health, and Established Investigator awards (G. A. Z., S. M. P.) from
the
American
Heart
Association.
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