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(Received for publication, October 17, 1996)
From the Departments of Biological Chemistry and Molecular and
Medical Pharmacology, and the Molecular Biology Institute, UCLA Center
for the Health Sciences, Los Angeles, California 90095
ABSTRACT Aggregation of IgE cell surface receptors on
MMC-34 cells, a murine mast cell line, induces the synthesis and
secretion of prostaglandin D2 (PGD2). Synthesis
and secretion of PGD2 in activated MMC-34 cells occurs in
two stages, an early phase that is complete within 30 min after
activation and a late phase that reaches a maximum about 6 h after
activation. The early and late phases of PGD2 generation
are mediated by prostaglandin synthase 1 (PGS1) and prostaglandin
synthase 2 (PGS2), respectively. Arachidonic acid, the substrate for
both PGS1 and PGS2, is released from membrane phospholipids by the
activation of phospholipases. We now demonstrate that in activated mast
cells (i) secretory phospholipase A2 (PLA2) mediates the release of arachidonic acid for early,
PGS1-dependent synthesis of PGD2; (ii)
secretory PLA2 does not play a role in the late,
PGS2-dependent synthesis of PGD2; (iii)
cytoplasmic PLA2 mediates the release of arachidonic acid
for late, PGS2-dependent synthesis of PGD2; and
(iv) a cytoplasmic PLA2-dependent step precedes
secretory PLA2 activation and is necessary for optimal PGD2 production by the secretory
PLA2/PGS1-dependent early pathway.
INTRODUCTION Mast cells, an important cell type in allergic diseases, are
widely distributed throughout vascularized tissue and epithelia. Activation of mast cells by aggregation of high affinity IgE receptors causes degranulation, releasing stored mediators of inflammation such
as histamine and serotonin. Mast cell activation also induces the
synthesis and release of leukotrienes and prostaglandin D2 (PGD2). Ligand stimulation in most cells elicits a
relatively slow production of prostaglandins, peaking only after 4-6 h
(1, 2). In contrast, PGD2 synthesis in activated mast cells
occurs in two stages, a rapid, early phase and a late, delayed phase (3, 4).
Prostaglandin production is regulated by both phospholipases
A2 (PLA2)1 and
prostaglandin synthases (PGS). PLA2 enzymes release
arachidonic acid from membrane phospholipids. Free arachidonate is
converted to prostaglandin H2 (PGH2), a common
precursor for all prostanoids, by prostaglandin synthases. Several
PLA2 enzymes have been implicated in arachidonate release
following ligand stimulation of various cell types (5, 6). Many cells
also express two distinct prostaglandin synthases: PGS1, a primarily
constitutively expressed form, and PGS2, an inducible PGS expressed
following appropriate ligand stimulation in different cell types (1,
2). Experiments using antisense oligonucleotide inhibition (7) and
NS-398, a PGS2-specific inhibitor (1, 8), demonstrated that
ligand-induced prostaglandin production in fibroblasts and macrophages
requires induced PGS2 expression, despite the presence of active PGS1
enzyme. In contrast, the rapid, early phase of PGD2
synthesis in activated mast cells is mediated by pre-existing PGS1 (3,
4). The second, delayed phase of PGD2 synthesis in
activated mast cells is similar to prostaglandin production in growth
factor-induced fibroblasts and endotoxin-induced macrophages, requiring
activation-induced PGS2 expression (3, 4).
Following activation, mast cells secrete a low molecular weight
PLA2, sPLA2 (9). Fonteh et al. (9)
suggest that the sPLA2 released following activation plays
a role in eicosanoid biosynthesis by activated mast cells. Activation
of mast cells also induces the activation, translocation, and
expression of cytoplasmic cPLA2 (10-13). In this report we
investigate the roles of sPLA2 and cPLA2 in
early, PGS1-dependent PGD2 synthesis and late,
PGS2-dependent PGD2 synthesis following mast
cell activation by aggregation of high affinity IgE receptors.
EXPERIMENTAL PROCEDURES Mouse MMC-34 cells (14) were grown in RPMI
1640 medium (ICN, Cleveland, OH) supplemented with 10% fetal calf
serum (Gemini Bioproducts Inc., Calabasas, CA).
Murine IgE and monoclonal anti-IgE were purchased
from Pharmingen (San Diego, CA). PGD2 assay kits were from
Amersham Corp. (UK); aminopropyl solid phase columns No. 9070 (100 mg/ml) were from Burdick and Jackson (Muskegon, MI).
[3H]Arachidonate-labeled Escherichia coli
suspension was from DuPont NEN. Methyl arachidonylfluorophosphonate
(MAFP) was obtained from Cayman Chemical Co. (Ann Arbor, MI). The
cytosolic phospholipase A2 assay kit used for the data
shown in Fig. 10, right panel, was also from Cayman. NS-398
was a gift from Taisho Corp (Japan). Monoclonal antibody F10 (mAbF10)
directed against recombinant PLA2 (9), recombinant
sPLA2, and SB203347, a specific inhibitor of
sPLA2 (15), were the gifts of Dr. Lisa Marshall (SmithKline Beecham Pharmaceuticals, King of Prussia, PA). Recombinant
cPLA2 was the gift of Dr. Michael H. Gelb (University of
Washington, Seattle).
Cell cultures were treated
with IgE and anti IgE as described in figure legends and text. Medium
was collected by centrifugation and analyzed for PGD2 using
the Amersham kit.
For the assay
of cPLA2 activity from extracts of activated mast cells
(Fig. 10, right panel), we used the cPLA2 kit
from Cayman Chemical Co., according to the manufacturer's
instructions. Briefly, samples of mast cell extracts (see legend to
Fig. 10 for details) were incubated with
arachidonylthiophosphatidylcholine (ATPC), the substrate. Enzymatic
hydrolysis of ATPC releases free thiol, which is then converted into
5-thio-2-nitrobenzoic acid by Ellman's reagent
[5,5 RESULTS The early phase of PGD2 synthesis, completed
within the first 10-30 min after mast cell activation, is mediated by
PGS1 (3, 4). The late phase of PGD2 production, mediated by
PGS2, does not peak until 4-6 h after activation (3, 4). We began our studies on the relationships between phospholipases and prostaglandin synthases in mast cells by analyzing the effect of inhibiting sPLA2 activity on PGD2 production after
activation by aggregation of IgE receptors. If mast cells are treated
with the sPLA2 inhibitor SB203347 (15), the early burst of
PGD2 synthesis is completely blocked (Fig.
1), suggesting that sPLA2 mediates the early
burst of PGD2 production in response to activation by
cross-linking of IgE receptors.
It is possible that the inhibitory effects of SB203347 described above
might be due to inactivation of PGS1, since this enzyme is necessary
for the early burst of PGD2 production following mast cell
activation (3, 4). To examine this question, we asked whether mast
cells activated in the presence of SB203347 can utilize exogenous
arachidonic acid as substrate. Cells activated in the presence of
SB203347, although unable to produce the early burst of prostaglandin
from endogenous arachidonate, are capable of converting exogenous
arachidonate to PGD2 (Fig. 2). The PGS1 present in these cells is, therefore, not inactivated and is
enzymatically active in the presence of SB203347. The data in Fig. 1
and Fig. 2 suggest that SB203347 blocks the early burst of
PGD2 synthesis in activated mast cells by preventing
sPLA2 from mobilizing arachidonic acid from phospholipids
and providing substrate for PGS1.
As an alternative means of demonstrating the role of sPLA2
in prostaglandin synthesis in activated mast cells, we examined the
effect of a monoclonal antibody to this enzyme on PGD2
production. In this experiment NS-398, a specific inhibitor of PGS2
(18), was added to prevent late phase, PGS2-dependent
PGD2 production. Treatment of MMC-34 mast cells with either
(i) a monoclonal antibody, mAbF10, against sPLA2 (9) or
(ii) the specific sPLA2 inhibitor, SB203347, blocked
activation-induced PGD2 production (Fig. 3, left panel), again suggesting that early,
PGS1-dependent PGD2 production in activated
mast cells requires sPLA2.
To confirm that the inhibition of PGD2 synthesis in
response to either SB203347 or mAbF10 is due to a reduction in
sPLA2 activity, the culture media from this experiment were
also analyzed for secreted phospholipase activity (Fig. 3, right
panel). Activated MMC-34 cells accumulated increased
PLA2 activity in the medium. Accumulation of this
PLA2 activity, like PGD2 accumulation, was blocked by treatment with the sPLA2 inhibitors mAbF10 or
SB203347. We conclude that the sPLA2 released by activated
mast cells is required for early, PGS1-dependent
PGD2 synthesis.
The late phase of PGD2 synthesis in activated
mast cells appears to be unaffected by the sPLA2 inhibitor
SB203347 at 1 µM concentration (Fig. 1). To more
accurately assess the level of late phase, PGS2-dependent
PGD2 accumulation in SB203347-treated MMC-34 cells
following activation, we included in this experiment (Fig. 1) a set of
cells in which PGS1 activity was eliminated by preincubation with
aspirin. We have shown that aspirin preincubation and washing
irreversibly inhibits both PGS1 activity and the early phase of
PGD2 synthesis in MMC-34 cells, without interfering with either (IgE + anti-IgE)-induced PGS2 induction or the
PGS2-dependent late phase of PGD2 production
(3). PGD2 production in activated mast cells in which
sPLA2 activity was inhibited by SB203347 is identical to
that observed in cells in which the PGS1 activity was blocked by
aspirin preincubation (Fig. 1), suggesting that sPLA2, like
PGS1 (3), plays no role in the late phase of PGD2 synthesis
in activated mast cells.
To demonstrate that the late burst of PGD2 synthesis
occurring in the presence of SB203347 was not due to insufficient
levels of inhibitor, we carried out an SB203347 dose-response analysis in which we measured PGD2 production at 6 h. Higher
concentrations of SB203347 were unable to further inhibit
PGD2 accumulation (Fig. 4), confirming that
the late phase of PGD2 synthesis in activated mast cells
does not require sPLA2 activity.
To extend these observations, we demonstrated that inhibiting
sPLA2 activity with either sPLA2-directed
monoclonal antibody mAbF10 or the sPLA2 inhibitor SB203347
does not increase the inhibition of total (i.e. early phase
and late phase) PGD2 production in activated mast cells
beyond that seen by aspirin pretreatment alone (Fig. 5,
lanes 3-5). Inhibition of PGS1 alone (lane 3) or inhibition of both PGS1 and sPLA2 (lanes 4 and
5) result in the same reduction in total PGD2
production at 6 h by activated MMC-34 cells. We conclude that
sPLA2 activity, like PGS1 activity, is required for the
early phase of PGD2 production in activated mast cells but
not for the late phase of PGD2 production.
The late phase of PGD2 production following IgE + anti-IgE
activation of MMC-34 cells is mediated entirely by PGS2 and can be
completely suppressed by the PGS2-specific nonsteroidal
anti-inflammatory drug NS-398 (3). In contrast, NS-398 has no effect on
the PGS1-dependent early phase of PGD2
production in activated mast cells (3). Although NS-398 treatment
blocks only a portion (i.e. the late phase) of total
PGD2 production in activated MMC-34 cells (Fig. 5,
lane 6), the combination of either SB203347 or mAbF10, both of which inhibit the early phase of PGD2 production, with
NS-398 can completely suppress mast cell PGD2 production
(Fig. 5, lanes 7 and 8).
Our data suggest that
sPLA2 does not play a role in late,
PGS2-dependent PGD2 production in activated
mast cells (Figs. 1, 4, and 5). Another phospholipase must, therefore,
provide arachidonate for the late phase of PGD2 production.
The enzyme most likely to fulfill this role is the type IV cytoplasmic
PLA2, or cPLA2, enzyme (see "Discussion").
MAFP has been reported to preferentially inhibit cPLA2 and
to have relatively little effect on sPLA2 (19). We also
find that MAFP preferentially inhibits recombinant cPLA2, whereas SB203347 preferentially inhibits recombinant sPLA2
(Fig. 6). We used MAFP to determine whether
cPLA2 might play a role in either early
PGS1-dependent PGD2 synthesis or late
PGS2-dependent PGD2 synthesis following mast
cell activation.
When 10 µM MAFP is added at the same time that IgE
receptors are aggregated on MMC-34 cells, the early phase of
PGD2 accumulation is not substantially inhibited (Fig.
7, left panel). In contrast, the late phase
of PGD2 accumulation is suppressed to an extent similar to
that observed with NS-398, the PGS2-specific inhibitor. These data
suggest that the late PGS2-dependent phase of
PGD2 production in activated mast cells requires
cPLA2 activity to provide arachidonic acid as substrate for
PGS2. To demonstrate that the degree of inhibition of late phase
PGD2 production following activation is maximal at the 10 µM concentration of MAFP used, we carried out a
dose-response curve for MAFP (Fig. 7, right panel). Even at
a concentration 5-fold greater than that used in the time course
experiment, no additional inhibition of late phase PGD2 accumulation by MAFP was observed.
MAFP might exert its effect on the late phase of PGD2
accumulation in part by inhibiting the induction and/or enzymatic
activity of PGS2. To investigate this question, we examined the ability of MAFP-treated, activated MMC-34 cells to utilize exogenous
arachidonic acid as substrate for PGD2 synthesis. Untreated
MMC-34 cells do not produce PGD2 from endogenous
arachidonic acid but can convert exogenous arachidonate to
PGD2 because they express constitutive PGS1 (Fig.
8). Treatment with aspirin inactivates PGS1 and prevents unstimulated MMC-34 cells from converting exogenous arachidonate to
PGD2 (Fig. 8). If MMC-34 cells are first pretreated with
aspirin, then activated by aggregation of IgE receptors, they produce
PGD2 from endogenous arachidonic acid during a 6-h
incubation, as a result of the activity of cPLA2 and the
induction of PGS2 (Fig. 8, left panel). MAFP can completely
prevent this PGD2 production from endogenous sources of
arachidonic acid in cells activated following pretreatment with aspirin
(Fig. 8, left panel). However, these same cells are able to
produce PGD2 from exogenous arachidonate, demonstrating
that induction of functional PGS2 occurs in the presence of MAFP in
response to mast cell activation (Fig. 8, right panel). Thus
MAFP inhibition of the late, PGS2-dependent component of
PGD2 production in activated mast cells is not due to
either inhibition of PGS2 production or to inhibition of PGS2 enzyme
activity.
Balsinde and
Dennis (20) have recently provided compelling evidence that
cPLA2 activation is necessary for, and precedes, sPLA2 activation in murine macrophages stimulated with
endotoxin and platelet activating factor. In these experiments, MAFP
treatment was able to block a substantial proportion of the
sPLA2-dependent production of arachidonic acid
in macrophages (20). However, this inhibition required preincubation of
cells with MAFP prior to ligand activation. To investigate the role of
cPLA2 in the early sPLA2-dependent
phase of PGD2 production as well as in the late
sPLA2-independent phase of PGD2 production in
activated mast cells, we first compared the effect of (i) MAFP
preincubation on total PGD2 production in activated mast
cells with (ii) PGD2 production when MAFP was added at the
time of aggregation of IgE receptors. If MAFP is added at the time of
activation, the inhibition of PGD2 accumulation at 6 h
is identical to that observed with NS-398, the inhibitor of PGS2
activity (Fig. 9, top panel, lanes 4 and
5). These data are consistent with our conclusions that cPLA2 and PGS2 mediate the late phase of PGD2
production in activated mast cells. However, if MMC-34 cells are
preincubated with MAFP prior to IgE receptor aggregation, an additional
increment of PGD2 accumulation is inhibited (Fig. 9,
top panel, lane 6), suggesting that cPLA2 may
also play a role in the early
sPLA2/PGS1-dependent phase of PGD2
production in activated mast cells.
To more completely characterize the role of cPLA2 on the
early sPLA2/PGS1-dependent phase of
PGD2 production in activated mast cells, we examined the
time- and dose-dependent influence of MAFP. Maximal
inhibition of the early phase of PGD2 production in
activated mast cells occurred at a concentration of 10 µM
MAFP (Fig. 9, middle panel) and was complete within 15 min
of preincubation (Fig. 9, lower panel). These data, like
those of Balsinde and Dennis (20), suggest that cPLA2
activation must precede sPLA2 activation for optimal early
phase PGD2 production in response to mast cell IgE receptor
aggregation.
We analyzed the
abilities of the sPLA2 inhibitor, SB203347, and the
cPLA2 inhibitor, MAFP, to inhibit the phospholipase
activity present in the culture media and in the homogenates of
activated MMC-34 cells. The bulk of the phospholipase activity present
in the media from activated mast cells is inhibited by SB203347. Little
or no phospholipase activity secreted into the culture media following
activation is inhibitable by MAFP (Fig. 10, left panel). In contrast, the bulk of the phospholipase activity in extracts prepared from activated mast cells is inhibited by MAFP. None
of the phospholipase activity remaining associated with cells after
activation is inhibited by SB203347 (Fig. 10, right
panel).
MAFP can also inhibit iPLA2, the calcium-independent
phospholipase A2 (21). To determine whether
iPLA2 activity is present in these mast cell extracts, we
examined the level of phospholipase activity in the extracts in the
presence of EGTA. Greater than 80% of the MAFP-inhibitable
phospholipase A2 activity present in the activated mast
cell extract is calcium-dependent (Fig. 10, right
panel; compare lanes 2 and 4).
DISCUSSION Aggregation of mast cell IgE receptors results in two phases of
PGD2 production, an early burst completed within 10-30 min and a second phase peaking after 5-6 h (3, 4). Pharmacologic studies
demonstrated that the early PGD2 burst is due solely to activity of pre-existing PGS1 enzyme, while delayed PGD2
production in activated mast cells requires induced PGS2 synthesis (3, 4). Arachidonic acid, released from membrane phospholipids by
PLA2, is a substrate for both PGS enzymes. Mast cells
contain at least two distinct arachidonic acid precursor pools, whose arachidonate products remain segregated after release from cellular phospholipids (9, 22, 23). Moreover, mast cells have at least three
distinct PLA2 isoforms (6, 24). The temporal separation of
PGD2 production by PGS1 and PGS2 in activated mast cells
suggested that distinct phospholipid arachidonate pools and distinct
phospholipases might provide arachidonate to the two prostaglandin
synthase enzymes.
The inability of either antibody to sPLA2 or
SB203347, the sPLA2 inhibitor, to reduce
PGS2-dependent PGD2 production demonstrates that sPLA2 plays no role in the late phase of
PGD2 synthesis. MAFP was initially described as a
cytoplasmic type IV cPLA2 inhibitor, with no effect on type
II sPLA2 (19). The late, PGS2-dependent phase
of PGD2 production in activated mast cells is completely blocked by MAFP, suggesting that cPLA2 is required to
provide arachidonate for this second component of PGD2
production. MAFP, however, also inhibits a cytosolic,
calcium-independent PLA2 (iPLA2) (21). When
assayed previously, iPLA2 could not be detected in bone
marrow-derived murine mast cells (25). In our experiments, we found
only a small fraction of MAFP-inhibitable phospholipase A2
activity present in extracts of activated mast cells to be calcium-independent (Fig. 10). In addition, specific iPLA2
inhibition in stimulated P388D1 macrophages enhances,
rather than inhibits, arachidonate release (20). MAFP inhibition of
late, PGS2-dependent PGD2 production in
activated mast cells is, therefore, likely to be due to type IV
cPLA2 inactivation. cPLA2 and PGS2 appear to be
metabolically coupled in activated mast cells for the delayed phase of
PGD2 synthesis. However, conclusive proof of this
hypothesis will require the specific suppression of cPLA2
synthesis by antisense cPLA2, development of pharmacologic
agents that more specifically inhibit cPLA2, or mast cells
derived from animals in which the cPLA2 gene has been
disrupted.
Previous experiments suggested that sPLA2 plays
a role in PGD2 production following mast cell activation
(9). The ability, demonstrated here, of both a monoclonal antibody to
sPLA2 (9) and a specific sPLA2 inhibitor,
SB203347 (15), to completely block early, PGS1-dependent
PGD2 production following mast cell activation supports
this conclusion and demonstrates that sPLA2 and PGS1 are
metabolically coupled. sPLA2 activity is required for the
early phase of PGD2 production in activated mast cells. The
low molecular weight, secreted PLA2 from mast cells has not been molecularly characterized. Three related murine genes encoding low
molecular weight, secreted PLA2 enzymes of related
structure are known, each with a cell type-specific distribution
pattern (26, 27). It will be of great interest to identify the
PLA2 isoform(s) secreted by activated mast cells, since
this enzyme(s) may represent an important target in mast cell
eicosanoid production.
Balsinde and Dennis (20) have demonstrated, in activated
P388D1 macrophages, that "a functionally active
cPLA2 appears to be necessary for sPLA2 to
act." Their results prompted us to examine the effects of
preincubation of mast cells with MAFP on the early, sPLA2/PGS1-dependent phase of PGD2
synthesis in activated mast cells. An MAFP-sensitive step is required
for one-half to two-thirds of the early phase of PGD2
production following the activation of mast cells by aggregation of
their IgE receptors (Fig. 9). Since we have verified the inability of
MAFP to block the enzymatic activity of either recombinant
sPLA2 (Fig. 6) or the sPLA2 activity present in
mast cell supernatants (Fig. 10), we conclude that the MAFP-sensitive
step in early PGD2 production in response to aggregation of
mast cell IgE receptors is likely to be mediated by cPLA2. Like the data of Balsinde and Dennis (20), our results suggest that a
cPLA2-mediated function is required prior to the action of
sPLA2. It should be emphasized that, although there exists a cPLA2-dependent event required for the early
phase of PGD2 synthesis in activated mast cells,
cPLA2 does not release arachidonic acid that is then
available for PGD2 production by PGS1. Inhibition of
sPLA2 by either SB203347 or mAbF10, which do not inhibit
cPLA2, prevents all PGD2 production in the
early phase of mast cell activation (Figs. 1, 2, 3).
In most cells, PGS1 is associated with the endoplasmic
reticulum (1, 2). In contrast, biochemical and ultrastructural analysis
suggested that mast cell granules contain phospholipids (28),
sPLA2 (29), and PGS1 (30). Degranulation and exposure of
granule contents to high extracellular calcium following aggregation of
IgE receptors may activate sPLA2, leading to production of arachidonic acid substrate for PGS1 and the synthesis of the early PGD2 burst. Additional investigation of sPLA2
and PGS1 localization in resting and activated mast cells will be of
great interest, now that two temporal phases of mast cell
PGD2 production have been demonstrated.
PGS2 is detected in both the endoplasmic reticulum and the nuclear
envelope of mitogen-stimulated fibroblasts (31). Subcellular PGS2
localization has not been reported in mast cells. Following mast cell
activation, cPLA2 moves from the cytoplasm to the nuclear envelope (13). Ligand treatment also stimulates translocation of
cPLA2 to the nuclear fraction in Chinese hamster ovary
cells (32) and in macrophages (33). Assuming PGS2 is also localized predominantly at the nuclear envelope in mast cells after
activation-induced synthesis, the data are consistent with the
following model. Activated mast cells translocate cPLA2 to
the nuclear envelope, where this enzyme releases arachidonate from
phospholipids. Newly synthesized PGS2, also present in the nuclear
envelope, utilizes this arachidonate to catalyze the late phase of
PGD2 production. Mast cell PGS1 may not be able to use the
arachidonate produced by cPLA2 for several reasons. It
seems most likely that physical sequestration of PGS1 in mast cells may
make cPLA2-dependent arachidonate, produced at
the nuclear envelope, inaccessible to PGS1. Alternatively, PGS1 may be
inactivated by a suicide reaction (34) during the early
PGD2 synthesis phase, prior to adequate activation-induced cPLA2 translocation.
Identification of the PLA2 and PGS isoforms that mediate
the two phases of PGD2 production in activated mast cells
may permit development of additional pharmacologic agents that can
discriminate between these two PGD2 production pathways. In
addition to mediating PGS1-dependent PGD2
production by mast cells following activation, sPLA2
released by activated mast cells can also initiate trans-cellular prostaglandin production by mobilizing arachidonate in a distal cell as
substrate for PGS1 in that cell (16). Specific inhibition of mast cell
sPLA2 may, therefore, be an important pharmacologic target
for several modes of prostaglandin production in chronic and acute
inflammatory responses.
FOOTNOTES Acknowledgments We thank Raymond Basconcillo and Arthur
Catapang for technical assistance and the members of the Herschman lab
for helpful discussions. We also thank Lisa Marshall (SmithKline
Beecham) for the gifts of mAbF10, recombinant sPLA2, and
SB203347 and Michael Gelb (University of Washington) for recombinant
cPLA2.
REFERENCES
Volume 272, Number 6,
Issue of February 7, 1997
pp. 3231-3237
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
Fig. 10.
SB203347 and MAFP inhibit distinct
components of phospholipase A2 activity in activated mast
cells. MMC-34 mast cells were cultured and activated by
aggregation of IgE receptors as described in the legend to Fig. 1.
Left panel, 1 h after addition of anti-IgE the cells
were separated from the culture media by centrifugation, and the
supernatant from the activated cells was split into three samples. One
sample was left untreated. SB203347 was added to a final concentration
of 1 µM to one sample. MAFP was added to a final
concentration of 10 µM to a second sample. After
incubation at 37 °C for 30 min, each sample was assayed for
phospholipase A2 activity. Right panel, 4 h
after addition of anti-IgE the cells were separated from the culture
media by centrifugation. The cell pellet was washed twice in
phosphate-buffered saline and resuspended in sonication buffer (100 mM Tris-HCl, pH 7.4, 100 mM NaCl, 25 µg/ml
each of aprotinin, pepstatin A, and leupeptin, and 100 µg/ml
phenylmethylsulfonyl fluoride). The cells were sonicated for 10 s
at 50% duty cycle (Heat Systems, Ultrasonics, Inc). After
centrifugation, the lysate was divided into four samples, each
containing extract from 106 cells. One sample was left
untreated. SB203347, MAFP, or EGTA (0.25 mM) were added to
the remaining samples, as shown in the figure. Extract samples were
incubated at 37 °C for 30 min and then assayed for phospholipase
activity as described under "Experimental Procedures."
[View Larger Version of this Image (20K GIF file)]
-dithiobis(2-nitrobenzoic acid)]. 5-Thio-2-nitrobenzoic acid
concentration is determined by spectrophotometric analysis at 412 nm.
All other phospholipase assays were performed as described previously
(16, 17). Briefly, supernatants were incubated in a reaction mixture
(150 µl) containing 25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl2, and 0.1 µCi
of [3H]arachidonate-labeled E. coli membranes
for 1 h at 37 °C. Free arachidonic acid was separated by
elution of the sample over aminopropyl solid phase silica columns and
quantitated by scintillation counting.
Fig. 1.
sPLA2 mediates early
PGS1-dependent PGD2 synthesis but not late
PGS2-dependent PGD2 synthesis in activated mast
cells. MMC-34 mast cells were plated in 12-well culture dishes at
a density of 106 cells/ml. Mast cells were activated by
aggregating their IgE receptors, using sequential treatment with IgE
followed by anti-IgE. IgE (1 µg/ml) was added to all wells. Two h
later cells were washed and replated. Anti-IgE (1 µg/ml) (
) alone
or anti-IgE and SB203347 (1 µM) (
) were added to the
indicated cultures. In order to irreversibly inactivate PGS1, aspirin
was added to the indicated cultures (
) prior to anti-IgE activation.
For this purpose, IgE was added to cells at time 0. Ninety min later
aspirin (200 µM) was added. Thirty min later the cells
were washed to remove the aspirin and replated. The aspirin-treated
cells, in which PGS1 was inactivated, were then treated with anti-IgE
to activate PGD2 synthesis. Culture media were collected by
centrifugation at the times indicated and assayed for PGD2.
All analyses were performed on triplicate culture wells. Data are
expressed as averages, ± standard deviations.
[View Larger Version of this Image (17K GIF file)]
Fig. 2.
SB203347 does not inhibit constitutive PGS1
enzyme activity. MMC-34 cells were activated by aggregating their
IgE receptors. SB203347 (1 µM) was added to the indicated
culture at the time anti-IgE was added. One h after activation the
culture media were removed and saved for analysis of PGD2
produced from endogenous arachidonic acid (left panel). The
cells were washed twice with phosphate-buffered saline and incubated at
37 °C with exogenous arachidonic acid (10 µM). After
10 min the supernatants were collected by centrifugation and assayed
for PGD2 accumulation (right panel). All
analyses were performed on triplicate culture wells. Data are expressed
as averages, ± standard deviations.
[View Larger Version of this Image (18K GIF file)]
Fig. 3.
Both SB203347 and a monoclonal antibody for
sPLA2 inhibit the early phase of PGD2
production in activated MMC-34 cells and the appearance of
phospholipase activity in culture media. MMC-34 mast cells were
plated in 12-well culture dishes at a density of 106
cells/ml. Mast cells were activated by aggregating their IgE receptors,
using sequential treatment with IgE followed by anti-IgE. IgE (1 µg/ml) was added to the indicated wells. Two h later cells were
washed and replated. Anti-IgE (1 µg/ml), mAbF10 (10 µg/ml), and
SB203347 (1 µM) were added as indicated. NS-398 (1 µM) was added to all cultures, to inhibit activity of any
induced PGS2. Supernatants were collected by centrifugation from all
cultures 1 h after IgE addition and assayed both for
PGD2 (left panel) and for phospholipase activity
(right panel). All analyses were performed on triplicate
culture wells. Data are expressed as averages, ± standard
deviations.
[View Larger Version of this Image (18K GIF file)]
Fig. 4.
Increased concentrations of SB203347, the
sPLA2 inhibitor, do not inhibit the late phase of
PGD2 accumulation in activated MMC-34 cells. MMC-34
mast cells were activated by aggregating their IgE receptors, using
sequential treatment with IgE followed by anti-IgE. SB203347 was added,
at the concentrations indicated in the figure, at the time of anti-IgE
addition. Supernatants were collected by centrifugation 6 h after
anti-IgE and SB203347 addition, and assayed for PGD2. All
analyses were performed on triplicate culture wells. Data are expressed
as averages, ± standard deviations.
[View Larger Version of this Image (15K GIF file)]
Fig. 5.
Inhibition of sPLA2 and PGS2 can
completely suppress PGD2 production by activated mast
cells. MMC-34 mast cells were cultured and activated by
aggregation of IgE receptors as described in the legend to Fig. 1.
Lane 1, MMC-34 cells were treated with IgE alone. Lane
2, cells were treated with IgE for two h, washed and further
treated with anti-IgE to activate the cells. In lanes 3, 4, and 5, aspirin was added prior to anti-IgE activation, in order to irreversibly inactivate PGS1. For this purpose, IgE was added
to cells at time 0. Ninety min later aspirin was added. Thirty min
later the cells were washed to remove the aspirin, and replated. The
aspirin-treated cells, in which PGS1 was inactivated, were then treated
with anti-IgE to activate the late phase of PGD2 synthesis
(3rd lane), or with anti-IgE + mAbF10 (4th lane), or anti-IgE + SB203347 (5th lane). In the 6th and
8th lanes, PGS2-dependent PGD2
production was blocked by NS-398, and the effect of the various inhibitors was examined following activation by aggregation of IgE
receptors. MMC-34 cells were treated with IgE for 2 h, washed, plated, and further incubated with anti-IgE + NS-398 (6th
lane), anti-IgE + NS-398 + mAbF10 (7th lane), or
anti-IgE + NS-398 + SB203347 (8th lane). Reagent
concentrations were the same as those indicated in the legends to the
previous figures. Supernatants were isolated by centrifugation 6 h
after anti-IgE addition and analyzed for PGD2. All analyses
were performed on triplicate culture wells. Data are expressed as
averages, ± standard deviations.
[View Larger Version of this Image (19K GIF file)]
Fig. 6.
MAFP preferentially inhibits recombinant
cPLA2 and SB203347 preferentially inhibits recombinant
sPLA2. Recombinant sPLA2 (2 ng)
(left panel) or recombinant cPLA2 (20 ng)
(right panel) were incubated with vehicle, SB203347 (1 µM), or MAFP (10 µM) for 10 min at
37 °C. Phospholipase A2 reactions were initiated by
adding 0.1 µCi of [3H]arachidonic acid-labeled E. coli
membranes. Incubation was continued for 60 min. Free arachidonic acid
was separated and analyzed as described previously (3). Data are expressed as percent of vehicle control phospholipase A2
activity.
[View Larger Version of this Image (21K GIF file)]
Fig. 7.
MAFP, when added at the time of activation,
inhibits the late phase of PGD2 production in mast cells.
Left panel, MMC-34 mast cells were cultured and activated by
aggregation of IgE receptors as described in the legend to Fig. 1. At
the time of activation by addition of anti-IgE (), MAFP (10 µM) () or NS-398 (1 µM) () was added
to the indicated cultures. Culture media were collected by
centrifugation at the times indicated and assayed for PGD2. Right panel, MAFP was added at the concentrations indicated
in the figure at the time of anti-IgE addition. Supernatants were collected by centrifugation 6 h after anti-IgE and MAFP addition and assayed for PGD2. All analyses were performed on
triplicate culture wells. Data are expressed as averages, ± standard
deviations.
[View Larger Version of this Image (14K GIF file)]
Fig. 8.
MAFP does not inhibit PGS2 enzyme
activity. MMC-34 mast cells were activated by aggregation of IgE
receptors, following inactivation of endogenous PGS1 by aspirin
preincubation (see legend to Fig. 1). One set of culture wells was
activated in the presence of MAFP (10 µM), added at the
same time as anti-IgE. Six h after addition of anti-IgE, the culture
media were isolated by centrifugation and saved for PGD2
analysis (left panel). The pelleted cells were washed twice
with phosphate-buffered saline and incubated at 37 °C with exogenous
arachidonic acid (10 µM). After 10 min the media were
collected by centrifugation and assayed for PGD2
accumulation (right panel). All analyses were performed on
triplicate culture wells. Data are expressed as averages, ± standard
deviations.
[View Larger Version of this Image (17K GIF file)]
Fig. 9.
MAFP, a cPLA2 inhibitor, also
modulates early PGD2 production in activated mast cells.
Top panel, MMC-34 cells were activated by aggregation of IgE
receptors as described in the legend to Fig. 1. At the time of
activation by addition of anti-IgE, either 1 µM SB203347
(3rd lane), 10 µM MAFP (4th lane),
or 1 µM NS-398 (5th lane) was added to
indicated cultures. One set of cultures (6th lane) received
10 µM MAFP 30 min prior to initiation of activation by
the addition of anti-IgE. For this treatment cells were exposed to IgE
for 2 h. Cells were washed, and MAFP was added in complete medium.
Thirty min later anti-IgE was added. Supernatants were collected from
all cultures 6 h after anti-IgE addition and analyzed for
PGD2. Middle panel, MAFP was added, at the
concentrations indicated in the figure, 15 min prior to the initiation
of activation of MMC-34 mast cells by the addition of anti-IgE.
Supernatants were collected by centrifugation 1 h after anti-IgE
addition and assayed for PGD2. Lower panel,
MMC-34 cells were pretreated with 10 µM MAFP, for the
times indicated, prior to activation of MMC-34 mast cells by the
addition of anti-IgE. Supernatants were collected by centrifugation
1 h after anti-IgE addition and assayed for PGD2. All
analyses were performed on triplicate culture wells. Data are expressed
as averages, ± standard deviations.
[View Larger Version of this Image (17K GIF file)]
To whom correspondence should be addressed: 341A Molecular Biology
Institute, UCLA, Los Angeles, CA 90095. Tel.: 310-825-8735; Fax:
310-825-1447; E-mail: harvey{at}lbes.medsch.ucla.edu.
1
The abbreviations used are: PLA2,
phospholipase A2; sPLA2, secretory
phospholipase A2; cPLA2, cytoplasmic
phospholipase A2; iPLA2, cytosolic,
calcium-independent PLA2; PGS1, prostaglandin synthase 1;
PGS2, prostaglandin synthase 2; ATPC,
arachidonylthiophosphatidylcholine; MAFP, methyl
arachidonylfluorophosphonate.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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