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INTRODUCTION |
Arachidonic acid (AA)1
mobilization and the generation of prostaglandins by major
immunoinflammatory cells such as macrophages and mast cells usually
occur in two phases. The immediate phase, which takes minutes and is
elicited by Ca2+-mobilizing agonists such as
platelet-activating factor (PAF), is characterized by a burst of AA
liberation. In some cells such as P388D1 macrophages (1, 2)
and MMC-34 mast cells (3), this burst is mainly produced by a secretory
phospholipase A2 (sPLA2) but is strikingly
regulated by the cytosolic Group IV phospholipase A2
(cPLA2).
The delayed phase of prostaglandin production is accompanied by the
continuous supply of AA over long incubation periods spanning several
hours. There is some discrepancy about the identity of the
PLA2 isoform(s) involved in the delayed phase. Despite this phase being independent of a Ca2+ increase, the
cPLA2 has often been suggested to be critically involved
(3-5). However, other studies have suggested the quantitatively more
important role of the sPLA2, an enzyme that is dramatically induced during long term incubation of the cells with a variety of
stimuli (4-6). There is, however, agreement that COX-2, another enzyme
whose expression is augmented dramatically after long term stimulation,
is absolutely required for long term PGE2 production, irrespective of the constitutive presence of COX-1 (7-9).
Using a new clone of the P388D1 macrophage-like cells
termed P388D1/MAB, we provide herein evidence for the
involvement of Group V sPLA2 in delayed PGE2
production. Furthermore, our results suggest that Group V
sPLA2 expression is dependent upon the activation of Group
IV cPLA2.
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EXPERIMENTAL PROCEDURES |
Materials--
Mouse P388D1 macrophage-like cells
were obtained from the American Type Culture Collection (Rockville,
MD). Iscove's modified Dulbecco's medium (endotoxin <0.05 ng/ml) was
from Whittaker Bioproducts (Walkersville, MD). Fetal bovine serum was
from Hyclone Laboratories (Logan, UT). Nonessential amino acids were
from Irvine Scientific (Santa Ana, CA).
[5,6,8,9,11,12,14,15-3H]Arachidonic acid (specific
activity, 100 Ci/mmol) was from NEN Life Science Products, and
1-palmitoyl-2-[14C]palmitoyl-sn-glycero-3-phosphocholine
(specific activity, 54 mCi/mmol) was from Amersham Pharmacia Biotech.
PAF, LPS (Escherichia coli 0111:B4), and
actinomycin D were from Sigma. Methylarachidonyl fluorophosphonate
(MAFP) and NS-398 were from Biomol (Plymouth Meeting, PA). Antibodies
against murine COX isoforms were generously provided by Dr. W. L. Smith (Department of Biochemistry, Michigan State University, East
Lansing, MI). The antibody against Group IV cPLA2 was
generously provided by Dr. Ruth Kramer (Lilly). The sPLA2
inhibitor, 3-(3-acetamide 1-benzyl-2-ethylindolyl-5-oxy)propanesulfonic acid (LY311727), was generously provided by Dr. Edward Mihelich (Lilly). cDNA probes for Groups V and IIA sPLA2s were
synthesized as described previously (11). cDNA probes for murine
glyceraldehyde-3-phosphate dehydrogenase were from Cayman (Ann Arbor, MI).
Cell Culture and Labeling Conditions--
P388D1
cells were maintained at 37 °C in a humidified atmosphere at 90%
air and 10% CO2 in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum, 2 mM glutamine,
100 units/ml penicillin, 100 µg/ml streptomycin, and nonessential amino acids. P388D1 cells were plated at
106/well, allowed to adhere overnight, and used for
experiments the following day. All experiments were conducted in
serum-free Iscove's modified Dulbecco's medium. When required,
radiolabeling of the P388D1 cells with [3H]AA
was achieved by including 0.5 µCi/ml [3H]AA during the
overnight adherence period (20 h). Labeled AA that had not been
incorporated into cellular lipids was removed by washing the cells four
times with serum-free medium containing 1 mg/ml albumin.
Measurement of PGE2 Production and Extracellular
[3H]AA Release--
The cells were placed in serum-free
medium for 30 min before the addition of LPS for different periods of
time. Afterward, the supernatants were removed and cleared of detached
cells by centrifugation, and PGE2 was quantitated using a
specific radioimmunoassay (PersPective Biosystems, Framingham, MA). For
[3H]AA release experiments, cells labeled with
[3H]AA were used, and the incubations were performed in
the presence of 0.5 mg/ml bovine serum albumin. The supernatants were
removed, cleared of detached cells by centrifugation, and assayed for
radioactivity by liquid scintillation counting. The standard LPS/PAF
stimulation protocol for immediate responses has been described
previously (1). Briefly, the cells were incubated for 1 h with 200 ng/ml LPS followed by a 10-min incubation with 100 nM PAF.
Western Blot Analyses--
The cells were overlaid with a buffer
consisting of 10 mM Hepes, 0.5% Triton X-100, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, 20 µM aprotinin,
pH 7.5. Samples from cell extracts (10 µg for cPLA2, 200 µg for COX) were separated by SDS-polyacrylamide gel electrophoresis
(10% acrylamide gel) and transferred to Immobilon-P (Millipore). For
cPLA2 mobility shift studies, 24-cm acrylamide gels were
run. Nonspecific binding was blocked by incubating the membranes with
5% nonfat milk in phosphate-buffered saline for 1 h. Membranes
were then incubated with anti-cPLA2, anti-COX-1, or
anti-COX-2 antisera and treated with horseradish peroxidase-conjugated
protein A (Amersham Pharmacia Biotech). Bands were detected by enhanced
chemiluminescence (ECL, Amersham Pharmacia Biotech).
Northern Blot Analyses--
Total RNA was isolated from
unstimulated or LPS-stimulated cells by the TriZOL reagent method (Life
Technologies, Inc.), exactly as indicated by the manufacturer. Fifteen
µg of RNA were electrophoresed in a 1% formaldehyde/agarose gel and
transferred to nylon filters (Hybond, Amersham Pharmacia Biotech) in
10× SSC buffer. Hybridizations were performed in QuickHyb solution
(Stratagene) following the manufacturer's instructions.
32P-Labeled probes for Group IIA or
glyceraldehyde-3-phosphate dehydrogenase were co-incubated with the
filters for 1 h at 66 °C followed by three washes with 2× SSC
containing 0.1% SDS at room temperature for 30 min. A final wash was
carried out at 60 °C for 30 min with 1× SSC containing 0.1% SDS.
For Group V sPLA2, hybridizations were performed in
ExpressHyb solution (CLONTECH) following the manufacturer's instructions. The 32P-labeled probes were
co-incubated with the filters for 1 h at 66 °C followed by two
washes with 2× SSC containing 0.05% SDS for 15 min: the first at room
temperature and the second at 40 °C. Afterward the filters were
washed twice more with 0.1× SSC containing 0.1% SDS for 15 min at
room temperature. Bands were visualized by autoradiography.
Phospholipase A2 Assay--
Aliquots (50-100 µl)
of supernatants from LPS-treated cells were assayed for
PLA2 activity as follows. The assay mixture (500 µl)
consisted of 100 µM
1-palmitoyl-2-[14C]palmitoyl-sn-glycero-3-phosphocholine
substrate (2000 cpm/nmol), 10 mM CaCl2, 100 mM KCl, 25 mM Tris-HCl, pH 8.5. Reactions
proceeded at 40 °C for 30 min, after which
[14C]palmitate release was determined by a modified Dole
procedure (10).
Antisense Inhibition Studies in P388D1
Cells--
Transient transfection of P388D1 cells with
antisense oligonucleotide, ASGV-2, or its sense counterpart, SGV-2,
plus LipofectAMINE was carried out as described (11). Briefly,
P388D1 cells were transfected with oligonucleotide (125 nM) in the presence of 5 µg/ml LipofectAMINE (Life
Technologies, Inc.) under serum-free conditions for 8 h prior to
treating the cells with or without 100 ng/ml LPS for 10 h after
transfection (11). Antisense oligonucleotide ASGV-2 (5'-GGA CUU GAG UUC
UAG CAA GCC-3') is complementary to nucleotides 64-84 of the mouse
Group V PLA2 gene. SGV-2 (5'-GGC UUG CUA GAA CUC AAG
UCC-3') is the sense complement of ASGV-2.
For Group VI iPLA2 antisense experiments, a protocol
identical to that reported elsewhere was followed (12).
Data Presentation--
Assays were carried out in duplicate or
triplicate. Each set of experiments was repeated three times with
similar results. Unless otherwise indicated, the data presented are
from representative experiments.
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RESULTS |
AA Release in a Novel P388D1 Cell Clone
(MAB)--
Stimulation of murine P388D1 macrophages with
nanomolar amounts of the receptor agonist PAF results in very little AA
mobilization. However, preincubation of the cells with LPS prior to
stimulation with PAF increases the release of AA by these cells well
above unstimulated levels, the relative magnitude of the response being dependent on the cell batch (13, 14). We have now selected by limit
dilution a clone of the P388D1 cells termed MAB, which shows a remarkably higher [3H]AA release response to
LPS/PAF when compared with the ATCC batch of P388D1 cells
from which the MAB clone was obtained (Fig.
1). More interestingly, in addition to an
immediate response to LPS/PAF (Fig.
2A), cells from the MAB clone
also exhibited a delayed [3H]AA release response,
spanning several hours, to LPS alone (Fig. 2A). The dose
response of the effect of LPS on long term [3H]AA release
is shown in Fig. 2B. The maximal effect was observed at a
LPS dose as low as 10 ng/ml.

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Fig. 1.
AA release in a new P388D1 cell
clone, MAB. [3H]AA release in LPS/PAF-treated
(closed bars) or untreated (open bars) was
assayed in cells from the ATCC or the MAB clone as indicated. The cells
were incubated with 200 ng/ml LPS for 1 h followed by a 10-min
incubation with 100 nM PAF.
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Fig. 2.
LPS-stimulated long term [3H]AA
metabolism in P388D1 macrophages. A, time
course of [3H]AA release in response to LPS/PAF (the
latter was added 1 h after the former, and the incubations
proceeded for the time indicated) (closed triangles), LPS
alone (closed circles), or neither (open
circles). B, dose response of the LPS effect (20-h
incubation). C, the time course of PGE2
production by cells treated with (closed circles) or without
(open circles) 100 ng/ml LPS.
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Prostaglandin Production by P388D1/MAB Cells--
Fig.
2C shows the time course of PGE2 production by
LPS in these cells as measured by radioimmunoassay, which corresponded well with the [3H]AA mobilization response. Thus,
LPS-induced PGE2 barely increased above controls within the
first 3 h of treatment, rising afterward, and reaching a plateau
after 12 h.
The effect of LPS on the protein levels of the two COX isoenzymes these
cells express (2) was assessed by immunoblot. Expression of COX-1 did
not change along the time course of LPS activation (data not shown),
whereas COX-2 levels noticeably increased with maximal expression
between 12 and 18 h (Fig.
3A). Interestingly, LPS-induced COX-2 expression almost parallels PGE2
generation (cf. Figs. 2C and 3A),
suggesting that COX-2 is the enzyme responsible for LPS-induced
PGE2 synthesis. Indeed, the COX-2-specific inhibitor NS-398
(15) completely blocked LPS-induced PGE2 production (Fig. 3B). Therefore, LPS-delayed PGE2 generation
depends exclusively on COX-2, irrespective of the continued presence of
COX-1.

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Fig. 3.
LPS-stimulated long term PGE2
production. A, the effect of 100 ng/ml LPS on COX-2
protein levels at the indicated times (h) as measured by immunoblot.
B, the effect of NS-398 on LPS-induced PGE2
production. The cells were treated with (closed bars) or
without (open bars) 5 µM NS-398 for 20 min
before the addition of LPS for 18 h.
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cPLA2 Involvement in LPS-induced Long Term
Responses--
Expression of the Group IV cPLA2 protein in
P388D1/MAB cells was constitutive and did not change after
exposure to LPS. To address the possible involvement of
cPLA2 in LPS-induced AA mobilization in
P388D1/MAB cells, experiments were conducted with MAFP, an inhibitor that has previously been shown to block the immediate, cPLA2-dependent [3H]AA release in
LPS/PAF-treated macrophages (1). As shown in Fig.
4, MAFP strongly blocked the LPS-induced
long term [3H]AA release response. MAFP has recently been
observed to inhibit the Group VI iPLA2 in addition to the
cPLA2 (10). Therefore, it could be possible that part of
the MAFP effects reported herein resulted from inhibition of the
iPLA2 in addition to any effect on the cPLA2.
We have recently described the inhibition of iPLA2 expression in P388D1 cells by antisense RNA
oligonucleotides (12). Using this technique, we have been able to
significantly inhibit iPLA2 expression, assayed both by
protein content by immunoblot and activity by a specific in
vitro assay (12). Antisense RNA inhibition of the
iPLA2 under identical conditions as those shown previously
(12) showed no reduction in AA release in response to LPS (not shown).
Therefore these data make it likely that the above reported effects of
MAFP on the response are because of the inhibition of the
cPLA2.

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Fig. 4.
Effect of MAFP and LY311727 on LPS-induced AA
release. The cells were treated with 25 µM MAFP
(closed triangles), 25 µM LY311727
(closed squares), or neither (closed circles) for
20 min before the addition of LPS. Open circles denote
control incubations, i.e. those that received neither LPS
nor inhibitors. The inhibitors alone did not change the control
release.
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Role of sPLA2--
LPS-induced long term
[3H]AA release was also noticeably blocked by the
selective sPLA2 inhibitor LY311727 (17), indicating that in
addition to the cPLA2, a sPLA2 is also involved
in the process (Fig. 4). PGE2 production by LPS was also
inhibited by LY311727 by about 90%. Although originally described as a
selective Group II sPLA2 inhibitor (17), we have recently
shown that this compound is also a potent Group V sPLA2
inhibitor (18).
Our previous work (11) has demonstrated that P388D1 cells
express measurable message levels for Group V sPLA2, both
under unstimulated and LPS/PAF-stimulated conditions. However, message levels for Groups IIA sPLA2 or IIC sPLA2 were
undetectable even by reverse transcriptase-polymerase chain reaction
(11). As shown in Fig. 5A, an
antisense oligonucleotide specific for Group V sPLA2
(ASGV-2) strongly blocked PGE2 production in LPS-treated cells, whereas its sense control (SGV-2) had no effect. Moreover, mRNA analyses by Northern blot at long times of stimulation with LPS confirmed the presence of mRNA for Group V sPLA2
(Fig. 5, B and C) but not for Group IIA
sPLA2 (data not shown). The signal for Group V
sPLA2 markedly increased after LPS stimulation, reaching a
peak at approximately 10 h.

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Fig. 5.
Group V sPLA2 involvement in AA
release. A, the effect of a specific Group V antisense
oligonucleotide (AGV-2) or its sense control (SGV-2) on
PGE2 production in cells treated without (open
bars) or with (closed bars) 100 ng/ml LPS for 10 h. None denotes incubations that received no
oligonucleotide. B, the effect of LPS on Group V
sPLA2 message levels as determined by Northern blot. Total
RNA from cells incubated with (+) or without ( )
100 ng/ml LPS for the indicated periods of time was isolated and
analyzed by Northern blot with probes specific for Group V
sPLA2 or glyceraldehyde-3-phosphate dehydrogenase
(G3PDH). C, densitometric analysis of the Group V
sPLA2 signals normalized for the glyceraldehyde-3-phosphate
dehydrogenase signal in each lane.
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PLA2 activity measurements in the supernatants of
LPS-stimulated cells revealed a time-dependent increase in
activity (Fig. 6), which correlated well
with the time course of Group V sPLA2 mRNA induction
(cf. Figs. 5B and 6). Extracellular
PLA2 activity was decreased if the experiments were
conducted in the presence of the RNA synthesis inhibitor actinomycin D
(Fig. 7A). This increased extracellular activity was found to correspond to that of Group V
sPLA2 by the following criteria: (i) it was completely
inhibited by the sPLA2 inhibitor LY311727 (Fig.
7B) and (ii) it was decreased in supernatants from cells
treated with an antisense RNA oligonucleotide specific for Group V
sPLA2, ASGV-2 (11) (Fig. 7C).

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Fig. 6.
Effect of MAFP on the appearance of
sPLA2 activity in the supernatants of P388D1
cells and the effect of MAFP. The cells, pretreated with
(closed triangles) or without (closed circles) 25 µM MAFP for 20 min, were challenged with (closed
symbols) or without (open circles) 100 ng/ml LPS for
the indicated times. Afterward, supernatants were collected and assayed
for PLA2 activity. The amount of PLA2 activity
detected in supernatants of cells not treated with LPS (open
circles) was not changed whether the cells were pretreated or not
with MAFP.
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Fig. 7.
Effect of different treatments on the
appearance of PLA2 activity in supernatants from
LPS-treated cells. A, the effect of actinomycin D is
shown. PLA2 activity in the supernatants from cells treated
with LPS plus the indicated concentrations of actinomycin D for 18 h is indicated. Control denotes incubations carried out
without either LPS or actinomycin D. B, blockage by LY311727
of the PLA2 activity of supernatants from LPS-treated or
untreated cells. An aliquot of the culture medium of cells treated
without (Control) or with LPS for 18 h was incubated
with or without 25 µM LY311727 for 20 min at 40 °C and
then assayed for PLA2 activity. C, the effect of
a specific Group V antisense oligonucleotide (AGV-2) or its sense
control (SGV-2) on PLA2 activity in the supernatants from
LPS-treated or untreated cells.
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Role of cPLA2 in sPLA2 Activation--
Our
previous studies have indicated that the immediate AA release triggered
by LPS/PAF in these cells involves the sequential action of both
cPLA2 and sPLA2, with the activity of the
latter being dependent on previous activation of the former (1, 2). Thus we sought to investigate if a similar cross-talk exists between the two enzymes during long term stimulation conditions. We found that
no increased PLA2 activity beyond what was observed in the basal state could be found in supernatants from cells treated with MAFP
(Fig. 6). In addition, the cPLA2 inhibitor markedly decreased the LPS-induced expression of Group V sPLA2
mRNA (Fig. 8).

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Fig. 8.
The effect of 25 µM MAFP on Group V sPLA2
expression from LPS-treated (100 ng/ml, 18 h) or untreated
cells.
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DISCUSSION |
A striking hallmark of the immunoinflammatory response is the
generation of oxygenated derivatives of AA such as the prostaglandins. The response of major prostaglandin-secreting cells such as macrophages and mast cells to proinflammatory stimuli is generally biphasic (4).
The first phase is completed within minutes after the addition of the
stimulus, whereas the second phase usually takes several hours (4).
Using the murine macrophage-like cell line P388D1, we have
been studying the molecular mechanisms responsible for AA mobilization
and prostaglandin production in response to LPS/PAF. When primed by
LPS, these cells will respond to Ca2+-mobilizing stimuli
such as PAF by generating a rapid burst of free AA, part of which is
converted to prostaglandins such as PGE2. Strikingly, this
process is completed within a few minutes after the addition of PAF
(19). No free AA or prostaglandins are produced after the immediate
phase is completed, not even after several hours of cell exposure to
LPS/PAF (13). Such a behavior, which is abnormal for a macrophage cell,
has prevented us from studying the molecular mechanisms responsible for
delayed prostaglandin production in macrophages. In an attempt to
overcome this problem, we subcloned the P388D1 cells by
limit dilution, and selecting on the basis of high responsivity to
LPS/PAF, we obtained a clone termed MAB, which shows enhanced
sensitivity to LPS/PAF in the immediate phase (min) and exhibits a
delayed response (h) to LPS alone.
Using the MAB clone, we have characterized the LPS-induced delayed
prostaglandin production in terms of the role played by distinct
PLA2 enzymes and their coupling with downstream COX enzymes during LPS signaling. Our previous work on the immediate response of
the cells to LPS/PAF highlighted the very important role played by the
novel Group V sPLA2 as the provider of most of the free AA
directed to PGE2 biosynthesis (11). Herein, several lines of evidence suggest that Group V sPLA2 also behaves as a
major provider of AA for the delayed phase of PGE2
production in LPS-treated cells. First, delayed [3H]AA
release and PGE2 production correspond with the induction of Group V sPLA2 mRNA and enhanced secretion of a
sPLA2-like activity to the supernatants, with no change in
the constitutive levels of cPLA2 and no detectable
induction of Group IIA sPLA2. Second, delayed
PGE2 production is strongly blunted by LY311727, a
selective sPLA2 inhibitor. Third, an antisense
oligonucleotide specific for Group V sPLA2 (11) suppresses
Group V sPLA2 activity and inhibits delayed
PGE2 production. Our conclusions in this regard fully agree
with recent works by Kudo and co-workers (20, 21) that were published
while this manuscript was under review. By using transfection
techniques, Kudo and co-workers (20, 21) have also documented the
importance of Group V sPLA2 in delayed AA release and
PGE2 production.
Our data have also implicated the cPLA2 as an important
step in LPS signaling by enabling the subsequent action of the
sPLA2. Thus the cPLA2 inhibitor MAFP (1)
markedly blocked both long term [3H]AA release and Group
V sPLA2 mRNA induction. Collectively, these results
suggest an intriguing cross-talk between the cPLA2 and the
Group V sPLA2 for the delayed phase of prostaglandin
production in macrophages. This is a very interesting concept because
cross-talk appears to exist as well between these two enzymes during
the immediate phase of prostaglandin production (1, 2). In the immediate phase, cPLA2 activation generates a rapid and
early burst of free AA inside the cell that enables sPLA2
activation by an as yet unidentified mechanism (1, 2). In the delayed phase, cPLA2 activation influences sPLA2
apparently by regulating sPLA2 mRNA levels.
Cross-talk between cPLA2 and sPLA2 in the
immediate phase of prostaglandin production was also found to take
place in mast cells (3) when the same protocol originally used in
macrophages (1) was employed. Furthermore, a recent study by Kuwata
et al. (22) about fibroblasts suggests that cross-talk
between cPLA2 and sPLA2 in the delayed phase
could also constitute a general mechanism of activation. Using a
different cPLA2 inhibitor, arachidonyl trifluoromethyl
ketone, Kuwata et al. (22) found that cPLA2 inhibition blocked sPLA2 expression in fibroblasts, leading
to reduced PGE2 generation. The study by Kuwata et
al. (22) is interesting not only because it supports the possible
universality of cross-talk between cPLA2 and
sPLA2 but because the sPLA2 expressed by rat
fibroblasts is a Group IIA enzyme, not Group V. This lends further
support to the emerging notion that Group IIA and Group V
sPLA2 may be functionally redundant (23). In addition,
Kuwata et al. (22) were able to show that overcoming
cPLA2 inhibition by exogenous AA partially restored the
Group IIA sPLA2 expression. These results suggest that the
AA mobilized by cPLA2 is responsible for cross-talk between
cPLA2 and sPLA2 (22). This is again reminiscent of what happens in the immediate phase of activation, wherein the
cPLA2-derived AA is also responsible for cross-talk between cPLA2 and sPLA2, albeit by different mechanisms
(1, 2). Unfortunately, inhibition by MAFP of Group V sPLA2
expression and activity could not be reversed in our macrophage studies
with LPS alone by supplementing the medium with exogenous AA (up to 100 µM). This was not unexpected because P388D1
cells manifest an extraordinarily high capacity to import free AA from
exogenous sources and incorporate it into membranes (19, 24, 25), which
is much higher than that of most other cells (26). Thus, the half-life
of the free AA in the cell would be too short to adequately mimic the
low but continued production of AA-derived cPLA2 upon long
term LPS exposure.
A model has recently emerged suggesting differential actions of COX-1
and COX-2 by virtue of differential coupling to distinct PLA2s (2, 3, 6, 8, 20, 21, 27). Thus, depending on whether
cPLA2 or sPLA2 is the provider of free AA,
either COX-1 or COX-2 would be responsible for PGE2
release. However, which PLA2 form couples to which COX
isoform appears to depend strongly on cell type. We have recently
demonstrated that the immediate, PAF receptor-mediated phase of
PGE2 production in LPS-primed macrophages involves
sPLA2 coupling to COX-2 (2). The current results support a
similar kind of coupling for the delayed PGE2 production in LPS-treated cells. Identical coupling has been suggested by Arm and
co-workers (6) for the delayed phase of PGE2 generation in
mast cells. These results raise another interesting concept regarding
the regulation of PGE2 during both phases of activation. As
is the case for AA release (Fig. 2A), we have observed that the amount of PGE2 generated during the
Ca2+-dependent short term stimulation is
comparable to the amount produced in the late phase. It follows from
this comparison that although the effector enzymes involved in the
response are the same (i.e. cPLA2,
sPLA2, COX-2), the regulatory mechanisms differ. Thus, in
the short phase at low levels of COX-2, it appears that the dramatic
burst in AA release is what determines the amount of PGE2
produced. In contrast, in the delayed phase at comparably lower AA
availability, it appears that both the induction of large amounts of
COX-2 protein and of the AA provider, Group V sPLA2, determine the amount of PGE2 produced.
It is important to note, however, that our results have not excluded
that a minor fraction of the long term PGE2 produced in
response to LPS could arise from the AA generated by the
cPLA2. Should this be the case, some
cPLA2/COX-2 coupling may exist as well, similar to what has
been suggested by Reddy and Herschman (3) for delayed PGD2
production in mast cells and by Murakami et al. (5) in cells
derived from Group IIA-deficient mice. The striking feature of the
current work is that although COX-1 is present in active form in the
P388D1 cells (2), it appears to be spared from the process
of long term PGE2 production. This finding remains
unexplained but has recently been recognized in other cell types as
well (6, 8, 22). Recent work by Spencer et al. (16) showed
no differences in the distribution of COX-1 versus COX-2
among subcellular fractions in a variety of cells. Thus subcellular
compartmentalization may not be the cause for COX-1 not being utilized
during LPS signaling. Other putative explanations may include the
existence of COX-selective regulatory components, selective coupling to
terminal PG synthases, or kinetic differences in AA utilization by the
two isoforms.
In summary, we have established a subclone of P388D1 cells,
MAB, that displays long term responsiveness to LPS in terms of PGE2 generation. We have confirmed (11) that these cells
express Group V sPLA2, not Group IIA sPLA2, and
found that (i) Group V sPLA2 is a key enzyme in long term
AA mobilization as well and (ii) Group V sPLA2 is
functionally coupled to COX-2. Furthermore, our results have suggested
that cPLA2 plays a key role in long term AA mobilization,
at least partly by regulating the expression of Group V
sPLA2.