(Received for publication, June 5, 1995; and in revised form, October 25, 1995)
From the
Prostaglandins mediate many biological processes. Arachidonic
acid, the common precursor for all prostaglandins, is released from
membrane phospholipids by both secretory and cytoplasmic forms of
phospholipase A. Free arachidonate is converted to
prostaglandin H
, the common precursor to all prostanoids,
by prostaglandin synthase. Both mitogen-induced prostaglandin synthesis
in fibroblasts and endotoxin-induced prostaglandin synthesis in
macrophages require expression of the inducible prostaglandin
synthase-2; arachidonate released in these contexts is unavailable to
prostaglandin synthase-1 constitutively present in fibroblasts or
macrophages.
In contrast to the results for fibroblasts and
macrophages, prostaglandin synthesis by activated mast cells is
mediated by prostaglandin synthase-1. Mast cell activation also
provokes release of secretory phospholipase A
(sPLA
). We now demonstrate that sPLA
released
from activated mast cells can mobilize arachidonate from distal Swiss
3T3 cells. This arachidonate is then used by prostaglandin synthase-1
present in 3T3 cells for prostaglandin synthesis. We thus distinguish
two pathways for prostaglandin synthesis: (i) an intracellular pathway
by which arachidonate released following ligand stimulation is made
available only to prostaglandin synthase-2, and (ii) a transcellular
pathway by which sPLA
of proximal cells mobilizes, in
distal cells, arachidonate available to prostaglandin synthase-1.
Molecular and pharmacologic approaches to modulating
prostaglandin-mediated events will differ for these two pathways.
The prostanoids (prostaglandins, thromboxanes, and
prostacyclins) mediate a wide variety of physiological processes,
including ovulation, hemostasis, platelet aggregation, kidney water
balance, and immune responses(1) . Prostanoid production is
regulated both by release of arachidonic acid from membrane lipid
stores by phospholipase A and by conversion of arachidonic
acid to prostaglandin H
(PGH
), (
)the
common precursor for all prostanoids, by cyclooxygenase/prostaglandin
synthase (PGS)(2) .
Arachidonic acid can be released from
cellular membranes either by the 14 kilodalton secretory phospholipase
A (sPLA
), or by the 85 kilodalton intracellular
isoform of the enzyme, cytoplasmic phospholipase A
(cPLA
)(3) . Two forms of prostaglandin
synthase, the rate-limiting enzyme in the conversion of free arachidonic acid to prostanoids, also exist. Most cells contain a
constitutively-expressed prostaglandin synthase, PGS1, but can also
express an inducible form of the enzyme, PGS2, in response to
stimulation by appropriate ligands. Ligand-induced PGS2 gene expression
has been observed in fibroblasts, macrophages, endothelial cells,
epithelial cells, ovarian granulosa cells, neurons, and smooth muscle
cells(4) . Aspirin and other nonsteroidal anti-inflammatory
drugs exert their antipyretic, analgesic, and anti-inflammatory actions
by inhibiting the activity of the prostaglandin
synthases(5, 6) .
Experiments utilizing antisense
oligonucleotide inhibition demonstrate that both mitogen-stimulated
PGE production in fibroblasts and endotoxin-stimulated
PGE
production in macrophages require the induced
expression and activity of the PGS2 gene and protein, despite the
presence of constitutive, enzymatically active PGS1(7) . In
contrast to the requirement that PGS2 enzyme be synthesized for
prostaglandin production following activation by many ligands in
fibroblasts and macrophages, PGD
synthesis occurs rapidly
in activated mast cells. Moreover, the rapid PGD
production
in mast cells activated by aggregation of IgE receptors is dependent
only on the presence and activity of constitutive PGS1 (8) .
Following activation, mast cells degranulate to release preformed
ligands such as histamine and serotonin. In addition, activation
releases stored sPLA from mast cells(9) . Activated
mast cells can use arachidonic acid released by sPLA
for
prostaglandin synthesis(9) . In this report we demonstrate that
sPLA
released from activated MMC-34 mast cells can
stimulate PGS1-dependent synthesis of PGE
in co-cultured
Swiss 3T3 cells. These results provide evidence for distinct pathways
for sPLA
mediated, PGS1-dependent transcellular
prostaglandin production and for mitogen-stimulated, PGS2-dependent
intracellular prostaglandin production.
Figure 1:
N. naja sPLA stimulates PGE
synthesis in Swiss 3T3 cells.
Confluent Swiss 3T3 cells in 12-well culture dishes were treated either
with N. naja sPLA
(2 units/ml; closed
circles) or with TPA (50 ng/ml; open circles). At the
times shown, media were collected and assayed for PGE
. Data
are the averages of triplicate determinations,
±S.D.
Figure 2:
PGE production in Swiss 3T3
cells following sPLA
and TPA treatment occurs via distinct
prostaglandin synthase isoforms. 3T3 cells in 12-well culture dishes
were either untreated (lane 1), treated with N. naja sPLA
for 1 h (lane 2), pretreated for 30 min
with 200 µM aspirin, washed with fresh medium, and then
treated with N. naja sPLA
for 1 h (lane
3), treated with sPLA
and 1 µM NS-398 for
1 h (lane 4), treated with 50 ng/ml TPA for 6 h (lane
5), pretreated for 30 min with aspirin, washed with fresh medium,
and then treated with TPA for 6 h (lane 6), or treated with
TPA + NS-398 for 6 h (lane 7). Media were collected and
assayed for PGE
. Data are averages of triplicate
determinations, ±S.D.
Production of
PGE by TPA-treated cells is abolished by the PGS2-specific
inhibitor NS-398 (Fig. 2). In contrast, inactivaton of PGS
by aspirin pretreatment and washing has no effect on subsequent
TPA-induced PGE
induction in 3T3 cells. We conclude that
sPLA
-induced PGE
synthesis in 3T3 cells is
mediated by preexisting PGS1 enzyme, while TPA-induced PGE
synthesis in 3T3 cells requires synthesis and activity of PGS2
enzyme.
If
MMC-34 mast cells are simply co-cultured with 3T3 cells, little
PGE is present in the medium (Fig. 3, lane
1). If IgE receptors are aggregated on mast cells by treatment
with IgE followed by anti-IgE, PGD
is produced as expected
(data not shown), but no production of PGE
is observed (Fig. 3, lane 2). When Swiss 3T3 cells are treated with
IgE followed by anti-IgE, there is also no significant increase in
PGE
levels observed in the medium, since 3T3 cells do not
have IgE receptors (Fig. 3, lane 3). However,
substantial production of PGE
is observed in MMC-34 mast
cell/Swiss 3T3 co-cultures following addition of IgE + anti-IgE (Fig. 3, lane 4). The PGE
present in these
co-cultures after stimulation with IgE + anti-IgE must come from
the 3T3 cells, since activation of mast cells alone by IgE +
anti-IgE does not produce PGE
. Activation-induced PGE
production in the co-cultures must, therefore, be due to a mast
cell-mediated effect on the 3T3 cells. Medium from activated mast cells
can also stimulate PGE
production when applied to 3T3 cells (Fig. 3, lane 5). We conclude that a secreted
intermediate from activated mast cells mediates PGE
production in co-cultured Swiss 3T3 cells.
Figure 3:
Activation of mast cells induces PGE synthesis in MMC-34 mast cell/Swiss 3T3 cell co-cultures. MMC-34
mast cells and Swiss 3T3 cells were grown to confluence (2
10
cells per well, for both cell types). Although the
MMC-34 cells are not attached tightly to the plastic, they settle to
the bottom of the wells during culture. For co-culture experiments, the
medium from the Swiss 3T3 cells was replaced with the medium and cells
from the wells containing the MMC-34 cells. First lane; Swiss 3T3 cells
and MMC-34 mast cells were co-cultured for 1 h with no additions. Media
were collected and assayed for prostaglandins. Second lane; MMC-34
cells were incubated with IgE (1 µg/ml) for 2 h, then treated with
anti-IgE (1 µg/ml). One hour later prostaglandins in the medium
were assayed. Lane 3, 3T3 cells were incubated with IgE for 2
h, then incubated with anti-IgE for 1 h, and prostaglandins were
assayed. Lane 4, MMC-34 cells were treated with IgE for 2 h.
The media and MMC-34 cells were then transferred to confluent wells of
3T3 cells. Anti-IgE was added to the co-cultures, and 1 h later the
supernatants were assayed for prostaglandins. Lane 5, MMC-34
mast cells were incubated with IgE for 2 h, then treated with anti-IgE
for 1 h. The medium from activated MMC-34 cells (indicated by the asterisk) was collected by centrifugation and added to Swiss
3T3 cells. One hour later medium was again collected, and
prostaglandins were assayed. All experiments were performed in
triplicate. Data are the averages, ±S.D. PGD
present
in the supernatants from activated mast cells did not interfere with
the PGE
determinations (data not
shown).
Figure 4:
Both
phospholipase A activity and fibroblast PGE
inducing activity of activated mast cell supernatants are
inhibited by a monoclonal antibody to recombinant PLA
and
by SB 203347. Supernatants were prepared from control MMC-34 cells and
from cells treated with IgE (2 h) + anti-IgE (1 h). Activated
supernatants (indicated by the asterisk) were treated with mAb
F10 (10 µg/ml), a control mAb preparation, or SB 203347 (1
µM) as indicated in the figure. Samples (0.1 ml) of each
supernatant were assayed for phospholipase activity as described under
``Experimental Procedures.'' Phospholipase activity is
expressed as arachidonic acid (a.a.) released. Samples (0.9
ml) of the same supernatants were placed on confluent cultures of Swiss
3T3 cells. Media were collected after 1 h and assayed for
PGE
. Data are the averages of triplicate determinations,
±S.D.
It seems likely that the sPLA released following
activation of mast cells is responsible for the production of PGE
in the mast cell/fibroblast co-cultures. We used mAb F10, a
monoclonal antibody directed against recombinant
sPLA
(9) , and SB 203347, a recently described
inhibitor of sPLA
enzyme activity(16) , to
determine whether the sPLA
released by activated MMC-34
mast cells is responsible for PGE
production by 3T3 cells
in the mast cell/fibroblast co-cultures. Recombinant sPLA
(rPLA
), like the N. naja sPLA
and the supernatants from activated mast cells, can induce
PGE
production in 3T3 cells (Fig. 5). Both mAb F10
and SB 203347 inhibit PGE
induction in 3T3 cells in
response to rPLA
treatment (Fig. 5). mAb F10 and SB
203347 block 72% and 74% of the phospholipase activity of the
rPLA
protein used in the experiment illustrated in Fig. 5(data not shown). The amount of rPLA
activity
(150 pmol of [
H]arachidonic acid released/min/ml)
used in this experiment was chosen to resemble that found in the
supernatants of activated MMC-34 mast cells (Fig. 4).
Figure 5:
The ability of recombinant PLA to induce PGE
production in fibroblasts is inhibited
by mAb F10 and by SB 203347. The enzymatic activity of rPLA
was determined as described under ``Experimental
Procedures.'' Samples of rPLA
with an activity of 150
pmol of arachidonic acid released/min were treated with mAb F10 (10
µg), control mAb, or SB 203347 (1 µM). The treated and
untreated rPLA
samples were incubated with confluent
cultures of Swiss 3T3 cells, in fresh medium. Media were collected
after 1 h and assayed for PGE
. Data are the averages of
triplicate determinations, ±S.D.
The
PLA enzymatic activity released into MMC 34 mast cell
supernatants following IgE + Anti-IgE activation can be
neutralized by mAb F10, and inhibited by SB 203347 (Fig. 4, left panel). In contrast, a control mAb has no effect on
PLA
activity in activated mast cell supernatants.
Similarly, addition of either the anti-rPLA
mAb F10 or the
sPLA
inhibitor SB 203347 block the ability of supernatants
from activated MMC 34 mast cells to induce PGE
production
by 3T3 cells (Fig. 4, right panel). These data
demonstrate that the sPLA
released by mast cells following
activation is essential for transcellular prostaglandin synthesis in
3T3 cells.
To investigate
more extensively the source of arachidonic acid substrate in the distal
3T3 cells engaged in transcellular prostaglandin production, we labeled
the membranes of either proximal MMC-34 mast cells or distal Swiss 3T3
cells with [H]arachidonic acid, and examined
radioactive PGE
synthesis in [IgE +
anti-IgE]-treated cultures of each cell type individually, and in
co-cultures. No radioactive PGE
is produced following
stimulation of [
H]arachidonic acid-labeled 3T3
cells (Fig. 6). Although radioactive arachidonate is released by
the medium change that accompanied this treatment (eluting from the
chromatogram at a later time; data not shown), this arachidonate cannot
be converted to PGE
, because no PGS2 is
present(7) . No radioactive PGE
is produced
following activation of [
H]arachidonic
acid-labeled MMC-34 mast cells, since mast cells make PGD
,
but not PGE
. When co-cultures of MMC-34 mast cells and
[
H]arachidonic acid-labeled Swiss 3T3 cells are
stimulated with IgE + anti-IgE, synthesis of radioactive PGE
is observed. In contrast, when co-cultures containing
[
H]arachidonate-labeled MMC-34 cells and
unlabeled Swiss 3T3 cells are treated with IgE + anti-IgE, no
radioactive PGE
is produced. We conclude (i) that
arachidonic acid released from activated mast cells in co-culture is
not available as substrate for 3T3 cells (presumably because of
sequestration by plasma proteins) and (ii) that arachidonic acid
present in membranes of 3T3 cells is released by mast cell sPLA
following activation, and is utilized by PGS1 present in the 3T3
cells for PGE
production.
Figure 6:
Arachidonic acid from 3T3 cells is used
for PGE production in activated co-cultures. Swiss 3T3
cells and MMC-34 mast cells were labeled with
[
H]arachidonic acid as described in
``Experimental Procedures.'' Labeled 3T3 cells (top
panel) or MMC-34 cells (second panel) were treated with
IgE, followed by anti-IgE for 1 h before media were collected. For
co-culture experiments, unlabeled MMC-34 cells (third panel)
or labeled MMC-34 (bottom panel) cells were treated with IgE,
washed, and added to 3T3 cultures. All co-culture experiments were in
medium containing 0.5% serum. The asterisk (*) denotes the
cell population labeled with [
H]arachidonate.
Anti-IgE was added, and media were collected at 1 h. All samples were
subjected to HPLC, and radioactivity was determined by scintillation
counting. Each experimental condition was performed in duplicate.
Elution profiles indicating times of elution for radioactive compounds
are shown for two cultures for all conditions. The initial peak at
3-10 min contains unidentified polar compounds. Arachidonic acid
elutes at 30-38 min (not shown).
We suggest that there are two distinct routes of prostanoid
biosynthesis in 3T3 fibroblasts (Fig. 7). For the first route,
ligand-activated intracellular phospholipase(s) releases arachidonic
acid that is not available to PGS1; this arachidonate must be converted
to PGH by PGS2 enzyme, following ligand-activated PGS2 gene
expression(7) . Activation of an intracellular, cytoplasmic
PLA
is likely to be required for this pathway. The second,
transcellular route of prostanoid synthesis occurs via release of
sPLA
by proximal cells. This sPLA
enzyme can
then act as a paracrine mediator of arachidonate release in distal 3T3
cells, leading to transcellular prostaglandin production by
constitutive PGS1.
Figure 7:
Two routes of PGE production
in Swiss 3T3 cells. L, ligand; R, receptor; AA, arachidonic acid; PGS, prostaglandin synthase; PLA
, phospholipase
A
.
The proposed alternative pathways of
prostaglandin synthesis are illustrated in Fig. 7. The
14-kilodalton form of PLA remains in the cytoplasm and/or
is secreted following cellular activation(3) . In contrast,
following ligand stimulation, the 85-kilodalton isoform of
PLA
becomes phosphorylated, and then associates with cell
membranes(3) . Recent studies have suggested that, following
ligand stimulation, the 85-kilodalton PLA
molecule becomes
associated with the nuclear membrane(17) . Until recently, both
PGS1 and PGS2 enzymes were thought to be associated with the
endoplasmic reticulum (18, 19) . However, confocal
immunofluorescent microscopy studies suggest that PGS2, unlike PGS1,
associates with the nuclear membrane (20) . The recent
demonstrations that one isoform of PLA
(85-kilodalton
cPLA
) and one isoform of PGS (PGS2) can preferentially
associate with the nuclear membrane suggests a potential spatial
explanation for the distinct routes of prostaglandin production
observed in fibroblasts.
Inhibition of prostaglandin synthesis,
either by interfering with phospholipase-mediated release of
membrane-bound arachidonic acid or by interfering with prostaglandin
synthase-mediated conversion of free arachidonate to PGH,
is a major pharmacologic goal. With the discovery of alternative
PLA
and prostaglandin synthase isoforms, new targets for
specific pharmacologic intervention of prostaglandin synthesis have
emerged. Demonstration that PGS2 is induced in inflammatory responses
(reviewed in (4) ) and that PGS1 and PGS2 have restricted pools
of precursor arachidonate (7, 8) has increased
interest in identifying isoform-specific inhibitors of prostaglandin
synthase and phospholipase A
as therapeutic agents. The
goal of identifying PGS2-specific inhibitors is to produce NSAIDs
without ulcerogenic or nephrotoxic side effects. A number of lead
NSAIDs that are substantially more effective in inhibiting PGS2
activity relative to PGS1 have now been described(21) . Our
data demonstrating that both a transcellular route of prostaglandin
synthesis and an intracellular route of prostaglandin synthesis exist,
and utilize alternative PGS and PLA
isoforms, suggests that
combinatorial use of specific phospholipase and prostaglandin synthase
inhibitors may be necessary to achieve suppression of prostaglandin
production in cases where substantial sPLA
production
occurs.
How general might transcellular prostanoid production by the
proximal sPLA/distal PGS1 pathway be? The first
considerations are (i) the range of cells that produce and secrete
sPLA
, and (ii) the range of cells that respond to
sPLA
administration by PGS1-mediated prostanoid production.
sPLA
is present in inflammatory fluids, e.g. synovial fluid, and in inflammatory tissue exudates(22) .
Activated mast cells(9) , platelets (23) , and
neutrophils (24) rapidly release sPLA
.
Cytokine-induced sPLA
synthesis and release occurs in
astrocytes (25) and mesangial cells(26) . sPLA
induction is also part of the septic shock and liver acute phase
responses(22) . With regard to distal cell responsiveness to
sPLA
, antisense sPLA
oligonucleotides reduce
both endotoxin-stimulated arachidonic acid release and prostaglandin
synthesis in P388D
macrophage-like cells (27) .
Extracellular sPLA
has also been implicated in
prostaglandin production in mesangial cells (28) and
endothelial cells(29) .
Co-cultivation of alveolar
epithelial cells and macrophages labeled respectively with
[C]arachidonate and
[
H]arachidonate has demonstrated that proximal
macrophages can serve as a source of substrate arachidonic acid for
prostaglandin synthesis by distal epithelial cells(30) . In
contrast, we find that the arachidonic acid produced by activated mast
cells, the proximal cells in our co-cultures that engage in
transcellular prostaglandin synthesis, is not available to the
distal 3T3 cell prostaglandin synthase. Although sPLA
is
postulated to at least in part mediate antigen-induced prostaglandin
synthesis in mast cells (8) and endotoxin-induced prostaglandin
synthesis in P388D
macrophage-like cells (27) in an
autocrine fashion, evidence for sPLA
as a transcellular
mediator of prostaglandin synthesis has not previously been described.
Although previous reports of transcellular prostanoid synthesis are
rare, there is a substantial literature demonstrating transcellular
production of the other class of eicosanoids, the leukotrienes.
LTA, the product of 5`-lipoxygenase, can be transformed to
LTB
by LTA
hydrolase or conjugated to
glutathione by LTC
synthase. 5`-Lipoxygenase is restricted
to myeloid cells(31) . One might, therefore, expect that these
are the only cells that would produce leukotrienes. However, many cell
types unable to express 5`-lipoxygenase nevertheless produce
LTB
. This occurs because (i) such cells express
LTA
hydrolase and (ii) LTA
can be released from
proximal myeloid cells and used as substrate by LTA
hydrolase present in distal cells. Erythrocytes(32) ,
endothelial cells(33) , and epithelial cells (34) engage in transcellular LTB
production.
LTC
synthase has been demonstrated in platelets,
endothelial cells, and epidermal cells, in addition to myeloid cells.
Transcellular synthesis of LTC
from LTA
has
been documented for platelets(35) , endothelial
cells(33, 34) , epithelial cells(36) , and
keratinocytes (37) . It should be emphasized that substrate is
passed between cells for transcellular leukotriene synthesis.
Phospholipases have not been reported to act as transcellular mediators
of leukotriene sythesis.