(Received for publication, October 21, 1994; and in revised form, January 31, 1995)
From the
This study examines the regulatory effects of two cytokines,
stem cell factor (SCF) and interleukin-3, and a glucocorticoid,
dexamethasone, on lipid mediator generation in mouse bone
marrow-derived mast cells (BMMC). Treatment of BMMC with SCF induced a
modest, dose-dependent increase in three eicosanoids, thromboxane
B, prostaglandin D
, and leukotriene
B
. These increases were accompanied by a marked elevation
in cytosolic PLA
(cPLA
). Dexamethasone blocked
the induction of cPLA
levels and the elevation in
leukotriene B
induced by SCF. By contrast, the combination
of SCF and dexamethasone dramatically increased (5-8-fold) the
capacity by BMMC to produce prostanoid products. This increase in
prostanoid products was mirrored by an increase in prostaglandin G/H
synthase I (PGHS-I) levels. Dexamethasone, alone, had no effect on
PGHS-I, cPLA
, or prostanoid levels. Moreover, neither SCF
or dexamethasone, alone or in combination, influenced prostaglandin G/H
synthase II (PGHS-II) levels. In contrast to SCF, interleukin-3 alone
or in combination with dexamethasone had no effect on prostanoid
synthesis or PGHS-I or II levels. To better understand the SCF and
dexamethasone effect, PGHS-I and PGHS-II mRNA expression were examined
by Northern analysis. PGHS-I mRNA was markedly induced (maximal levels
at 5 h) by the combination of SCF and dexamethasone. PGHS-II mRNA was
undetectable in either control or SCF/dexamethasone-treated BMMC.
Neither SCF or dexamethasone, alone, altered mRNA for either PGHS
isotype. Taken together, these studies reveal that PGHS-I may be
critical to prostanoid formation in mast cells exposed to cytokines and
glucocorticoids. Moreover, they suggest that synergistic induction of
PGHS-I could represent a novel mechanism for the anti-inflammatory
action of glucocorticoids.
Mast cells play a pivotal role in inflammatory reactions by
secreting a wide variety of potent inflammatory mediators (for reviews
see (1) and (2) ). These mediators include metabolites
of AA ()such as leukotrienes, prostaglandins, and
thromboxane (for review, see (3) ). The formation of AA
metabolites by mast cells is initiated by the release of AA from
membrane phospholipid stores during immunologic activation of the cell
and is catalyzed by the enzyme phospholipase A
(PLA
). Several forms of PLA
have been
described (for reviews, see (4) and (5) ). A
relatively high molecular weight PLA
(cPLA
) has
been isolated from the cytosolic fraction of several cell
types(6, 7, 8, 9, 10) . In
addition, there is a family of low molecular weight PLA
enzymes that are released upon activation of a variety of cell
types(11, 12, 13, 14, 15) .
Both secretory and cytosolic forms of PLA
have been
reported in mast cells(16, 17) . Studies indicate that
levels of cPLA
are regulated by cytokines and growth
factors in several cell types. For example, IL-1
, epidermal growth
factor, and transforming growth factor
2 induce the synthesis of
cPLA
and AA metabolites in mesangial cells(18) ,
while interferon
causes the synthesis of cPLA
in
bronchial epithelial cells(19) . IL-1
and IL-1
cause
the accumulation of cPLA
and prostaglandin E
formation in lung and synovial fibroblasts,
respectively(20, 21) . Similarly, tumor necrosis
factor
increases cPLA
in epithelial carcinoma cells (22) . In most cases, these cytokine-induced increases in
cPLA
can be blocked by the antiinflammatory drug
dexamethasone(18, 20, 22) .
Once released
from membrane phospholipids, AA can be metabolized by lipoxygenase
activity to hydroxyeicosatetraenoic acids and leukotrienes or to
prostaglandins and thromboxanes through the action of prostaglandin G/H
synthase (PGHS)(3, 23) . Two forms of PGHS, PGHS-I and
PGHS-II, have been described in several cell
types(24, 25, 26, 27, 28) .
Like cPLA, levels of PGHS-II are induced in a number of
cell types in response to cytokines and priming agents(24, 29, 30, 31, 32, 33) and,
in some cases, this increase has been correlated with elevated
prostanoid synthesis(29, 34, 35) . As with
cPLA
, dexamethasone has been shown to block the induction
of PGHS-II by cytokines in some cell
types(30, 31, 32, 33, 36) .
While most studies in the scientific literature point to PGHS-II as the
inducible form and PGHS-I as the constitutive form of PGHS, there are
reports that the expression of PGHS-I and not PGHS-II is modulated in
some cell types(29, 37, 38, 39) .
For example, transforming growth factor
increases PGHS-I mRNA and
prostacyclin production in bovine endothelial cells without any effect
on PGHS-II mRNA(39) . Similarly, increased PGD
synthesis by monocytoid THP-1 cells treated with phorbol ester
correlates with increases in PGHS-I mRNA and protein levels, while
PGHS-II is unaffected(38) . Taken together, these studies point
to complex mechanisms through which cPLA
and PGHS are
regulated in mammalian cells.
The cytokines IL-3 and SCF exert
regulatory effects on the development and function of mast cells. IL-3
is required for the proliferation and differentiation of mast cell
precursors(40) , while SCF, a ligand for the c-kit receptor, is an essential factor in mast cell
maturation(41, 42) . When provided to mast cells in vitro for short periods of time, both IL-3 and SCF can
induce the release of inflammatory
mediators(43, 44, 45) . In the current study,
we have utilized IL-3, SCF, and dexamethasone to modulate the
expression of cPLA and PGHS and the formation of AA
metabolites. We report that PGHS-I may be critical to prostanoid
formation in mast cells treated with cytokines and glucocorticoids.
BMMC were placed
in media without WEHI supplement for 24 h prior to treatment with
cytokine or dexamethasone. This was found to be necessary to reduce
basal levels of cPLA protein in BMMC. BMMC were then
treated with 1-100 ng/ml SCF or 0-10 ng/ml IL-3 for 24 h.
The range of SCF and IL-3 concentrations used in this study was chosen
to include doses previously reported to induce proliferation or
differentiation of BMMC(41, 42, 47) . Some
cultures received dexamethasone (final concentration 0.1-10
µM) 2 h prior to addition of the cytokine. For ionophore
challenge, BMMC were removed from culture, washed twice in HBSS, and
placed at a concentration of 2
10
/ml in HBSS
containing 1 mM calcium. BMMC were then stimulated with
ionophore A23187 (final concentration 1 µM) for 5 min at
37 °C.
Immediately following activation with A23187, BMMC were
pelleted by centrifugation (500 g for 5 min at 4
°C), and supernatant fluids were added to 4 volumes of ethanol
containing 10 ng each of
H
PGD
,
H
TxB
, and
H
LTB
and 100 ng of
H
AA as internal standards. Solvents were
evaporated under a stream of nitrogen and the eicosanoids and fatty
acids converted to methoxime-pentafluorobenzylester-trimethylsilyl
ether derivatives as described previously(48) .
BMMC cultured for 24 h in WEHI-free media released
arachidonic acid and synthesized TxB, PGD
, and
LTB
when stimulated with calcium ionophore A23187 (Fig. 1). Treatment of BMMC with 1, 10, or 100 ng/ml SCF for an
additional 24 h induced a modest, dose-dependent increase in all three
eicosanoids when compared to cells not treated with SCF. Stimulated
SCF-treated cells produced quantities of unmetabolized AA similar to
levels produced by identically challenged BMMC that had not been
exposed to SCF. To determine the effect of glucocorticoids on AA
release and eicosanoid generation in BMMC, cells were pretreated with
various concentrations of dexamethasone (0.1-10 µM)
for 2 h followed by exposure to SCF (100 ng/ml) for 24 h. When given in
conjunction with SCF, dexamethasone caused a 5-8-fold increase in
the synthesis of PGD
by BMMC when compared to untreated
cells or cells treated with SCF alone (Fig. 1). Similarly, there
was a significant elevation in the amount of another PGHS product,
TxB
, produced by cells treated with SCF and dexamethasone.
In contrast, the combination of dexamethasone with SCF induced no
significant changes in levels of LTB
produced by stimulated
BMMC.
Figure 1:
Effect of SCF and dexamethasone on the
production of eicosanoids by stimulated BMMC. BMMC were treated with
SCF or SCF plus dexamethasone for 24 h and stimulated with 1 µM A23187 for 5 min. Eicosanoids released into supernatant fluid were
analyzed by GC/MS as described under ``Experimental
Procedures.'' Leukotriene B and prostanoids are
illustrated in the top and bottompanels,
respectively. Shown are the means ± S.E.; n =
4.
To identify mechanism(s) responsible for the increased
production of PGHS products released by BMMC, we examined the effects
of SCF, dexamethasone, and combined SCF/dexamethasone treatment on
levels of cPLA and PGHS in BMMC. Treatment of BMMC with SCF
(1-100 ng/ml) for 24 h resulted in an increase in cPLA
as evidenced by Western blot analysis (Fig. 2, upperpanel). A major immunoreactive band at approximately 85
kDa and a larger minor immunoreactive band were observed. Digital image
analysis showed a dose-dependent increase in cPLA
as
function of SCF concentration. Maximal levels of cPLA
were
observed at 100 ng/ml SCF. The increase in cPLA
induced by
a maximal dose of SCF was partially blocked by dexamethasone at
concentrations ranging from 0.1 to 10 µM.
Figure 2:
Effect of
SCF and dexamethasone on cPLA and PGHS-I protein levels in
BMMC. Cell lysates from BMMC treated with SCF or SCF plus dexamethasone
for 24 h were subjected to Western blotting analysis using an
anti-cPLA
antibody (upperpanel). Blots
were then stripped and reprobed with an anti-PGHS 1 antibody (lowerpanel). The relative optical density of the bands,
normalized for the darkest band on each gel, is shown to facilitate
comparisons. Shown are representative blots from three or more
experiments.
Preliminary detection of PGHS-I in BMMC was performed using a polyclonal antibody that has some cross-reactivity to PGHS-II. SCF treatment of BMMC induced the appearance of a 70-kDa protein band, consistent with the previously reported molecular mass of PGHS-I. Subsequent analysis utilizing a monoclonal antibody with high specificity for PGHS-I confirmed the identity of this band as PGHS-I (data not shown). Combined treatment of BMMC with SCF (100 ng/ml) and dexamethasone (0.1-100 µM) induced a pronounced increase in PGHS-I above that seen with SCF alone (Fig. 2, lowerpanel). Maximal induction of PGHS-I levels was detected in BMMC that received SCF (100 ng/ml) and the lowest (0.1 µM) concentration of dexamethasone. Using a specific antipeptide antibody, PGHS-II was not found under basal conditions and remained undetectable following treatment of BMMC with SCF or SCF in combination with dexamethasone (data not shown).
To determine
whether glucocorticoid alone could induce PGHS-I in BMMC, cells were
treated for 24 h with varying concentrations of dexamethasone and
eicosanoid production as well as cPLA, and PGHS levels were
examined. There were no significant changes in eicosanoids and AA
production by stimulated BMMC treated with dexamethasone alone (Table 1). Treatment with dexamethasone alone reduced basal
cPLA
levels, while basal levels of PGHS-I were unchanged
relative to untreated BMMC (data not shown). Once again, PGHS-II was
undetectable in resting or dexamethasone-treated BMMC.
To ascertain
whether the synergistic effect of dexamethasone and cytokine treatment
was specific to SCF, eicosanoid production and levels of cPLA and PGHS were measured in BMMC treated with IL-3. IL-3 in
concentrations ranging from 0.1 to 10 ng/ml had no significant effect
on the production of PGD
, TxB
, or LTB
by stimulated BMMC (Fig. 3). In sharp contrast to the
effect of SCF, a combination of IL-3 and dexamethasone did not result
in increased production of PGD
, nor did it increase the
release of other eicosanoids (Fig. 3). As seen with SCF, there
was an increase in cPLA
levels in BMMC treated with IL-3 (Fig. 4, upperpanel). Furthermore, the
increase in cPLA
induced by IL-3 (10 ng/ml) was
partially blocked by dexamethasone (Fig. 4, upperpanel). Compared to untreated BMMC, levels of
PGHS-I were unchanged in cells that received 10 ng/ml IL-3. Unlike the
synergistic effect of SCF and dexamethasone, treatment of BMMC with
IL-3 (10 ng/ml) and dexamethasone resulted in a small decrease in
PGHS-I levels relative to levels detected in BMMC that received IL-3
alone (Fig. 4, lowerpanel). There was no
detectable PGHS-II in BMMC treated with IL-3 alone or IL-3 in
combination with dexamethasone.
Figure 3:
Effect of IL-3 and dexamethasone on the
production of eicosanoids by stimulated BMMC. BMMC were treated with
IL-3 or IL-3 plus dexamethasone for 24 h and stimulated with 1
µM A23187 for 5 min. Eicosanoids released into supernatant
fluids were analyzed by GC/MS as described under ``Experimental
Procedures.'' Leukotriene B and prostanoids are
illustrated in the top and bottom panels, respectively. Shown are the
mean ± S.E., n = 4.
Figure 4:
Effect of IL-3 and dexamethasone on
cPLA and PGHS-I protein levels in BMMC. Cell lysates from
BMMC treated with IL-3 or IL-3 plus dexamethasone for 24 h were
subjected to Western blotting analysis using an anti-cPLA
antibody (upperpanel). Blots were then
stripped and reprobed with an anti-PGHS-I antibody (lowerpanel). The relative optical density of the bands,
normalized for the darkest band on each gel, is shown to facilitate
comparisons. Shown are representative blots from three or more
experiments.
To determine whether the observed increase in PGHS-I protein with SCF and dexamethasone was mirrored by an elevation in mRNA for PGHS-I, total RNA was isolated and analyzed by Northern analysis using cDNA probes specific for PGHS-I and PGHS-II mRNA. PGHS-II mRNA was undetectable in BMMC treated with SCF or dexamethasone either alone or in combination (data not shown). PGHS-I mRNA was detected at rest, and basal levels were not significantly altered by the addition of SCF or dexamethasone. In contrast, PGHS-I mRNA was markedly induced by the combination of SCF and dexamethasone (Fig. 5). Increases in PGHS-I mRNA were observed at 2 h, peaked at 5 h, and returned to base-line levels by 24 h. These findings correlated with changes observed in total PGHS-I protein content and eicosanoid production in BMMC following cotreatment with SCF and dexamethasone.
Figure 5: Effect of SCF and dexamethasone on PGHS-I mRNA levels in BMMC. Total RNA was isolated from BMMC treated with SCF or SCF plus dexamethasone for different periods of time. Northern analysis was performed as described utilizing cDNA probes for PGHS-I and glyceraldehyde-3-phosphate dehydrogenase. Shown are representative blots from four separate experiments.
Recently, inhibitors that specifically block PGHS-II
but not PGHS-I have become available. To determine whether specific
inhibition of PGHS-II would block the increase in PGD after
SCF and dexamethasone treatment, the selective inhibitor NS-398 was
used. NS-398 (at concentrations 30 - fold higher than IC
for PGHS-II) failed to attenuate the SCF/dexamethasone-induced
increase in PGD
synthesis (SCF/dexamethasone, 131.0
± 41 pmol versus SCF/dexamethasone + NS-398 (1
µM), 106 ± 48). These data support the findings
described above, which suggest that induction of PGHS-I, and not
PGHS-II, is responsible for increases in PGD
synthesis by
BMMC treated with SCF and dexamethasone.
The central findings of the current study are that SCF
induces the expression of cPLA and PGHS-I in BMMC. The
increase in the levels of these two enzymes is accompanied by modest
increases in the production of eicosanoids. Although the
glucocorticoid, dexamethasone, blocked the cytokine-induced elevation
in cPLA
, it has the paradoxical effect of dramatically
increasing the capacity of SCF-treated mast cells to produce
prostanoids. This increase in prostanoid synthesis by BMMC correlated
with marked induction of PGHS-I without any effect on PGHS-II. Taken
together, these studies suggest that PGHS-I may be critical to
prostanoid formation in glucocorticoid-treated mast cells.
Cytokines
have been reported to increase the expression of cPLA in a
variety of cell
types(18, 19, 20, 21, 22) .
Some of these studies have also shown that glucocorticoids such as
dexamethasone can inhibit this
induction(18, 20, 22) . Consistent with these
previously published reports, in the present study we found that both
SCF and IL-3 increased levels of cPLA
and that
dexamethasone blocked this induction in BMMC. It is interesting to note
that both SCF and IL-3 promoted the appearance of two immunoreactive
bands of cPLA
. Recent studies demonstrate that
phosphorylation of cPLA
often accompanies cell activation
and this phosphorylation changes the electrophoretic properties of
cPLA
(50) . It is possible that SCF and IL-3
increase cPLA
expression and this cPLA
is
phosphorylated during cell activation.
To date, most studies which
have examined the regulation of PGHS isoforms have pointed to PGHS-II
as the inducible form of this enzyme in mammalian
cells(29, 30, 31, 32, 33, 51) .
However, there appear to be specific cell types in which PGHS-I, and
not PGHS-II, is induced (29, 37, 38, 39) by certain stimuli.
Murakami and colleagues have recently demonstrated that both PGHS-I and
PGHS-II can be induced by various cytokine combinations in
BMMC(52) . Their data further suggest that AA which is
metabolized to PGD in response to cytokine stimulation
utilizes PGHS-II while AA which forms PGD
in response to
IgE cross-linking utilized PGHS-I. The current data indicate that SCF
alone caused a moderate increase in PGHS-I which correlated with small
increases in prostanoid products (in response to ionophore A23187), and
that SCF-treatment of BMMC failed to induce PGHS-II. Glucocorticoids
have been demonstrated to block the induction of PGHS-II expression (29, 34, 53) . However, a stimulatory effect
of dexamethasone on overall cellular PGHS activity has been observed in
amnion epithelial cells (54) . Our data demonstrate a dramatic
elevation of PGHS-I (>10-fold increase) protein, mRNA and
prostanoids (>5-fold increase) in SCF-treated mast cells incubated
with low concentrations (0.1 µM) of dexamethasone. To our
knowledge, this is the first study to demonstrate an increase in PGHS-I
levels by combined dexamethasone and cytokine treatment. Future studies
will be necessary to determine whether other enzymes (such as PGD
synthase) are regulated by this combination.
The mechanism(s) through which dexamethasone potentiates the SCF-induced increase of PGHS-I protein in BMMC are not known. Our studies reveal that SCF or dexamethasone alone have only a modest influence on PGHS-I. The synergistic effect of dexamethasone on the production of prostanoids and PGHS-I expression, appears to be specific to SCF, in that it was not observed with IL-3, another cytokine known to exert effects on mast cells. One possible explanation for the differential effect of these two cytokines may be found in the differences between the SCF and IL-3 receptors. For example, SCF is a ligand for the c-kit receptor, a member of the type III tyrosine kinase receptor family(55) .
The mechanistic basis for the anti-inflammatory
effects of glucocorticoid compounds such as dexamethasone, is still
incompletely understood. Reduction of PLA and PGHS gene
expression, through both transcriptional and post-transcriptional
events, has been proposed as a mechanism that leads to decreased AA
release and diminished prostanoid
production(56, 57, 58, 59, 60, 61) .
Our data demonstrating that dexamethasone blunts SCF and IL-3-induced
increases in cPLA
levels in BMMC are consistent with this
hypothesis. In contrast, the potentiation of cytokine-induced increases
in prostanoid synthesis, a previously unreported effect of
dexamethasone, would appear to be a paradoxical effect of this
anti-inflammatory drug. PGD
, the main prostanoid product
synthesized by mast cells, has pro-inflammatory effects such as
bronchoconstriction and vasodilation(62) . On the other hand,
PGD
is an inhibitor of platelet aggregation which appears
to function by increasing cyclic AMP levels(63, 64) .
If the synergistic increase in PGHS-I induced by dexamethasone and SCF
treatment occurs in other cell types, it is possible that additional
prostanoids with anti-inflammatory effects, such as PGE
,
may be produced. In this regard, synergistic induction of PGHS-I could
represent a novel mechanism for the anti-inflammatory action of
glucocorticoids.