(Received for publication, August 11, 1994; and in revised form, October 4, 1994)
From the
We have examined the cytokine regulation of IgEdependent
prostaglandin (PG) D generation in mouse mast cells by
assessing the changes in the levels of the transcript, translated
protein, and activity of the enzymes involved in the synthesis of
PGD
from endogenous arachidonic acid. When mouse mast
cells, derived by culture of bone marrow cells with WEHI-3
cell-conditioned medium as a source of interleukin (IL)-3 (BMMC), were
cultured in recombinant c-kit ligand (KL), sensitized with
IgE, and stimulated with antigen, PGD
generation increased
3-fold; when KL was combined with IL-3, IL-9, or IL-10, PGD
generation increased 6-8-fold above that produced by the
cells cultured in IL-3 alone. The increased IgE-dependent PGD
generation by BMMC was apparent after 1 day of culture, reached a
maximum after 2-4 days of culture, and was dose-dependent for KL
and for each of the accessory cytokines. IgE-dependent generation of
leukotriene C
increased 2-fold after the cells were
cultured with KL and was not increased by the addition of IL-3, IL-9,
or IL-10. Assays for steady-state transcripts by RNA blotting, for
protein by SDS-PAGE/immunoblotting, and for function by enzymatic
activities revealed that KL alone stimulated the increased expression
of cytosolic phospholipase A
(cPLA
),
prostaglandin endoperoxide synthase (PGHS)-1, and the terminal enzyme,
hematopoietic PGD
synthase, without a change in expression
of 5-lipoxygenase. IL-3, IL-9, and IL-10 each enhanced the KL-induced
expression of PGHS-1. In contrast, transcripts for PGHS-2, which were
detected transiently after the cells had been cultured for 5 h in KL
+ IL-3, were not expressed during the period of subsequent
increase in IgE-dependent PGD
generation. These findings
demonstrate that KL up-regulates expression of cPLA
,
PGHS-1, and hematopoietic PGD
synthase, leading to a
relatively selective increase in IgE-dependent production of PGD
from endogenously released arachidonic acid in BMMC, and they
provide the first example of cytokine regulation of hematopoietic
PGD
synthase.
Mast cells are highly specialized effector cells of the immune
system that, when activated, release diverse types of biologically
active molecules including amines, proteoglycans, proteases,
eicosanoids, platelet-activating factor, and
cytokines(1, 2, 3, 4) . There are at
least two distinct populations of mast cells in mice, connective tissue
mast cells (CTMC) ()and mucosal mast cells (MMC). Bone
marrow-derived mast cells developed in WEHI-3 cell-conditioned medium
as a source of interleukin (IL)-3 (BMMC) represent a relatively
immature population of mast cells that reconstitutes both CTMC and MMC
in mast cell-deficient mice of the WBB6F1/J-W/W
strain(5) . Serosal mast cells, generally studied as a
CTMC surrogate, respond to Fc
receptor I (Fc
RI)-dependent
activation with preferred generation of the cyclooxygenase pathway
product, prostaglandin (PG) D
, whereas rat MMC and mouse
BMMC generate leukotriene (LT) C
via the 5-lipoxygenase
pathway in preference to
PGD
(1, 6, 7, 8) .
The
initial step in arachidonic acid metabolism is the release of free
arachidonic acid from cell membrane phospholipids by cytosolic
phospholipase A (cPLA
), which is activated by
translocation from the cytosol to a cell membrane compartment in
response to an increase in cytoplasmic Ca
concentration (9, 10, 11) .
cPLA
, which is ubiquitously and constitutively expressed in
mammalian cells, undergoes increased expression during cellular
responses to several
cytokines(12, 13, 14, 15) .
Prostaglandin endoperoxide synthase (PGHS, or cyclooxygenase), which
occurs in two isoforms, catalyzes the oxygenation of arachidonic acid
to PGG
, which is reduced to PGH
by the
hydroperoxidase activity of the same enzyme(16) . PGHS-1 is
constitutively expressed in a wide range of cells and
tissues(17, 18, 19) , whereas PGHS-2 is
induced in response to growth factors and proinflammatory
cytokines(20, 21, 22, 23, 24, 25, 26) .
PGH
is metabolized by specific synthases, each of which has
a restricted distribution, to individual prostanoids. PGD
synthases, which catalyze the conversion of PGH
to
PGD
, exist in two forms, each with a molecular mass of 26
kDa(27, 28) . The brain enzyme is glutathione
(GSH)-independent(27) , whereas the hematopoietic enzyme, first
described in rat spleen, is GSH-dependent(28, 29) .
The presence of hematopoietic PGD
synthase in rat CTMC was
shown by GSH dependence and immunochemical identity(30) .
5-Lipoxygenase, which is activated by a Ca
-dependent
translocation to the perinuclear membrane(31) , catalyzes the
sequential metabolism of arachidonic acid to
5-hydroperoxyeicosatetraenoic acid and then to
LTA
(32, 33) . An 18-kDa perinuclear
membrane protein, termed 5-lipoxygenase activating protein, presents
arachidonic acid to
5-lipoxygenase(31, 34, 35) . LTA
is processed to LTC
by a microsomal LTC
synthase with restricted substrate
specificity(36, 37) .
The tissue-specific elements
that direct the mast cell phenotype to respond to IgE-mediated
activation with preferential generation of PGD or LTC
are unknown. Only two cytokines, c-kit ligand (KL) and
IL-3, develop and/or maintain nontransformed BMMC in vitro. We
now report that one of them, KL, a stromal cytokine that also regulates
a variety of mast cell
functions(38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56) ,
primes BMMC for increased Fc
RI-mediated production of PGD
in a dose- and time-dependent manner. KL primes the cells by
eliciting increases in steady-state transcription, immunodetected
protein, and function of each enzyme (cPLA
, PGHS-1, and
hematopoietic PGD
synthase) in the post-receptor
biosynthetic pathway to PGD
generation after IgE-dependent
activation of the cells. IL-9 and IL-10, cytokines known to act as
accessory growth factors for mast
cells(48, 57, 58) , as well as IL-3, all
augment the priming effect of KL but only through a further increase in
the expression of PGHS-1. The up-regulation of hematopoietic PGD
synthase in BMMC by KL may also contribute to a mechanism for the
preferred metabolism of endogenous arachidonic acid to PGD
in CTMC.
Rabbit antiserum to human cPLA, which cross-reacts with
mouse cPLA
, and a human cPLA
cDNA (10) were provided by J. D. Clark, Genetics Institute,
Cambridge, MA. Rabbit antiserum to sheep PGHS-1 was provided by W. L.
Smith, Michigan State University, East Lansing, MI. Affinity-purified
rabbit polyclonal antibody to mouse PGHS-2 was purchased from Cayman
Chemical, Ann Arbor, MI. Mouse PGHS-1 (17) and PGHS-2 (21, 23) cDNA probes were provided by J. Trzaskos,
Merck DuPont, Wilmington, DE. Rabbit antiserum to rat PGD
synthase has been described previously (28, 29, 30) . The specificity of this
antibody has previously been demonstrated by immunoblotting after one-
and two-dimensional electrophoresis of crude rat spleen
lysates(28, 29, 30) . Although hematopoietic
PGD
synthase has GSH-S-transferase activity and
its N-terminal amino acid sequence has homology to various
GSH-S-transferase isozymes (29) , the antibody does
not cross-react with other GSH-S-transferase isozymes or with
the brain form of PGD
synthase(28) . Rabbit
antiserum to human 5-lipoxygenase and a human 5-lipoxygenase cDNA (33) were provided by J. F. Evans, Merck Frosst, Quebec,
Canada.
BMMC were washed
once with enriched medium; resuspended at a cell density of 1
10
cells/ml in the enriched medium supplemented with 100
units/ml IL-3 alone, with 100 ng/ml KL alone, with 100 ng/ml KL in
combination with 100 units/ml IL-3, 100 units/ml IL-9, or 10 units/ml
IL-10, or with 50% WEHI-3 cell-conditioned medium; and then cultured
for various periods. The concentrations of each cytokine chosen were
those that gave maximal priming of IgE-dependent lipid mediator
generation and release. After 7 days of culture, the number of mast
cells increased 4.9 ± 1.6-fold (mean ± S.E., n = 6) with KL alone, 3.7 ± 0.9-fold (n = 6) with IL-3 alone, and 5.3 ± 0.7-fold (n = 3) with 50% WEHI-3 cell-conditioned medium. During 7 days
of culture with KL in combination with IL-3, IL-9, or IL-10, the number
of BMMC increased 22.5 ± 5.5-fold (n = 6), 13.1
± 1.0-fold (n = 5), and 6.7 ± 0.9-fold (n = 4), respectively. When BMMC were cultured in
cytokines for more than 2 days, their density was adjusted to 1
10
cells/ml 2 days before harvest; the cell density when
harvested ranged from 2 to 3
10
cells/ml.
cPLA activity was assessed by the hydrolysis of
1-acyl-2-[
C]arachidonoyl-phosphatidylcholine (30
µCi/µmol) (Amersham) to liberate
[
C]arachidonic acid as described
previously(65) . Briefly, a 50-µl sample of the BMMC lysate
was adjusted to a final volume of 125 µl containing 4 mM CaCl
, 100 mM Tris-HCl, pH 8.0, 1 mM dithiothreitol (Sigma), and 10 µM 1-acyl-2-[
C]arachidonoyl-phosphatidylcholine,
and was incubated for 30 min at 37 °C. Dithiothreitol was included
to inhibit the activity of secretory group II
PLA
(9) . The reaction was stopped by the addition
of 625 µl of Dole's reagent (65) . Free
[
C]arachidonic acid was extracted in n-heptane and counted in a liquid
-scintillation counter
(Beckman, Palo Alto, CA).
PGHS activity was measured by the
conversion of [H]arachidonic acid (100
mCi/µmol) (Amersham) to [
H]PGH
. A
sample of the cell lysate was adjusted to 100 µl containing 100
mM Tris-HCl, pH 8.0, 1 mM phenol (Sigma), 2
µM hematin (Oxford Biochemical Research, Oxford, MI), 1
mMp-chloromercuribenzenesulfonic acid (Sigma), and
20 µM [
H]arachidonic acid. The
sample was incubated for 2 min at 25 °C, and the reaction was
stopped by the addition of 300 µl of diethyl ether, methanol, 1 M citric acid (30:4:1, v/v) precooled to -20 °C. A
100-µl portion of the ether phase was separated by thin layer
chromatography at -20 °C with a solvent system of diethyl
ether/methanol/acetic acid (90:2:0.1, v/v). The zone on the silica gel
corresponding to PGH
was determined by comparison with the
mobility of synthetic standards. The PGH
zone and the
remaining zones were scraped into vials, and the radioactivity was
counted in a liquid scintillation counter. PGH
formation
was calculated from the ratio of the radioactivity in the PGH
zone to the total radioactivity recovered, with the specific
activity of the substrate known(28) .
PGD synthase was assayed by measuring the conversion of
[
H]PGH
to
[
H]PGD
.
[
H]PGH
was obtained as described (28) by incubating 20 µM [
H]arachidonic acid (100 mCi/µmol) with
250 units/ml sheep PGHS-1 (Oxford Biochemical Research) in 100 mM Tris-HCl, pH 8.0, containing 1 mM phenol, 2 µM hematin and 1 mMp-chloromercuribenzenesulfonic
acid for 2 min at 25 °C. [
H]PGH
was extracted by adding diethyl ether, methanol, 1 M citric acid (30:4:1, v/v). The solvent was evaporated, and
[
H]PGH
was dissolved in diethylene
glycol dimethyl ether. The final concentration of
[
H]PGH
was calculated by measurement
of radioactivity in a liquid
-scintillation counter under the
assumption that its specific radioactivity was the same as that of the
arachidonic acid used as substrate. A portion of the cell lysate was
centrifuged for 1 h at 100,000
g at 4 °C to
separate soluble and particulate fractions. The 100,000
g pellet was reconstituted in an equal amount of lysis buffer. Equal
volumes of total cell lysate, the 100,000
g supernatant, and the 100,000
g pellet were
incubated for 90 s at 25 °C with 20 µM [
H]PGH
in 100 mM Tris-HCl, pH 8.0, with and without 1 mM GSH (Sigma).
Products were extracted in diethyl ether, methanol, 1 M citric
acid (30:4:1, v/v), and separated on thin layer chromatography plates,
and PGD
formation was calculated from the ratio of the
radioactivity in the PGD
zone to the total radioactivity
recovered(28) . Arachidonic acid, PGB
,
PGH
, PGD
, and PGE
used as synthetic
thin layer chromatography standards were purchased from Cayman
Chemical.
The activity of each enzyme was expressed as picomoles of substrate converted to product/min of reaction time per million cell equivalents, and was calculated by measurement of the radioactivity associated with the final product, with the specific activity of the substrate being known.
Figure 1:
Time
course of the differences in mediator release from BMMC sensitized with
IgE and activated with antigen after treatment with various cytokines.
BMMC were cultured for the indicated periods with 100 units/ml IL-3 (open squares), 100 ng/ml KL (closed squares), 100
ng/ml KL + 100 units/ml IL-3 (closed circles), 100 ng/ml
KL + 100 units/ml IL-9 (closed triangles), or 100 ng/ml
KL + 10 units/ml IL-10 (open circles). Cells were then
sensitized with IgE anti-TNP and activated with TNP-bovine serum
albumin for 10 min as described under ``Experimental
Procedures.'' Portions of the supernatants of the stimulated cells
were assayed for their content of -hexosaminidase,
PGD
, and LTC
.
-Hexosaminidase was also
quantitated in pellets after freeze-thawing, and the net percent
release of
-hexosaminidase is shown. Values represent means
± S.E. of seven independent experiments. Beta-HEX,
-hexosaminidase.
The dependence of
priming for mediator release on the concentration of KL was examined
after 2 days of culture (Fig. 2). A full dose-dependent
enhancement of PGD generation was demonstrated with KL from
0 to 25 ng/ml in the presence of IL-3. BMMC cultured for 2 days with KL
alone at concentrations below 25 ng/ml KL, or with IL-9 or IL-10 at
concentrations below 6 ng/ml KL did not consistently maintain
viability. Thus, the dose-dependent effect of KL was limited over those
doses that were evaluated in combination with either IL-9 or IL-10, and
KL alone had no further effect over the threshold dose for viability.
LTC
generation after 2 days of culture was also maximum
with 25 ng/ml KL alone and increased less than 2-fold with or without
accessory cytokines (Fig. 2). The reduction of
-hexosaminidase release in BMMC cultured with KL, KL + IL-9,
or KL + IL-10 relative to cells cultured with KL + IL-3 was
not dependent on the concentration of KL (Fig. 2).
Figure 2:
Dose-dependent effect of KL on
IgE-dependent mediator release from BMMC after 2 days of culture. BMMC
were cultured for 2 days with various concentrations of KL in the
absence (closed squares) or presence of 100 units/ml IL-3 (closed circles), 100 units/ml IL-9 (closed
triangles), or 10 units/ml IL-10 (open circles), and then
sensitized with IgE anti-TNP and activated with TNP-bovine serum
albumin. Portions of the supernatants of the stimulated cells were
assayed for their content of -hexosaminidase, PGD
, and
LTC
.
-Hexosaminidase was also quantitated in the cell
pellets, and net percent release of
-hexosaminidase is shown.
Values represent means ± S.E. of four independent experiments. Beta-HEX,
-hexosaminidase.
The
maximal generation of both PGD and LTC
by BMMC
in response to IgE and antigen after culture for 2 days with KL alone
and KL with various accessory cytokines increased significantly
relative to culture with IL-3 alone, with a preferential fold increase
in PGD
relative to LTC
for each cytokine
combination (Table 1).
Figure 3:
Expression of the enzymes involved in the
metabolism of arachidonic acid to PGD, assessed by
SDS-PAGE/immunoblotting and RNA blotting, after treatment of BMMC for 2
days with various combinations of cytokines. A, effect of
cytokines on expression of protein for each enzyme as visualized by
SDS-PAGE/immunoblotting. Samples of cell lysates (10
cell
equivalents for cPLA
, PGHS-1, and 5-lipoxygenase (5-LO); 5
10
cell equivalents for PGHS-2;
and 10
cell equivalents for hematopoietic PGD
synthase (PGDS)) were analyzed by SDS-PAGE/immunoblotting with
antibodies specific for each enzyme, as described under
``Experimental Procedures.'' BMMC treated with 100 ng/ml KL
and 10 units/ml IL-10 for 5 h were used as a positive control for
PGHS-2. A representative result of at least three independent
experiments is shown. B, effect of cytokines on the expression
of steady-state transcripts of cPLA
, PGHS-1,
5-lipoxygenase, and
-actin in 10 µg of RNA. The blots were
probed with
P-labeled cDNAs encoding cPLA
,
PGHS-1, 5-lipoxygenase, and
-actin, and were exposed to Kodak
XAR-5 films for 7, 2, 5, and 1 days, respectively. A representative
result of three independent experiments is
shown.
The expression over time of
cPLA, PGHS-1, PGHS-2, and hematopoietic PGD
synthase was studied in BMMC cultured with KL + IL-3 (Fig. 4). cPLA
protein increased modestly by 6 h,
and reached a maximum by 2 days (Fig. 4A). After 4 days
of culture, the levels of cPLA
protein decreased, although
the levels after 7 days of culture were still higher than those in
starting BMMC. There was a small (1.7 ± 0.2-fold on day 2, n = 3, p < 0.05 versus day 0) increase
in steady-state levels of the 3.4-kilobase cPLA
transcript
at day 2 which persisted to day 4 (Fig. 4B). The
expression of PGHS-1 protein was increased 1 day after the start of the
culture, was near maximum by day 2, and plateaued at 4-7 days (Fig. 4A), with a concomitant increase in steady-state
levels of the 2.8-kilobase PGHS-1 transcript (Fig. 4B).
The 4.8-kilobase PGHS-2 transcript was detected transiently in BMMC
cultured with KL + IL-3 and was undetectable by 1 day (Fig. 4B). PGHS-2 protein was not detectable at any
point (Fig. 4A). By comparison, PGHS-2 protein was
detectable in BMMC cultured with KL + IL-10 after 5 h of culture (Fig. 4A). The level of immunoreactive PGD
synthase increased after 1 to 2 days of culture, and reached a
maximum by 4 days in BMMC cultured with KL + IL-3 (Fig. 4A).
Figure 4:
Time
course of the expression of enzymes involved in the metabolism of
arachidonic acid to PGD, assessed by
SDS-PAGE/immunoblotting and RNA blotting, after treatment of BMMC with
KL + IL-3. A, time-dependent changes in the expression of
proteins as visualized by SDS-PAGE/immunoblotting. The same number of
cell equivalents (10
for cPLA
and PGHS-1; 5
10
for PGHS-2; and 10
for hematopoietic
PGD
synthase (PGDS)) were applied to each lane. A
representative result of at least three independent experiments is
shown. BMMC treated with 100 ng/ml KL and 10 units/ml IL-10 for 5 h and
RBL-2H3 cells were used as positive controls for PGHS-2 and PGD
synthase, respectively. B, time-dependent changes in
steady-state transcripts of cPLA
, PGHS-1, PGHS-2, and
-actin in 10 µg of RNA. The blots were probed with
P-labeled cDNA for cPLA
, PGHS-1, PGHS-2, and
-actin, and were exposed to Kodak XAR-5 films for 7, 2, 7, and 1
days, respectively. A representative result of four independent
experiments is shown.
The dependence of the induction of
cPLA, PGHS-1, and PGD
synthase on the
concentration of KL was examined after 2 days culture of BMMC with
various concentrations of KL in the presence of IL-3. As assessed by
both RNA blotting and immunoblotting, the expression of
cPLA
, PGHS-1, and PGD
synthase increased in a
dose-dependent fashion, reaching a maximum at 25 ng/ml KL (data not
shown).
Biochemical assays were performed to confirm that the
increases in expression of immunoreactive enzymes were accompanied by
functional enzymatic activity. cPLA activity, measured by
release of [
C]arachidonic acid from
1-acyl-2-[
C]arachidonoyl-phosphatidylcholine,
was increased 2-3-fold after culture of BMMC with KL as compared
with culture with IL-3 alone (Table 2). The addition of IL-3,
IL-9, or IL-10 to the cells cultured with KL did not significantly
enhance cPLA
activity. PGHS activity, determined by the
conversion of [
H]arachidonic acid to
[
H]PGH
, increased approximately
2-fold in BMMC cultured with KL alone and increased 4-8-fold in
BMMC cultured with KL in the presence of IL-3, IL-9, or IL-10 (Table 2). PGD
synthase activity in BMMC was assayed
by quantitating the conversion of [
H]PGH
to [
H]PGD
and was further
characterized by its GSH dependence and subcellular localization (Table 3). PGD
synthase activity increased more than
10-fold in BMMC cultured with KL + IL-3 compared to BMMC cultured
with IL-3 alone and was absolutely dependent on the presence of GSH (Table 3). The GSH-dependent PGD
synthase activity of
BMMC cultured with KL + IL-3 and of RBL-2H3 cells was confined to
the 100,000
g supernatant.
The cell- and tissue-derived factors that regulate
arachidonic acid metabolism in mast cells toward IgE-dependent
PGD generation are unknown. Although previous studies
showed that co-culture of BMMC with mouse 3T3 fibroblasts in the
presence of WEHI-3 cell-conditioned medium resulted in granule
maturation toward a CTMC phenotype, accompanied by augmented
IgE-dependent PGD
generation(38, 39) ,
these studies did not investigate changes at individual enzymatic steps
or the participation of particular cytokines. We have more recently
reported on the capacity of a particular combination of cytokines, KL
+ IL-10 + IL-1
, to elicit PGD
generation
directly from BMMC and to subsequently prime the same cells for
augmented IgE-dependent PGD
generation(66) . The
cytokine-initiated PGD
generation was associated in time
with the transient expression of PGHS-2, and studies with specific
inhibitors of PGHS-2 confirmed the separate linking of this isoform
with transmembrane activation by cytokines. The expression of PGHS-2
failed to prime IgE-dependent PGD
generation, which was
considered to be linked to PGHS-1. There was no examination of the
contribution of enzymes other than the PGHS isoforms to the priming
event, and the profile of cytokines studied was limited. We now report
that IL-3 and IL-9 can be substituted for IL-10 in the priming of BMMC
for Fc
RI-mediated PGD
production. Of particular note
is that priming by KL is associated with the increased expression of
each enzyme in the post-receptor biosynthetic pathway for PGD
generation, namely cPLA
, PGHS-1, and hematopoietic
PGD
synthase, as assessed by RNA blot analysis (not
assessed for hematopoietic PGD
synthase because the cDNA
has not been cloned), SDS-PAGE/immunoblotting for protein and
biochemical assays of enzymatic activity. Only the expression of PGHS-1
is increased further by the added presence of IL-3, IL-9, or IL-10.
When BMMC were cultured with KL alone, there was a 3-fold increase
in PGD generation in response to IgE sensitization and
antigen stimulation (Table 1), which was maximal after 2-4
days (Fig. 1). BMMC cultured with KL in combination with IL-3,
IL-9, or IL-10, which regulate the growth, differentiation, and
maturation of mast
cells(61, 62, 67, 68, 69) ,
exhibited a 6-8-fold increase in PGD
generation
compared with BMMC maintained in IL-3 alone. Other cytokines, including
IL-1
, IL-6, tumor necrosis factor-
, interferon-
,
transforming growth factor-
1, and nerve growth factor, had no
effect alone or in combination with KL (data not shown), indicating
that the accessory cytokines were selective. Priming of IgE-dependent
PGD
generation by BMMC cultured with KL + IL-3 was
dependent upon the concentration of KL with a plateau at 25 ng/ml (Fig. 2). Although the dose-dependent studies were limited when
KL was used alone or in combination with IL-9 or IL-10 due to the loss
of cell viability below the threshold requirement, maximal priming of
PGD
generation was again observed at 25-50 ng/ml of
KL. Priming of BMMC for PGD
synthesis is therefore distinct
from the priming of human skin (54) and lung (55) mast
cells for histamine release, which occurred within minutes of exposure
to concentrations of KL below 1 ng/ml. Priming of IgE-dependent
PGD
generation was also dependent upon the concentration of
IL-3, IL-9, and IL-10 when the concentration of KL was fixed (data not
shown). LTC
synthesis increased less than 2-fold during
BMMC culture with KL and was not influenced by the presence of
accessory cytokines. Thus, after 2 days culture with concentrations of
each cytokine that were optimal for maximal IgE-dependent eicosanoid
generation, the ratio of PGD
/LTC
synthesis
increased about 2-fold to 0.36 in BMMC cultured with KL alone, as
compared to 0.2 for those cultured with IL-3 alone, and increased about
3.5-fold to 0.68 in BMMC cultured with KL + IL-10 (Table 1).
In contrast to the IgE-mediated increase in PGD and
LTC
synthesis after BMMC were cultured in KL alone or with
accessory cytokines, the same cells showed a progressive loss over days
of their capacity to undergo IgE-mediated exocytosis (Fig. 1).
Only cells cultured with KL + IL-3 or with IL-3 alone maintained
their ability to degranulate substantially; however, the diminution of
exocytosis with KL alone or combined with other cytokines was not
dependent on the dose of KL (Fig. 2). Thus, diminished
IgE-dependent degranulation appears to be due to an absence of IL-3. In
contrast, the increase in IgE-dependent PGD
generation was
dependent on both the concentration of KL (Fig. 2) and the
concentration of accessory cytokines (data not shown). Thus, exocytosis
and eicosanoid synthesis may be independently regulated in BMMC,
possibly due to their dependence upon separate post-receptor pathways,
and in certain circumstances degranulation may not be the optimal
marker of mast cell activation.
In order to identify the biochemical
steps leading to increased PGD synthesis in
cytokine-treated BMMC, the changes in expression of the individual
enzymes involved in post-receptor metabolism of arachidonic acid to
PGD
were assessed in terms of steady-state levels of mRNA,
expressed protein, and activity of each enzyme. Although mast cells
express both secretory group II PLA
and arachidonic
acid-selective cPLA
(65) , cPLA
likely
plays the dominant role in IgE-dependent lipid mediator
production(70, 71) . SDS-PAGE/immunoblot analysis and
assay of enzymatic activity demonstrated an increase in immunoreactive
and functional cPLA
in KL-treated BMMC. Accessory cytokines
did not enhance the effect of KL on the expression of mRNA and protein
for cPLA
(Fig. 3) or on cPLA
activity (Table 2). The expression of cPLA
protein in BMMC
reached a maximum after 2 days of culture (Fig. 4A)
coincident with the plateau for increased IgE-dependent generation of
PGD
(Fig. 1). Increased expression of cPLA
protein and function was accompanied by only minimal increases in
cPLA
transcripts, suggesting significant
post-transcriptional regulation of its expression in KL-treated BMMC.
Increased expression of cPLA
in fibroblasts, HeLa cells,
and monocytes in response to IL-1
, tumor necrosis factor
, or
macrophage colony-stimulating factor was also linked to enhanced
generation of eicosanoids after cell stimulation with a second agonist
that raised the intracellular Ca
concentration(12, 13, 14, 15, 70) .
Macrophage colony-stimulating factor, which increases mRNA and
functional protein for cPLA
in human
monocytes(15) , and KL belong to the same family of growth
factors based on homology between their own structures and between the
structures of their respective tyrosine kinase receptors, c-fms and c-kit(72) .
With regard to the second step
in PGD synthesis, conversion of arachidonic acid to
PGH
, RNA blot analysis, SDS-PAGE/immunoblot analysis, and
assay of PGHS enzymatic activity demonstrated that expression of PGHS-1
in BMMC cultured with KL increased and was further augmented by the
addition of IL-3, IL-9, or IL-10. The increase in PGHS-1 transcript in
BMMC cultured with KL + IL-3 was time-dependent, reaching a
maximum (4-fold) at 1 day; it was followed by increased expression of
PGHS-1 protein, which reached a maximum by 2 days (Fig. 4, A and B). Although PGHS-2 mRNA was transiently induced (Fig. 4B), immunoreactive PGHS-2 protein was not
detectable (Fig. 4A). The lack of immunodetectable
PGHS-2 in BMMC cultured with KL + IL-3 was due neither to
methodologic error nor to unresponsiveness of BMMC as demonstrated by
the expression of PGHS-2 protein after the cells were cultured for 5 h
with KL + IL-10 (Fig. 4A) as we have shown (66) . Furthermore, the expression and disappearance of PGHS-2
steady-state mRNA preceded the augmented IgE-dependent PGD
synthesis. Investigations that evaluate PGHS activity by
measuring the generation of the final product after supplying
arachidonic acid to intact cells or cell homogenates reflect the
combined activity of PGHS and the terminal enzyme for eicosanoid
generation and do not recognize changes occurring in each enzyme
separately. In the present study, both the time-dependent changes in
PGHS-1 protein (Fig. 4A) and the increased expression
of immunoreactive and functional PGHS-1 in response to accessory
cytokines, acting only at the PGHS-1 step (Fig. 3A, Table 2), suggest that PGHS-1 plays a critical role in regulating
antigen-dependent PGD
synthesis by IgE-sensitized BMMC.
Thus, the constitutive isoform with presumptive physiologic
functions(17, 18, 19) , rather than the
inducible isoform with putative proinflammatory
functions(20, 21, 22, 23, 24, 25, 26) ,
is the intermediate enzyme species in mast cell-dependent
Fc
RI-mediated events in the microenvironment. These data also
provide unequivocal evidence that cytokine priming of a cell for
increased prostanoid synthesis by a particular transmembrane signal is
linked to increased expression of PGHS-1 and not PGHS-2, and that the
optimal cytokine combination for induction of PGHS-1 involves accessory
cytokines that do not elicit further induction of expression of
cPLA
and the terminal biosynthetic enzyme.
The final
step in the generation of PGD from arachidonic acid in mast
cells is the metabolism of PGH
by the terminal enzyme,
hematopoietic PGD
synthase(28, 29, 30) . The
hematopoietic form of PGD
synthase, first described in rat
spleen, is distinguished from the brain enzyme immunochemically and by
its dependence on GSH(28) . Although the N-terminal amino acid
sequence of rat hematopoietic PGD
synthase was shown to be
similar to cytosolic GSH transferases(29) , its cDNA has not
been cloned. There are no previous data on the cytokine regulation of
the expression of this terminal enzyme of arachidonic acid metabolism.
In the present study, an immunoreactive protein was recognized in BMMC
that has the same mobility on SDS-PAGE as rat hematopoietic PGD
synthase from RBL-2H3 cells. Expression of this immunoreactive
26-kDa protein increased during cell culture in KL and was not affected
by the co-presence of accessory cytokines (Fig. 3). Furthermore,
the effect of KL was on a GSH-dependent cytosolic PGD
synthase in BMMC (Table 3). Thus, both immunochemical and
biochemical criteria indicate that the hematopoietic form of PGD
synthase is present in mouse BMMC and its expression is increased
by stimulation of the cells with KL. The time course of the increase in
immunoreactive PGD
synthase protein (Fig. 4) was
consistent with the change in the profile of IgE-dependent PGD
synthesis (Fig. 1), indicating that hematopoietic
PGD
synthase may be a further regulatory step leading to
increased IgE-dependent PGD
generation.
Priming of
IgE-dependent PGD generation after culture of BMMC with KL
was accompanied by a less than 2-fold increase in LTC
generation, although the increment in LTC
generation
was comparable to the increment in PGD
generation in
absolute terms (Table 1). The increment in IgE and
antigen-dependent LTC
generation was maximal by 1-2
days and was not affected by the addition of accessory cytokines (Fig. 1). Thus, the kinetics and the cytokine dependence of
LTC
generation were similar to those of cPLA
protein expression. The expression of 5-lipoxygenase was
unchanged by any of the culture conditions (Fig. 3). The
possible contributions of other factor(s), such as 5-lipoxygenase
activating protein and LTC
synthase, were not assessed. The
human cDNA for LTC
synthase has recently been
cloned(37) , but the mouse cDNA and antibodies are not yet
available. It seems likely that increased expression of cPLA
contributes to the KL-primed increase in eicosanoid generation
after cell sensitization with IgE and stimulation with antigen, without
selectivity for either the cyclooxygenase or 5-lipoxygenase pathway.
The effect of cytokines on the differentiation and maturation of
mast cells has previously been reported in terms of the expression of
granule-associated markers. BMMC cultured with KL counterstain with
safranin, synthesize heparin proteoglycan and more
histamine(46) , and express transcripts for mMCP-4, a protease
normally expressed in CTMC (47, 48) . The increase in
IgE-dependent PGD generation by KL is compatible with the
notion that KL leads to maturation of BMMC toward a CTMC-like
phenotype. However, although IL-3, IL-9, and IL-10 further augment
KL-primed IgE-dependent generation of PGD
, IL-3 suppresses
KL-induced granule maturation in terms of the appearance of
safranin-positive granules, heparin biosynthesis, and expression of
mMCP-4(47) ; and IL-9 and IL-10 each induce the expression of
the MMC-associated proteases mMCP-1 and
-2(48, 67, 68, 69) . Thus, the
precise combination of factors in the microenvironment that lead to
complete maturation of BMMC to a CTMC phenotype has yet to be
elucidated, and separate regulatory mechanisms may exist that govern
the maturation of granule constituents as compared to those that
regulate lipid mediator generation.
In conclusion, a detailed
examination of steady-state mRNA and the expression of protein and
enzymatic activity revealed that the priming of BMMC by KL for
enhancement of IgE-dependent eicosanoid generation is a result of
increased expression of several enzymes, including cPLA,
PGHS-1, and hematopoietic PGD
synthase. The increase in
expression of cPLA
contributes to the rise in IgE-dependent
generation of both PGD
and LTC
, whereas the
increased expression of PGHS-1 and PGD
synthase contributes
to the enhancement of IgE-dependent PGD
generation in
preference to LTC
generation. The accessory cytokines,
IL-3, IL-9, and IL-10, modulate IgE-dependent PGD
generation by augmenting the KL-induced expression and activity
of PGHS-1. The expression of the terminal enzyme for eicosanoid
biosynthesis, hematopoietic PGD
synthase, is up-regulated
by KL but is not affected by any of the hematopoietic cytokines
studied.