RasGRP4 Regulates the Expression of Prostaglandin D2
in Human and Rat Mast Cell Lines*
Lixin
Li
,
Yi
Yang
, and
Richard L.
Stevens
§
From the
Department of Medicine, Brigham and Women's
Hospital and Department of Medicine, Harvard Medical School,
Boston, Massachusetts 02115
Received for publication, November 12, 2002, and in revised form, December 6, 2002
 |
ABSTRACT |
Mast cells (MCs) are a major source of
prostaglandin (PG) D2 in connective tissues, and the
expression of this eicosanoid has been linked to asthma and other
inflammatory disorders. While it is known that the surface receptor
c-kit controls PGD2 expression in MCs by
regulating the levels of a synthase that converts PGH2 to
PGD2, the intracellular signaling proteins that act
downstream of c-kit in this cyclooxygenase pathway have not
been identified. We recently cloned a new cation-dependent,
guanine nucleotide exchange factor/phorbol ester receptor (designated
RasGRP4) that is required for the efficient expression of granule
proteases in the human MC line HMC-1. GeneChip analysis of ~12,600
transcripts in RasGRP4
and RasGRP4+ HMC-1
cells revealed a >100-fold difference in the levels of hematopoietic
PGD2 synthase mRNA. No other transcript in the
eicosanoid pathway was influenced by RasGRP4 in a comparable manner. As
assessed by SDS-PAGE immunoblot analysis, RasGRP4+ HMC-1
cells contained substantial amounts of PGD2 synthase
protein. RasGRP4+ MCs also produced ~15-fold more
PGD2 than did RasGRP4
MCs when both cell
populations were activated by calcium ionophore. The induced transcript
is therefore translated, and substantial amounts of functional
PGD2 synthase accumulate in RasGRP4+ MCs. In
support of the conclusion that RasGRP4 controls PGD2 expression in MCs, inhibition of RasGRP4 expression in the rat MC line
RBL-2H3 using a siRNA approach resulted in low levels of
PGD2 synthase protein.
 |
INTRODUCTION |
Activated human and rodent mast cells
(MCs)1 generate and release
substantial amounts of prostaglandin (PG) D2 (1), and many
of the vasodilation and hemodynamic problems that occur in patients
with systemic mastocytosis are thought to be caused by the excessive
production of this eicosanoid. PGD2 is a
neuromodulator/sleep-inducing factor in the central nervous system. In
peripheral tissues, PGD2 inhibits platelet aggregation (2)
but activates eosinophils. PGD2 is a potent chemotactic
factor for eosinophils (3), and PGD2-treated eosinophils
increase their calcium mobilization, actin polymerization, and surface
expression of CD11b (4, 5). This eicosanoid also enhances the rate of
apoptosis of eosinophils if these granulocytes are cultured for ~20 h
in the absence of a viability-enhancing cytokine such as interleukin
(IL) 5 (6). Pulmonary MCs play important roles in the initiation and/or
progression of asthma, and substantial amounts of PGD2 are
released into the lungs during asthma attacks (7, 8). The observation
that patients with asthma undergo bronchoconstriction when they inhale PGD2 (9) documents the pathologic consequences of high
levels of PGD2 in the lung. PGD2 exerts its
biological actions via two seven-transmembrane, G protein-coupled
receptors (designated PTGDR/DP and GPR44/CRTH2) (10-12). Targeted
disruption of the PTGDR gene in the mouse leads to a marked reduction
in antigen-induced airway reactivity to acetylcholine (13), thereby
supporting the earlier inhalation studies in humans and dogs that
implicated an adversarial role for PGD2 in the lung.
In the cyclooxygenase pathway that ultimately leads to PGD2
expression, liberated arachidonic acid is converted to PGG2
and then to PGH2. PG endoperoxide H synthase (PGHS) 1 (also
known as cyclooxygenase 1) and PGHS-2 (also known as cyclooxygenase 2)
are both able to carry out this two-step biosynthetic process. The
resulting precursor eicosanoid is then metabolized by terminal synthases to form PGD2, PGE2,
PGF2
, PGI2/prostacylin, and thromboxane
A2. Two PGD2 synthases have been identified in
mice, rats, and humans (14, 15). The brain enzyme is a
glutathione-independent member of the lipocalin family of proteins. The
distinct hematopoietic enzyme that is expressed in MCs (16) is a
sigma-class, glutathione S-transferase family member.
PGH2 can be metabolized inside cells to thromboxane
A2 and to a variety of PGs. Thus, the amount of
PGD2 produced by an Fc
RI- or calcium ionophore-activated
MC is determined in a large part by the amount of PGD2
synthase protein in the cell. MCs are heterogeneous in terms of what
eicosanoids they produce. c-kit is a member of the type III
receptor tyrosine kinase family. PGD2-expressing MCs
contain abundant amounts of c-kit on their surfaces, and
Murakami et al. (17) noted that c-kit ligand (KL)
somehow regulates the levels of PGD2 synthase in mouse MCs.
To a lesser extent, IL-3 and IL-10 also influence the expression of
PGD2 synthase in MCs. Treatment of human megakaryocytic
cell lines with phorbol esters results in a 2-5-fold increase in the
levels of PGD2 synthase mRNA (18, 19). While these
findings suggest that one or more diacylglycerol/phorbol
ester-responsive proteins play an important role in the expression of
PGD2 synthase in hematopoietic cells, the intracellular
proteins that act downstream of c-kit and other membrane
receptors to control the levels of PGD2 synthase in MCs have not been identified.
We recently cloned a new member of the Ras guanine nucleotide-releasing
protein (RasGRP) family of intracellular signaling proteins (20). In
contrast to the other three members of its family, RasGRP4 normally is
restricted to mature MCs and their circulating progenitors. RasGRP4
functions as a cation-dependent, guanine nucleotide
exchange factor. It also is a diacylglycerol/phorbol ester receptor
that appears to act downstream of c-kit. The hRasGRP4 gene
resides on chromosome 19q13.1 (20) in the vicinity of a site that has
been linked to bronchial hyperresponsiveness (21, 22). RasGRP1 is
essential for the final stages of T-cell development (23). Although
human MCs do not express RasGRP1, RasGRP2, or RasGRP3, transfection
studies carried out with the RasGRP4-defective HMC-1 cell line derived
from a patient with a MC leukemia suggests that RasGRP4 is required for
the final stages of MC development (20). Thus, at least two members of
the RasGRP family of signaling proteins appear to control cellular
differentiation and maturation. We previously noted that RasGRP4
influences the storage of varied neutral proteases in the secretory
granules of a MC line. We now report that RasGRP4 also controls what
eicosanoids this immune cell produces.
 |
EXPERIMENTAL PROCEDURES |
Transcript Analysis of RasGRP4
and
RasGRP4+ HMC-1 Cells--
RasGRP4+ and
RasGRP4
HMC-1 cells (20) were cultured in enriched medium
(Iscove's modified Dulbecco's medium (BioWhittaker) containing 10%
heat-inactivated fetal calf serum (Sigma), 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 10 µM monothioglycerol (Sigma) with or
without 200-500 µg/ml G418) in the absence of human cytokines. Total
RNA was isolated from the two populations of cells with TRIzol
(Invitrogen), and comparative transcript profiling was carried out at
the Gene Array Technology Center (Brigham and Women's Hospital,
Boston, MA) with HG-U95A GeneChips (Affymetrix, Santa Clara, CA) and
the experimental protocol recommended by Affymetrix. Each GeneChip
contains ~12,600 probe sets. In these analyses, 8 µg of total RNA
from RasGRP4
and RasGRP4+ HMC-1 cells were
reverse-transcribed using the GeneChip T7-oligo(dT) promoter primer
kit. Biotinylated complementary RNAs, generated from the resulting
cDNAs, were fragmented and incubated with the GeneChips for 16 h. The resulting GeneChips were incubated with streptavidin-phycoerythrin staining solution. The obtained signals were
then amplified by sequentially incubating the GeneChips with goat IgG,
biotinylated goat anti-streptavidin antibody, and staining solution.
Hybridization to the array was quantified with a Hewlett-Packard gene
array laser scanner. In separate studies, the generated RT-PCR products
were subjected to gel electrophoresis to confirm that they were derived
from the authentic PGD2 synthase transcript.
Quantitation of PGD2 Synthase mRNA Levels in
Cells by Real-time RT-PCR--
The GeneChip data obtained with the
PGD2 synthase probe set were confirmed by real-time RT-PCR.
The PCR primers and fluorogenic probes for measuring PGD2
synthase mRNA levels were designed with the use of "Primer
Express" (Applied Biosystems, CA). TaqMan's 18 S rRNA control
reagents were used to normalize RNA levels in each HMC-1 sample.
Fluorescent probes were selected such that their Tm
was ~10 °C higher than the matching primer pair. Each high
performance liquid chromatography-purified fluorescent probe
contained a 6-carboxyfluorescein (FAM) reporter dye covalently attached at its 5' end and a black hole quencher 1 quencher dye covalently attached at its 3' end. The forward primer
5'-GGGCAGAGAAAAAGCAAGATGT-3', the reverse primer
5'-CCCCCCTAAATATGTGTCCAAG-3', and the dual-labeled fluorescent probe
5'-(FAM)-CAATGAGCTGCTCACGTATAATGCGCC-(BHQ-1)-3' were used to
quantitate PGD2 synthase mRNA levels in these assays. Reactions were carried out using an iCycler IQ real-time detection system (Bio-Rad). SuperScript one-step RT-PCR with Platinum
Taq kits (Invitrogen) were used. Each 50-µl reaction
contained 200 ng of total RNA, 5 mM MgSO4, 500 nM forward and reverse primers, and 200 nM
fluorescent probe. Samples were analyzed in triplicate. Negative-control reactions were carried out on replicate samples that
had not been subjected to the reverse transcriptase step. Additional
negative-control reactions were carried out in wells lacking HMC-1
cellular RNA. The reaction conditions were as follows: 15 min at
50 °C and 5 min at 95 °C, followed by 45 two-temperature cycles
(15 s at 95 °C and 1 min at 60 °C). The standard curve method
(24, 25) was used to analyze the obtained data.
SDS-PAGE Immunoblot Analysis--
RasGRP4
and
RasGRP4+ HMC-1 cells were analyzed for their expression of
four enzymes that participate in the cyclooxygenase and/or
5-lipoxygenase pathways. Both populations of cells were resuspended at
a density of 4 × 105 cells/ml in enriched medium
alone or medium supplemented with varied combinations of IL-3 (10 ng/ml), IL-4 (10 ng/ml), IL-10 (10 ng/ml) (Chemicon, Temecula, CA),
IL-13 (10 ng/ml), KL (50 ng/ml), and tumor necrosis factor
(TNF-
; 20 ng/ml) (R & D Systems, Minneapolis, MN). After 5 days of
cytokine exposure, the cultured cells were washed with
phosphate-buffered saline, boiled in SDS sample buffer containing
-mercaptoethanol, and subjected to gel electrophoresis. The resolved
proteins were blotted onto polyvinylidene difluoride membranes
(Bio-Rad) and probed with rabbit antibodies that recognize human
PGHS-1, 5-lipoxygenase, PGE2 synthase, and hematopoietic-type PGD2 synthase (Cayman Chemical, Ann
Arbor, MI). The latter antibody also recognizes rat hematopoietic-type PGD2 synthase. After each blot was washed three times with
Tris-buffered saline containing 0.1% Tween 20, the treated blot was
exposed to Tris-buffered saline (15 ml) containing 5% nonfat milk,
0.1% Tween 20, 0.5% goat serum, and a 1:1000 dilution of a stock
solution of horseradish peroxidase-conjugated goat anti-rabbit IgG
(Bio-Rad) for 1 h at room temperature. Immunoreactive proteins
were visualized using a chemiluminescence kit (Genotech, St. Louis, MO)
and BioMax MR film (Eastman Kodak Co.).
Calcium Ionophore Activation of RasGRP4
and
RasGRP4+ HMC-1 Cells--
RasGRP4
and
RasGRP4+ HMC-1 cells were washed, suspended at a
concentration of 106 cells/ml in calcium/magnesium-free
phosphate-buffered saline, and stimulated with 0.5 µM
calcium ionophore A23187 (Sigma) at 37 °C for 30 min as done in
other eicosanoid studies of MCs (26). The generated eicosanoids
PGD2, PGE2, and leukotriene C4
(LTC4) in the supernatants were quantitated using the
relevant ELISA kits (Cayman Chemical). Each reaction was read at 450 nm using an ELISA plate reader (Molecular Device). Data are given as
mean ± S.D. Significance was defined as p < 0.05 by the Student's t test.
siRNA-mediated Inhibition of RasGRP4 Expression in RBL-2H3
Cells--
A siRNA approach similar to that described by Elbashir
et al. (27) was used to evaluate the consequences of
decreased expression of RasGRP4 in the rat MC line RBL-2H3. The coding
sequence of rat RasGRP4 (28) was scanned to identify a gene-specific
21-nucleotide sequence downstream of an "AA" sequence that
possesses a 55% GC content. A BLAST search confirmed that the selected
sequence (corresponding to residues 27-47 in GenBankTM
accession number AF465263) is not present in another transcript in GenBankTM data bases. The RasGRP4-specific
oligonucleotide 5'-GUCUCAUCAGGAAUGCUCUGGdTdT-3' and its corresponding
oligonucleotide 5'-CCAGAGCAUUCCUGAUGAGACdTdT-3' were synthesized
and purified (Dharmacon Research, Lafayette, CO) and then
annealed to form the final siRNA duplex with its TT overhangs. The
resulting siRNA duplex was introduced into RBL-2H3 cells (line
CRL-2256; American Type Culture Collection, Manassas, VA) using a
liposome transfection approach. Liposome/siRNA complexes were formed at
room temperature using 3 µl of 20 µM siRNA, 2 µl of
LipofectAMINETM 2000 (Invitrogen), and 100 µl of Opti-MEM
I serum-free culture medium (Invitrogen). The resulting solution was
added dropwise to each culture dish containing ~5 × 104 adherent MCs. The cells were incubated 3-4 h at
37 °C. One ml of serum-enriched medium was then added, and the cells
were cultured for an additional 24-48 h. The transiently transfected
cells were harvested, and the levels of PGD2 synthase and
-actin protein were measured using the above SDS-PAGE immunoblot
approach. In these assays, each protein blot was incubated ~17 h with
anti-PGD2 synthase antibody and then for 1 h with the
anti-
-actin antibody (Sigma) before final development.
 |
RESULTS AND DISCUSSION |
All nontransformed rodent and human MCs that have been examined to
date preferentially metabolize arachidonic acid via the cyclooxygenase
pathway to PGD2 rather than to PGE2.
Nevertheless, Macchia et al. (29) discovered that HMC-1
cells produce ~20-fold more PGE2 than PGD2.
This surprising finding allowed us to use the
c-kit+ HMC-1 cell line to further elucidate the
intracellular signaling pathways that control PGD2
production in MCs. Transcript analysis (Fig.
1) revealed that the failure of HMC-1
cells to generate large amounts of PGD2 is a consequence of
a low rate of transcription of the PGD2 synthase gene
and/or a high rate of catabolism of its transcript.

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Fig. 1.
Evaluation of PGD2 synthase
mRNA levels in RasGRP4 and
RasGRP4+ HMC-1 cells. A, real-time RT-PCR
was used to quantitate the levels of PGD2 synthase mRNA
in RasGRP4 HMC-1 cells (lane 1) and in HMC-1
cells that were transfected with a RasGRP4-expressing construct on
three separate occasions over a six month period (lanes
2-4). The PGD2 synthase (PGDS) mRNA
levels in each sample were normalized to that of 18S rRNA. The RNA used
in the GeneChip experiment (Table I) was obtained from the first
transfection experiment (lane 2). B, a separate
semiquantitative RT-PCR approach also was used to evaluate the levels
of PGD2 synthase mRNA in these same four populations of
HMC-1 cells. Gel electrophoresis confirmed that the 100-bp product
generated in each case corresponds to PGD2 synthase. Size
markers are shown on the left.
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|
The RasGRP4 transcript was initially cloned from IL-3-developed mouse
bone marrow-derived MCs (mBMMCs). While all mouse, rat, and human MCs
appear to express RasGRP4 mRNA and/or protein, the amount of
RasGRP4 protein in a mouse peritoneal MC greatly exceeds that in a
mBMMC as assessed by SDS-PAGE immunoblot
analysis.2 Calcium ionophore-
or Fc
RI-activated mBMMCs produce ~25-fold more LTC4
than PGD2, whereas peritoneal MCs activated in a similar manner produce >40-fold more PGD2 than LTC4
(1, 26). The cumulative data raised the possibility that RasGRP4
regulates arachidonic acid metabolism in MCs. Thus, we evaluated
whether or not RasGRP4 controls PGD2 and/or
LTC4 expression in HMC-1 and RBL-2H3 cells.
Comparative transcript analysis of RasGRP4
and
RasGRP4+ HMC-1 cells using an Affymetrix GeneChip approach
revealed a dramatic difference in the steady-state levels of the
transcript that encodes hematopoietic PGD2 synthase in the
two populations of cells (Table I).
RasGRP4+ HMC-1 cells contained >100-fold more
PGD2 synthase mRNA than did the starting population of
HMC-1 cells that express nonfunctional forms of RasGRP4. No transcript
was induced to a comparable level, including the transcripts that
encode brain-type PGD2 synthase and LTC4
synthase. Table I shows profile data relating to the levels of the
transcripts that encode different proteins that participate in
arachidonic acid metabolism. The PGD2 synthase GeneChip
data were confirmed by real-time RT-PCR (Fig. 1A) and by
semiquantitative RT-PCR (Fig. 1B) analyses in three separate populations of RasGRP4-expressing cells. In a control experiment, HMC-1
cells transfected with the expression vector pcDNA3.1 lacking the
RasGRP4 cDNA contained barely detectable amounts of
PGD2 synthase transcript (data not shown).
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Table I
Comparative expression of the transcripts that encode 20 proteins that
participate in arachidonic acid metabolism in cells
|
|
Because the levels of a transcript do not always correlate with the
levels of its translated product, an SDS-PAGE immunoblot approach was
used to compare the levels of PGD2 synthase protein in
RasGRP4
and RasGRP4+ HMC-1 cells. The amount
of PGD2 synthase protein in RasGRP4
HMC-1
cells was nearly below detection (Fig.
2). In contrast, RasGRP4+
HMC-1 cells contained substantial amounts of an intracellular 25-kDa
protein that was recognized by the anti-PGD2 synthase
antibody. The induced PGD2 synthase transcript is therefore
translated and the appropriately sized biosynthetic enzyme accumulates
in the transfectants. As assessed by SDS-PAGE immunoblot analysis,
RasGRP4 did not induce HMC-1 cells to increase their accumulation of
PGHS1, PGE2 synthase, or 5-lipoxgenase protein (Fig. 2).
Thus, RasGRP4 induces a selective accumulation of PGD2
synthase mRNA and protein in this MC line.

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Fig. 2.
PGD2 synthase, PGE2
synthase, PGHS-1, and 5-lipoxygenase protein levels in
RasGRP4 and RasGRP4+
HMC-1 cells. SDS-PAGE immunoblots, prepared from the lysates of
RasGRP4 and RasGRP4+ HMC-1 cells, were probed
with antibodies specific for human PGD2 synthase
(PGDS), PGHS-1, PGE2 synthase (PGES),
and 5-lipoxygenase. Recombinant human PGD2 synthase
(rhPGDS) was used in the former analysis as a positive
control.
|
|
As assessed by ELISA, calcium ionophore-activated RasGRP4+
HMC-1 cells produced 12-20-fold more PGD2
(p < 0.05) than did RasGRP4
HMC-1 cells
(Fig. 3). The levels of
PGE2 and LTC4 were modestly increased and
decreased, respectively, in the calcium ionophore-treated RasGRP4+ cells. However, the variations in the amounts of
these eicosanoids were not statistically significant. The fact that
HMC-1 cells express nonfunctional forms of RasGRP4 indicates that
RasGRP4 is not essential in the early stages of MC development,
including the c-kit/KL-mediated proliferation of its
progenitors. Nevertheless, the observation that HMC-1 cells are unable
to granulate (20) and to produce substantial amounts of
PGD2 (Fig. 3) implies that RasGRP4 is required for the
efficient expression of the cassette of genes that encode a number of
the granule and lipid mediators of MC. The siRNA data obtained from
transiently transfected RBL cells (Fig.
4) support this conclusion. RBL cells
contain PGD2 synthase protein, and these rat MCs (30)
produce substantial amounts of PGD2 when exposed to calcium
ionophore (31). RBL-2H3 cells also contain RasGRP4
mRNA.2 Thus, a siRNA approach was used to evaluate the
consequences of decreased expression of RasGRP4 in RBL-2H3 cells. As
noted in Fig. 4, inhibition of RasGRP4 expression in the MC line
resulted in a transient (12-48 h) inhibition of PGD2
synthase expression. As far as we are aware, no one has examined
eicosanoid production in transgenic mice that lack RasGRP1 or in
cultured cells that have been induced to express varied forms of the
other RasGRP family members. Nevertheless, the finding that RasGRP4
regulates PGD2 expression in two populations of cultured
MCs raises the possibility that RasGRP1, RasGRP2, and/or RasGRP3
regulate eicosanoid production in other cell types.

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Fig. 3.
Generation of PGD2,
PGE2, and LTC4 in calcium ionophore-activated
RasGRP4 and RasGRP4+
HMC-1 cells. RasGRP4 and RasGRP4+ HMC-1
cells were exposed to calcium ionophore A23187 for 30 min. The amounts
of PGD2 (left bars), PGE2
(middle bars), and LTC4 (right bars)
generated in each experiment were determined by separate ELISAs.
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Fig. 4.
PGD2 synthase levels in control
and siRNA-treated RBL-2H3 cells. SDS-PAGE immunoblots, prepared
from the lysates of RBL-2H3 cells before and after these cells were
transfected with a RasGRP4-specific siRNA for 48 h, were probed
for with anti-PGD2 synthase (PGDS) and
anti- -actin antibodies. Similar data were obtained in a second siRNA
experiment.
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Earlier in vitro studies suggested that KL is required for
maximal expression of PGD2 synthase in mouse MCs. HMC-1
cells are able to proliferate in the absence of exogenous human
cytokines, because these transformed cells possess an activating
mutation in c-kit (32). The inability of HMC-1 cells to
produce large amounts of PGD2 supports the conclusion that
RasGRP4 acts downstream of c-kit. Murakami et al.
(17) identified a number of cytokines that influence the KL-mediated
expression of PGD2 synthase in cultured mouse MCs either in
a positive or negative manner. As assessed by GeneChip analysis (data
not shown), HMC-1 cells express the transcripts that encode the surface
receptors for IL-4, IL-10, IL-13, and KL. This MC line also expresses
three distinct receptors that recognize TNF-
and its family members.
RasGRP4
and RasGRP4+ HMC-1 cells were
therefore cultured for 5 days in the presence of varied combinations of
IL-3, IL-4, IL-10, IL-13, KL, and TNF-
. None of these cytokines were
able to induce PGD2 synthase expression in
RasGRP4
HMC-1 cells (data not shown). In addition, none
of these cytokines were able to inhibit the expression of
PGD2 synthase in RasGRP4+ HMC-1 cells. These
data imply that RasGRP4 is the dominant intracellular signaling protein
that controls PGD2 expression in MCs no matter what
extracellular cytokine environment this immune cell encounters in tissues.
 |
ACKNOWLEDGEMENT |
We thank Dr. Richard Pratt (Brigham and
Women's Hospital, Boston, MA) for helpful discussions regarding
GeneChip and real-time RT-PCR analyses.
 |
FOOTNOTES |
*
This work was supported by Grants AI-23483, HL-36110, and
HL-63284 from the National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Brigham and Women's
Hospital, Dept. of Medicine, Smith Bldg., Rm. 616B, 1 Jimmy Fund Way,
Boston, MA 02115. Tel.: 617-525-1231; Fax: 617-525-1310; E-mail:
rstevens@rics.bwh.harvard.edu.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.C200635200
2
L. Li, Y. Yang, and R. L. Stevens,
unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
MC, mast
cell;
mBMMC, mouse bone marrow-derived MC;
IL, interleukin;
LT, leukotriene;
PG, prostaglandin;
PGHS, PG endoperoxide H synthase;
RT, reverse transcriptase;
RBL, rat basophilic leukemia;
TNF-
, tumor necrosis factor
;
KL, c-kit ligand;
ELISA, enzyme-linked immunosorbent assay;
siRNA, small interfering RNA.
 |
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