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* Department of Medicine and
Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599;
Department of Medicine, Division of Nephrology, Duke University, and Durham Veterans Affairs Medical Centers, Durham, NC 27710;
Genetic Technologies, Pfizer Global Research and Development, Groton, CT 06340; and
¶ Institut National de la Santé et de la Recherche Médicale, Institut Pasteur de Lille, Lille, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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i,
PGE2 activation of the EP3 receptor leads to
both inhibition of adenylate cyclase and increased intracellular
Ca2+. The magnitude of increase in intracellular
Ca2+ induced by EP3 activation is similar to
that observed after activation of cells with IgE and Ag. Although PGE
alone is not sufficient to initiate BMMC degranulation, stimulation of
cells with PGE along with PMA induces degranulation. These actions are
mediated by the EP3 receptor through signals involving
Ca2+ mobilization and/or decreased cAMP levels.
Accordingly, these studies identify PGE2/EP3 as
a proinflammatory signaling pathway that promotes mast cell
activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI receptor-bound IgE or when IgG- or IgE-Ag complexes bind
to Fc receptors.
The signaling pathway leading to degranulation of mast cells after
engagement of the FcRI receptor by Ag and Ab has been extensively
characterized (2, 3, 4). Engagement of the receptor leads to
phosphorylation of tyrosine kinases, activation of phospholipase C, an
increase in diacylglycerol, and mobilization of internal
Ca2+ stores by inositol triphosphate. This
is followed by activation of protein kinase C by diacylglycerol
accompanied by an increase in mitogen-activated protein kinase activity
and increased Ca2+ influx from external stores.
The increase in intracellular Ca2+ is required
for the release of preformed mediators from mast cell granules
(5). In addition, eicosanoids, such as the leukotriene
B4
(LTB4),4
and various cytokines, such as IL-6, can be released independently of
these degranulation events.
Although stimulation of Fc receptors is sufficient to induce mast cell degranulation, isolated stimulation of other receptors does not cause degranulation, yet their activation may modify mast cell function during an inflammatory response. PGE2 is one example of an agent that has not been shown to trigger degranulation yet has long been recognized as an important modulator of mast cell function. Because production of eicosanoids, including PGE2, occurs in virtually every inflammatory and allergic response and because a wide range of cell types, including macrophages, fibroblasts, and epithelial cells, synthesize PGE2, these actions may have wide-ranging influences (6, 7, 8). However, the mechanism by which PGE2 modifies mast cell function is complex, and both stimulatory and inhibitory effects of PGE2 on mast cell function have been described.
The biological actions of PGE2 are mediated by
four different G-protein-coupled receptors: EP1,
EP2, EP3, and
EP4 (9). Each of these receptors is
unique in its tissue distribution, pharmacology, and signal
transduction properties. For example, EP2 and
EP4 receptors signal by coupling to
Gs proteins, increasing intracellular cAMP
levels. In contrast, stimulation of the EP1
receptor is coupled to phospholipase C and increases in intracellular
Ca2+ (10). The major signaling
pathway described for the EP3 receptor is
mediated by Gi and leads to a reduction in
intracellular cAMP levels. However, several EP3
receptor isoforms generated by alternative splicing from the single
EP3 receptor gene have been identified, and the
intracellular signals used by various EP3
isoforms may differ.
The existence of multiple EP receptors coupled to different intracellular signals provides a molecular basis for the diverse physiological actions of PGE2. However, the roles of the individual EP receptor subtypes in mediating specific actions of PGE2 in various cell types and tissues, including mast cells, have not been well characterized. This is due in part to the lack of subtype-specific EP agonists and antagonists. Accordingly, using mouse lines deficient in the EP1, EP2, EP3, or EP4 receptors, we examined the modulation of mast cell functions and signaling by PGE2. We find that the actions of PGE2 in mast cells are mediated primarily through the EP3 receptor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation of mice deficient in EP1, EP2, EP3, and EP4 receptors has been previously reported (11, 12, 13, 14). All mice used were at least 8 wk old and were bred and maintained in specific pathogen-free animal facilities at the University of North Carolina (Chapel Hill, NC), in accordance with the Institutional Animal Care and Use Committee guidelines.
Preparation of bone marrow-derived mast cells (BMMCs)
Bone marrow was isolated from the femurs of 2- to 3-mo-old mice and placed in culture. Cells were grown for at least 4 wk in the presence of mouse IL-3-supplemented culture medium to select for pure populations of mast cells as described previously (15). Briefly, bone marrow-derived cells were grown in RPMI 1640 medium supplemented with 8% FCS, 8% mouse IL-3 culture supplement (Collaborative Biomedical Products, Bedford, MA), 20 mM HEPES, 4 mM L-glutamine, 0.08 U/ml penicillin, 0.08 mg/ml streptomycin, 800 µM nonessential amino acids, 800 µM sodium pyruvate, 0.04 mg/ml gentamicin, and 92 µM 2-ME (BMMC culture medium). Cell cultures were maintained in a constant environment (humidified 37°C, 5% CO2). To deplete adherent cells such as macrophages and monocytes from culture, cells in suspension were transferred to fresh dishes a few days after harvesting and then weekly at a concentration of 105-106 cells/ml. Cells cultured in this manner were examined visually after fixation with Carnoy’s fixative and toluidine blue staining. Although the percentage of mast cells of all cultures was not tested before each experiment, we have since examined populations of cells cultured from two different EP3-/- mice, two wild-type mice, and one culture derived from each of the following: an EP4-/-, an EP2-/-, and an EP1-/- mouse. Cultured cells were comprised of 98% toluidine blue positive staining granulated cells, regardless of the genotype.
RNA analysis of EP receptor expression
Total RNA from cultured BMMCs was obtained using RNAzol B
(Teltest, Friendswood, TX) according to the manufacturer’s
instructions. Total RNA (20 µg) was fractionated by gel
electrophoresis under denaturing conditions (1.2% agarose/1.1%
formaldehyde). The contents of the gel were then transferred for
Northern blot analysis. Hybridization to
[32P]cytidine 5'-triphosphate-labeled
cDNA probe (EP receptor or 
-actin) was performed at 68°C for
1 h using a QuickHyb solution (Stratagene, La Jolla, CA). Washes
(42°C, 15 min) were performed twice in 2x SSC/0.1% SDS
and once in 0.2x SSC/0.1% SDS. RT-PCR analysis to detect the presence
of the EP2 receptor was conducted as described
previously (12). Briefly, cDNA was synthesized from BMMC
RNA by reverse transcription according to the manufacturer’s protocol,
and PCR amplification was conducted using the following primers:
EP2-1F (5'-GTGGCCCTGGCTCCCGAAAGTC-3') and
EP2-2R (5'-GGCAAGGAGCATATGGCGAAGGTG-3'). The
presence of EP3 receptor isoforms was analyzed by
RT-PCR analysis using isoform-specific primers as previously described
(16).
Measurement of hexosaminidase release
BMMCs from wild-type and EP1-,
EP2-, EP3-, and
EP4-deficient mice were loaded with murine
DNP-specific IgE mAb (clone SPE-7 from Sigma-Aldrich, St. Louis, MO)
overnight at a concentration of 100 ng/ml per million cells. They were
washed twice with Siraganian buffer (119 mM NaCl, 5 mM KCl, 25 mM
PIPES, 5.6 mM glucose, 1 mM CaCl2, 0.4 mM
MgCl2, and 0.1% BSA, pH 7.2) to eliminate any
excess Ab. BMMCs were then resuspended (5 x 105
cells per 75 µl) in Siraganian buffer, transferred to 96-well
microtiter plates, and preincubated for 15 min at 37°C. Cells were
then exposed to 25 µl of prewarmed PGE1 or
PGE2 (Cayman Chemicals, Ann Arbor, MI), PMA, or
PGE1 and PMA solutions for 20 min, followed by
addition of 100 µl of Ag (PMA and DNP-human serum albumin
(HSA); Sigma-Aldrich). In some experiments, Ag was added
immediately after PGE1 or
PGE2 treatment, as noted in Fig. 2
B.
Plates were then incubated for 30 min (humidified 37°C, 5%
CO2) followed by centrifugation at 500 x
g for 5 min (4°C). A 100-µl aliquot of the supernatant
was then taken and the mast cell pellets with the remaining
supernatants were lysed with 100 µl of 0.2% Triton X-100. The
supernatant and an aliquot of the cell lysate were then transferred to
a well containing 100 µl of 1 mM
p-nitrophenyl-N-acetyl--D-glucosaminide
(Sigma-Aldrich) in citrate buffer (0.1 M citric acid, 0.1 M sodium
citrate, pH 4.5). The reactions were then incubated for 1 h
(humidified 37°C, 5% CO2), then halted with
the addition of 100 µl of 0.1 M
Na2CO3/0.1 M
NaHCO3. The absorbance was read at 405 nm to
measure hexosaminidase activity. Data are expressed as the percentage
of released hexosaminidase relative to the total cellular
hexosaminidase content.
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As described above, BMMCs were loaded with IgE overnight. After a 30-min preincubation with 10 µM indomethacin to inhibit endogenous prostaglandin formation, cells were washed twice in BMMC culture medium. Cells were then resuspended at 5 x 105 cells in 100 µl of fresh medium, plated in microtiter plates, and treated with Ag (50 ng/ml), PGE1 (10 µM), or Ag and PGE1 in a final volume of 200 µl. BMMCs were then incubated for 7 h (humidified 37°C, 5% CO2). At the end of the incubation period, cells were centrifuged at 500 x g for 5 min (4°C). Supernatants were carefully transferred to new wells and aliquots were stored at -80°C until assayed. Samples were diluted 10-fold in BMMC culture medium to fit within the standard curve. IL-6 content was determined by enzyme immunoassay (EIA) (PerSeptive Biosystems, Framingham, MA) according to the manufacturer’s protocol.
Measurement of LTB4 production
As described above, BMMCs were loaded with IgE overnight. BMMCs were washed two times and resuspended in fresh culture medium, and 5 x 105 cells in 100 µl of medium were transferred to 96-well tissue culture plates, treated with Ag (50 ng/ml), PGE1 (10 µM), or Ag and PGE1, and incubated at 37°C for 20 min. After the incubation period, cells were centrifuged at 500 x g for 5 min (4°C). Supernatants were then transferred to new wells and stored at -80°C until assayed by EIA according to the manufacturer’s protocol (Cayman Chemicals).
Measurement of intracellular calcium concentrations ([Ca2+]i)
BMMCs were loaded with murine anti-DNP IgE by overnight incubation with the mAb as described above. Cells were incubated in the presence of 10 µM indomethacin (Sigma-Aldrich) for 30 min and then washed twice in calcium buffer (135 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, 10 mM HEPES, 0.1% BSA, and 10 µM indomethacin, pH 7.4). After washing, cells were resuspended in calcium buffer (106 cells/ml) and loaded with 10 µM fura 2-acetoxymethyl ester (fura 2-AM; Molecular Probes, Eugene, OR) for 45 min (humidified 37°C, 5% CO2). Cells were then washed in calcium buffer and transferred to a cuvette. Fluxes in intracellular free [Ca2+] in response to the indicated treatments of Ag and PGE2 were recorded with a luminescence spectrometer (LS50B; PerkinElmer, Wellesley, MA) at excitation wavelengths of 340/380 nm and an emission wavelength of 510 nm. The fluorescence ratio signal was calibrated by adding 1 µM ionomycin (Sigma-Aldrich) and the addition of 5 mM EGTA to determine the maximal and minimal fluorescence, respectively. Free cytosolic Ca2+ concentrations were then calculated using a Kd of fura 2 for Ca2+ of 224 nM according to the method previously reported (17). Stock solutions of PGE2 and indomethacin were prepared in 100% ethanol. Fura 2-AM and ionomycin were dissolved in DMSO.
Measurement of intracellular cAMP levels ([cAMP]i)
BMMCs were washed two times with Tyrode’s solution (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 0.2 mM NaH2PO4, 12 mM NaHCO3, 5.5 mM glucose) and resuspended at 2 x 106 cells per 200 µl of solution. Cells were then transferred to a sample vial and incubated at 37°C with stirring for 1 min and then treated with PGE1, PGE2, prostacyclin (PGI2), or vehicle control for 1 min. Each treatment was conducted on three different cell samples. Purification and EIA analysis of [cAMP]i were then conducted according to the manufacturer’s protocol (Amersham Pharmacia, Piscataway, NJ). The triplicate data points were then used to calculate baseline levels of [cAMP]i and the change in [cAMP]i, with results expressed as the percent of the control baseline level.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BMMCs were cultured from EP1-deficient
(EP1R-/-), EP2-deficient
(EP2R-/-), EP3-deficient
(EP3R-/-), EP4-deficient
(EP4R-/-), and wild-type control mice. Total RNA was prepared from these
cells and the level of expression of the PG receptors was examined by
Northern analysis. The EP1 receptor was easily
detected in control BMMCs (Fig. 1, left). EP2 mRNA could not be detected
in either the normal or the mutant cell lines by Northern analysis of
total RNA; however, RT-PCR analysis of the RNA indicated low levels of
the EP2 receptor transcript in BMMCs (data not
shown). Expression of the EP3 receptor was easily
detected in wild-type BMMCs but not in the cells derived from
the EP3-deficient animals (Fig. 1, center). To determine which EP3
receptor splice variants are expressed by BMMCs, RT-PCR analysis was
conducted using primer sets specific for each of the isoforms. After
gel electrophoresis and ethidium bromide staining, PCR products
corresponding to EP3 and
EP3 isoforms were easily detected (data not
shown). However, expression of the EP3
isoform
was not observed. In contrast, all three EP3
isoforms were easily detected on similar analysis of RNA prepared from
mouse kidney. By Northern analysis, high levels of
EP4 expression were easily detected in the BMMCs
(Fig. 1, right).
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Degranulation of BMMCs was monitored by measuring the release of
hexosaminidase into the culture supernatant. PGE2
and PGE1 bind with equal affinity to the four
known EP receptors. However, PGE1 has a higher
affinity than PGE2 for the IP prostacyclin
receptor (18). Incubation of mast cells with either
PGE1 or PGE2 alone had no
effect on hexosaminidase release by wild-type mast cells (Fig. 2
). By contrast, in the presence of
IgE-Ag receptor complexes, PGE1 and
PGE2 significantly augmented release of
hexosaminidase in a dose-dependent manner, even at very low doses of
1 x 10-8 M and across the range of Ag
concentrations that were tested (Fig. 2A). No measurable
difference in the relative effectiveness of PGE1
and PGE2 was observed. To reconcile the differing
results of previous studies, which have found that PGs can inhibit or
potentiate mast cell mediator release, it has been suggested that the
effect might depend on the timing of the addition of this lipid
mediator, relative to exposure of the cells to Ag (19).
Therefore, we examined the ability of PGE1 and
PGE2 to modulate degranulation of BMMCs when
added either immediately before Ag or 20 min before stimulation with
Ag. As can be seen in Fig. 2, under both experimental conditions,
exposure to either PGE1 or
PGE2 enhanced mast cell degranulation. To
identify the EP receptor subtype that mediates the enhanced
degranulation, we compared the effects of PGE1 or
PGE2 in mast cell cultures prepared from each of
the EP-deficient mouse lines. No difference in the response of the four
different EP-deficient mast cell cultures to IgE and Ag alone was
observed, suggesting that these PGE2 receptors
are not required for the development of these cells or for the ability
of these cells to be activated via the FcR1 receptor (Fig. 3). We next examined the ability of
PGE1 or PGE2 to augment
this IgE-mediated degranulation. The enhancement of mast cell
degranulation by PGE2 continued to be observed on
examination of mast cells prepared from
EP1R-/-
mice (Fig. 3A). Similarly, PGE2
continued to augment degranulation of BMMCs that lacked the
EP2 receptor (Fig. 3B). Examination of
BMMCs prepared from
EP4R-/-
mice also responded in a similar manner to control populations when
exposed to both Ag and PGE1 (Fig. 3D).
In stark contrast, the enhanced response to IgE-Ag after
PGE1 treatment was completely abrogated in
EP3-deficient BMMCs (Fig. 3C). These
results demonstrate that the enhancement of FcRI-mediated
degranulation by PGs is mediated by EP3
receptors.
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Activation of mast cells also stimulates cytokine release. Thus,
we next determined whether PGs could modify release of cytokines and
whether this effect was also mediated by the EP3
receptor. As described above, mast cells were incubated with IgE and
then treated with Ag alone or Ag plus PGE1. Seven
hours later, the supernatant was harvested and IL-6 levels were
determined. After Ag stimulation, high levels of IL-6 were released
from mast cells (Fig. 4) and this IL-6
release was significantly enhanced in the wild-type mast cells by the
treatment with PGE1. In contrast,
PGE1 failed to enhance the release of IL-6 in the
EP3-deficient mast cells. Similar to wild-type
control BMMCs, IL-6 release by EP2- and
EP4-deficient BMMCs was enhanced upon exposure to
PGE1 after IgE-Ag/FcRI receptor complex
formation (data not shown).
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EP3 receptor signaling in BMMCs
Based on our finding that mast cells express multiple
EP3 receptor isoforms and the diverse signaling
pathways used by these isoforms, we explored EP3
receptor signaling in BMMCs by comparing
[cAMP]i of wild-type and
EP3-deficient cells after incubation with
PGE1 and PGE2. Treatment of
wild-type BMMCs with PGE1 significantly increased
[cAMP]i. In contrast, exposure of the cells to
PGE2 did not significantly alter levels of cAMP
in wild-type cells (Fig. 5A).
Because PGE1, but not PGE2,
may also activate the Gs-coupled IP receptor,
we examined the effect of PGI2 on BMMCs. IP
receptor activation in mast cells causes a marked stimulation of cAMP
levels (Fig. 5A). To obtain maximum stimulation of adenylate
cyclase we used PGE1 in subsequent experiments to
determine the impact of loss of the EP3 receptor
on [cAMP]i. Stimulation of cAMP levels by
PGE1 was markedly enhanced in
EP3R-/-
compared with wild-type BMMCs, suggesting that
EP3 receptors couple to
Gi proteins in BMMCs (Fig. 5B).
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A, left).
Moreover, the magnitude of the change in
[Ca2+]i induced by
PGE2 is similar to that elicited by Ag treatment
(Fig. 6B). This increase in
[Ca2+]i in response to
PGE2 is completely dependent on expression of the
EP3 receptor because PGE2
had no effect on [Ca2+]i
in EP3-deficient BMMCs (Fig. 6A,
right). We next examined whether PGE2
could enhance IgE-Ag-induced Ca2+ mobilization.
In wild-type cells, this increase in
[Ca2+]i is substantially
enhanced after exposure to both PGE2 and Ag (Fig. 6B, top right). In contrast, after addition of
Ag, PGE2 does not further augment
[Ca2+]i in
EP3R-/-
mast cells (Fig. 6B, bottom). We confirmed these
observations by treating IgE-loaded BMMCs sequentially with Ag and 2
min later with PGE2. After stimulation with Ag, a
further increase in
[Ca2+]i levels is seen in
wild-type cells. This response was not observed in the
EP3-deficient mast cells (data not shown). Both
PGE1 and PGE2 had similar
effects on [Ca2+]i (data
not shown).
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Although PGE alone stimulates
[Ca2+]i flux to a similar
degree as IgE-Ag, it fails to effect a mediator release. This suggests
that additional signals are required to trigger mast cell
degranulation. Similarly, whereas activation of protein kinase C (PKC)
modulates mast cell degranulation, treatment of mast cells with
PKC-activating PMA alone does not result in mast cell degranulation.
Thus, we next determined whether PGE1 and PMA
could act synergistically to induce degranulation. Consistent with
previous studies, treatment of wild-type or
EP3R-/-
BMMCs with PMA alone did not alter hexosaminidase release (Fig. 7) or
[Ca2+]i (data not shown).
However, in wild-type BMMCs treated with PMA,
PGE1 caused a dose-dependent increase in BMMC
degranulation. These actions of PGE1 are mediated
through the EP3 receptor, because mediator
release was not enhanced in EP3-deficient mast
cells treated with PMA and PGE1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression analysis and pharmacological studies have indicated that
mast cells express a number of prostanoid receptors, including several
PGE2 receptors and the
Gs-coupled prostacyclin receptor.
Pharmacological studies are consistent with the expression of
EP3 and EP4 on
IL-3-dependent mast cell lines and on rat peritoneal mast cells
(20, 21, 22). Expression analysis of MC/9 mouse mast cell line
revealed the expression of EP1,
EP3, and EP4
(19). We show here that BMMCs express high levels of the
Gs-coupled EP4 receptor.
Although we could not detect expression of the
EP2 receptor by Northern analysis, RT-PCR studies
indicate that mast cells do express this receptor. This finding is
consistent with the cloning of the mouse EP2
receptor from a mouse mastocytoma cell line. The relatively low
expression of the EP2 receptor in comparison to
EP4 in virtually all tissues that coexpress these
two receptors has been noted previously (22). In addition
to these Gs-coupled receptors, we show that
BMMCs express high levels of the EP3 receptor,
including both the EP3 and
EP3 isoforms. Consistent with previous
reports, we detected high levels of EP1 receptor
expression. Activation of this receptor has been shown to result in an
increase in [Ca2+]i.
We show that treatment of BMMCs with PGE1 or PGE2 alone does not induce degranulation of BMMCs. However, addition of PGE to BMMCs stimulated with IgE and Ag potentiates degranulation of these cells. This is consistent with the findings reported by Leal-Berumen et al. (23), using rat peritoneal mast cells, and with results obtained using BMMCs and IL-3-dependent mast cell lines (19, 20). Our results contrast sharply, however, with other studies that showed that PGE2 could inhibit the release of mediators from both rat peritoneal mast cells (24, 25) and human lung mast cells (26, 27). It is likely that at least some of these differences reflect the use of different mast cell populations in these studies. As discussed below, these observations suggest that the impact of PGE2 on mast cell function may be dependent on the maturity, tissue type, and perhaps activation state of the cell.
The studies reported here also show that PGE1 can enhance the production of IL-6 by IgE-Ag-stimulated mast cells. Unlike previous reports, we failed to observe an increase in production of IL-6 by cells stimulated by PGE alone. Stimulation of rat peritoneal mast cells with PGE1 or PGE2 was reported to induce a significant increase in the release of IL-6 from 3 to 18 h after treatment (23). In similar studies using BMMCs, no significant increase in IL-6 release was observed at early time points on comparison of medium-treated and PGE2-treated BMMCs (19). However, in these same studies IL-6 production in response to PGE2 alone was noted at later time points. We did not observe substantial release of IL-6 in the response to PGE1 or PGE2 alone, even at these later time points (data not shown).
The enhancement of Ag-stimulated degranulation of BMMCs by PGE1 and PGE2 is perhaps surprising in light of our demonstration that these cells express both the EP4 and the EP2 receptors and of the established coupling of these receptors to stimulatory G proteins in all cell types examined to date. Agents that stimulate an increase in [cAMP]i have been shown to inhibit mast cell degranulation (28), and an increase in [cAMP]i due to PGE1 addition is believed to precede the inhibition of histamine release from rat peritoneal mast cells in response to stimulation of IgE receptors (27). There are a number of possible explanations for the observed lack of inhibition of BMMC degranulation after treatment with PGE1 and the similarity of the response of EP4- or EP2-deficient mast cells compared with wild-type cells. It is possible, for instance, that although these cells express very high levels of EP4 receptor mRNA, this does not correspond to high numbers of EP4 receptors at the cell surface of BMMCs. Alternatively, the coupling of the EP4 receptors to adenylate cyclase might be inefficient in these immature mast cells, or rapid desensitization of this receptor in BMMCs might reduce their impact on cell physiology. Alteration of the EP4/EP2 inhibitory pathways as mast cells mature after migration to various tissues might alter the response of mast cells to PGE2, resulting in a primarily inhibitory function for this lipid mediator. This later interpretation provides an explanation for the inhibitory response to PGE2 observed in a number of studies that used mature tissue mast cells.
The observed enhancement of Ag-mediated BMMC degranulation upon treatment with PGE1 or PGE2 is consistent with the activation of the EP3 and/or the EP1 receptor, because both of these receptors have been shown to mediate increases in [Ca2+]i and this pathway is critical to the degranulation of mast cells. Using EP3 receptor-deficient BMMCs, we show that the PGE2-mediated enhancement of mast cell degranulation is entirely due to activation of this receptor. Similarly, the ability of PGE to enhance IL-6 production is abolished in EP3-deficient mast cells. These results are consistent with earlier pharmacological studies that implicated either the EP3 or the EP1 receptor in the activation of mast cells and/or mast cell lines (19, 20). When EP1 receptor-deficient mice were tested directly, they exhibited a degranulation response that was sensitive to PG potentiation. Together with the complete absence of a PGE response in the EP3-deficient mast cells, this suggests that the EP1 receptor is not likely involved in this response in BMMCs.
To further examine the mechanism by which activation of the
EP3 receptor can modify mast cell function, we
have examined the impact of loss of EP3 receptor
expression on cAMP accumulation and on alterations in
[Ca2+]i. Transfection of
both the EP3 and EP3
receptors into Chinese hamster ovary (CHO) cells has been reported to
inhibit forskolin-induced cAMP accumulation (29).
We show here that treatment of BMMCs with PGE1
leads to an increase in [cAMP]i and that,
consistent with the activation of Gi, this
accumulation of [cAMP]i is enhanced in the
EP3 receptor-deficient cells.
In addition to their ability to inhibit cAMP accumulation, both the
EP3 and the EP3
isoforms have been shown to stimulate an increase in
[Ca2+]i upon transfection
into CHO cells. This increase in
[Ca2+]i was pertussis
toxin-sensitive, consistent with the coupling of both of these isoforms
to Gi proteins (30). We report
that treatment of BMMCs with PGE1 or
PGE2 resulted in a rapid increase in
[Ca2+]i. Furthermore, we
show that this response is completely dependent on the expression of
the EP3 receptor. Therefore, it is likely that in
BMMCs, similar to the CHO cells, the EP3 and
the EP3 are coupled to
Gi proteins, whose
G subunits can mediate increases in
[Ca2+]i by activation of
phospholipase C. It is likely that this in turn is responsible for the
potentiation of mast cell degranulation observed on treatment with
PGE2. However, we cannot rule out the possibility
that PGE/EP3 contribute to this response, at
least in part, by inhibition of adenylate cyclase activity and
[cAMP]i accumulation. This seems less likely
given the observation that the loss of EP4 and
EP2 receptors had no effect on mast cell
degranulation.
There are a number of possible explanations for the inability of PGE to initiate mast cell degranulation, despite the substantial increase in [Ca2+]i. First, it is possible that subtle differences in the magnitude, duration, and/or the stores of Ca2+ released lead to the differential effect on mast cell activation. Alternatively, it is possible that although activation of the EP3 receptor leads to changes in Ca2+ sufficient for mast cell degranulation, activation of additional pathways is necessary to bring about these physiological changes. It has been suggested that activation of PKC plays an important role in mast cell degranulation. To determine whether the failure of mast cells to degranulate in response to PGE2 alone is related to its inability to activate the PKC pathway, we examined mast cell degranulation in EP3-deficient and wild-type BMMCs treated with PMA alone and PMA together with PGE1. PMA treatment of BMMCs did not result in any changes in [Ca2+]i. The ability of EP3 receptor activation to act synergistically with PMA to bring about degranulation of mast cells suggests that PKC activity can contribute to mast cell activation and that the failure of PGE2 to activate this pathway prevents PGE2 alone from initiating mast cell degranulation in these cells.
We cannot formally rule out the possibility that other cell types might contribute to the observed differences between the responses of the BMMCs derived from the various EP-deficient mouse lines. Because the number of mast cells present was not determined for every preparation of cells (only representative cultures of each genotype) used in the reported studies, we cannot rule out the possibility that differences in the number of mast cells or presence of contaminating populations contribute to the observed difference in the response of wild-type and EP3-/- populations to PGE2. However, the fact that the baseline response to IgE and Ag was similar in all of the EP-deficient cells (only the ability of PGE to modulate this response was altered in the EP3-deficient cells) suggests that loss of these receptors does not alter the development and/or growth of BMMCs under these culture conditions.
The studies reported here, together with those of other investigators,
begin to develop a model that unifies the often "opposing" actions
of prostanoids on mast cell physiology. The effect of
PGE2 is determined by both the expression profile
of the various prostanoid receptors and by the activity of the
intracellular pathways to which they are coupled. It is easy to
envision a model in which this contributes to the ability of mast cells
to modulate their responses in both a temporal and spatial manner. For
example, early during an inflammatory process,
PGE2 produced upon local tissue damage leads to
heightened production of cytokines necessary for the recruitment of
inflammatory cells. This could lead to altered expression or activity
of the various EP receptors on the mast cells, with a shift from the
GI- to the Gs-linked
receptors and restoration of homeostasis as the inflammation is
resolved. It is also possible that different EP receptor pathways
dominate in the various mast cell populations. This would contribute to
the adaptation of mast cells to the particular organ in which it is
located. The generation of mouse lines with deficiencies in a single or
combination of receptors and the study of these animals in model
systems in which a role for mast cells has been defined should help us
answer these questions in the future.
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Footnotes |
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2 M.N. and M.S. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Beverly H. Koller, University of North Carolina, Cystic Fibrosis Foundation Center, 7027 Thurston-Bowles Building, Chapel Hill, NC 27599-7248. E-mail address: treawouns{at}aol.com
4 Abbreviations used in this paper: LTB4, leukotriene B4; BMMC, bone marrow-derived mast cell; HSA, human serum albumin; EIA, enzyme immunoassay; [Ca2+]i, intracellular calcium concentration; fura 2-AM, fura 2-acetoxymethyl ester; [cAMP]i, intracellular cAMP level; PGI2, prostacyclin; LT, leukotriene; PKC, protein kinase C; CHO, Chinese hamster ovary.
Received for publication November 8, 2001. Accepted for publication August 14, 2002.
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). In
parallel, samples received Ag together with various amounts of
PGE1 or PGE2 (
, 1 x 10-8
M; 
, 1 x 10-7 M; •, 1 x 10-6
M; and 
, 1 x 10-5 M). The experiments shown in
B differ only in that the PGE2 was added 20
min before the addition of Ag. Thirty minutes after the addition of Ag,
degranulation was assessed by measurement of the fraction of
hexosaminidase released into the supernatant. Addition of
PGE1 or PGE2 enhanced hexosaminidase release by
BMMCs in response to Ag in a dose-dependent manner.
, 1 x 10-9 M; 
