©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Selective Induction of Prostaglandin G/H Synthase I by Stem Cell Factor and Dexamethasone in Mast Cells (*)

(Received for publication, October 21, 1994; and in revised form, January 31, 1995)

James M. Samet (1) Mary Beth Fasano (2) Alfred N. Fonteh (1) Floyd H. Chilton (1) (3)(§)

From the  (1)Sections on Pulmonary and Critical Care Medicine and (2)Infectious Diseases, and the Departments of Internal Medicine and (3)Biochemistry, Bowman Gray School of Medicine, Winston-Salem, North Carolina 27157

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

This study examines the regulatory effects of two cytokines, stem cell factor (SCF) and interleukin-3, and a glucocorticoid, dexamethasone, on lipid mediator generation in mouse bone marrow-derived mast cells (BMMC). Treatment of BMMC with SCF induced a modest, dose-dependent increase in three eicosanoids, thromboxane B(2), prostaglandin D(2), and leukotriene B(4). These increases were accompanied by a marked elevation in cytosolic PLA(2) (cPLA(2)). Dexamethasone blocked the induction of cPLA(2) levels and the elevation in leukotriene B(4) induced by SCF. By contrast, the combination of SCF and dexamethasone dramatically increased (5-8-fold) the capacity by BMMC to produce prostanoid products. This increase in prostanoid products was mirrored by an increase in prostaglandin G/H synthase I (PGHS-I) levels. Dexamethasone, alone, had no effect on PGHS-I, cPLA(2), or prostanoid levels. Moreover, neither SCF or dexamethasone, alone or in combination, influenced prostaglandin G/H synthase II (PGHS-II) levels. In contrast to SCF, interleukin-3 alone or in combination with dexamethasone had no effect on prostanoid synthesis or PGHS-I or II levels. To better understand the SCF and dexamethasone effect, PGHS-I and PGHS-II mRNA expression were examined by Northern analysis. PGHS-I mRNA was markedly induced (maximal levels at 5 h) by the combination of SCF and dexamethasone. PGHS-II mRNA was undetectable in either control or SCF/dexamethasone-treated BMMC. Neither SCF or dexamethasone, alone, altered mRNA for either PGHS isotype. Taken together, these studies reveal that PGHS-I may be critical to prostanoid formation in mast cells exposed to cytokines and glucocorticoids. Moreover, they suggest that synergistic induction of PGHS-I could represent a novel mechanism for the anti-inflammatory action of glucocorticoids.


INTRODUCTION

Mast cells play a pivotal role in inflammatory reactions by secreting a wide variety of potent inflammatory mediators (for reviews see (1) and (2) ). These mediators include metabolites of AA (^1)such as leukotrienes, prostaglandins, and thromboxane (for review, see (3) ). The formation of AA metabolites by mast cells is initiated by the release of AA from membrane phospholipid stores during immunologic activation of the cell and is catalyzed by the enzyme phospholipase A(2) (PLA(2)). Several forms of PLA(2) have been described (for reviews, see (4) and (5) ). A relatively high molecular weight PLA(2) (cPLA(2)) has been isolated from the cytosolic fraction of several cell types(6, 7, 8, 9, 10) . In addition, there is a family of low molecular weight PLA(2) enzymes that are released upon activation of a variety of cell types(11, 12, 13, 14, 15) . Both secretory and cytosolic forms of PLA(2) have been reported in mast cells(16, 17) . Studies indicate that levels of cPLA(2) are regulated by cytokines and growth factors in several cell types. For example, IL-1beta, epidermal growth factor, and transforming growth factor beta2 induce the synthesis of cPLA(2) and AA metabolites in mesangial cells(18) , while interferon causes the synthesis of cPLA(2) in bronchial epithelial cells(19) . IL-1alpha and IL-1beta cause the accumulation of cPLA(2) and prostaglandin E(2) formation in lung and synovial fibroblasts, respectively(20, 21) . Similarly, tumor necrosis factor alpha increases cPLA(2) in epithelial carcinoma cells (22) . In most cases, these cytokine-induced increases in cPLA(2) can be blocked by the antiinflammatory drug dexamethasone(18, 20, 22) .

Once released from membrane phospholipids, AA can be metabolized by lipoxygenase activity to hydroxyeicosatetraenoic acids and leukotrienes or to prostaglandins and thromboxanes through the action of prostaglandin G/H synthase (PGHS)(3, 23) . Two forms of PGHS, PGHS-I and PGHS-II, have been described in several cell types(24, 25, 26, 27, 28) . Like cPLA(2), levels of PGHS-II are induced in a number of cell types in response to cytokines and priming agents(24, 29, 30, 31, 32, 33) and, in some cases, this increase has been correlated with elevated prostanoid synthesis(29, 34, 35) . As with cPLA(2), dexamethasone has been shown to block the induction of PGHS-II by cytokines in some cell types(30, 31, 32, 33, 36) . While most studies in the scientific literature point to PGHS-II as the inducible form and PGHS-I as the constitutive form of PGHS, there are reports that the expression of PGHS-I and not PGHS-II is modulated in some cell types(29, 37, 38, 39) . For example, transforming growth factor beta increases PGHS-I mRNA and prostacyclin production in bovine endothelial cells without any effect on PGHS-II mRNA(39) . Similarly, increased PGD(2) synthesis by monocytoid THP-1 cells treated with phorbol ester correlates with increases in PGHS-I mRNA and protein levels, while PGHS-II is unaffected(38) . Taken together, these studies point to complex mechanisms through which cPLA(2) and PGHS are regulated in mammalian cells.

The cytokines IL-3 and SCF exert regulatory effects on the development and function of mast cells. IL-3 is required for the proliferation and differentiation of mast cell precursors(40) , while SCF, a ligand for the c-kit receptor, is an essential factor in mast cell maturation(41, 42) . When provided to mast cells in vitro for short periods of time, both IL-3 and SCF can induce the release of inflammatory mediators(43, 44, 45) . In the current study, we have utilized IL-3, SCF, and dexamethasone to modulate the expression of cPLA(2) and PGHS and the formation of AA metabolites. We report that PGHS-I may be critical to prostanoid formation in mast cells treated with cytokines and glucocorticoids.


EXPERIMENTAL PROCEDURES

Materials

Tissue culture media and reagents were purchased from Life Technologies, Inc. Deuterated eicosanoid standards (^2H(4)PGD(2), ^2H(4)TxB(2), and ^2H(4)LTB(4)) and arachidonic acid (5,6,8,9,11,12,14,15-^2H-AA) were obtained from Biomol Research Laboratories (Plymouth Meeting, PA). Calcium ionophore A23187, horseradish-peroxidase conjugated anti-rabbit IgG antibodies, dexamethasone (9a-fluoro-16a-methylprednisolone), phosphate-buffered saline (PBS), Hanks' buffered salt solution (HBSS), human serum albumin, Tween 20, Nonidet P-40, N-tosyl-L-phenylalanine chloromethylketone, diisopropyl fluorophosphate, quercitin, pepstatin, leupeptin, NaF, Na(3)VO(4), and common laboratory reagents were obtained from Sigma. Supplies for Western analysis including precast gels, SDS/Tris/glycine running buffer and nitrocellulose were purchased from Bio-Rad. Chemiluminescence reagents for Western blot detection were from Amersham Corp. Methoxylamine HCl (MOX in pyridine), acetonitrile, pentafluorobenzylbromide (20% in acetonitrile), diisopropyl ethylamine (20% in acetonitrile), and N,O-bis(trimethylsilyl)trifluoroacetamide were bought from Pierce. HPLC-grade solvents were purchased from Fisher. Recombinant rat SCF (SCF) was a generous gift from Amgen, Inc. (Thousand Oaks, CA). Murine IL-3 (Escherichia coli-expressed) was obtained from R & D Systems (Minneapolis, MN). A polyclonal anti-peptide antibody specific to the unique 18-amino acid sequence of PGHS-II was provided by Dr. David DeWitt (Michigan State University). Antibodies to PGHS-I include a nonspecific polyclonal anti-PGHS antibody purchased from Sigma and a monoclonal anti-PGHS-I antibody raised against purified ram seminal vesicles kindly provided by Dr. Stephen Prescott (University of Utah). Purified rabbit anti-cPLA(2) antibody was kindly provided by Hoffman LaRoche. The 1.7-kilobase pair mouse PGHS-1 cDNA probe was purchased from Oxford Biomedical Research, Inc (Oxford, MI). The 1.156-kilobase pair mouse PGHS-2 cDNA probe was a gift from Dr. David DeWitt of Michigan State University (East Lansing, MI). NS-398 was purchased from Cayman Chemical Co.

Mast Cell Culture, Treatment with Cytokines, and Activation

Mouse bone marrow-derived mast cells (BMMC) were obtained from CBA/J mice (Jackson Laboratories, Bar Harbor, ME) femurs as described previously (46) and grown in 60% RPMI 1640 culture medium containing 10% fetal calf serum (v/v), 50 µM 2-mercaptoethanol, 2 mML-glutamine, 25 µg/ml gentamicin, and 40% WEHI-conditioned media as a source of IL-3. Culture media was changed twice per week and placed in new flasks weekly in order to remove adherent cells. By the end of 3 weeks of culture, this procedure routinely yielded cultures that were >95% mast cells with a viability > 90%(46) .

BMMC were placed in media without WEHI supplement for 24 h prior to treatment with cytokine or dexamethasone. This was found to be necessary to reduce basal levels of cPLA(2) protein in BMMC. BMMC were then treated with 1-100 ng/ml SCF or 0-10 ng/ml IL-3 for 24 h. The range of SCF and IL-3 concentrations used in this study was chosen to include doses previously reported to induce proliferation or differentiation of BMMC(41, 42, 47) . Some cultures received dexamethasone (final concentration 0.1-10 µM) 2 h prior to addition of the cytokine. For ionophore challenge, BMMC were removed from culture, washed twice in HBSS, and placed at a concentration of 2 times 10^6/ml in HBSS containing 1 mM calcium. BMMC were then stimulated with ionophore A23187 (final concentration 1 µM) for 5 min at 37 °C.

Immediately following activation with A23187, BMMC were pelleted by centrifugation (500 times g for 5 min at 4 °C), and supernatant fluids were added to 4 volumes of ethanol containing 10 ng each of ^2H(4)PGD(2), ^2H(4)TxB(2), and ^2H(4)LTB(4) and 100 ng of ^2H(8)AA as internal standards. Solvents were evaporated under a stream of nitrogen and the eicosanoids and fatty acids converted to methoxime-pentafluorobenzylester-trimethylsilyl ether derivatives as described previously(48) .

GC/MS of Eicosanoid Products

Derivatized eicosanoids and fatty acids were extracted with hexane and analyzed by negative ion chemical ionization(49) . Carboxylate anions for AA (m/z 303), ^2H(8)AA (m/z 311), LTB(4) (m/z 479), ^2H(4)LTB(4) (m/z 483), PGD(2) (m/z 524), ^2H(4)PGD(2) (m/z 528), TxB(2) (m/z 614), and ^2H(4)TxB(2) (m/z 618) were analyzed in the single ion monitoring mode on a Hewlett-Packard 5890 series II gas chromatograph interfaced to a 5989A quadrupole mass spectrometer.

Western Analyses of cPLA(2), PGHS-I, and PGHS-II

BMMC (1 times 10^6) were pelleted in microcentrifuge tubes and lysed for 10 min on ice with 50 µl of a lysis buffer (1% Nonidet P-40, 100 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM EDTA, 1 mM Na(3)VO(4) and 50 mM NaF, 100 µMN-tosyl-L-phenylalanine chloromethylketone, 100 µM quercitin, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 5 mM diisopropyl fluorophosphate). Nuclei were removed by centrifugation (13,000 times g in a microcentrifuge for 10 min at 4 °C), and the resulting supernatant fluids were mixed 1:1 with 2 times loading buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol and 10% mercaptoethanol) and boiled for 5 min. Proteins were then separated by SDS-PAGE on 4-20% polyacrylamide gels using SDS/Tris/glycine buffer. Proteins were electrotransferred onto nitrocellulose, and the resulting blot was blocked with 5% nonfat milk in 0.05% Tween and PBS for 1 h. Blots were then incubated overnight with anti-cPLA(2) antibody (1 µg/ml) in 1% blocking solution. After washing (in PBS-Tween), the blots were incubated with a secondary antibody (anti-rabbit IgG conjugated to horseradish peroxidase). Immunodetection was performed with Amersham ECL reagents. Detection on film was accomplished using horseradish peroxidase-conjugated anti-rabbit IgG and enhanced chemiluminescence reagents. Blots were stripped using 100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl, pH 6.8, at 50 °C for 30 min and reprobed using polyclonal anti-PGHS (1:1000), monoclonal anti-PGHS-I (1:15,000), or polyclonal anti PGHS-II (1:1000) antibodies as described previously(24) . Film images were digitized and band intensity analyzed using a Millipore Digital Bioimaging System (Bedford, MA).

RNA Isolation and Northern Analysis

Total RNA was isolated from BMMC using RNazol (Tel Test Inc., Friendswood, TX) as directed by the manufacturer. Five µg of total RNA were electrophoresed through formaldehyde-containing agarose gels, transferred onto GeneScreen Plus (DuPont NEN) membranes using capillary blotting, and cross-linked by UV irradiation. The cDNA probes were labeled by a nick translation system (DuPont NEN) as directed by the manufacturer. Membranes were prehybridized in Quikhyb (Stratagene, La Jolla, CA) for 15 min at 68 °C. The cDNA probe was added directly to the hybridization solution at a concentration of 1.25 times 10^6 cpm/ml and incubated at 68 °C for 1 h. Excess probe was removed by two 15-min washes in 1 times SSC, 0.1% SDS at 25 °C followed by one 15-min wash in 0.2 times SSC, 0.1% SDS at 65 °C. The signals were detected by exposing the blot to x-ray film with intensifying screens at -80 °C. The PGHS-I and PGHS-II levels were normalized against levels of human glyceraldehyde-3-phosphate dehydrogenase.

Statistical Analyses

Results are expressed as the mean ± standard error. Multi-group comparisons were tested using one-way analysis of variance of non-transformed or log-transformed values. Posthoc analyses were done using the Tukey-Kramer multiple group comparison test; p leq 0.05 was considered significant.


RESULTS

BMMC cultured for 24 h in WEHI-free media released arachidonic acid and synthesized TxB(2), PGD(2), and LTB(4) when stimulated with calcium ionophore A23187 (Fig. 1). Treatment of BMMC with 1, 10, or 100 ng/ml SCF for an additional 24 h induced a modest, dose-dependent increase in all three eicosanoids when compared to cells not treated with SCF. Stimulated SCF-treated cells produced quantities of unmetabolized AA similar to levels produced by identically challenged BMMC that had not been exposed to SCF. To determine the effect of glucocorticoids on AA release and eicosanoid generation in BMMC, cells were pretreated with various concentrations of dexamethasone (0.1-10 µM) for 2 h followed by exposure to SCF (100 ng/ml) for 24 h. When given in conjunction with SCF, dexamethasone caused a 5-8-fold increase in the synthesis of PGD(2) by BMMC when compared to untreated cells or cells treated with SCF alone (Fig. 1). Similarly, there was a significant elevation in the amount of another PGHS product, TxB(2), produced by cells treated with SCF and dexamethasone. In contrast, the combination of dexamethasone with SCF induced no significant changes in levels of LTB(4) produced by stimulated BMMC.


Figure 1: Effect of SCF and dexamethasone on the production of eicosanoids by stimulated BMMC. BMMC were treated with SCF or SCF plus dexamethasone for 24 h and stimulated with 1 µM A23187 for 5 min. Eicosanoids released into supernatant fluid were analyzed by GC/MS as described under ``Experimental Procedures.'' Leukotriene B(4) and prostanoids are illustrated in the top and bottompanels, respectively. Shown are the means ± S.E.; n = 4.



To identify mechanism(s) responsible for the increased production of PGHS products released by BMMC, we examined the effects of SCF, dexamethasone, and combined SCF/dexamethasone treatment on levels of cPLA(2) and PGHS in BMMC. Treatment of BMMC with SCF (1-100 ng/ml) for 24 h resulted in an increase in cPLA(2) as evidenced by Western blot analysis (Fig. 2, upperpanel). A major immunoreactive band at approximately 85 kDa and a larger minor immunoreactive band were observed. Digital image analysis showed a dose-dependent increase in cPLA(2) as function of SCF concentration. Maximal levels of cPLA(2) were observed at 100 ng/ml SCF. The increase in cPLA(2) induced by a maximal dose of SCF was partially blocked by dexamethasone at concentrations ranging from 0.1 to 10 µM.


Figure 2: Effect of SCF and dexamethasone on cPLA(2) and PGHS-I protein levels in BMMC. Cell lysates from BMMC treated with SCF or SCF plus dexamethasone for 24 h were subjected to Western blotting analysis using an anti-cPLA(2) antibody (upperpanel). Blots were then stripped and reprobed with an anti-PGHS 1 antibody (lowerpanel). The relative optical density of the bands, normalized for the darkest band on each gel, is shown to facilitate comparisons. Shown are representative blots from three or more experiments.



Preliminary detection of PGHS-I in BMMC was performed using a polyclonal antibody that has some cross-reactivity to PGHS-II. SCF treatment of BMMC induced the appearance of a 70-kDa protein band, consistent with the previously reported molecular mass of PGHS-I. Subsequent analysis utilizing a monoclonal antibody with high specificity for PGHS-I confirmed the identity of this band as PGHS-I (data not shown). Combined treatment of BMMC with SCF (100 ng/ml) and dexamethasone (0.1-100 µM) induced a pronounced increase in PGHS-I above that seen with SCF alone (Fig. 2, lowerpanel). Maximal induction of PGHS-I levels was detected in BMMC that received SCF (100 ng/ml) and the lowest (0.1 µM) concentration of dexamethasone. Using a specific antipeptide antibody, PGHS-II was not found under basal conditions and remained undetectable following treatment of BMMC with SCF or SCF in combination with dexamethasone (data not shown).

To determine whether glucocorticoid alone could induce PGHS-I in BMMC, cells were treated for 24 h with varying concentrations of dexamethasone and eicosanoid production as well as cPLA(2), and PGHS levels were examined. There were no significant changes in eicosanoids and AA production by stimulated BMMC treated with dexamethasone alone (Table 1). Treatment with dexamethasone alone reduced basal cPLA(2) levels, while basal levels of PGHS-I were unchanged relative to untreated BMMC (data not shown). Once again, PGHS-II was undetectable in resting or dexamethasone-treated BMMC.



To ascertain whether the synergistic effect of dexamethasone and cytokine treatment was specific to SCF, eicosanoid production and levels of cPLA(2) and PGHS were measured in BMMC treated with IL-3. IL-3 in concentrations ranging from 0.1 to 10 ng/ml had no significant effect on the production of PGD(2), TxB(2), or LTB(4) by stimulated BMMC (Fig. 3). In sharp contrast to the effect of SCF, a combination of IL-3 and dexamethasone did not result in increased production of PGD(2), nor did it increase the release of other eicosanoids (Fig. 3). As seen with SCF, there was an increase in cPLA(2) levels in BMMC treated with IL-3 (Fig. 4, upperpanel). Furthermore, the increase in cPLA(2) induced by IL-3 (10 ng/ml) was partially blocked by dexamethasone (Fig. 4, upperpanel). Compared to untreated BMMC, levels of PGHS-I were unchanged in cells that received 10 ng/ml IL-3. Unlike the synergistic effect of SCF and dexamethasone, treatment of BMMC with IL-3 (10 ng/ml) and dexamethasone resulted in a small decrease in PGHS-I levels relative to levels detected in BMMC that received IL-3 alone (Fig. 4, lowerpanel). There was no detectable PGHS-II in BMMC treated with IL-3 alone or IL-3 in combination with dexamethasone.


Figure 3: Effect of IL-3 and dexamethasone on the production of eicosanoids by stimulated BMMC. BMMC were treated with IL-3 or IL-3 plus dexamethasone for 24 h and stimulated with 1 µM A23187 for 5 min. Eicosanoids released into supernatant fluids were analyzed by GC/MS as described under ``Experimental Procedures.'' Leukotriene B(4) and prostanoids are illustrated in the top and bottom panels, respectively. Shown are the mean ± S.E., n = 4.




Figure 4: Effect of IL-3 and dexamethasone on cPLA(2) and PGHS-I protein levels in BMMC. Cell lysates from BMMC treated with IL-3 or IL-3 plus dexamethasone for 24 h were subjected to Western blotting analysis using an anti-cPLA(2) antibody (upperpanel). Blots were then stripped and reprobed with an anti-PGHS-I antibody (lowerpanel). The relative optical density of the bands, normalized for the darkest band on each gel, is shown to facilitate comparisons. Shown are representative blots from three or more experiments.



To determine whether the observed increase in PGHS-I protein with SCF and dexamethasone was mirrored by an elevation in mRNA for PGHS-I, total RNA was isolated and analyzed by Northern analysis using cDNA probes specific for PGHS-I and PGHS-II mRNA. PGHS-II mRNA was undetectable in BMMC treated with SCF or dexamethasone either alone or in combination (data not shown). PGHS-I mRNA was detected at rest, and basal levels were not significantly altered by the addition of SCF or dexamethasone. In contrast, PGHS-I mRNA was markedly induced by the combination of SCF and dexamethasone (Fig. 5). Increases in PGHS-I mRNA were observed at 2 h, peaked at 5 h, and returned to base-line levels by 24 h. These findings correlated with changes observed in total PGHS-I protein content and eicosanoid production in BMMC following cotreatment with SCF and dexamethasone.


Figure 5: Effect of SCF and dexamethasone on PGHS-I mRNA levels in BMMC. Total RNA was isolated from BMMC treated with SCF or SCF plus dexamethasone for different periods of time. Northern analysis was performed as described utilizing cDNA probes for PGHS-I and glyceraldehyde-3-phosphate dehydrogenase. Shown are representative blots from four separate experiments.



Recently, inhibitors that specifically block PGHS-II but not PGHS-I have become available. To determine whether specific inhibition of PGHS-II would block the increase in PGD(2) after SCF and dexamethasone treatment, the selective inhibitor NS-398 was used. NS-398 (at concentrations 30 - fold higher than IC for PGHS-II) failed to attenuate the SCF/dexamethasone-induced increase in PGD(2) synthesis (SCF/dexamethasone, 131.0 ± 41 pmol versus SCF/dexamethasone + NS-398 (1 µM), 106 ± 48). These data support the findings described above, which suggest that induction of PGHS-I, and not PGHS-II, is responsible for increases in PGD(2) synthesis by BMMC treated with SCF and dexamethasone.


DISCUSSION

The central findings of the current study are that SCF induces the expression of cPLA(2) and PGHS-I in BMMC. The increase in the levels of these two enzymes is accompanied by modest increases in the production of eicosanoids. Although the glucocorticoid, dexamethasone, blocked the cytokine-induced elevation in cPLA(2), it has the paradoxical effect of dramatically increasing the capacity of SCF-treated mast cells to produce prostanoids. This increase in prostanoid synthesis by BMMC correlated with marked induction of PGHS-I without any effect on PGHS-II. Taken together, these studies suggest that PGHS-I may be critical to prostanoid formation in glucocorticoid-treated mast cells.

Cytokines have been reported to increase the expression of cPLA(2) in a variety of cell types(18, 19, 20, 21, 22) . Some of these studies have also shown that glucocorticoids such as dexamethasone can inhibit this induction(18, 20, 22) . Consistent with these previously published reports, in the present study we found that both SCF and IL-3 increased levels of cPLA(2) and that dexamethasone blocked this induction in BMMC. It is interesting to note that both SCF and IL-3 promoted the appearance of two immunoreactive bands of cPLA(2). Recent studies demonstrate that phosphorylation of cPLA(2) often accompanies cell activation and this phosphorylation changes the electrophoretic properties of cPLA(2)(50) . It is possible that SCF and IL-3 increase cPLA(2) expression and this cPLA(2) is phosphorylated during cell activation.

To date, most studies which have examined the regulation of PGHS isoforms have pointed to PGHS-II as the inducible form of this enzyme in mammalian cells(29, 30, 31, 32, 33, 51) . However, there appear to be specific cell types in which PGHS-I, and not PGHS-II, is induced (29, 37, 38, 39) by certain stimuli. Murakami and colleagues have recently demonstrated that both PGHS-I and PGHS-II can be induced by various cytokine combinations in BMMC(52) . Their data further suggest that AA which is metabolized to PGD(2) in response to cytokine stimulation utilizes PGHS-II while AA which forms PGD(2) in response to IgE cross-linking utilized PGHS-I. The current data indicate that SCF alone caused a moderate increase in PGHS-I which correlated with small increases in prostanoid products (in response to ionophore A23187), and that SCF-treatment of BMMC failed to induce PGHS-II. Glucocorticoids have been demonstrated to block the induction of PGHS-II expression (29, 34, 53) . However, a stimulatory effect of dexamethasone on overall cellular PGHS activity has been observed in amnion epithelial cells (54) . Our data demonstrate a dramatic elevation of PGHS-I (>10-fold increase) protein, mRNA and prostanoids (>5-fold increase) in SCF-treated mast cells incubated with low concentrations (0.1 µM) of dexamethasone. To our knowledge, this is the first study to demonstrate an increase in PGHS-I levels by combined dexamethasone and cytokine treatment. Future studies will be necessary to determine whether other enzymes (such as PGD synthase) are regulated by this combination.

The mechanism(s) through which dexamethasone potentiates the SCF-induced increase of PGHS-I protein in BMMC are not known. Our studies reveal that SCF or dexamethasone alone have only a modest influence on PGHS-I. The synergistic effect of dexamethasone on the production of prostanoids and PGHS-I expression, appears to be specific to SCF, in that it was not observed with IL-3, another cytokine known to exert effects on mast cells. One possible explanation for the differential effect of these two cytokines may be found in the differences between the SCF and IL-3 receptors. For example, SCF is a ligand for the c-kit receptor, a member of the type III tyrosine kinase receptor family(55) .

The mechanistic basis for the anti-inflammatory effects of glucocorticoid compounds such as dexamethasone, is still incompletely understood. Reduction of PLA(2) and PGHS gene expression, through both transcriptional and post-transcriptional events, has been proposed as a mechanism that leads to decreased AA release and diminished prostanoid production(56, 57, 58, 59, 60, 61) . Our data demonstrating that dexamethasone blunts SCF and IL-3-induced increases in cPLA(2) levels in BMMC are consistent with this hypothesis. In contrast, the potentiation of cytokine-induced increases in prostanoid synthesis, a previously unreported effect of dexamethasone, would appear to be a paradoxical effect of this anti-inflammatory drug. PGD(2), the main prostanoid product synthesized by mast cells, has pro-inflammatory effects such as bronchoconstriction and vasodilation(62) . On the other hand, PGD(2) is an inhibitor of platelet aggregation which appears to function by increasing cyclic AMP levels(63, 64) . If the synergistic increase in PGHS-I induced by dexamethasone and SCF treatment occurs in other cell types, it is possible that additional prostanoids with anti-inflammatory effects, such as PGE(2), may be produced. In this regard, synergistic induction of PGHS-I could represent a novel mechanism for the anti-inflammatory action of glucocorticoids.


FOOTNOTES

*
This work was supported by Grants AI 24985, HL-50395, and RR 07122-0352 from the National Institutes of Health and a grant from the American Lung Association (to M. B. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Section on Pulmonary and Critical Care Medicine, Bowman Gray School of Medicine, Winston-Salem, NC 27157. Tel.: 910-716-3923; Fax: 910-716-7277.

(^1)
The abbreviations used are: AA, arachidonic acid; SCF, stem cell factor; IL-3, interleukin-3; PGHS, prostaglandin G/H synthase; cPLA(2), cytosolic PLA(2); LTB(4), leukotriene B(4); PGD(2), prostaglandin D(2); TxB(2), thromboxane B(2); BMMC, bone marrow-derived mast cell; PBS, phosphate-buffered saline; HBSS, Hanks' buffered salt solution.


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