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The Journal of Immunology, 2002, 168: 1397-1404.
Copyright © 2002 by The American Association of Immunologists

Regulation of Prostaglandin Endoperoxide Synthase-2 and IL-6 Expression in Mouse Bone Marrow-Derived Mast Cells by Exogenous But Not Endogenous Prostanoids1

Bruno L. Diaz*,{dagger}, Hiroshi Fujishima*,{dagger}, Yoshihide Kanaoka*,{dagger}, Yoshihiro Urade§ and Jonathan P. Arm2,*,{dagger},{ddagger}

* Department of Medicine, Harvard Medical School, Boston, MA 02115; {dagger} Division of Rheumatology, Immunology, and Allergy, and {ddagger} Partners’ Asthma Center, Brigham and Women’s Hospital, Boston, MA 02115; and § Osaka Bioscience Institute, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mouse bone marrow-derived mast cells (BMMC), stimulated with stem cell factor, IL-1{beta}, and IL-10, secrete IL-6 and demonstrate a delayed phase of PGD2 generation that is dependent upon the induced expression of PG endoperoxide synthase (PGHS)-2. We have examined the potential for exogenous prostanoids, acting in a paracrine fashion, and endogenous prostanoids, acting in an autocrine fashion, to regulate PGHS-2 induction and IL-6 secretion in mouse BMMC. Exogenous PGE2, which acts through G protein-coupled receptors, and 15-deoxy-{Delta}12,14-PGJ2, which is a ligand for peroxisome proliferator-activated receptor (PPAR){gamma}, elicited a 2- to 3-fold amplification of PGHS-2 induction, delayed-phase PGD2 generation, and IL-6 secretion in response to stem cell factor, IL-1{beta}, and IL-10. The effect of PGE2 was reproduced by the E prostanoid (EP)1 receptor agonist 17-trinor-PGE2, and the EP1/EP3 agonist, sulprostone, but not the EP2 receptor agonist, butaprost. Although BMMC express PPAR{gamma}, the effects of 15-deoxy-{Delta}12,14-PGJ2 were not reproduced by the PPAR{gamma} agonists, troglitazone and ciglitazone. PGHS-2 induction, but not IL-6 secretion, was impaired in cPLA2-deficient BMMC. However, there was no impairment of PGHS-2 induction in BMMC deficient in hematopoietic PGD synthase or PGHS-1 in the presence or absence of the PGHS-2 inhibitor, NS-398. Thus, although exogenous prostanoids may contribute to amplification of the inflammatory response by augmenting PGD2 generation and IL-6 secretion from mast cells, endogenous prostanoids do not play a role.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mast cells are critical effector cells of the immune response. They have been implicated in diverse inflammatory states including allergic inflammation (1, 2), rheumatoid arthritis (3), fibrosis (4), and septic peritonitis (5). Mast cells contribute to the inflammatory response through the release of preformed mediators such as histamine and serine proteases, eicosanoids derived from the oxidative metabolism of arachidonic acid, and cytokines (2).

We have previously reported the biphasic generation of eicosanoids from mouse bone marrow-derived mast cells (BMMC).3 Stimulation of BMMC by cross-linking of Fc{epsilon}RI (6), by interaction of stem cell factor (SCF) with its receptor, c-kit (7), or by the calcium ionophore, A23187 (8), elicits secretory granule exocytosis and an immediate phase of leukotriene C4 and PGD2 generation. These events are complete within 10 min. The immediate phase of PGD2 generation requires the action of constitutively expressed PG endoperoxide synthase (PGHS)-1 (7). We (9, 10) and others (11, 12) described a second, delayed phase of PGD2 generation from cultured mouse mast cells. The delayed phase, elicited by SCF in combination with IL-1{beta} and IL-10 (9) or by Ag activation after sensitization with hapten-specific IgE (10, 12), is characterized by the generation of PGD2 in the absence of leukotrienes. Importantly and distinctively, delayed-phase PGD2 generation depends on the induced expression of PGHS-2 (7, 9, 12). Cross-linking of Fc{epsilon}RI on mast cells also elicits de novo synthesis and release of various cytokines including IL-3, GM-CSF, TNF-{alpha}, and IL-6, as well as certain chemokines (13, 14, 15, 16, 17). Activation of BMMC with SCF, IL-10, and IL-1{beta} elicits not only delayed-phase PGD2 generation and PGHS-2 induction but also the de novo generation of IL-6 (18).

The first step in prostanoid generation is the release of arachidonic acid from cell membrane phospholipids by phospholipase A2. We have previously demonstrated that cytokine-dependent induction of PGHS-2 is impaired in mice with gene disruption of cytosolic PLA2-{alpha} (cPLA2-{alpha}) (19). The products of cPLA2-{alpha} regulating PGHS-2 expression were not identified and the effect of gene disruption of cPLA2-{alpha} on cytokine-dependent IL-6 secretion was not examined. PGD2 is the major prostanoid produced by mast cells, which express the hematopoietic form of PGD synthase (hPGDS) (20, 21). It acts through one of two cell surface G protein-coupled receptors, the D prostanoid (DP) receptor (22) or the chemoattractant receptor-homologous molecule expressed on Th2 cells (23). 15-deoxy-{Delta}12,14-PGJ2 (15dPGJ2), a metabolite of PGD2, is a ligand for the nuclear peroxisome proliferator-activated receptor (PPAR){gamma}, which may modulate PGHS-2 induction (24, 25) and IL-6 secretion (26). PGE2 acts through a family of cell surface G protein-coupled receptors. While not a major product of mast cells, it is produced at sites of inflammation and has been reported to regulate both PGHS-2 induction (27, 28) and IL-6 secretion (29, 30). These observations suggest that the induction of PGHS-2 and IL-6 secretion by mast cells may be modulated in an autocrine fashion by endogenous 15dPGJ2 or in a paracrine fashion by exogenous PGE2. Therefore, we examined the effects of PGE2 and 15dPGJ2 on cytokine-dependent induction of PGHS-2 and IL-6 secretion in mouse mast cells. We then tested the hypothesis that endogenous prostanoids regulate cytokine-dependent induction of PGHS-2 and IL-6 secretion in these cells using BMMC derived from mice with disruption of the gene for PGHS-1 or hPGDS and the PGHS-2 inhibitor, NS-398.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Recombinant mouse SCF and IL-10 were expressed in baculovirus and their concentrations were determined as previously described (9). The following reagents were purchased: recombinant mouse IL-1{beta} (R&D Systems, Minneapolis, MN); mouse monoclonal IgG1 against rat PGHS-2 that cross-reacts with mouse and human PGHS-2 (clone 33; BD Transduction Laboratories, Lexington, KY); mouse monoclonal IgG1 against mouse/human PPAR{gamma} (clone E-8; Santa Cruz Biotechnology, Santa Cruz, CA); the PGHS-2 inhibitor, NS-398 (Cayman Chemicals, Ann Arbor, MI); the 5-lipoxygenase inhibitor, AA-861 (Biomol, Plymouth Meeting, PA); the FLAP antagonist, MK-886, and the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) (Biomol). Troglitazone was a gift from Dr. P. Sarraf (Dana-Farber Cancer Institute, Boston, MA) and was diluted in DMSO (the final DMSO concentration was 0.01% or less).

Culture and activation of BMMC

Bone marrow cells from male BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) were cultured in 50% enriched medium (RPMI 1640 containing 100 U/ml penicillin, 100 µg/ml streptomycin, 10 µg/ml gentamicin, 2 mM L-glutamine, 0.1 mM nonessential amino acids, and 10% FBS)/50% WEHI-3 cell (American Type Culture Collection, Manassas, VA)-conditioned medium. After 4 wk, >99% of the cells were BMMC with characteristic metachromatic mast cell granules as assessed by staining with toluidine blue. For activation, BMMC were washed and resuspended at 106 cells/ml in 50% WEHI-3 cell-conditioned medium supplemented with 100 ng/ml SCF, 10 U/ml IL-10, and 5 ng/ml IL-1{beta}. Control cells were maintained in 50% WEHI-3 cell-conditioned medium alone. Cells were washed after 1 h of activation to remove the products of the immediate phase of eicosanoid generation and were resuspended in medium with fresh cytokines. After a further 7 h BMMC were centrifuged at 230 x g for 10 min. Supernatants were assayed for PGD2 and IL-6 by ELISA (Cayman Chemicals and Endogen (Woburn, MA) respectively). Cell pellets were analyzed for expression of PGHS-2 by RNA blotting and/or SDS-PAGE/immunoblotting.

To assess the role of eicosanoids in amplifying the delayed generation of PGD2 and the induction of PGHS-2 prostanoids, PPAR{gamma} ligands, or NS-398, a specific inhibitor of PGHS-2, were added to the BMMC suspension after the wash at 1 h. Inhibitors of the lipoxygenase pathway were added 5 min before activation. Alternatively, delayed-phase PGD2 generation and PGHS-2 induction were analyzed in BMMC derived from mice with homozygous disruption of the following genes: cPLA2-{alpha} (cPLA2-/-; provided by Dr. J. V. Bonventre, Harvard Medical School, Boston, MA) (19, 31), hPGDS-/- (Y. Kanaoka and Y. Urade, manuscript in preparation), and PGHS-1 (PGHS-1-/-; provided by Dr. R. Langenbach, National Institute of Environmental Health Science, Research Triangle Park, NC) (32); and from strain-matched homozygous wild-type (+/+) mice.

RNA blot analysis

RNA was extracted in Tri-Reagent (Molecular Research Center, Cincinnati, OH). A total of 5 µg of RNA was loaded per lane and resolved in 1.2% agarose/formaldehyde gels and transferred to Immobilon-N (Millipore, Bedford, MA) as described (9). RNA blots were hybridized with cDNA probes encoding mouse IL-6 (18) and mouse PGHS-2 (9), which were labeled with [32P]dCTP by random priming (Amersham, Arlington Heights, IL).

SDS-PAGE/immunoblot analysis

SDS-PAGE immunoblotting was performed as previously described (9, 33), modified for use of 10% polyacrylamide Bis-Tris gels according to the manufacturer’s instructions (NOVEX, San Diego, CA). Lysates from 1.5 x 105 cells were applied to each gel lane. Primary mouse Abs against PGHS-2 and PPAR{gamma} were used at 1 µg/ml. HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was used at a 1/5000 dilution. The protein bands were visualized with a chemiluminescent technique (ECL Western blot analysis system; Pierce, Rockford, IL). The resulting membranes were exposed to Kodak MR film (Kodak, Rochester, NY) for 10–120 s. It should be noted that when exposing a membrane containing lysates of BMMC treated with SCF, IL-10, IL-1{beta}, and prostanoids, the lanes containing lysates of cells cultured with SCF, IL-10, and IL-1{beta} alone were underexposed, appearing to have little expression of PGHS-2, to prevent overexposure of lanes containing lysates of BMMC treated with prostanoids. However, the level of PGHS-2 induction with SCF, IL-10, and IL-1{beta} was comparable between experiments and was similar to previously published data.

RT-PCR analysis

RNA extracted from resting BMMC was used as a template for RT-PCR using OneStep RT-PCR kit (Qiagen, Valencia, CA). One microgram of RNA was used per 50-µl reaction according to the manufacturer’s instructions. A total of 10 µl of product was loaded per lane of a 2% agarose gel. PCR products were purified using High Pure PCR Product Purification kit (Roche, Indianapolis, IN) and sequenced using the PCR primers. The sequences of the primers (Oligos Etc., Bethel, ME) and the sizes of the amplified products were as follows: E prostanoid (EP)1, 5'-CTGCTGGTGTTGGTGGTGTTG-3' and 5'-CTGGGCACATTCAGAGGTGAC-3' (282 bp); EP2, 5'-AGGACTTCGATGGCAGAGGAGAC-3' and 5'-CAGCCCCTTACACTTCTCCAATG-3' (410 bp); EP3, 5'-CCGGGCACGTGGTGCTTCAT-3' and 5'-TAGCAGCAGATAAACCCAGG-3' (437 bp); EP4, 5'-TTCCGCTCGTGGTGCGAGTGTTC-3' and 5'-GAGGTGGTGTCTGCTTGGGTCAG-3' (423 bp); DP, 5'-TGTGCTCGTGTGTGGCTTGAC-3' and 5'-GCACGAACTTCCCAAAACCAGC-3' (340 bp).

Statistical analysis

Data were analyzed by Student’s t test. Differences were considered significant for p < 0.05. Results are expressed as means ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exogenous PGE2 augments cytokine-dependent PGHS-2 induction and IL-6 secretion

When BMMC were incubated with 100 ng/ml SCF, 10 U/ml IL-10, and 5 ng/ml IL-1{beta}, there was a delayed phase of PGD2 generation, which was maximal at 8–24 h. This was accompanied by a time-dependent induction of steady-state transcripts for PGHS-2, which peaked at 5 h, and the de novo expression of PGHS-2 protein, which was maximal at 8 h, as previously reported (9). There was no induction of PGHS-2 or delayed-phase PGD2 generation in cells maintained in 50% WEHI-3 cell-conditioned medium. The addition of 1 µg/ml PGE2 at the time of cytokine-dependent activation led to increased delayed-phase PGD2 generation and increased expression of PGHS-2 steady-state transcripts and protein with no change in the kinetics of their expression (data not shown). Therefore, dose response studies of PGE2 were performed at 5 h for analysis of PGHS-2 steady-state transcripts and at 8 h for PGHS-2 protein and delayed-phase PGD2 generation. The addition of PGE2 to BMMC activated with SCF, IL-10, and IL-1{beta} elicited a dose-dependent amplification of PGHS-2 induction and delayed-phase PGD2 generation (Fig. 1Go). Cytokine-dependent delayed-phase PGD2 generation increased from 6.96 ± 0.76 ng/106 cells in the absence of PGE2 to 14.42 ± 1.72 ng/106 cells in the presence of 1 µg/ml PGE2 (p < 0.01).



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  		FIGURE 1. Dose-dependent effect of PGE2 on cytokine-dependent delayed-phase PGD2 generation and PGHS-2 induction. BMMC were incubated for 5–8 h in 50% WEHI-3 cell-conditioned medium supplemented with SCF, IL-10, and IL-1{beta}. Control cells were maintained in 50% WEHI-3 cell-conditioned medium alone. Cells were washed at 1 h to remove immediate phase products, and fresh medium and cytokines, with or without PGE2 (1–1000 ng/ml) were added. Delayed-phase PGD2 generation at 8 h (mean ± SEM, n = 3) was determined on cell-free supernatants by ELISA (A); PGHS-2 protein (B) and transcripts (C) were analyzed on cell pellets at 8 and 5 h, respectively. PGD2 generation in response to cytokines alone is indicated by a dashed horizontal line in A. Representative blots from three independent experiments are shown.

 
Treatment of BMMC with SCF, IL-10, and IL-1{beta} also elicited a time-dependent induction of transcripts for IL-6 that peaked at 3–5 h and that was followed by secretion of IL-6 into the culture medium that was maximal at 24 h as previously reported (18). The addition of 1 µg/ml PGE2 at the time of cytokine-dependent activation led to increased secretion of IL-6 with no change in the kinetics of its secretion (data not shown). Therefore, dose response studies to PGE2 were performed at 5 h for analysis of steady-state transcripts encoding IL-6 and at 8 h for secretion of IL-6 into the culture medium. Cytokine-dependent IL-6 secretion increased from 3217 ± 203 pg/106 cells in the absence of PGE2 to 8028 ± 194 pg/106 cells in the presence of 1 µg/ml PGE2 (Fig. 2Go) (p < 0.01), with no discernable increase in steady-state transcripts for IL-6 compared with BMMC stimulated with SCF, IL-10, and IL-1{beta} in the absence of PGE2.



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  		FIGURE 2. Dose-dependent effect of PGE2 on cytokine-dependent IL-6 secretion. BMMC were incubated for 5–8 h in 50% WEHI-3 cell-conditioned medium supplemented with SCF, IL-10, and IL-1{beta}. Control cells were maintained in 50% WEHI-3 cell-conditioned medium alone. Cells were washed at 1 h to remove immediate phase products, and fresh medium and cytokines, with or without PGE2 (1–1000 ng/ml), were added. IL-6 production at 8 h (mean ± SEM, n = 3) was determined on cell-free supernatants by ELISA (A); IL-6 transcripts (B) were analyzed on cell pellets at 5 h. IL-6 secretion in response to cytokines alone is indicated by a dashed horizontal line in A. Representative blots of three independent experiments are shown.

 
The effects of PGE2 are mediated through the activation of a family of G protein-coupled receptors, EP1 through EP4, respectively. To determine which EP receptor(s) were likely involved in the amplification of cytokine-dependent PGHS-2 induction and IL-6 secretion we used RT-PCR to examine the expression of EP receptors in BMMC. Transcripts for EP1, EP3, and EP4 but not EP2 could be detected in BMMC using RT-PCR (Fig. 3GoA). Control positive reactions using RNA extracted from mouse spleen showed amplification of all EP receptors under the same conditions (data not shown). We then examined the effects of EP receptor agonists on cytokine-dependent PGHS-2 induction and IL-6 secretion. Addition of 17-trinor-PGE2, an EP1 agonist, or sulprostone, an EP1/EP3 agonist, to BMMC at the time of cytokine-dependent activation elicited an increase in PGHS-2 induction (Fig. 3Go, B and C), delayed-phase PGD2 generation, and IL-6 secretion (Table IGo). Butaprost, an EP2 agonist, was without effect (Fig. 3GoD and Table IGo). RT-PCR also revealed the presence of transcripts for DP, a PGD2 receptor. However, the selective DP agonist, BW245C, had no effect on cytokine-dependent PGD2 generation or IL-6 secretion (Table IGo).



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  		FIGURE 3. Expression and function of prostanoid receptors in BMMC. A, RNA from BMMC cultured in 50% WEHI cell-conditioned medium was used as template for a one-step RT-PCR using primers specific for the EP1, EP2, EP3, EP4, and DP receptors. A 100-bp ladder (Life Technologies) was used to size the PCR products; the 600-bp band is indicated. B–D, BMMC were incubated for 8 h in 50% WEHI-3 cell-conditioned medium supplemented with SCF, IL-10, and IL-1{beta}. Control cells were maintained in 50% WEHI-3 cell-conditioned medium alone. Cells were washed at 1 h, and fresh medium and cytokines with or without 17-trinor PGE2 (B), sulprostone (C), or butaprost (D) were added at the indicated doses. PGHS-2 protein was detected by SDS-PAGE/immunoblot analysis.

 

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  		Table I. Effect of prostanoid receptor agonists on IL-6 production and cytokine-dependent delayed-phase generation of PGD2 by BMMC1

 
Exogenous 15dPGJ2 augments cytokine-dependent PGHS-2 induction and IL-6 secretion

The lack of effect of BW245C suggested that PGD2, the major prostanoid product of mast cells, is unlikely to amplify cytokine-dependent PGHS-2 induction and IL-6 secretion through an autocrine effect at the DP receptor. Nevertheless, 15dPGJ2, a metabolite of PGD2, is a ligand for PPAR{gamma}. Therefore, we examined the effect of 15dPGJ2 on cytokine-dependent PGHS-2 induction, delayed-phase PGD2 generation, and IL-6 secretion. The addition of 15dPGJ2 elicited a dose-dependent amplification of cytokine-dependent PGHS-2 induction and delayed-phase PGD2 generation (Fig. 4Go), which increased from 6.96 ± 0.76 ng/106 cells in the absence of 15dPGJ2 to 21.52 ± 2.52 ng/106 cells in the presence of 1 µg/ml 15dPGJ2 (p < 0.01). 15dPGJ2 also elicited a dose-dependent increase in cytokine-dependent IL-6 secretion from 3217 ± 203 pg/106 cells in the absence of 15dPGJ2 to 5341 ± 515 pg/106 cells in the presence of 1 µg/ml 15dPGJ2 (Fig. 5Go) (p < 0.01), accompanied by no discernable increase in steady-state transcripts for IL-6.



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  		FIGURE 4. Dose-dependent effect of 15dPGJ2 on cytokine-dependent delayed-phase PGD2 generation and PGHS-2 induction. BMMC were incubated for 5–8 h in 50% WEHI-3 cell-conditioned medium supplemented with SCF, IL-10, and IL-1{beta}. Control cells were maintained in 50% WEHI-3 cell-conditioned medium alone. Cells were washed at 1 h to remove immediate phase products, and fresh medium and cytokines, with or without 15dPGJ2 (1–1000 ng/ml), were added. Delayed-phase PGD2 generation at 8 h (mean ± SEM, n = 3) was determined on cell-free supernatants by ELISA (A); PGHS-2 protein (B) and transcripts (C) were analyzed on cell pellets at 8 and 5 h, respectively. PGD2 generation in response to cytokines alone is indicated by a dashed horizontal line in A. Representative blots of three independent experiments are shown.

 


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  		FIGURE 5. Dose-dependent effect of 15dPGJ2 on cytokine-dependent IL-6 secretion. BMMC were incubated for 5–8 h in 50% WEHI-3 cell-conditioned medium supplemented with SCF, IL-10, and IL-1{beta}. Control cells were maintained in 50% WEHI-3 cell-conditioned medium alone. Cells were washed at 1 h to remove immediate phase products, and fresh medium and cytokines, with or without 15dPGJ2 (1–1000 ng/ml), were added. IL-6 production at 8 h (mean ± SEM, n = 3) was determined on cell-free supernatants by ELISA (A); IL-6 transcripts (B) were analyzed in cell pellets at 5 h. IL-6 secretion in response to cytokines alone is indicated by a dashed horizontal line in A. Representative blots of three independent experiments are shown.

 
A comparison of the dose response curves to each prostanoid revealed that PGE2 (Figs. 1Go and 2Go) was active at lower concentrations than 15dPGJ2 (Figs. 4Go and 5Go), eliciting amplification of cytokine-dependent PGHS-2 induction, delayed-phase PGD2 generation, and IL-6 secretion at 1 ng/ml, the lowest concentration tested. The response to 15dPGJ2 had not reached a plateau at 1 µg/ml. Nevertheless, at 1 µg/ml 15dPGJ2 elicited a greater amplification of PGHS-2 induction and delayed-phase PGD2 generation than did PGE2. In contrast, PGE2 elicited a greater amplification of IL-6 secretion than did 15dPGJ2. Neither prostanoid elicited an effect in the absence of cytokine activation.

In contrast to PGE2, which acts at G protein-coupled receptors, 15dPGJ2 is an endogenous ligand for the nuclear receptor, PPAR{gamma}. SDS-PAGE immunoblotting revealed the expression of PPAR{gamma} in BMMC, which was modestly increased 8 h after cytokine-dependent activation (Fig. 6GoA). However, the selective PPAR{gamma} agonist, troglitazone, only marginally increased the cytokine-dependent induction of PGHS-2 (Fig. 6GoB), and 10 µg/ml troglitazone inhibited delayed-phase PGD2 generation (Fig. 6GoC). Troglitazone had no effect on cytokine-dependent IL-6 generation (Fig. 6GoD). Similar data were obtained with the PPAR{gamma} agonist, ciglitazone (data not shown). Thus, the effects of 15dPGJ2 cannot be explained solely by its action at PPAR{gamma}.



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  		FIGURE 6. A PPAR{gamma} agonist does not mimic the effects of 15dPGJ2. A, PPAR{gamma} expression by BMMC was determined by SDS-PAGE immunoblot analysis; a lysate of Jurkat cells was used as a positive control. The effects of troglitazone on cytokine-dependent PGHS-2 expression (B), delayed-phase PGD2 generation (C), and IL-6 production (D) at 8 h (mean ± SEM, n = 3) are shown.

 
Role of endogenous prostanoids in cytokine-dependent PGHS-2 induction and IL-6 secretion

The cytokine-dependent induction of PGHS-2 in BMMC derived from cPLA2-/- mice is impaired by ~70% (19) (Fig. 7GoA) but can be restored by addition of exogenous PGE2 or arachidonic acid (19). Having demonstrated an effect of exogenous prostanoids on cytokine-dependent induction of PGHS-2 and IL-6 secretion in BMMC, we investigated the role of endogenous prostanoids using genetic and pharmacological approaches. Cytokine-dependent PGHS-2 induction was intact in PGHS-1-/- BMMC (Fig. 7GoB), demonstrating that endogenous prostanoids produced in the immediate phase of eicosanoid generation do not amplify PGHS-2 induction. Furthermore, NS-398 did not attenuate cytokine-dependent PGHS-2 induction in BALB/c BMMC (Fig. 7GoC) or in PGHS-1-/- BMMC (Fig. 8Go), excluding a role for autocrine prostanoid-dependent amplification of PGHS-2. PGD2 is the major biosynthetic product of the prostanoid pathway in BMMC (34). Consistent with the results in PGHS-1-/- BMMC and in BMMC treated with NS-398, cytokine-dependent PGHS-2 induction was intact in hPGDS-/- BMMC (Fig. 7GoD), in which both immediate and delayed phases of PGD2 generation were absent (data not shown). These results exclude a role for endogenous PGD2 or its metabolite, 15dPGJ2, in cytokine-dependent PGHS-2 induction. Cytokine-dependent IL-6 generation was not significantly attenuated in cPLA2-/- BMMC, PGHS-1-/- BMMC, or hPGDS-/- BMMC, or in the presence of NS-398 (Fig. 7Go, E–H), excluding a role for endogenous eicosanoids in amplifying cytokine-dependent IL-6 secretion.



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  		FIGURE 7. Role of endogenous prostanoids in cytokine-dependent PGHS-2 induction and IL-6 secretion. BMMC derived from mice with disruption of the genes for cPLA2-{alpha} (A and E), PGHS-1 (B and F), or hPGDS (D and H), or their wild-type littermates, or BALB/c BMMC treated with the indicated doses of NS-398 were incubated for 8 h in 50% WEHI-3 cell-conditioned medium supplemented with SCF, IL-10, and IL-1{beta}. PGHS-2 protein expression (A–D) and IL-6 production (E–H) (mean ± SEM, n = 3) were analyzed. Results from two to three independent experiments are shown.

 


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  		FIGURE 8. Role of endogenous eicosanoids in cytokine-dependent PGHS-2 induction. PGHS-1-deficient BMMC were incubated for 8 h in 50% WEHI-3 cell-conditioned medium supplemented with SCF, IL-10, and IL-1{beta}, and were treated with inhibitors of FLAP (10 µM MK-886), 5-LO (10 µM AA-861), or PGHS-2 (100 ng/ml NS-398); or with a combination of NS-398 and 30 µM NDGA. PGHS-2 expression was assessed by SDS-PAGE/immunoblot analysis.

 
Besides PGD2, the other major product of arachidonic acid generated by mast cells is the 5-LO product, leukotriene C4 (7, 8). To explore the role of endogenous lipoxygenase products in the cytokine-dependent induction of PGHS-2, the effects of 10 µM AA-861, a 5-LO inhibitor, 10 µM MK-886, a FLAP inhibitor, and 30 µM NDGA, a nonspecific lipoxygenase inhibitor, were evaluated (Fig. 8Go). None of these agents attenuated cytokine-dependent PGHS-2 induction in PGHS-1-/- BMMC (Fig. 8Go) or in BALB/c BMMC (data not shown). Indeed, blockade of both cyclooxygenase and lipoxygenase pathways through the addition of 100 ng/ml NS-398 and 30 µM NDGA to PGHS-1-/- BMMC augmented PGHS-2 induction in response to SCF, IL-10, and IL-1{beta} (Fig. 8Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Prostanoids may modulate the inflammatory response through diverse mechanisms including amplification of PGHS-2 expression and cytokine generation. For example, PGE2, which signals through a family of G protein-coupled receptors (35), has been shown to amplify the induction of PGHS-2 in LPS-stimulated mouse RAW 264.7 macrophages (28), the human PC-3 prostatic carcinoma cell line (36), LPS-stimulated microglial cells (37), and mouse MC3T3-E1 osteoblasts (38). Various resident cells and inflammatory leukocytes including fibroblasts, endothelial cells, and monocytes/macrophages generate PGE2, which has pleiotropic effects on the immune system (39, 40). It contributes to pain and inflammation (41) and acts as a "third" signal for Th2 lymphocyte differentiation (42, 43). Its role in inflammation is supported by the efficacy of PGHS-2 inhibitors in inflammatory arthritis (44, 45) and the attenuation of carrageenan-induced footpad inflammation and adjuvant arthritis in rats by neutralizing Abs to PGE2 (46). Therefore, we evaluated the effects of exogenous PGE2 on cytokine-dependent PGHS-2 induction in BMMC. Addition of PGE2 alone to BMMC did not induce expression of PGHS-2. However, PGE2 augmented cytokine-dependent induction of PGHS-2 in a dose-dependent manner, augmenting induction of both transcripts and protein (Fig. 1Go). PGE2 was active at nanograms per milliliter concentrations, which are likely to be found at sites of inflammation (47, 48).

PGE2 has been reported to amplify IL-6 generation in rat peritoneal mast cells (49) and more recently in mouse BMMC in response to Fc{epsilon}RI cross-linking (50). Therefore, we determined whether cytokine-dependent IL-6 induction was regulated by prostanoids in mouse BMMC. PGE2 alone did not induce IL-6 generation from BMMC but elicited a 2- to 3-fold amplification of cytokine-dependent IL-6 generation (Fig. 2Go). This was not accompanied by a detectable increase in steady-state transcripts for IL-6, indicating an action of PGE2 at a posttranscriptional level. Amplification of IL-6 generation by PGE2 has been described in various other cell types including mouse macrophages (29) and the mouse J774 macrophage cell line stimulated with LPS and UTP (30). The relevance of these findings for inflammatory responses in vivo is suggested by the observation that PGHS-2-dependent PGE2 generation amplified IL-6 generation by alveolar macrophages in a model of acute lung injury (51) and in pristane-induced peritonitis (52).

The principal prostanoid product of mast cells is PGD2. PGD2 acts at the DP receptor to relax vascular smooth muscle, leading to vasodilatation and inhibition of platelet aggregation, thus facilitating plasma leakage at sites of inflammation (22). PGD2 may be particularly relevant in allergic inflammation and asthma. PGD2 is a bronchoconstrictor (53). Gene disruption of the DP receptor led to attenuation of pulmonary Th2 cytokine generation and allergen-induced airway hyperresponsiveness in a mouse model of asthma (54). Furthermore, PGD2 acts at the newly described G protein-coupled receptor, the chemoattractant receptor-homologous molecule expressed on Th2 cells, to elicit chemotaxis of Th2 cells, eosinophils, and basophils (23). IL-6 is produced by mast cells in response to cytokine stimulation (18) or cross-linking of the high-affinity FcR for IgE (16) and is an important modulator of inflammation (55, 56). It is notable that mast cells are the major source of IL-6 in the nasal mucosa in allergic rhinitis (57) and in the bronchial mucosa in asthma (58). Mice with transgenic expression of IL-6 exhibit airway remodeling with subepithelial fibrosis and myofibroblast proliferation (59). Although PGE2 is a bronchodilator and inhibits cysteinyl leukotriene generation and bronchoconstriction in aspirin-induced asthma (60), disruption of the EP2 receptor in mice led to attenuation of Th2 cytokine generation and pulmonary allergic inflammation (61). The observation that PGE2 amplifies PGHS-2 induction, PGD2 generation, and IL-6 secretion by mast cells suggests that it may amplify the allergic inflammatory response and airway remodeling through a paracrine action.

PGE2 exerts its effects through a family of G protein-coupled receptors, EP1 through EP4. Alternate splicing of EP3 mRNA leads to further diversity of EP receptors. EP2 and EP4 act through elevation of cAMP, whereas EP1 raises intracellular Ca2+. EP3 may either elevate or lower intracellular cAMP levels depending on the isoform of the receptor that is expressed (35). There is limited data on EP receptor expression by mast cells. RT-PCR revealed transcripts for EP1, EP3, and EP4, but not EP2 in mouse BMMC (Fig. 3GoA). The observation that the effects of PGE2 were reproduced by sulprostone, an EP1/EP3 agonist, and by 17-trinor PGE2, an EP1 agonist, but not by butaprost, an EP2 agonist (Table IGo and Fig. 3Go, B–D), suggests that PGE2 acts through the EP1 receptor to elicit amplification of PGHS-2 and IL-6 induction in BMMC. This is supported by the observation that forskolin, which elevates intracellular cAMP levels, failed to amplify cytokine-dependent PGHS-2 induction or IL-6 generation by BMMC (data not shown). The action of PGE2 through the EP1 receptor in BMMC is in agreement with the findings of Gomi and colleagues (50), who reported that PGE2 augmented IgE-dependent IL-6 generation in mouse BMMC through the EP1 receptor. This is in contrast to the amplification of PGHS-2 induction in LPS-stimulated RAW 264.7 macrophages by PGE2, which was mediated through EP2 and cAMP (28).

15dPGJ2 is a metabolite of PGD2 (62), the principal prostanoid product of mast cells (34), and is a ligand for the nuclear receptor PPAR{gamma} (63, 64). PPAR ligands have been reported to regulate PGHS-2 expression, inducing PGHS-2 in immortalized mouse liver cells (65) and human epithelial cells (25) but inhibiting its induction in LPS-stimulated differentiated U937 cells (24). 15dPGJ2 and other PPAR{gamma} ligands may also modulate cytokine generation, down-regulating the production of TNF-{alpha}, IL-6, and IL-1{beta} in human monocytes stimulated with PMA (26). In a previous study we observed that induction of PGHS-2 is attenuated in BMMC derived from mice with disruption of the gene for cPLA2 (19). Therefore, we postulated that 15dPGJ2, derived from endogenous PGD2, might modulate PGHS-2 induction and IL-6 secretion in BMMC. We indeed found that 15dPGJ2 augmented cytokine-dependent induction of PGHS-2, delayed-phase PGD2 generation, and IL-6 production in mouse BMMC (Figs. 4Go and 5Go). As with PGE2, 15dPGJ2 amplified cytokine-dependent induction of PGHS-2 transcripts but not IL-6 transcripts, suggesting posttranscriptional regulation of IL-6. The action of 15dPGJ2 in BMMC suggested that prostanoids might modulate PGHS-2 induction and cytokine generation in mast cells not only through cell surface G protein-coupled receptors but also through nuclear PPARs. Although BMMC were found to express PPAR{gamma} (Fig. 6GoA), troglitazone (Fig. 6Go, B–D) and ciglitazone (data not shown) elicited minimal augmentation of either PGHS-2 induction or IL-6 generation at concentrations up to 10 µM. It is possible that higher concentrations of these agonists may be required to elicit an effect. However, the observation that 10 µM troglitazone modestly attenuated delayed-phase PGD2 generation in the face of a small increase in PGHS-2 induction suggests that 15dPGJ2 may be acting at a site distinct from PPAR{gamma}. It is notable that the suppression of inducible NO synthase in microglial cells by 15dPGJ2 appeared to be independent of its action as a PPAR{gamma} ligand (66). Furthermore, 15dPGJ2 was shown to down-regulate LPS-induced inducible NO synthase expression in RAW 264.7 cells in a PPAR{gamma}-independent fashion through inhibition of NF-{kappa}B signaling (67). The mechanism of action of 15dPGJ2 in up-regulating cytokine-dependent PGHS-2 expression and IL-6 generation in mouse BMMC would also appear to be independent of PPAR{gamma}, though the mechanism has yet to be determined.

To investigate whether endogenous prostanoids regulate cytokine-dependent PGHS-2 induction and IL-6 generation by BMMC we turned to gene disruption and pharmacological approaches. In contrast to the effect of disrupting the gene for cPLA2-{alpha} (19), disruption of the gene for PGHS-1 or the gene for hPGDS had no effect on cytokine-dependent PGHS-2 induction (Fig. 7Go). Thus, the PGHS-1-dependent immediate phase generation of PGD2 does not amplify delayed-phase PGHS-2 induction. Addition of the PGHS-2 inhibitor, NS-398, to cytokine-stimulated BALB/c BMMC (Fig. 7GoC) or to PGHS-1-/- BMMC (Fig. 8Go) was also without effect, ruling out "feed-forward" amplification of PGHS-2, which has been described in MC3T3-E1 osteoblasts (27). While PPAR{gamma} ligands have diverse effects in cytokine gene regulation, adipogenesis, and glucose homeostasis (68, 69), their endogenous ligands have not been defined. Our observations in BMMC that 15dPGJ2 acts independently of PPAR{gamma} (Fig. 6Go) and that endogenous PGD2 metabolites do not regulate IL-6 production or PGHS-2 induction in BMMC fail to provide support for the hypothesis that 15dPGJ2 is a functional endogenous PPAR{gamma} ligand, at least in this limited setting.

Finally, addition of lipoxygenase inhibitors or the FLAP inhibitor, MK-886, to PGHS-1-/- BMMC did not attenuate cytokine-dependent PGHS-2 induction (Fig. 8Go). Thus, endogenous eicosanoids appear not to regulate PGHS-2 induction in BMMC. This suggests that arachidonic acid itself, platelet-activating factor, or lysophospholipids may be the products of cPLA2-{alpha} responsible for PGHS-2 amplification in BMMC. The observation that the combined inhibition of PGHS-2 and lipoxygenases in PGHS-1-/- BMMC amplified cytokine-dependent PGHS-2 induction (Fig. 8Go) favors a role for arachidonic acid, which we have shown can overcome the effect of cPLA2 disruption in BMMC (19). While cytokine-dependent PGHS-2 induction is attenuated in cPLA2-/- BMMC, endogenous products of cPLA2-{alpha} are not required for cytokine-dependent IL-6 generation (Fig. 7Go).

In conclusion, we have demonstrated that exogenous prostanoids amplify proinflammatory PGHS-2 induction and IL-6 generation by mast cells, providing a mechanism for amplification of local inflammatory responses. Endogenous eicosanoids appear not to contribute to the induced expression of either gene, although arachidonic acid may amplify PGHS-2 induction in BMMC.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant HL36110; by American Cancer Society Grant RPG-97-001-01-BE (to J.P.A.); by a post-doctoral fellowship from Coordenaçao de Aperfeiçoamento de Pessoal de Nível Superior, Brazil, and a Sepracor Young Investigator Research Award grant from the American Academy of Allergy, Asthma, and Immunology (to B.L.D.); by an International Research Grant from the Japan Eye Bank and by the Kowa Life Science Foundation (to H.F.); and by grants from the program for Core Research for Evolutional Science and Technology of the Japan Science and Technology Corporation, a grant-in-aid for Scientific Research (B) (12558078) of the Ministry of Education, Culture, Sports, Science and Technology of Japan, and an Applied Research Pilot Project for the Industrial Use of Space promoted by National Space Development Agency of Japan and the Japan Space Utilization Promotion Center (to Y.U.). Back

2 Address correspondence and reprint requests to Dr. Jonathan P. Arm, Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital, Smith Research Building, Room 638B, 1 Jimmy Fund Way, Boston, MA 02115. E-mail address: jarm{at}rics.bwh.harvard.edu Back

3 Abbreviations used in this paper: BMMC, bone marrow-derived mast cell; SCF, stem cell factor; PGHS, PG endoperoxide synthase; cPLA2-{alpha}, cytosolic PLA2-{alpha}; hPGDS, hematopoietic PGD synthase; 15dPGJ2, 15-deoxy-{Delta}12,14-PGJ2; PPAR, peroxisome proliferator-activated receptor; NDGA, nordihydroguaiaretic acid; DP, D prostanoid; EP, E prostanoid. Back

Received for publication June 7, 2001. Accepted for publication November 26, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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