2 Couple to Different Transmembrane Stimuli to Generate ...

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and by a grant from the Hyde and Watson Foundation. The costs of publication ...... Meade, E. A., Smith, W. L., and DeWitt, D. L. (1993) J. Biol. Chem. 268,. Natl.
Vol. 269, No. 35, Issue of September 2, pp. 22269-22275, 1994 Printed in U.S.A.

JOURNAL OF B r o w c r c ~ CHEMI~TRY ~ 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. THE

Prostaglandin Endoperoxide Synthase-1 and -2 Couple to Different Transmembrane Stimuli to Generate ProstaglandinD2 in Mouse Bone Marrow-derived Mast Cells* (Received forpublication, May 20, 1994, and in revised form, June 21, 1994)

Makoto MurakamiS, RyojiMatsumoto, K. Frank Austen, and Jonathan P. A r m § From the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115 and the Department of Rheumatology and Immunology, Brigham and Women's Hospital, Boston, Massachusetts 02115

constitutive enzyme, PGHS-1, encoded by a 2.8-kilobase transcript, serves certainphysiologic housekeeping functions, such a s thrombogenic thromboxane 4 generation by platelets, antithrombogenic PGI, production by endothelial cells, generation of cytoprotective prostanoids in the gastricmucosa, and regulation of renal bloodflowby prostanoids (2-5). In contrast, expression of PGHS-2, encoded by a 4.4-kilobase transcript, is induced in such cells as macrophages, fibroblasts, vascular endothelial cells, and vascular smooth muscle cells by proinflammatory stimuli such as interleukin (ILI-lp, tumor necrosis factor a , and endotoxin or in response to growth factors, such as platelet-derived growthfactor. PGHS-2 is thoughtto contribute to the generation of prostanoids at sitesof inflammation (6-12). There are, however, no examples of separate stimulus-specific regulation of PGHS-1 and PGHS-2 in the same cell at a time when both proteins are expressed and functional. BALB/cJ mouse bone marrow-derived mast cells (BMMC), obtained by culture of bone marrow cells with a source of IL-3, are a pleotropic progenitor population that reconstitutes distinct tissue-related mature mast cell subclasses in mast celldeficient mice of the WBB6F1/J-W/N7" strain (13). addition In to IL-3, other cytokines, such as the ligand for c-kit (kit ligand, K L ) alone, or nerve growthfactor, IL-9, or IL-10 in combination viability of BMMC and with IL-3or KL, support the growth and regulate their phenotypic diversity in vitro (14-19). Activation of BMMC by cross-linking of Fce receptor type I (FceRI)leads to exocytosis of preformed granule-associatedhistamine, serotonin, proteoglycans, and proteases and generation and release of newly synthesized lipid mediators and cytokines (20, 21). Arachidonic acid, released from cell membrane phospholipids Prostaglandin-endoperoxide synthase (PGHS,' cyclooxygen- of activated BMMC by the action of phospholipase 4, is mease) catalyzes the conversion of arachidonic acid, released by tabolized in the presence of 5-lipoxygenase-activating protein phospholipase 4,to prostaglandin (PG)H,, which is thenme- by 5-lipoxygenase and leukotriene C, synthase to leukotriene hematopoietic tabolized by one or more terminal synthases to a variety of C,, and by the sequentialaction of PGHS and the biologically active prostanoids(1).Studies have established the form of PGD, synthase to PGD, (22-25). Thus, this pleotropic presence of two distinct PGHS isoforms of about 70 kDa. The progenitor population of mast cells provides a nontransformed cell with which to analyze the separate induction and subse* This work was supported in part by Grants HL36110,AI22531, quent functional responsesof PGHS-1 andPGHS-2 to a transAI31599, AI23483, and RR05950 from the National Institutes of Health membrane stimulus of immediate onset, FceRI-mediated actiand by a grant from the Hyde and Watson Foundation. The costs of vation a s compared with that accompanying the cytokine publication of this article were defrayed in part by the payment of page charges. This article musttherefore be hereby marked "advertisement" response alone. We now report that PGHS-1 andPGHS-2, intermediate enin accordance with 18 U.S.C. Section 1734 solely to indicate this fact. zyme isoforms in arachidonic acid metabolism, are independ$ Supported by an International Human Frontier Science Program postdoctoral training grant. ently regulated in the same cell, BMMC, by cytokines that 8 Supported by a Burroughs Wellcome Developing Investigator regulate proliferation and/or function of mast cells. More imAward. To whom correspondence should be addressed: Dept. of Rheuportantly, the function of each PGHS isoform is segregated to matology and Immunology, Brigham and Women's Hospital, Harvard Medical School, 250 Longwood Ave., Boston, MA 02115. Tel.: 617-432- the activation stimuluswhile contributing PGH, to PGD, syn1335; Fax: 617-432-0979. thase for the generationof a common end product, PGD,, from The abreviations used are: PGHS, prostaglandin-endoperoxidesyn- the endogenously derived arachidonic acid. Thus, cytokine inthase; PG, prostaglandin;IL, interleukin;BMMC, bone marrow-derived mast cells; KL, c-kit ligand; mMCP, mouse mast cell protease; FceRI, duction of PGHS-2 leads to concomitant generation of PGD,, PGD, generation is mediated by Fce receptor type I; TNP, trinitrophenyl; BSA, bovine serum albumin; whereasIgE-dependent PAGE, polyacrylamide gel electrophoresis. PGHS-1 regardless of the presence of PGHS-2.

The view that the two isoforms of prostaglandin-endoperoxide synthase (cyclooxygenase), PGHS-1 and PGHS-2, mediatephysiologicandinflammatoryprocesses, respectively, implies separate pathways of arachidonic acid metabolismwith different benefits to the host. Functional segregation of these steps in endogenous arachidonic acid metabolism in a single cell in response to different stimuliis now demonstrated. When mouse bone marrow-derived mast cells developed in interleukin-3 (IL-3)-containingmediumwerecultured with c-kit ligand in combination with IL-10 and IL-lp, transient expression of PGHS-2 mRNA and protein occurred in a dose- and time-dependentfashion, accompanied by substantial release of prostaglandin D, (PGD,) into the culture medium from 2 to 10 h. In contrast, induction ofPGHS-2 did not mediate an increase in PGD, generation in response to stimulation with IgE and antigen.After a longer period of culture, from 24 to 48 h, the expression ofPGHS-1 increased, as did the increase in IgE/antigen-dependent generationof PGD,. Dexamethasone, which inhibited the induction of PGHS-2 but not PGHS-1, and a PGHS-2-selective inhibitor suppressed cytokine-induced PGD, generation but notIgE-dependent PGD, generation.Thus, at atime when bothPGHS-1 and PGHS-2 are presentin bone marrow-derived mastcells, they function independently by coupling to different stimulus-initiated pathways to PGD, generation from endogenouslyderivedarachidonic acid.

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Stimulus Coupling of PGHS-1 and PGHS-2in Mouse Mast Cells

(3,000 Ci/mmol; DuPont NEN) by random priming. All hybridizations EXPERIMENTAL PROCEDURES were performed at 43 "C for 24 h in 50% formamide (Life Technologies, Materials-Recombinant mouse IL-3 and IL-1p were purchased from Inc.), 0.75 M NaC1,75 m~ sodium citrate,0.1% SDS, 5mM EDTA, 10 m~ Genzyme, Boston, MA. Recombinant mouse IL-10 was obtained from sodium phosphate, pH 6.8, 2 x Denhardt's solution, 10% dextran sulculture supernatantsof COS-7 cells (AmericanType Culture Collection, fate, and 100 pg/ml salmon sperm DNA (Sigma). The RNA blots were Rockville, MD) transfected with plasmid (pCD-SRa) containingcDNA a washed twice with 150 mM NaC1, 15 mM sodium citrate, 1 mM EDTA, encoding mouse IL-10 (K. W. Moore, DNAX, CA) (26). Briefly, COS-7 0.1% SDS, and 10 mM sodium phosphate, pH 6.8, for 5 min eachfollowed cells (1x lo7cells) weremixed with 30 pg of plasmid DNAin RPMI 1640 by two washes at 65 "C in 30 mM NaC1, 3 mM sodium citrate, 1 mM V, 960 microfarads)(Biomedium and subjected to electroporation (200 EDTA, 0.1% SDS, and 10 m~ sodium phosphate, pH 6.8, for 15 min Rad). The cells were seeded a t 2.5 x lo6 cells/75-cm2flask (Corning, Inc.) each. The blots were visualized by autoradiography with Kodak XAR-5 in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplefilm and an intensifying screen at -80 "C. The relative amountof each mented with 10% fetal calf serum (Life Technologies, Inc.) and cultured transcript was estimated by quantitating the associated radioactivity for 4 days, and the supernatants were collected. The concentration of with a Betascope 603Blot Analyzer (Betagen, Waltham,MA). The -fold IL-10 in theCOS-7 supernatants was assayed in the presence of a fixed increase in steady-statemRNA was calculated as the ratio of radioacconcentration of IL-3 by incorporation of [3H]thymidine (Amersham tivity associated with a specific transcript in treated cells compared Corp.) into BMMC (27). Mouse KL cDNA (J. Flanagan, Harvard Medwith that in control cellsand was correctedfor changes in steady-state ical School, Boston, MA) (28) was inserted into the multiple cloning site levels of p-actin transcript to adjustfor differences in loading between of pVL1393 (Pharmigen, SanDiego, CA). The recombinant plasmid (2 lanes. pg) was co-transfected withBaculo Goldm Linearized BaculovirusDNA SDS-PAGEI Immunoblot Analysis-BMMC cultured with eachcyto(Pharmigen) into 3 x lo6 Sf9 cells (Invitrogen, San Diego, CA) with kine were washed once with 10mM phosphate buffer, pH 7.2, containing Ca2+-phosphate (29). The cells were cultured at 27 "C in TNM-FH insect 140 mM NaCl and 2 mM KC1 and then lysed with a buffer composed of medium (Invitrogen) supplemented with 10% fetal calf serum. After 7 50 mM Tris-HC1, pH 7.4, 0.1% SDS, 0.5% Nonidet P-40 (Boehringer days, the recombinant virus in the supernatant was selected by plaque Mannheim), 5 mM Na3V0, (Sigma),50 pg/ml leupeptin (Sigma),1.5 p~ assay and amplified. Sf9 cells (1x lo6 celldml) were infected with the pepstatin A (Sigma), and 1 mM phenylmethylsulfonyl fluoride (Sigma). recombinant virus and cultured for 7 days. The concentrationof KL in A sample of thelysatewasappliedtoSDS-polyacrylamide gels the supernatants was determined by [3H]thymidine incorporation into (Schleicher & Schuell) and electrophoresed. The separated proteins BMMC by comparison with authentic purified recombinantKL stand- were electroblotted onto nitrocellulose membranes (Bio-Rad) in a transard (Immunex, Seattle,WA). fer buffer consistingof 20 mM Tris, 150 mM glycine, and20% methanol Rabbit anti-sheep PGHS-1polyclonal antiserum was provided by W. with a Mini Trans-Blot electrophoretic transfercell (Bio-Rad)at 250 V L. Smith, Michigan State University, East Lansing, MI. Rabbit antifor 1.5 h. The membranes were then sequentially treated with the mousePGHS-2affinity-purified polyclonal antibodywaspurchased following: 5% nonfat milk in 10 mM Tris-HC1, pH 7.4, containing 150mM from Cayman Chemical, Ann Arbor, MI. Mouse PGHS-1 and PGHS-2 NaCl and 0.1% Tween 20 (Bio-Rad) (TBS-Tween) for 1 h; antibodies cDNA probes and thePGHS-2 inhibitor, NS-398(301, were provided by against PGHS-1 or PGHS-2 a t a dilution of 1:50,000 and 1:2,000, reJ. Trzaskos, Merck DuPont, Wilmington, DE. Dexamethasone and in- spectively, in TBS-Tween for 1 h; TBS-Tween for three washes; and domethacin were obtainedfrom Sigma. horseradishperoxidase-conjugatedgoatanti-rabbit IgG (Amersham Cytokine-induced PGD, Generation-Bone marrow cells from male Corp.) (1:2,000 dilution) in TBS-Tween for 1 h. After five washes, the BALB/cJ mice (Jackson Laboratory,Bar Harbor, ME) were cultured for protein bands were visualized witha chemiluminescent technique us3-6 weeks i n 50% enrichedmedium(RPMI1640containing100 ing an ECL Western blot analysis system (Amersham Corp.). The reunitdm1 penicillin,100 pg/ml streptomycin, 10 pg/ml gentamycin, 2mM sultingfilterswere exposed to Kodak XAR-5 film for 30-60 s for L-glutamine, 0.1 mM nonessential amino acids, 50 p~ 2-mercaptoetha- PGHS-1 and for 120-180 s for PGHS-2. nol, and 10% fetal calf serum), 50% WEHI-3 cell (American Type Culture Collection) conditioned medium. After 3 weeks, >97%of the cells in RESULTS culture wereBMMC as assessed by staining with toluidine blue or with alcian blue and safranin (20). Cytokine-induced PGD, Generation-Whereas BMMC develBMMC were washedonce with enriched medium, resuspended in theoped and maintained inIL-3-containing medium released 0.23 enriched medium supplemented with various combinations of recombi0.09 ng of PGDJlO' cells during 10 h of culture ( n = 51, nant cytokines at a cell density of 1 x lo6 celldml, and cultured for various periods. For culture with cytokines for 2 days, thecell density replicate cells cultured for the same interval withKL + IL-10, a t t h estart of the culture was adjusted so that the finalcell density was with KL + IL-lp, and withKL + IL-10 + IL-1p released 1.5 0.7 approximately 5 x lo6 celldml. The following concentrations of cyto- ( p < 0.001 uersus IL-3 alone; mean 2 S.E., n = 6), 2.5 2 0.2 ( p kines were used:KL, 100 ng/ml; IL-10,lO units/ml; IL-lp,5 ng/ml; and < 0.001 versus IL-3 alone, n = 41, and 15 2 5.4 ( p < 0.001 versus IL-3, 100 units/ml. After culture for various periods with cytokines, theIL-3 alone, n = 6) ng of PGDJlO' cells, respectively (Fig. lA). cells were sedimented at 120 x g at 4 "C for 5 min. The supernatants BMMC cultured with KL alone, with KL IL-3, with IL-3 were collected and assayed for PGD, by radioimmunoassay with comIL-10, and withIL-1p alone released 0.6 2 0.4 ( n = 6), 0.55 0.2 mercial kits (Amersham Corp.). IgE-dependent Activationof BMMC-BMMC that hadbeen cultured ( n = 31, < 0.15 ( n = 2), and < 0.15 (n = 2) ng of PGDdlO' cells, respectively ( p > 0.05 uersus IL-3 alone) (Fig. lA).SDS-PAGE/ with cytokines were sedimented at 120 x g at 4 "C for 5 min, resuspended a t 1 x lo7 celldml in enriched medium containing the same immunoblot analysis using anantibody specific to PGHS-2 recytokines usedfor culture, and sensitized with pg/ml 10 monoclonal IgE vealed that cytokine-directed PGD, generation appeared t o be anti-trinitrophenyl (TNP) for 30 min. After being washed twice with related to theinduction and magnitudeof PGHS-2 protein exTyrode's buffer containing 1.8mM Ca", 0.2 mM M P , 0.1% (w/v) gelatin pression (Fig. 1B). PGHS-2 protein was present most abun(Sigma), and 10 mM HEPES (Sigma) pH7.2 (Tyrode gelatin buffer),the dantly inBMMC treated for 5 h with KL + IL-10 + IL-lp, which cells were resuspended inTyrode gelatin buffer at 5 x 10' cells/ml. The provided the most PGD,; it was present inBMMC treated with cells were stimulated at 37 "C with 10 ng/ml TNP-conjugated bovine serum albumin (TNP.BSA), a concentration shown i n preliminary ex- KL + IL-10 or KL + IL-lp, which generated some PGD,, and it periments to beon a plateau for IgE-dependent activationof each popu- was notdetectable in BMMC cultured withKL, IL-lp, or IL-10, lation of cytokine-treated BMMC. After10min, thereactionwas either alone or in combination with IL-3. In contrast, none of stopped by centrifugation of the cells a t 120 x g for 5 min at 4 "C, and the cytokines, alone or in combination, changed the amount of the supernatants were assayed for PGD,. PGHS-1 protein during this 10-h culture period (Fig. 1B).AlRNA Blot Analysis-Total cellular RNA was extracted in guanithough PGHS-2 mRNA was only minimally detected in BMMC diniumthiocyanatewithTRI-Reagent(MolecularResearchCenter, Cincinnati, OH) (31) according to the manufacturer's instructions and cultured in IL-3 alone, BMMC exposed for 2 h to KL alone, to was quantitated by measurement of its optical density at 260 nm. KL + IL-10, t o KL + IL-lp, and t o KL + IL-10 + IL-1p exhibited Approximately equal amounts of total RNA were applied to each lane of increases in PGHS-2 mRNA of approximately 7-, 80-, 17-, and 1.2% formaldehyde-agarose gels, electrophoresed, and transferred to 170-fold (n= 4), respectively (Fig. 1C). PGHS-2 mRNA was not Immobilon-N (Millipore, Bedford, MA). The resulting blots were seinduced when BMMC were cultured withIL-10 or IL-1p in the quentially analyzed withcDNA probes for PGHS-1, PGHS-2, andp-acabsence of KL. Thus, those cytokine combinations providing tin that were labeled (Megaprime; Amersham Corp.) with [32PldCTP

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Stimulus Coupling of PGHS-1 and PGHS-2 A ,

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0 10 20 30 40 50 FIG.1.Cytokine stimulationof PGD,release and of PGHS protein and transcript expressionby BMMC. A, release of PGD, into Hours the medium duringa 10-h cultureof BMMC with various combinations FIG.2. Kinetics of cytokine effects on PGD,generation.A, time of cytokines. BMMC (1x IO6celldml) were cultured with the indicated course of cytokine-induced PGD, release and of PGHS-2 steady-state combinations of cytokines for10 h, anda portion of the supernatant was mRNA and protein induction. PGD, release into the culture medium taken for PGD, radioimmunoassay. The values represent meansf S.E. from BMMC treated withKL + IL-10 + I L 1 p (closed circles), KL + I L l O The cytokine concentrations used in these and subsequent experiments(open squares), KL (closed triangles), and IL-3 (open circles) was aswere as follows: KL, 100 ng/ml; IL-lp, 5 ng/ml; IL-3, 100 unitdml; and sessed over timeby radioimmunoassay. Values represent means= S.E. IL-10,lO unitslml.B, expression of PGHS-1 and PGHS-2 proteins. After of four independent experiments. Inset, time course of the changes in culture for 5 h with cytokines, a sample of BMMC cell lysate (lo5cell the expressionof PGHSS mRNA and protein in BMMC treated withKL equivalents for PGHS-1 and2.5 x lo5cell equivalents for PGHS-2) was + IL-10 + IL-1p as determined by RNA blottingand SDS-PAGE/ subjected to SDS-PAGE and immunoblotting as described under “Ex- immunoblotting. B, time courseof the changes in IgE-dependent PGD, perimental Procedures.” A representative blot of three independentex- release after pretreatmentof BMMC with cytokines. After culture with periments is shown.C, expression of PGHS-2 mRNA. After 2 h of cul- the same combinations of cytokines depicted in A for the indicated ture, equal amounts of RNA (10 pg) extracted from the cellswere periods, BMMC were sensitized with 10 pg/ml monoclonal IgE antielectrophoresed in 1.2% formaldehyde-agarose gels and transfered to TNP and stimulated with TNP.BSA a s described under “Experimental Immobilon-N. The resulting blotswere sequentiallyanalyzedwith Procedures.” A sample of the supernatant was assayed for PGD, by cDNA probes for PGHS-2 and p-actin, as described under ”Experimen- radioimmunoassay. Values represent means S.E. of four independent of experiments. Inset, time course of the changes in the expression of tal Procedures.”Arepresentative blot (inset) and the relative amount each transcript estimated by quantitating the associated radioactivity PGHS-1 mRNAand protein in BMMC treated withKL + ILlO + IL-lp. with a Betascope 603 Blot Analyzerare shown (means= S.E., n = 4). A representative blotof four independent experiments is shown.

the greatest abundance of translated protein a t 5 h and production of PGD, via the terminalenzyme PGD, synthase a t 10 h also provided the highest steady-state levels of the mRNA transcript a t 2 h. The time intervals selected to illustrate thestimulatory actions of the cytokine combinations depicted in Fig. 1were based upon preliminary kinetic studies. Kinetic experimentswith BMMC cultured inKL + IL-10 + IL-1P revealed that PGD, was detected after 2 h of culture, increased linearly to a maximum by 10 h, and then declined (Fig. 2 A ) . The half-life of a trace amount of radiolabeled PGD, added to the culturemedium was about 12 hand was not influenced by the presence of cytokines (data not shown). Culture of BMMC with KL + IL-10 + IL-1P resulted in the induction of PGHS-2 mRNA, which reached a maximum a t 2 h (a 170-fold increase compared with starting BMMC), declined rapidly thereafter, and was undetectable by 10 h (Fig. 2A, Inset). The transient induction of PGHS-2 mRNA was followed by the transient expression of PGHS-2 protein, which was detectable by 2 h, reached a maximum a t 5-10 h, and declined rapidly thereafter. The cells exhibited similar kinetics after treatmentwith KL + IL-10 in theabsence of IL-lP, although the amountsof PGHS-2 mRNA and protein produced

were less than those produced in the presence of IL-1P (data not shown). The deDendence of PGD, generation and PGHS-2 expression on the cokentration of aiindividual cytokine (KL, IL-10, or IL-1P) was assessed in BMMC cultured for 5 h, when PGHS-2 expression was maximum, with constant concentrations of two cytokines, and varying concentrations of the third(Fig. 3).The production of PGD, over the initial 5-h stimulation period depended on the concentrations of each cytokine and increased in association with the incremental expression of immunoreactive PGHS-2 protein. The expression of PGHS-1 protein did not change during the 5-h culture. ZgE-dependent PGDz Generation-To assess whether induction of PGHS-2 was associated with a change in IgE-dependent PGD, generation, replicate cells cultured with various cytokines were sensitized with IgE and activated with haptenspecific antigen for 10 min. IgE-dependent PGD, synthesis did not increase significantly during the initial 10-h culture, and IgE-dependent PGD, generation was significantly enhanced only after 24 h of culture with KL, particularly in thepresence of IL-10 (Fig. 2 B ) . IL-lP had no appreciable effect on KL + IL-10-primed enhancement of IgE-dependent PGD, generation

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Stimulus Coupling of PGHS-1 and PGHS-2

FIG. 3. Effects on PGD, generation and expression of both PGHS-2 and PGHS-1 proteins by culture of BMMC with various concentrations of one cytokine, KL, IL-10, or IL-lf?, with fixed concentrations of the other two cytokines. Top, after BMMC were culturedwithvariousconcentrations of the indicatedcytokines for 5 h, a sample of thesupernatantwasassayed for PGD, by radioimmunoassay. Values represent means -c S.E. of four independent experiments. ~ o t t o m , remaining cells were lysed and analyzed for PGHS-1 or PGHS-2 by SDS-PAGE/immunoblotting. shown. representative is Ablot

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(Fig. 2B). The amounts of PGD, generated by BMMC stimulated with IgE and antigen after h of culture 48 withIL-3 alone, with KL alone, and with KL + IL-10 were 4.7 2 0.7, 16.3 2 5.6 ( p < 0.001 versus IL-3 alone, n = 41, and 43.0 2 3.0 ( p < 0.001 versus IL-3 alone andKL alone, n = 4) ng/lOficells, respectively. The increase in IgE-dependent PGD, generation by BMMC cultured with KL + IL-10 + IL-1p was accompanied by a timedependent increase in both mRNA and protein for PGHS-1 (Fig. 2B, inset). The steady-state levels of PGHS-1 mRNA increased about 15-fold after 5 h of culture and then declined to a plateau with about a 6-fold higher steady-state level than that in BMMC cultured in IL-3 alone. PGHS-1 protein was constant during thefirst 5 h, and then increasedprogressively to 48 h (Fig. 2B, inset). The changes in PGHS-1 mRNA and protein were nearly identical after treatmentof the cells with KL + IL-10 in the absence of IL-lp (data not shown). It is noteworthy that there wasno significant enhancement of IgEdependent PGD, generation during 5-10 h of culture with KL + IL-10 + IL-lp, the interval duringwhich PGHS-2 was maximally expressed and cytokine-induced spontaneous PGD, generation was at the maximal rate(Fig. 2 A ) . Furthermore, when BMMC were cultured in KL +IL-10 + IL-1p for 48 h so that PGHS-1 was maximally induced and PGHS-2 expression was undetectable,therewas no appreciablecytokine-dependent PGD, release (