©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
c-Kit Ligand Mediates Increased Expression of Cytosolic Phospholipase A, Prostaglandin Endoperoxide Synthase-1, and Hematopoietic Prostaglandin D Synthase and Increased IgE-dependent Prostaglandin D Generation in Immature Mouse Mast Cells (*)

(Received for publication, August 11, 1994; and in revised form, October 4, 1994)

Makoto Murakami (§) Ryoji Matsumoto Yoshihiro Urade (1) K. Frank Austen Jonathan P. Arm (¶)

From the Department of Medicine, Harvard Medical School, and the Department of Rheumatology and Immunology, Brigham and Women's Hospital, Boston, Massachusetts 02115, Osaka Bioscience Institute, Osaka 565, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have examined the cytokine regulation of IgEdependent prostaglandin (PG) D(2) generation in mouse mast cells by assessing the changes in the levels of the transcript, translated protein, and activity of the enzymes involved in the synthesis of PGD(2) from endogenous arachidonic acid. When mouse mast cells, derived by culture of bone marrow cells with WEHI-3 cell-conditioned medium as a source of interleukin (IL)-3 (BMMC), were cultured in recombinant c-kit ligand (KL), sensitized with IgE, and stimulated with antigen, PGD(2) generation increased 3-fold; when KL was combined with IL-3, IL-9, or IL-10, PGD(2) generation increased 6-8-fold above that produced by the cells cultured in IL-3 alone. The increased IgE-dependent PGD(2) generation by BMMC was apparent after 1 day of culture, reached a maximum after 2-4 days of culture, and was dose-dependent for KL and for each of the accessory cytokines. IgE-dependent generation of leukotriene C(4) increased 2-fold after the cells were cultured with KL and was not increased by the addition of IL-3, IL-9, or IL-10. Assays for steady-state transcripts by RNA blotting, for protein by SDS-PAGE/immunoblotting, and for function by enzymatic activities revealed that KL alone stimulated the increased expression of cytosolic phospholipase A(2) (cPLA(2)), prostaglandin endoperoxide synthase (PGHS)-1, and the terminal enzyme, hematopoietic PGD(2) synthase, without a change in expression of 5-lipoxygenase. IL-3, IL-9, and IL-10 each enhanced the KL-induced expression of PGHS-1. In contrast, transcripts for PGHS-2, which were detected transiently after the cells had been cultured for 5 h in KL + IL-3, were not expressed during the period of subsequent increase in IgE-dependent PGD(2) generation. These findings demonstrate that KL up-regulates expression of cPLA(2), PGHS-1, and hematopoietic PGD(2) synthase, leading to a relatively selective increase in IgE-dependent production of PGD(2) from endogenously released arachidonic acid in BMMC, and they provide the first example of cytokine regulation of hematopoietic PGD(2) synthase.


INTRODUCTION

Mast cells are highly specialized effector cells of the immune system that, when activated, release diverse types of biologically active molecules including amines, proteoglycans, proteases, eicosanoids, platelet-activating factor, and cytokines(1, 2, 3, 4) . There are at least two distinct populations of mast cells in mice, connective tissue mast cells (CTMC) (^1)and mucosal mast cells (MMC). Bone marrow-derived mast cells developed in WEHI-3 cell-conditioned medium as a source of interleukin (IL)-3 (BMMC) represent a relatively immature population of mast cells that reconstitutes both CTMC and MMC in mast cell-deficient mice of the WBB6F1/J-W/W^v strain(5) . Serosal mast cells, generally studied as a CTMC surrogate, respond to Fc receptor I (FcRI)-dependent activation with preferred generation of the cyclooxygenase pathway product, prostaglandin (PG) D(2), whereas rat MMC and mouse BMMC generate leukotriene (LT) C(4) via the 5-lipoxygenase pathway in preference to PGD(2)(1, 6, 7, 8) .

The initial step in arachidonic acid metabolism is the release of free arachidonic acid from cell membrane phospholipids by cytosolic phospholipase A(2) (cPLA(2)), which is activated by translocation from the cytosol to a cell membrane compartment in response to an increase in cytoplasmic Ca concentration (9, 10, 11) . cPLA(2), which is ubiquitously and constitutively expressed in mammalian cells, undergoes increased expression during cellular responses to several cytokines(12, 13, 14, 15) . Prostaglandin endoperoxide synthase (PGHS, or cyclooxygenase), which occurs in two isoforms, catalyzes the oxygenation of arachidonic acid to PGG(2), which is reduced to PGH(2) by the hydroperoxidase activity of the same enzyme(16) . PGHS-1 is constitutively expressed in a wide range of cells and tissues(17, 18, 19) , whereas PGHS-2 is induced in response to growth factors and proinflammatory cytokines(20, 21, 22, 23, 24, 25, 26) . PGH(2) is metabolized by specific synthases, each of which has a restricted distribution, to individual prostanoids. PGD(2) synthases, which catalyze the conversion of PGH(2) to PGD(2), exist in two forms, each with a molecular mass of 26 kDa(27, 28) . The brain enzyme is glutathione (GSH)-independent(27) , whereas the hematopoietic enzyme, first described in rat spleen, is GSH-dependent(28, 29) . The presence of hematopoietic PGD(2) synthase in rat CTMC was shown by GSH dependence and immunochemical identity(30) . 5-Lipoxygenase, which is activated by a Ca-dependent translocation to the perinuclear membrane(31) , catalyzes the sequential metabolism of arachidonic acid to 5-hydroperoxyeicosatetraenoic acid and then to LTA(4)(32, 33) . An 18-kDa perinuclear membrane protein, termed 5-lipoxygenase activating protein, presents arachidonic acid to 5-lipoxygenase(31, 34, 35) . LTA(4) is processed to LTC(4) by a microsomal LTC(4) synthase with restricted substrate specificity(36, 37) .

The tissue-specific elements that direct the mast cell phenotype to respond to IgE-mediated activation with preferential generation of PGD(2) or LTC(4) are unknown. Only two cytokines, c-kit ligand (KL) and IL-3, develop and/or maintain nontransformed BMMC in vitro. We now report that one of them, KL, a stromal cytokine that also regulates a variety of mast cell functions(38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56) , primes BMMC for increased FcRI-mediated production of PGD(2) in a dose- and time-dependent manner. KL primes the cells by eliciting increases in steady-state transcription, immunodetected protein, and function of each enzyme (cPLA(2), PGHS-1, and hematopoietic PGD(2) synthase) in the post-receptor biosynthetic pathway to PGD(2) generation after IgE-dependent activation of the cells. IL-9 and IL-10, cytokines known to act as accessory growth factors for mast cells(48, 57, 58) , as well as IL-3, all augment the priming effect of KL but only through a further increase in the expression of PGHS-1. The up-regulation of hematopoietic PGD(2) synthase in BMMC by KL may also contribute to a mechanism for the preferred metabolism of endogenous arachidonic acid to PGD(2) in CTMC.


EXPERIMENTAL PROCEDURES

Materials

Recombinant mouse IL-3 was purchased from Genzyme (Boston, MA), and recombinant mouse IL-9 (57) was provided by C. Uyttenhove and J-C. Renauld of the Ludwig Institute for Cancer Research, Brussels, Belgium. Recombinant mouse KL was obtained from culture supernatants of COS-7 cells (American Type Culture Collection, Rockville, MD) transfected with plasmid (pCDNA1) containing a cDNA encoding the mouse soluble form of KL (J. Flanagan, Harvard Medical School, Boston, MA)(59) . Briefly, COS-7 cells (1 times 10^7 cells) were mixed with 30 µg of plasmid DNA in RPMI 1640 medium and subjected to electroporation (200 V, 960 microfarads) (Bio-Rad). The cells were seeded at 2.5 times 10^6 cells/75-cm^2 flask (Corning, Inc.) in Dulbecco's modified Eagle's medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal calf serum (Life Technologies, Inc.) and cultured for 4 days; the supernatants then were collected. Recombinant mouse IL-10 (58) was obtained from the supernatant of COS-7 cells transfected with plasmid (pCD-SRalpha) containing a cDNA encoding mouse IL-10 (DNAX Research Institute, Palo Alto, CA). Alternatively, KL, IL-9, and IL-10 were acquired through expression in baculovirus(60) . Each cDNA was inserted into pVL1393 (Pharmigen, San Diego, CA). The recombinant plasmid (2 µg) was co-transfected with Baculo Gold Linearized Baculovirus DNA (Pharmigen) into 3 times 10^6 Sf9 cells (Invitrogen, San Diego, CA) with Ca-phosphate. The cells were cultured at 27 °C in Grace's insect medium (Invitrogen) supplemented with lactalbumin hydrolysate, yeastolate, and 10% fetal calf serum. After 7 days, the recombinant virus in the supernatant was selected by plaque assay and amplified. Sf9 cells (1 times 10^6 cells/ml) were infected with the recombinant virus and cultured for 7 days. The concentrations of KL in the COS-7 and insect cell supernatants were determined by [^3H]thymidine (Amersham Corp.) incorporation into BMMC, with authentic purified recombinant KL (Immunex, Seattle, WA) used as a standard. The concentrations of IL-9 or IL-10 in the COS-7 cell supernatants or the Sf9 cell supernatants were determined by [^3H]thymidine incorporation into BMMC cultured in the co-presence of IL-3 with authentic recombinant IL-9 (Ludwig Institute for Cancer Research and the University Catholique de Louvainm) and IL-10 (DNAX) used as standards(61, 62) .

Rabbit antiserum to human cPLA(2), which cross-reacts with mouse cPLA(2), and a human cPLA(2) cDNA (10) were provided by J. D. Clark, Genetics Institute, Cambridge, MA. Rabbit antiserum to sheep PGHS-1 was provided by W. L. Smith, Michigan State University, East Lansing, MI. Affinity-purified rabbit polyclonal antibody to mouse PGHS-2 was purchased from Cayman Chemical, Ann Arbor, MI. Mouse PGHS-1 (17) and PGHS-2 (21, 23) cDNA probes were provided by J. Trzaskos, Merck DuPont, Wilmington, DE. Rabbit antiserum to rat PGD(2) synthase has been described previously (28, 29, 30) . The specificity of this antibody has previously been demonstrated by immunoblotting after one- and two-dimensional electrophoresis of crude rat spleen lysates(28, 29, 30) . Although hematopoietic PGD(2) synthase has GSH-S-transferase activity and its N-terminal amino acid sequence has homology to various GSH-S-transferase isozymes (29) , the antibody does not cross-react with other GSH-S-transferase isozymes or with the brain form of PGD(2) synthase(28) . Rabbit antiserum to human 5-lipoxygenase and a human 5-lipoxygenase cDNA (33) were provided by J. F. Evans, Merck Frosst, Quebec, Canada.

Treatment of BMMC with Cytokines

Bone marrow cells from male BALB/cJ mice (Jackson Laboratory, Bar Harbor, ME) were cultured for 3-6 weeks in 50% enriched medium (RPMI 1640 containing 100 units/ml penicillin, 100 µg/ml streptomycin, 10 µg/ml gentamycin, 2 mML-glutamine, 0.1 mM nonessential amino acids, 50 µM 2-mercaptoethanol, and 10% fetal calf serum), 50% WEHI-3 cell (American Type Culture Collection) conditioned medium as described(2) . After 3 weeks, >97% of the cells in culture were BMMC as assessed by staining with toluidine blue or with alcian blue and safranin.

BMMC were washed once with enriched medium; resuspended at a cell density of 1 times 10^5 cells/ml in the enriched medium supplemented with 100 units/ml IL-3 alone, with 100 ng/ml KL alone, with 100 ng/ml KL in combination with 100 units/ml IL-3, 100 units/ml IL-9, or 10 units/ml IL-10, or with 50% WEHI-3 cell-conditioned medium; and then cultured for various periods. The concentrations of each cytokine chosen were those that gave maximal priming of IgE-dependent lipid mediator generation and release. After 7 days of culture, the number of mast cells increased 4.9 ± 1.6-fold (mean ± S.E., n = 6) with KL alone, 3.7 ± 0.9-fold (n = 6) with IL-3 alone, and 5.3 ± 0.7-fold (n = 3) with 50% WEHI-3 cell-conditioned medium. During 7 days of culture with KL in combination with IL-3, IL-9, or IL-10, the number of BMMC increased 22.5 ± 5.5-fold (n = 6), 13.1 ± 1.0-fold (n = 5), and 6.7 ± 0.9-fold (n = 4), respectively. When BMMC were cultured in cytokines for more than 2 days, their density was adjusted to 1 times 10^5 cells/ml 2 days before harvest; the cell density when harvested ranged from 2 to 3 times 10^5 cells/ml.

IgE-dependent Activation of BMMC

BMMC were suspended at a concentration of 1 times 10^7 cells/ml in enriched medium supplemented with cytokines and were sensitized with 10 µg/ml monoclonal IgE anti-trinitrophenyl (TNP) for 30 min at 37 °C. After being washed with Tyrode's buffer containing 1.8 mM Ca, 0.2 mM Mg, 0.1% (w/v) gelatin (Sigma) and 10 mM HEPES (Sigma), pH 7.2 (Tyrode's gelatin buffer) twice, the cells were resuspended in Tyrode's gelatin buffer at a concentration of 5 times 10^6 cells/ml. The cells were stimulated at 37 °C with 10 ng/ml TNP-conjugated bovine serum albumin, a concentration shown in preliminary experiments to be on a plateau for IgE-dependent activation of each population of BMMC, as defined by both release of beta-hexosaminidase and generation of eicosanoids. After 10 min, the reaction was stopped by centrifugation of the cells at 120 times g for 5 min at 4 °C, and the supernatants were retained for assay of mediator release(8) . The cell pellets were suspended in Tyrode's gelatin buffer and disrupted by freeze-thawing. beta-Hexosaminidase, a marker of mast cell degranulation, was quantitated by spectrophotometric analysis of the hydrolysis of p-nitrophenyl-beta-D-2-acetamido-2-deoxyglucopyranoside (63) . The percent release of beta-hexosaminidase was calculated by the formula [S/(S + P)] times 100, where S and P are the beta-hexosaminidase contents of equal portions of supernatant and cell pellet, respectively. PGD(2) and LTC(4) were measured by radioimmunoassay according to the manufacturer's instructions (Amersham).

RNA Blot Analysis

Total cellular RNA was extracted in guanidinium thiocyanate with TRI-Reagent (Molecular Research Center, Cincinnati, OH) (64) according to the manufacturer's instructions, and was quantitated by measurement of its optical density at 260 nm. Approximately equal amounts of total RNA were applied to each lane of 1.2% formaldehyde-agarose gels, electrophoresed, and transferred to Immobilon-N (Millipore, Bedford, MA). The resulting blots were sequentially analyzed with cDNA probes for cPLA(2), PGHS-1, PGHS-2, 5-lipoxygenase, and beta-actin that were labeled (Megaprime; Amersham) with [P]dCTP (3,000 Ci/mmol; DuPont NEN, Boston, MA) by random priming. All hybridizations were performed at 43 °C for 24 h in 50% formamide (Life Technologies, Inc.), 0.75 M NaCl, 75 mM sodium citrate, 0.1% SDS, 1 mM EDTA, 10 mM sodium phosphate, pH 6.8, 2 times Denhardt's solution, 10% dextran sulfate, and 100 µg/ml salmon sperm DNA (Sigma). The RNA blots were washed twice with 150 mM NaCl, 15 mM sodium citrate, 1 mM EDTA, 0.2% SDS, and 10 mM sodium phosphate, pH 6.8, for 5 min each followed by two washes at 65 °C in 30 mM NaCl, 3 mM sodium citrate, 1 mM EDTA, 0.2% SDS, and 10 mM sodium phosphate, pH 6.8, for 15 min each. The blots were visualized by autoradiography with Kodak XAR-5 film and an intensifying screen at -80 °C. The relative amount of each transcript was estimated by quantitating the associated radioactivity with a Betascope 603 Blot Analyzer (Betagen, Waltham, MA). The fold-increase in steady-state mRNA was calculated as the ratio of radioactivity associated with a specific transcript in treated cells compared with that in untreated cells and was corrected for changes in steady-state levels of beta-actin transcript to adjust for differences in loading between lanes.

SDS-PAGE/Immunoblot Analysis

BMMC cultured with each cytokine were washed once with 10 mM phosphate buffer, pH 7.2, containing 140 mM NaCl and 2 mM KCl (phosphate-buffered saline) and then lysed in 50 mM Tris-HCl, pH 7.4, 0.1% SDS, 0.5% Nonidet P-40 (Boehringer Mannheim), 5 mM Na(3)VO(4) (Sigma), 50 µg/ml leupeptin (Sigma), 1.5 µM pepstatin A (Sigma), and 1 mM phenylmethylsulfonyl fluoride (Sigma). A portion of the lysate was applied to SDS-polyacrylamide gels (Schleicher & Schuell) and electrophoresed. The separated proteins were electroblotted onto nitrocellulose membranes (Bio-Rad) in a transfer buffer consisting of 20 mM Tris, 150 mM glycine, and 20% methanol with a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) at 250 V for 1.5 h. The membranes were then sequentially treated with the following: 5% non-fat milk in 10 mM Tris-HCl, pH 7.4, containing 150 mM NaCl and 0.1% Tween 20 (Bio-Rad) (TBS-Tween) for 1 h; antibodies against cPLA(2), PGHS-1, PGHS-2, hematopoietic PGD(2) synthase, or 5-lipoxygenase at a dilution of 1:2,000, 1:50,000, 1:2,000, 1:2,000, and 1:5,000, respectively, in TBS-Tween for 1 h; TBS-Tween for three washes; and horseradish peroxidase-conjugated goat anti-rabbit IgG (Amersham) (1:2,000 dilution) in TBS-Tween for 1 h. After 5 washes, the protein bands were visualized with a chemiluminescent technique using an enhanced chemiluminesence (ECL) Western blot analysis system (Amersham).

Measurement of Enzyme Activities

BMMC cultured with each cytokine were washed once with phosphate-buffered saline; resuspended in phosphate-buffered saline containing 50 µg/ml leupeptin, 1.5 µM pepstatin A, and 1 mM phenylmethylsulfonyl fluoride at a concentration of 1 times 10^7 cells/ml; and then sonicated on ice for 1 min pulses (60% work cycle, setting 6) with a Branson Sonifier (Branson Sonic Power Co., Danbury, CT). The resulting cells lysates were assayed for cPLA(2), PGHS, and PGD(2) synthase activities.

cPLA(2) activity was assessed by the hydrolysis of 1-acyl-2-[^14C]arachidonoyl-phosphatidylcholine (30 µCi/µmol) (Amersham) to liberate [^14C]arachidonic acid as described previously(65) . Briefly, a 50-µl sample of the BMMC lysate was adjusted to a final volume of 125 µl containing 4 mM CaCl(2), 100 mM Tris-HCl, pH 8.0, 1 mM dithiothreitol (Sigma), and 10 µM 1-acyl-2-[^14C]arachidonoyl-phosphatidylcholine, and was incubated for 30 min at 37 °C. Dithiothreitol was included to inhibit the activity of secretory group II PLA(2)(9) . The reaction was stopped by the addition of 625 µl of Dole's reagent (65) . Free [^14C]arachidonic acid was extracted in n-heptane and counted in a liquid beta-scintillation counter (Beckman, Palo Alto, CA).

PGHS activity was measured by the conversion of [^3H]arachidonic acid (100 mCi/µmol) (Amersham) to [^3H]PGH(2). A sample of the cell lysate was adjusted to 100 µl containing 100 mM Tris-HCl, pH 8.0, 1 mM phenol (Sigma), 2 µM hematin (Oxford Biochemical Research, Oxford, MI), 1 mMp-chloromercuribenzenesulfonic acid (Sigma), and 20 µM [^3H]arachidonic acid. The sample was incubated for 2 min at 25 °C, and the reaction was stopped by the addition of 300 µl of diethyl ether, methanol, 1 M citric acid (30:4:1, v/v) precooled to -20 °C. A 100-µl portion of the ether phase was separated by thin layer chromatography at -20 °C with a solvent system of diethyl ether/methanol/acetic acid (90:2:0.1, v/v). The zone on the silica gel corresponding to PGH(2) was determined by comparison with the mobility of synthetic standards. The PGH(2) zone and the remaining zones were scraped into vials, and the radioactivity was counted in a liquid scintillation counter. PGH(2) formation was calculated from the ratio of the radioactivity in the PGH(2) zone to the total radioactivity recovered, with the specific activity of the substrate known(28) .

PGD(2) synthase was assayed by measuring the conversion of [^3H]PGH(2) to [^3H]PGD(2). [^3H]PGH(2) was obtained as described (28) by incubating 20 µM [^3H]arachidonic acid (100 mCi/µmol) with 250 units/ml sheep PGHS-1 (Oxford Biochemical Research) in 100 mM Tris-HCl, pH 8.0, containing 1 mM phenol, 2 µM hematin and 1 mMp-chloromercuribenzenesulfonic acid for 2 min at 25 °C. [^3H]PGH(2) was extracted by adding diethyl ether, methanol, 1 M citric acid (30:4:1, v/v). The solvent was evaporated, and [^3H]PGH(2) was dissolved in diethylene glycol dimethyl ether. The final concentration of [^3H]PGH(2) was calculated by measurement of radioactivity in a liquid beta-scintillation counter under the assumption that its specific radioactivity was the same as that of the arachidonic acid used as substrate. A portion of the cell lysate was centrifuged for 1 h at 100,000 times g at 4 °C to separate soluble and particulate fractions. The 100,000 times g pellet was reconstituted in an equal amount of lysis buffer. Equal volumes of total cell lysate, the 100,000 times g supernatant, and the 100,000 times g pellet were incubated for 90 s at 25 °C with 20 µM [^3H]PGH(2) in 100 mM Tris-HCl, pH 8.0, with and without 1 mM GSH (Sigma). Products were extracted in diethyl ether, methanol, 1 M citric acid (30:4:1, v/v), and separated on thin layer chromatography plates, and PGD(2) formation was calculated from the ratio of the radioactivity in the PGD(2) zone to the total radioactivity recovered(28) . Arachidonic acid, PGB(2), PGH(2), PGD(2), and PGE(2) used as synthetic thin layer chromatography standards were purchased from Cayman Chemical.

The activity of each enzyme was expressed as picomoles of substrate converted to product/min of reaction time per million cell equivalents, and was calculated by measurement of the radioactivity associated with the final product, with the specific activity of the substrate being known.


RESULTS

IgE and Antigen-dependent Mediator Release from BMMC

The time course of the differences in mediator release from BMMC cultured with various combinations of cytokines and then sensitized with IgE and activated with antigen is shown in Fig. 1. A progressive reduction occurred in beta-hexosaminidase exocytosis from BMMC cultured with KL, KL + IL-9, and KL + IL-10 over 7 days of culture, but there was no appreciable change in beta-hexosaminidase release from BMMC cultured with IL-3 alone or with KL + IL-3. PGD(2) synthesis by BMMC primed with KL alone increased significantly at 1 day, reached a maximum at 2-4 days and increased further in the presence of IL-3, IL-9, or IL-10. In contrast, continued culture of BMMC in IL-3 did not lead to increased IgE-dependent generation of PGD(2) at any point. IgE-dependent generation of LTC(4) increased less than 2-fold to a maximum by 1-2 days and then declined after culture of BMMC with KL and its combinations. There was no change in LTC(4) generation by BMMC treated with IL-3 as a negative control.


Figure 1: Time course of the differences in mediator release from BMMC sensitized with IgE and activated with antigen after treatment with various cytokines. BMMC were cultured for the indicated periods with 100 units/ml IL-3 (open squares), 100 ng/ml KL (closed squares), 100 ng/ml KL + 100 units/ml IL-3 (closed circles), 100 ng/ml KL + 100 units/ml IL-9 (closed triangles), or 100 ng/ml KL + 10 units/ml IL-10 (open circles). Cells were then sensitized with IgE anti-TNP and activated with TNP-bovine serum albumin for 10 min as described under ``Experimental Procedures.'' Portions of the supernatants of the stimulated cells were assayed for their content of beta-hexosaminidase, PGD(2), and LTC(4). beta-Hexosaminidase was also quantitated in pellets after freeze-thawing, and the net percent release of beta-hexosaminidase is shown. Values represent means ± S.E. of seven independent experiments. Beta-HEX, beta-hexosaminidase.



The dependence of priming for mediator release on the concentration of KL was examined after 2 days of culture (Fig. 2). A full dose-dependent enhancement of PGD(2) generation was demonstrated with KL from 0 to 25 ng/ml in the presence of IL-3. BMMC cultured for 2 days with KL alone at concentrations below 25 ng/ml KL, or with IL-9 or IL-10 at concentrations below 6 ng/ml KL did not consistently maintain viability. Thus, the dose-dependent effect of KL was limited over those doses that were evaluated in combination with either IL-9 or IL-10, and KL alone had no further effect over the threshold dose for viability. LTC(4) generation after 2 days of culture was also maximum with 25 ng/ml KL alone and increased less than 2-fold with or without accessory cytokines (Fig. 2). The reduction of beta-hexosaminidase release in BMMC cultured with KL, KL + IL-9, or KL + IL-10 relative to cells cultured with KL + IL-3 was not dependent on the concentration of KL (Fig. 2).


Figure 2: Dose-dependent effect of KL on IgE-dependent mediator release from BMMC after 2 days of culture. BMMC were cultured for 2 days with various concentrations of KL in the absence (closed squares) or presence of 100 units/ml IL-3 (closed circles), 100 units/ml IL-9 (closed triangles), or 10 units/ml IL-10 (open circles), and then sensitized with IgE anti-TNP and activated with TNP-bovine serum albumin. Portions of the supernatants of the stimulated cells were assayed for their content of beta-hexosaminidase, PGD(2), and LTC(4). beta-Hexosaminidase was also quantitated in the cell pellets, and net percent release of beta-hexosaminidase is shown. Values represent means ± S.E. of four independent experiments. Beta-HEX, beta-hexosaminidase.



The maximal generation of both PGD(2) and LTC(4) by BMMC in response to IgE and antigen after culture for 2 days with KL alone and KL with various accessory cytokines increased significantly relative to culture with IL-3 alone, with a preferential fold increase in PGD(2) relative to LTC(4) for each cytokine combination (Table 1).



Expression of Enzymes Involved in Arachidonic Acid Metabolism

The expression by BMMC of cPLA(2), PGHS-1, PGHS-2, hematopoietic PGD(2) synthase, and 5-lipoxygenase after 2 days of culture with the various combinations of cytokines was examined by SDS-PAGE/immunoblotting with specific antibodies and by RNA blotting with specific probes. The expression of cPLA(2) protein (Fig. 3A) increased after 2 days of culture of BMMC with KL alone, relative to culture with IL-3 alone, and did not increase further when KL was combined with IL-3, IL-9, or IL-10. Increased expression of cPLA(2) protein was accompanied by minimal to modest increases in steady-state levels of its 3.4-kilobase transcript (Fig. 3B). The expression of PGHS-1 also increased after 2 days of culture with KL alone relative to culture with IL-3 alone, and increased further when KL was combined with IL-3, IL-9, or IL-10 (Fig. 3A). The increase in PGHS-1 protein was accompanied by increased steady-state levels of the 2.8-kilobase PGHS-1 transcript in BMMC cultured with KL alone (1.8 ± 0.4-fold, mean ± S.E., n = 3, p < 0.05 versus IL-3 alone); these levels increased further when KL was combined with IL-3 (4.7 ± 0.4-fold, n = 3, p < 0.01 versus IL-3, p < 0.05 versus KL), IL-9 (3.0 ± 0.5-fold, n = 3, p < 0.01 versus IL-3, p < 0.05 versus KL), or IL-10 (6.6 ± 1.8-fold, n = 4, p < 0.01 versus IL-3, p < 0.001 versus KL) (Fig. 3B). PGHS-2 expression could not be detected by immunoblotting (Fig. 3A) or by RNA blotting (data not shown) after 2 days of culture. The expression of mouse hematopoietic PGD(2) synthase in BMMC at 2 days was detected by co-migration using an antibody raised against the rat enzyme, which recognized a 26-kDa protein in rat mastocytoma RBL-2H3 cells (Fig. 3A). This protein was more abundant at 2 days in BMMC cultured with KL than in those maintained with IL-3 (Fig. 3A), and its expression was not increased further when KL was combined with IL-3, IL-9, or IL-10. Thus, KL alone or in combination with IL-3, IL-9, or IL-10 induced the expression of cPLA(2), PGHS-1, and hematopoietic PGD(2) synthase and primed BMMC for IgE-dependent PGD(2) generation. IL-3, IL-9, and IL-10 enhanced the KL-dependent increase in PGHS-1 expression and the KL-primed IgE-dependent PGD(2) generation, but did not alter the KL-induced expression of cPLA(2) and hematopoietic PGD(2) synthase. The expression of 5-lipoxygenase during cell culture with KL in the presence or absence of the accessory cytokines was unchanged relative to culture in IL-3 alone, as assessed by SDS-PAGE/immnoblotting (Fig. 3A) and RNA blotting (Fig. 3B).


Figure 3: Expression of the enzymes involved in the metabolism of arachidonic acid to PGD(2), assessed by SDS-PAGE/immunoblotting and RNA blotting, after treatment of BMMC for 2 days with various combinations of cytokines. A, effect of cytokines on expression of protein for each enzyme as visualized by SDS-PAGE/immunoblotting. Samples of cell lysates (10^5 cell equivalents for cPLA(2), PGHS-1, and 5-lipoxygenase (5-LO); 5 times 10^5 cell equivalents for PGHS-2; and 10^6 cell equivalents for hematopoietic PGD(2) synthase (PGDS)) were analyzed by SDS-PAGE/immunoblotting with antibodies specific for each enzyme, as described under ``Experimental Procedures.'' BMMC treated with 100 ng/ml KL and 10 units/ml IL-10 for 5 h were used as a positive control for PGHS-2. A representative result of at least three independent experiments is shown. B, effect of cytokines on the expression of steady-state transcripts of cPLA(2), PGHS-1, 5-lipoxygenase, and beta-actin in 10 µg of RNA. The blots were probed with P-labeled cDNAs encoding cPLA(2), PGHS-1, 5-lipoxygenase, and beta-actin, and were exposed to Kodak XAR-5 films for 7, 2, 5, and 1 days, respectively. A representative result of three independent experiments is shown.



The expression over time of cPLA(2), PGHS-1, PGHS-2, and hematopoietic PGD(2) synthase was studied in BMMC cultured with KL + IL-3 (Fig. 4). cPLA(2) protein increased modestly by 6 h, and reached a maximum by 2 days (Fig. 4A). After 4 days of culture, the levels of cPLA(2) protein decreased, although the levels after 7 days of culture were still higher than those in starting BMMC. There was a small (1.7 ± 0.2-fold on day 2, n = 3, p < 0.05 versus day 0) increase in steady-state levels of the 3.4-kilobase cPLA(2) transcript at day 2 which persisted to day 4 (Fig. 4B). The expression of PGHS-1 protein was increased 1 day after the start of the culture, was near maximum by day 2, and plateaued at 4-7 days (Fig. 4A), with a concomitant increase in steady-state levels of the 2.8-kilobase PGHS-1 transcript (Fig. 4B). The 4.8-kilobase PGHS-2 transcript was detected transiently in BMMC cultured with KL + IL-3 and was undetectable by 1 day (Fig. 4B). PGHS-2 protein was not detectable at any point (Fig. 4A). By comparison, PGHS-2 protein was detectable in BMMC cultured with KL + IL-10 after 5 h of culture (Fig. 4A). The level of immunoreactive PGD(2) synthase increased after 1 to 2 days of culture, and reached a maximum by 4 days in BMMC cultured with KL + IL-3 (Fig. 4A).


Figure 4: Time course of the expression of enzymes involved in the metabolism of arachidonic acid to PGD(2), assessed by SDS-PAGE/immunoblotting and RNA blotting, after treatment of BMMC with KL + IL-3. A, time-dependent changes in the expression of proteins as visualized by SDS-PAGE/immunoblotting. The same number of cell equivalents (10^5 for cPLA(2) and PGHS-1; 5 times 10^5 for PGHS-2; and 10^6 for hematopoietic PGD(2) synthase (PGDS)) were applied to each lane. A representative result of at least three independent experiments is shown. BMMC treated with 100 ng/ml KL and 10 units/ml IL-10 for 5 h and RBL-2H3 cells were used as positive controls for PGHS-2 and PGD(2) synthase, respectively. B, time-dependent changes in steady-state transcripts of cPLA(2), PGHS-1, PGHS-2, and beta-actin in 10 µg of RNA. The blots were probed with P-labeled cDNA for cPLA(2), PGHS-1, PGHS-2, and beta-actin, and were exposed to Kodak XAR-5 films for 7, 2, 7, and 1 days, respectively. A representative result of four independent experiments is shown.



The dependence of the induction of cPLA(2), PGHS-1, and PGD(2) synthase on the concentration of KL was examined after 2 days culture of BMMC with various concentrations of KL in the presence of IL-3. As assessed by both RNA blotting and immunoblotting, the expression of cPLA(2), PGHS-1, and PGD(2) synthase increased in a dose-dependent fashion, reaching a maximum at 25 ng/ml KL (data not shown).

Biochemical assays were performed to confirm that the increases in expression of immunoreactive enzymes were accompanied by functional enzymatic activity. cPLA(2) activity, measured by release of [^14C]arachidonic acid from 1-acyl-2-[^14C]arachidonoyl-phosphatidylcholine, was increased 2-3-fold after culture of BMMC with KL as compared with culture with IL-3 alone (Table 2). The addition of IL-3, IL-9, or IL-10 to the cells cultured with KL did not significantly enhance cPLA(2) activity. PGHS activity, determined by the conversion of [^3H]arachidonic acid to [^3H]PGH(2), increased approximately 2-fold in BMMC cultured with KL alone and increased 4-8-fold in BMMC cultured with KL in the presence of IL-3, IL-9, or IL-10 (Table 2). PGD(2) synthase activity in BMMC was assayed by quantitating the conversion of [^3H]PGH(2) to [^3H]PGD(2) and was further characterized by its GSH dependence and subcellular localization (Table 3). PGD(2) synthase activity increased more than 10-fold in BMMC cultured with KL + IL-3 compared to BMMC cultured with IL-3 alone and was absolutely dependent on the presence of GSH (Table 3). The GSH-dependent PGD(2) synthase activity of BMMC cultured with KL + IL-3 and of RBL-2H3 cells was confined to the 100,000 times g supernatant.






DISCUSSION

The cell- and tissue-derived factors that regulate arachidonic acid metabolism in mast cells toward IgE-dependent PGD(2) generation are unknown. Although previous studies showed that co-culture of BMMC with mouse 3T3 fibroblasts in the presence of WEHI-3 cell-conditioned medium resulted in granule maturation toward a CTMC phenotype, accompanied by augmented IgE-dependent PGD(2) generation(38, 39) , these studies did not investigate changes at individual enzymatic steps or the participation of particular cytokines. We have more recently reported on the capacity of a particular combination of cytokines, KL + IL-10 + IL-1beta, to elicit PGD(2) generation directly from BMMC and to subsequently prime the same cells for augmented IgE-dependent PGD(2) generation(66) . The cytokine-initiated PGD(2) generation was associated in time with the transient expression of PGHS-2, and studies with specific inhibitors of PGHS-2 confirmed the separate linking of this isoform with transmembrane activation by cytokines. The expression of PGHS-2 failed to prime IgE-dependent PGD(2) generation, which was considered to be linked to PGHS-1. There was no examination of the contribution of enzymes other than the PGHS isoforms to the priming event, and the profile of cytokines studied was limited. We now report that IL-3 and IL-9 can be substituted for IL-10 in the priming of BMMC for FcRI-mediated PGD(2) production. Of particular note is that priming by KL is associated with the increased expression of each enzyme in the post-receptor biosynthetic pathway for PGD(2) generation, namely cPLA(2), PGHS-1, and hematopoietic PGD(2) synthase, as assessed by RNA blot analysis (not assessed for hematopoietic PGD(2) synthase because the cDNA has not been cloned), SDS-PAGE/immunoblotting for protein and biochemical assays of enzymatic activity. Only the expression of PGHS-1 is increased further by the added presence of IL-3, IL-9, or IL-10.

When BMMC were cultured with KL alone, there was a 3-fold increase in PGD(2) generation in response to IgE sensitization and antigen stimulation (Table 1), which was maximal after 2-4 days (Fig. 1). BMMC cultured with KL in combination with IL-3, IL-9, or IL-10, which regulate the growth, differentiation, and maturation of mast cells(61, 62, 67, 68, 69) , exhibited a 6-8-fold increase in PGD(2) generation compared with BMMC maintained in IL-3 alone. Other cytokines, including IL-1beta, IL-6, tumor necrosis factor-alpha, interferon-, transforming growth factor-beta1, and nerve growth factor, had no effect alone or in combination with KL (data not shown), indicating that the accessory cytokines were selective. Priming of IgE-dependent PGD(2) generation by BMMC cultured with KL + IL-3 was dependent upon the concentration of KL with a plateau at 25 ng/ml (Fig. 2). Although the dose-dependent studies were limited when KL was used alone or in combination with IL-9 or IL-10 due to the loss of cell viability below the threshold requirement, maximal priming of PGD(2) generation was again observed at 25-50 ng/ml of KL. Priming of BMMC for PGD(2) synthesis is therefore distinct from the priming of human skin (54) and lung (55) mast cells for histamine release, which occurred within minutes of exposure to concentrations of KL below 1 ng/ml. Priming of IgE-dependent PGD(2) generation was also dependent upon the concentration of IL-3, IL-9, and IL-10 when the concentration of KL was fixed (data not shown). LTC(4) synthesis increased less than 2-fold during BMMC culture with KL and was not influenced by the presence of accessory cytokines. Thus, after 2 days culture with concentrations of each cytokine that were optimal for maximal IgE-dependent eicosanoid generation, the ratio of PGD(2)/LTC(4) synthesis increased about 2-fold to 0.36 in BMMC cultured with KL alone, as compared to 0.2 for those cultured with IL-3 alone, and increased about 3.5-fold to 0.68 in BMMC cultured with KL + IL-10 (Table 1).

In contrast to the IgE-mediated increase in PGD(2) and LTC(4) synthesis after BMMC were cultured in KL alone or with accessory cytokines, the same cells showed a progressive loss over days of their capacity to undergo IgE-mediated exocytosis (Fig. 1). Only cells cultured with KL + IL-3 or with IL-3 alone maintained their ability to degranulate substantially; however, the diminution of exocytosis with KL alone or combined with other cytokines was not dependent on the dose of KL (Fig. 2). Thus, diminished IgE-dependent degranulation appears to be due to an absence of IL-3. In contrast, the increase in IgE-dependent PGD(2) generation was dependent on both the concentration of KL (Fig. 2) and the concentration of accessory cytokines (data not shown). Thus, exocytosis and eicosanoid synthesis may be independently regulated in BMMC, possibly due to their dependence upon separate post-receptor pathways, and in certain circumstances degranulation may not be the optimal marker of mast cell activation.

In order to identify the biochemical steps leading to increased PGD(2) synthesis in cytokine-treated BMMC, the changes in expression of the individual enzymes involved in post-receptor metabolism of arachidonic acid to PGD(2) were assessed in terms of steady-state levels of mRNA, expressed protein, and activity of each enzyme. Although mast cells express both secretory group II PLA(2) and arachidonic acid-selective cPLA(2)(65) , cPLA(2) likely plays the dominant role in IgE-dependent lipid mediator production(70, 71) . SDS-PAGE/immunoblot analysis and assay of enzymatic activity demonstrated an increase in immunoreactive and functional cPLA(2) in KL-treated BMMC. Accessory cytokines did not enhance the effect of KL on the expression of mRNA and protein for cPLA(2) (Fig. 3) or on cPLA(2) activity (Table 2). The expression of cPLA(2) protein in BMMC reached a maximum after 2 days of culture (Fig. 4A) coincident with the plateau for increased IgE-dependent generation of PGD(2) (Fig. 1). Increased expression of cPLA(2) protein and function was accompanied by only minimal increases in cPLA(2) transcripts, suggesting significant post-transcriptional regulation of its expression in KL-treated BMMC. Increased expression of cPLA(2) in fibroblasts, HeLa cells, and monocytes in response to IL-1alpha, tumor necrosis factor alpha, or macrophage colony-stimulating factor was also linked to enhanced generation of eicosanoids after cell stimulation with a second agonist that raised the intracellular Ca concentration(12, 13, 14, 15, 70) . Macrophage colony-stimulating factor, which increases mRNA and functional protein for cPLA(2) in human monocytes(15) , and KL belong to the same family of growth factors based on homology between their own structures and between the structures of their respective tyrosine kinase receptors, c-fms and c-kit(72) .

With regard to the second step in PGD(2) synthesis, conversion of arachidonic acid to PGH(2), RNA blot analysis, SDS-PAGE/immunoblot analysis, and assay of PGHS enzymatic activity demonstrated that expression of PGHS-1 in BMMC cultured with KL increased and was further augmented by the addition of IL-3, IL-9, or IL-10. The increase in PGHS-1 transcript in BMMC cultured with KL + IL-3 was time-dependent, reaching a maximum (4-fold) at 1 day; it was followed by increased expression of PGHS-1 protein, which reached a maximum by 2 days (Fig. 4, A and B). Although PGHS-2 mRNA was transiently induced (Fig. 4B), immunoreactive PGHS-2 protein was not detectable (Fig. 4A). The lack of immunodetectable PGHS-2 in BMMC cultured with KL + IL-3 was due neither to methodologic error nor to unresponsiveness of BMMC as demonstrated by the expression of PGHS-2 protein after the cells were cultured for 5 h with KL + IL-10 (Fig. 4A) as we have shown (66) . Furthermore, the expression and disappearance of PGHS-2 steady-state mRNA preceded the augmented IgE-dependent PGD(2) synthesis. Investigations that evaluate PGHS activity by measuring the generation of the final product after supplying arachidonic acid to intact cells or cell homogenates reflect the combined activity of PGHS and the terminal enzyme for eicosanoid generation and do not recognize changes occurring in each enzyme separately. In the present study, both the time-dependent changes in PGHS-1 protein (Fig. 4A) and the increased expression of immunoreactive and functional PGHS-1 in response to accessory cytokines, acting only at the PGHS-1 step (Fig. 3A, Table 2), suggest that PGHS-1 plays a critical role in regulating antigen-dependent PGD(2) synthesis by IgE-sensitized BMMC. Thus, the constitutive isoform with presumptive physiologic functions(17, 18, 19) , rather than the inducible isoform with putative proinflammatory functions(20, 21, 22, 23, 24, 25, 26) , is the intermediate enzyme species in mast cell-dependent FcRI-mediated events in the microenvironment. These data also provide unequivocal evidence that cytokine priming of a cell for increased prostanoid synthesis by a particular transmembrane signal is linked to increased expression of PGHS-1 and not PGHS-2, and that the optimal cytokine combination for induction of PGHS-1 involves accessory cytokines that do not elicit further induction of expression of cPLA(2) and the terminal biosynthetic enzyme.

The final step in the generation of PGD(2) from arachidonic acid in mast cells is the metabolism of PGH(2) by the terminal enzyme, hematopoietic PGD(2) synthase(28, 29, 30) . The hematopoietic form of PGD(2) synthase, first described in rat spleen, is distinguished from the brain enzyme immunochemically and by its dependence on GSH(28) . Although the N-terminal amino acid sequence of rat hematopoietic PGD(2) synthase was shown to be similar to cytosolic GSH transferases(29) , its cDNA has not been cloned. There are no previous data on the cytokine regulation of the expression of this terminal enzyme of arachidonic acid metabolism. In the present study, an immunoreactive protein was recognized in BMMC that has the same mobility on SDS-PAGE as rat hematopoietic PGD(2) synthase from RBL-2H3 cells. Expression of this immunoreactive 26-kDa protein increased during cell culture in KL and was not affected by the co-presence of accessory cytokines (Fig. 3). Furthermore, the effect of KL was on a GSH-dependent cytosolic PGD(2) synthase in BMMC (Table 3). Thus, both immunochemical and biochemical criteria indicate that the hematopoietic form of PGD(2) synthase is present in mouse BMMC and its expression is increased by stimulation of the cells with KL. The time course of the increase in immunoreactive PGD(2) synthase protein (Fig. 4) was consistent with the change in the profile of IgE-dependent PGD(2) synthesis (Fig. 1), indicating that hematopoietic PGD(2) synthase may be a further regulatory step leading to increased IgE-dependent PGD(2) generation.

Priming of IgE-dependent PGD(2) generation after culture of BMMC with KL was accompanied by a less than 2-fold increase in LTC(4) generation, although the increment in LTC(4) generation was comparable to the increment in PGD(2) generation in absolute terms (Table 1). The increment in IgE and antigen-dependent LTC(4) generation was maximal by 1-2 days and was not affected by the addition of accessory cytokines (Fig. 1). Thus, the kinetics and the cytokine dependence of LTC(4) generation were similar to those of cPLA(2) protein expression. The expression of 5-lipoxygenase was unchanged by any of the culture conditions (Fig. 3). The possible contributions of other factor(s), such as 5-lipoxygenase activating protein and LTC(4) synthase, were not assessed. The human cDNA for LTC(4) synthase has recently been cloned(37) , but the mouse cDNA and antibodies are not yet available. It seems likely that increased expression of cPLA(2) contributes to the KL-primed increase in eicosanoid generation after cell sensitization with IgE and stimulation with antigen, without selectivity for either the cyclooxygenase or 5-lipoxygenase pathway.

The effect of cytokines on the differentiation and maturation of mast cells has previously been reported in terms of the expression of granule-associated markers. BMMC cultured with KL counterstain with safranin, synthesize heparin proteoglycan and more histamine(46) , and express transcripts for mMCP-4, a protease normally expressed in CTMC (47, 48) . The increase in IgE-dependent PGD(2) generation by KL is compatible with the notion that KL leads to maturation of BMMC toward a CTMC-like phenotype. However, although IL-3, IL-9, and IL-10 further augment KL-primed IgE-dependent generation of PGD(2), IL-3 suppresses KL-induced granule maturation in terms of the appearance of safranin-positive granules, heparin biosynthesis, and expression of mMCP-4(47) ; and IL-9 and IL-10 each induce the expression of the MMC-associated proteases mMCP-1 and -2(48, 67, 68, 69) . Thus, the precise combination of factors in the microenvironment that lead to complete maturation of BMMC to a CTMC phenotype has yet to be elucidated, and separate regulatory mechanisms may exist that govern the maturation of granule constituents as compared to those that regulate lipid mediator generation.

In conclusion, a detailed examination of steady-state mRNA and the expression of protein and enzymatic activity revealed that the priming of BMMC by KL for enhancement of IgE-dependent eicosanoid generation is a result of increased expression of several enzymes, including cPLA(2), PGHS-1, and hematopoietic PGD(2) synthase. The increase in expression of cPLA(2) contributes to the rise in IgE-dependent generation of both PGD(2) and LTC(4), whereas the increased expression of PGHS-1 and PGD(2) synthase contributes to the enhancement of IgE-dependent PGD(2) generation in preference to LTC(4) generation. The accessory cytokines, IL-3, IL-9, and IL-10, modulate IgE-dependent PGD(2) generation by augmenting the KL-induced expression and activity of PGHS-1. The expression of the terminal enzyme for eicosanoid biosynthesis, hematopoietic PGD(2) synthase, is up-regulated by KL but is not affected by any of the hematopoietic cytokines studied.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL36110, AI22531, AI31599, AI23483, and RR05950. 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.

§
Supported by an International Human Frontier Science Program postdoctoral training grant.

Supported by a Burroughs Wellcome Developing Investigator Award. To whom correspondence should be addressed: Dept. of Rheumatology and Immunology, Brigham and Women's Hospital, Harvard Medical School, 250 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1335; Fax: 617-432-0979.

(^1)
The abbreviations used are: CTMC, connective tissue mast cells; BMMC, bone marrow-derived mast cells; PG, prostaglandin; LT, leukotriene; KL, c-kit ligand; IL, interleukin; FcRI, Fc receptor type I; IgE, immunoglobulin E; cPLA(2), cytosolic phospholipase A(2); PGHS, prostaglandin endoperoxide synthase; MMC, mucosal mast cells; TNP, trinitrophenyl; GSH, glutathione; mMCP, mouse mast cell protease.


ACKNOWLEDGEMENTS

We thank C. Nwankwo (Harvard Medical School) for her technical assistance in preparing cDNA probes. We thank Drs. J. D. Clark, W. L. Smith, J. Trzaskos, and J. F. Evans for provision of antibodies and/or cDNA for cPLA(2), PGHS-1, PGHS-2, and 5-lipoxygenase, respectively. Mouse IL-10 and KL cDNA were generously provided by Drs. K. W. Moore (DNAX) and J. Flanagan, respectively. Mouse recombinant IL-9 and IL-9 cDNA were provided by Drs. C. Uyttenhove and J-C. Renauld.


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