©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interleukin-3 Regulates Development of the 5-Lipoxygenase/Leukotriene C Synthase Pathway in Mouse Mast Cells (*)

(Received for publication, July 5, 1995; and in revised form, August 8, 1995)

Makoto Murakami (§) K. Frank Austen Clifton O. Bingham , III Daniel S. Friend John F. Penrose (¶) 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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To study cytokine regulation of the 5-lipoxygenase (5-LO)/leukotriene (LT) synthase pathway we have developed mouse bone marrow-derived mast cells (BMMC) that minimally express each protein of the pathway by using a novel culture system, lacking interleukin (IL)-3. When mouse bone marrow cells were cultured for 5 weeks with 100 ng/ml c-kit ligand (KL) and 10 units/ml IL-10, a population of >95% mast cells was obtained. These cells generated 8.3 ± 4.5 ng of LTC(4)/10^6 cells and 8.1 ± 2.4 ng of prostaglandin (PG) D(2)/10^6 cells after IgE-dependent activation. When these BMMC were cultured for 2-5 weeks more with 100 units/ml IL-3 in the continued presence of KL and IL-10, the IgE-dependent generation of LTC(4) and PGD(2) increased to 212 ± 36 and 25.5 ± 8.6 ng/10^6 cells, respectively. The dramatic increase in the IgE-dependent generation of LTC(4) in response to IL-3 was accompanied by a concomitant increase in expression of 5-LO and 5-LO-activating protein and preceded the increased expression of cytosolic phospholipase A(2) and LTC(4) synthase. The recognition that IL-3 up-regulates the expression of each protein of the 5-LO pathway for the generation of LTC(4) contrasts with our recent finding that KL up-regulates the expression of cytosolic phospholipase A(2), prostaglandin endoperoxide synthase-1, and hematopoietic PGD(2) synthase and increases the IgE-dependent generation of PGD(2) in BMMC developed from bone marrow with IL-3. Thus, developmentally segregated regulation of the prostanoid and cysteinyl leukotriene pathways in lineage-related committed mast cell progenitors reveals the pleiotropism of this effector cell of allergic inflammation, a cytokine/growth factor basis for preferential expression of pathways of eicosanoid biosynthesis, and the particular role of IL-3 in regulating the expression of the proteins of the 5-LO/LTC(4) synthase pathway.


INTRODUCTION

There are at least two distinct populations of mast cells in rodents, connective tissue mast cells (CTMC) (^1)and mucosal mast cells (MMC), which are distinguished by different fixation and staining characteristics, different expression of certain secretory granule proteoglycans and neutral proteases, and preferential generation of certain eicosanoids from endogenous arachidonic acid released with cell activation(1) . Mouse and rat serosal mast cells, generally studied as a surrogate for CTMC, respond to IgE-dependent activation with preferred generation of the cyclooxygenase pathway product, prostaglandin (PG) D(2)(2) , whereas rat MMC generate leukotriene (LT) C(4) via the 5-lipoxygenase (5-LO) pathway in preference to PGD(2)(3) . Mouse bone marrow-derived mast cells (BMMC) developed in WEHI-3 cell-conditioned medium as a source of interleukin (IL)-3 represent a relatively immature population of committed mast cell progenitors that reconstitutes both CTMC and MMC in mast cell-deficient mice of the WBB6F1/J-W/W strain (4) . When these committed mast cell progenitors are maintained in IL-3 and activated by perturbation of their high affinity IgE receptor, FcRI, they generate LTC(4) in marked preference to PGD(2)(5) .

We have recently reported that the prostanoid pathway of IL-3-developed BMMC could be up-regulated 5-7-fold by stimulation with KL + IL-10 through induction of cytosolic phospholipase A(2) (cPLA(2)), prostaglandin endoperoxide synthase (PGHS)-1, and hematopoietic PGD(2) synthase(6) . However, there was no change in the expression of 5-LO, 5-LO-activating protein (FLAP), or LTC(4) synthase(6, 7) , implying previous maximal expression. To establish a selective role for IL-3 in the regulation of the 5-LO/LTC(4) synthase pathway it was first necessary to develop a new subclass of BMMC that had not been exposed to this cytokine during their development. We now report that a single growth factor, IL-3, up-regulates each of the proteins involved in the generation of cysteinyl leukotrienes from membrane-derived arachidonic acid in mouse mast cells activated by IgE and antigen.


EXPERIMENTAL PROCEDURES

Materials

Recombinant mouse KL, IL-3, IL-9, and IL-10 were expressed in baculovirus-infected Sf9 insect cells (Invitrogen, San Diego, CA), and their concentrations were determined as described previously(6, 8) . Recombinant mouse IL-4, granulocyte macrophage colony-stimulating factor (GM-CSF), transforming growth factor-beta(1) (TGF-beta(1)) (Genzyme, Boston, MA), and 2.5 S nerve growth factor (NGF) (Sigma) were purchased.

Rabbit antiserum to human cPLA(2) and a human cPLA(2) cDNA (9) were provided by J. D. Clark (Genetics Institute, Cambridge, MA), rabbit antiserum to sheep PGHS-1 (10) by W. L. Smith (Michigan State University, East Lansing, MI), cDNAs and rabbit antisera for human 5-LO (11) and for human FLAP (12) by J. F. Evans (Merck Frosst, Quebec, Canada), and a cDNA for mouse LTC(4) synthase (GenBank accession number U27195) by B. K. Lam (Harvard Medical School, Boston, MA). Rabbit antiserum to human LTC(4) synthase was described previously(13) .

Culture of BMMC

Bone marrow cells from male BALB/cJ mice (Jackson Laboratory, Bar Harbor, ME) were suspended in 75-cm^2 flasks (Corning, Corning, NY) at a concentration of 0.5-1 times 10^6 cells/ml of 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, and 10% fetal calf serum) supplemented with 100 ng/ml KL and 10 units/ml IL-10. During the first 3 weeks of culture non-adherent cells were pelleted and resuspended in fresh medium once a week at a concentration of 2-5 times 10^5 cells/ml. After 3 weeks, the non-adherent cells were transferred to a 6-well plate (Corning) at a concentration of 2 times 10^5 cells/ml and were passaged every 4 days for approximately 2 weeks until the population of cells was >95% BMMC as determined by staining with toluidine blue. These BMMC were then cultured in 25- or 75-cm^2 flasks for various periods in the continued presence of 100 ng/ml KL and 10 units/ml IL-10 with or without IL-3; the cells were resuspended in fresh medium at a concentration of 1-2 times 10^5 cells/ml every 4 days. In an initial dose-response experiment a concentration of 10-100 units/ml IL-3 was found to be optimal for induction of 5-LO, as assessed by SDS-PAGE/immunoblotting; a concentration of 100 units/ml IL-3 was therefore used in all subsequent experiments.

The histamine content of BMMC was measured by radioimmunoassay (Immunotech, Boston, MA). Cytospins of the cultured mast cells were stained with toluidine blue or with Alcian blue and safranin. Measurements of the size of the cells were then made, using a calibrated loop, from photographs taken on a Leica Dialux 20 microscope (Leica Inc., Deerfield, IL) with a times50 objective. For electron microscopy cell pellets were fixed in 2.5% glutaraldehyde prior to processing. Micrographs were taken on a JEOL 100 CX (JEOL, Peabody, MA) at 80 kV.

IgE-dependent Immediate Eicosanoid Generation

BMMC were sensitized for 30 min with 10 µg/ml monoclonal IgE anti-trinitrophenyl (TNP) and stimulated for 10 min at 37 °C with 100 ng/ml TNP-conjugated bovine serum albumin (TNP-BSA). beta-Hexosaminidase, PGD(2), and LTC(4) release were measured as described previously(6) .

RNA Blot Analysis

Total cellular RNA was extracted in guanidinium thiocyanate(14) , resolved in 1.2% formaldehyde-agarose, transferred to Immobilon-N (Millipore, Bedford, MA), and probed as described previously (6, 8) with cDNAs for cPLA(2), PGHS-1, PGHS-2, 5-LO, FLAP, and LTC(4) synthase that were labeled (Megaprime, Amersham Corp.) with [P]dCTP (3,000 Ci/mmol; DuPont NEN) by random priming. The RNA blots were also probed with a 24-mer oligonucleotide corresponding to a region within the mouse 18 S ribosomal RNA(15) .

SDS-PAGE/Immunoblot Analysis

The expression of each protein was assessed by SDS-PAGE/immunoblot analysis(6, 8) . The following concentrations of primary antibodies were used: cPLA(2), 1:2,000; PGHS-1, 1:50,000; 5-LO, 1:5,000; FLAP, 1:2,000; and LTC(4) synthase, 1:1,000. The immunoblots were visualized with a chemiluminescent ECL Western blot analysis system (Amersham Corp.).

[^3H]Thymidine Incorporation Assay

Portions of 2 times 10^4 BMMC were cultured for 72 h in 100 µl of enriched medium containing various combinations of cytokines in wells of a 96-well plate (Costar, Cambridge, MA). The following concentrations of cytokines were used: 100 ng/ml KL, 5 ng/ml IL-1beta, 100 units/ml IL-3, 1.5 ng/ml IL-4, 100 units/ml IL-5, 5 ng/ml IL-6, 50 units/ml IL-9, 10 units/ml IL-10, 100 units/ml GM-CSF, 500 ng/ml 2.5 S NGF, 500 units/ml tumor necrosis factor alpha, 100 units/ml interferon-, and 5 ng/ml TGF-beta(1). During the last 8 h of culture, 0.1 µCi of [^3H]thymidine (6.7 Ci/mmol; DuPont NEN) was added to each culture. The radiolabeled nuclei were harvested onto glass filter papers with an automated cell harvester (Tomec, Orange, CT), and their incorporation of radioactivity was measured by liquid scintillation counting.


RESULTS AND DISCUSSION

Phenotypic Characterization of BMMC Developed in KL + IL-10

It had previously been demonstrated that KL, like IL-3, would generate BMMC but that the purity of these cells was limited and their viability declined with continuing culture(16) . When bone marrow cells were cultured in KL with IL-10, an accessory growth factor for mast cells(17) , 65.4 ± 7.8% (mean ± S.E., n = 4) and 84.3 ± 5.5% (n = 4) of the cells were mast cells after 3 and 4 weeks of culture, respectively, as assessed by toluidine blue staining. After 5 weeks of culture, 97.0 ± 2.2% (n = 4) of the cells were mast cells as assessed by metachromatic staining of their granules with toluidine blue (Fig. 1A) and by staining with Alcian blue/safranin (Fig. 1B). BMMC developed in KL + IL-10 contained 1.5 ± 0.5 µg of histamine/10^6 cells (n = 5), and about half the cells counterstained with safranin (Fig. 1B), indicating heparin biosynthesis. The cells in cytospin preparations were 15 µm in diameter, with a cross-sectional area of 268 ± 1.0 µm^2 (mean ± S.D., n = 33); there was a central or eccentric nucleus, and the cytoplasm contained abundant granules. Electron micrographs revealed that the secretory granules in BMMC developed in KL + IL-10 contained masses of particulate electron dense material and small vesicles (Fig. 1D). Approximately 3-6 times 10^6 such BMMC were developed from 1 times 10^7 bone marrow cells cultured for 5 weeks in KL + IL-10. The maturity of these BMMC is indicated by their substantial metachromasia with toluidine blue, counterstaining with Alcian blue/safranin (Fig. 1, A and B), content of histamine, and electron density of their granule contents (Fig. 1D).


Figure 1: Light (A-C) and electron (D and E) microscopy of BMMC developed in KL + IL-10. Cytospins of mouse bone marrow cells cultured for 5 weeks with KL + IL-10 were stained with either toluidine blue (A) or with Alcian blue/safranin before (B) and 2 weeks after the addition of IL-3 to the culture (C). Cells in which nearly all the granules are safranin-positive are indicated (arrows). Electron microscopy of BMMC developed in KL + IL-10 before (D) and 2 weeks after the addition of IL-3 to the culture (E). Bars = 10 µm in A-C and 1 µm in D and E.



When BMMC developed in KL + IL-10 were sensitized with IgE anti-TNP and activated with TNP-BSA, they exocytosed 40.0 ± 5.4% beta-hexosaminidase and generated 8.3 ± 4.5 ng of LTC(4)/10^6 cells and 8.1 ± 2.4 ng of PGD(2)/10^6 cells (n = 4) (Fig. 2). As compared with BMMC developed from bone marrow with IL-3, those developed with KL + IL-10 responded to IgE-dependent activation with the generation of 2-fold more PGD(2) but only one-third the amount of LTC(4)(6) . Hence, BMMC developed from bone marrow with KL + IL-10 provided a new class of BMMC in which to seek cytokine up-regulation of the 5-LO pathway.


Figure 2: Time course of the IL-3-stimulated changes in IgE-dependent eicosanoid generation. BMMC developed with KL + IL-10 were cultured with (closedsymbols) or without (opensymbols) IL-3 in the continued presence of KL + IL-10. Cells were then sensitized with IgE anti-TNP and activated with TNP-BSA for 10 min, and their supernatants were assayed for PGD(2) (triangles) and LTC(4) (circles). Values represent means ± S.E. of four independent experiments.



Development of the 5-Lipoxygenase/Leukotriene C(4)Synthase Pathway by IL-3

When BMMC developed in KL + IL-10 were cultured for 1 week with 100 units/ml IL-3 in the continued presence of KL and IL-10, the number of cells increased 12.3 ± 2.8-fold compared with an increase of only 1.5 ± 0.4-fold in cells cultured for 1 week more in KL + IL-10 alone (n = 3; p < 0.05). After 2 weeks of culture with IL-3, most of the BMMC contained granules that stained with Alcian blue but did not counterstain with safranin (Fig. 1C). Morphologically the cells nearly doubled in size, the cross-sectional area increasing to 556 ± 3.6 µm^2 (mean ± S.D., n = 45), while the size of the nucleus remained constant (Fig. 1C). Electron micrographs revealed that the secretory granules of these cells contained small vesicles (50-80 nm) and sparse electron dense material within clear matrices bound by the granule limiting membrane (Fig. 1E), suggesting that the intense IL-3-driven proliferation favored a less mature phenotype.

One week after the addition of IL-3 to BMMC cultured in KL + IL-10, IgE-dependent beta-hexosaminidase release doubled to 86.4 ± 11.4% (n = 4) but did not increase further at 4 weeks. IgE-dependent LTC(4) generation increased 15-fold after 1 week of culture with IL-3 and increased 25-fold to 212.0 ± 35.5 ng/10^6 cells by 2-4 weeks (n = 4, p < 0.01 versus cells maintained in KL + IL-10 without IL-3) (Fig. 2). In contrast, IgE-dependent PGD(2) generation of these cells increased only 3-fold to 25.5 ± 8.6 ng/10^6 cells after 1-4 weeks of culture with IL-3 (n = 4, p < 0.05) (Fig. 2). Thus the ratio of IgE-dependent generation of LTC(4) to PGD(2) increased from 1.0 to 8.3 in response to treatment with IL-3.

In analyzing the basis for this remarkable up-regulation of LTC(4) generation, the changes in expression of the four sequentially acting proteins involved in metabolism of arachidonic acid to LTC(4) were assessed in terms of steady-state levels of mRNA and expressed protein by RNA blot and SDS-PAGE/immunoblot (Fig. 3), respectively. The initial step in IgE-dependent arachidonic acid metabolism is the liberation of arachidonic acid from membrane phospholipids by cPLA(2)(18) . cPLA(2) protein, which was barely detectable during the first 2 weeks after addition of IL-3 to BMMC cultured concomitantly with KL + IL-10, increased from 3 to 5 weeks (Fig. 3A). The increase in expression of cPLA(2) lagged behind the increases in the IgE-dependent generation of LTC(4) and PGD(2), which were apparent by 1-2 weeks, suggesting that this step did not limit the capacity for eicosanoid generation, perhaps because KL represents an alternative cytokine for expression of cPLA(2)(6) . The increased expression of cPLA(2) protein was accompanied by only minimal change in cPLA(2) transcripts (Fig. 3B), indicating, as in response to KL(6) , a significant post-transcriptional regulation of its expression. The time course of weeks for IL-3-dependent cPLA(2) up-regulation in BMMC developed in KL + IL-10 is unlike the response observed within hours to days in IL-1-stimulated fibroblasts(19) , tumor necrosis factor alpha-stimulated HeLa cells(20) , and even KL-treated BMMC that had been developed in IL-3(6) .


Figure 3: Time course of the IL-3-stimulated expression of proteins involved in the metabolism of arachidonic acid to LTC(4). BMMC developed with KL + IL-10 for 5 weeks were cultured for the indicated periods with IL-3 in the continued presence of KL + IL-10. A, 1 times 10^5 cell eq were analyzed for expression of each protein by SDS-PAGE/immunoblotting; B, 10 µg of total RNA were assessed for steady-state transcripts of cPLA(2), 5-LO, FLAP, LTC(4) synthase (LTC(4)S), and mouse 18 S ribosomal RNA. A representative result of three independent experiments is shown.



FLAP, an integral nuclear envelope protein, presents arachidonic acid to translocated 5-LO(12) , which then sequentially catalyzes the conversion of arachidonic acid to 5-hydroperoxyeicosatetraenoic acid and LTA(4)(21) . IL-3-induced expression of 5-LO protein in BMMC developed with KL + IL-10 was evident within 4 days and increased progressively to reach a plateau by 2-3 weeks with accompanying changes in 5-LO transcripts (Fig. 3). FLAP protein increased in parallel with 5-LO protein over time. Steady-state transcripts for FLAP increased only modestly (Fig. 3), suggesting significant post-transcriptional regulation of FLAP expression. The time course of the up-regulation of the 5-LO and FLAP proteins (Fig. 3A) paralleled that of increased IgE-dependent LTC(4) generation (Fig. 2). Up-regulation of 5-LO and/or FLAP has been observed during differentiation of HL60 cells toward granulocytic cells in response to treatment with dimethyl sulfoxide(22) , during differentiation of human monocytic cells to macrophage-like cells in response to vitamin D(3) and TGF-beta(1) or TGF-beta(2)(23) , and in human neutrophils stimulated with GM-CSF(24) . That neither GM-CSF (Fig. 4) nor TGF-beta (data not shown) altered the expression of the 5-LO pathway in the BMMC developed with KL + IL-10, although GM-CSF is a proliferation cofactor and TGF-beta is anti-proliferative, indicates different regulatory cytokines for this pathway within the hematopoietic lineage.


Figure 4: Selectivity of cytokine-stimulated expression of the proteins involved in the metabolism of arachidonic acid to LTC(4). BMMC developed with KL + IL-10 were cultured for 3 weeks with 100 units/ml IL-3, 1.5 ng/ml IL-4, 100 units/ml IL-9, 500 ng/ml NGF, or 100 units/ml GM-CSF in the continued presence of KL + IL-10. 1 times 10^5 cell eq were analyzed for the expression of each protein by SDS-PAGE/immunoblotting. A representative result of three independent experiments is shown.



LTC(4) synthase, also an integral nuclear envelope protein sharing 31% amino acid identity with FLAP(25) , conjugates LTA(4) to reduced glutathione to form LTC(4). Although LTC(4) synthase is induced in U937 cells during differentiation into monocyte/macrophage-like cells in response to treatment with dimethyl sulfoxide (26) and in RBL-1 cells treated with retinoic acid (27) as assessed by an increase in enzyme activity, no information exists on the transcriptional or translational regulation of this terminal enzyme. The expression of LTC(4) synthase protein, which was detectable only after 4-5 weeks of culture with IL-3, was preceded by a significant increase in steady-state transcripts for this enzyme that was detectable at 1 week (Fig. 3). The plateau in LTC(4) biosynthesis at 2 weeks (Fig. 2) implies that the FLAP/5-LO step is more likely than LTC(4) synthase to be rate-limiting.

None of the other cytokines tested elicited any change in expression of the enzymes of the 5-LO/LTC(4) synthase pathway, revealing a strict specificity for IL-3 (Fig. 4). IL-4, IL-9, GM-CSF, and NGF, which acted as accessory cytokines for proliferation as assessed by a 1.5-2.5-fold net increase in [^3H]thymidine incorporation similar to that with IL-3 addition in the continued presence of KL + IL-10 (data not shown), were without effect on the 5-LO pathway (Fig. 4). That GM-CSF in combination with KL + IL-10 also did not up-regulate the 5-LO/LTC(4) synthase pathway proteins suggests that the selectivity for IL-3 is independent of a common beta subunit shared by the receptors for IL-3 and GM-CSF(28) .

Only two cytokines, KL (also known as stem cell factor) and IL-3 (initially termed multi-colony-stimulating factor), are capable of developing mast cell-committed progenitors from bone marrow in vitro and of stimulating their proliferation in the absence of other growth factors. That the initial enzyme of arachidonic acid metabolism, cPLA(2), which is shared by the cyclooxygenase and the 5-LO pathways, is regulated by both KL and IL-3 implies the early expression of both routes of eicosanoid biosynthesis in committed mast cell progenitors. KL, a tissue-derived cytokine essential for the development of both mouse mast cell subclasses in vivo(29, 30) , up-regulates the expression of the enzymes in the cyclooxygenase pathway leading to PGD(2) generation in BMMC that were derived in IL-3. IL-10 acts with KL to further up-regulate PGHS-1 with incremental IgE-dependent PGD(2) production(6) . IL-3 is implicated in the in vivo development over 2-3 weeks of helminthic infection of MMC that predominantly generate cysteinyl leukotrienes(31) . When BMMC are initially developed from bone marrow cells with KL + IL-10 and then treated with IL-3, the major up-regulation of eicosanoid metabolism occurs in the cysteinyl leukotriene generating 5-LO pathway ( Fig. 2and Fig. 3). Irrespective of whether or not these in vitro findings are directly related to in vivo tissue-specific mast cell phenotypes, these studies reveal that the early acting cytokines, KL and IL-3, respectively, up-regulate each protein in separate pathways for prostanoid and cysteinyl leukotriene biosynthesis in committed mast cell progenitors.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL36110, AI22531, AI31599, AI07306, and RR05950 and by a grant from The Hyde and Watson Foundation. 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 an Arthritis Foundation fellowship.

**
Supported by a Burroughs Wellcome developing investigator award. To whom correspondence should be addressed: Dept. of Rheumatology and Immunology, Brigham and Women's Hospital and Harvard Medical School, The Seeley G. Mudd Bldg., Rm. 628, 250 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1335; Fax: 617-432-0979.

(^1)
The abbreviations used are: CTMC, connective tissue mast cells; IL, interleukin; BMMC, bone marrow-derived mast cells; PG, prostaglandin; PGHS, prostaglandin endoperoxide synthase; FcRI, Fc receptor type I; KL, c-kit ligand; LT, leukotriene; cPLA(2), cytosolic phospholipase A(2); TNP, trinitrophenyl; BSA, bovine serum albumin; MMC, mucosal mast cells; FLAP, 5-lipoxygenase-activating protein; 5-LO, 5-lipoxygenase; GM-CSF, granulocyte-macrophage colony-stimulating factor; TGF-beta, transforming growth factor-beta; NGF, nerve growth factor; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank C. Nwankwo and R. Sanchez (Harvard Medical School) for their technical assistance.


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