(Received for publication, July 5, 1995; and in revised form, August 8, 1995)
From the
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/10
cells and 8.1 ± 2.4 ng of
prostaglandin (PG) D
/10
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
and PGD
increased to 212 ± 36 and 25.5 ± 8.6 ng/10
cells, respectively. The dramatic increase in the IgE-dependent
generation of LTC
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
and LTC
synthase. The recognition that IL-3
up-regulates the expression of each protein of the 5-LO pathway for the
generation of LTC
contrasts with our recent finding that KL
up-regulates the expression of cytosolic phospholipase A
,
prostaglandin endoperoxide synthase-1, and hematopoietic PGD
synthase and increases the IgE-dependent generation of PGD
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
synthase pathway.
There are at least two distinct populations of mast cells in
rodents, connective tissue mast cells (CTMC) ()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) , whereas rat MMC generate leukotriene (LT)
C
via the 5-lipoxygenase (5-LO) pathway in preference to
PGD
(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, Fc
RI, they generate LTC
in marked preference to PGD
(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 (cPLA
), prostaglandin endoperoxide synthase (PGHS)-1,
and hematopoietic PGD
synthase(6) . However, there
was no change in the expression of 5-LO, 5-LO-activating protein
(FLAP), or LTC
synthase(6, 7) , implying
previous maximal expression. To establish a selective role for IL-3 in
the regulation of the 5-LO/LTC
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.
Rabbit antiserum to human cPLA and a human
cPLA
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
synthase (GenBank accession number
U27195) by B. K. Lam (Harvard Medical School, Boston, MA). Rabbit
antiserum to human LTC
synthase was described
previously(13) .
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 50 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.
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% -hexosaminidase and generated 8.3
± 4.5 ng of LTC
/10
cells and 8.1
± 2.4 ng of PGD
/10
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
but only one-third the amount of
LTC
(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 (triangles) and LTC
(circles). Values represent means ± S.E. of four
independent experiments.
One week
after the addition of IL-3 to BMMC cultured in KL + IL-10,
IgE-dependent -hexosaminidase release doubled to 86.4 ±
11.4% (n = 4) but did not increase further at 4 weeks.
IgE-dependent LTC
generation increased
15-fold after 1
week of culture with IL-3 and increased
25-fold to 212.0 ±
35.5 ng/10
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
generation of these cells increased only 3-fold to 25.5
± 8.6 ng/10
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
to PGD
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 generation, the changes in
expression of the four sequentially acting proteins involved in
metabolism of arachidonic acid to LTC
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
(18) . cPLA
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
lagged behind the increases in the IgE-dependent
generation of LTC
and PGD
, 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
(6) .
The increased expression of cPLA
protein was accompanied by
only minimal change in cPLA
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
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
-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. 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
10
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
, 5-LO, FLAP,
LTC
synthase (LTC
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(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
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
and
TGF-
or TGF-
(23) , and in
human neutrophils stimulated with GM-CSF(24) . That neither
GM-CSF (Fig. 4) nor TGF-
(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-
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. 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
10
cell eq were
analyzed for the expression of each protein by SDS-PAGE/immunoblotting.
A representative result of three independent experiments is
shown.
LTC synthase,
also an integral nuclear envelope protein sharing 31% amino acid
identity with FLAP(25) , conjugates LTA
to reduced
glutathione to form LTC
. Although LTC
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
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
biosynthesis at 2 weeks (Fig. 2) implies that the FLAP/5-LO step is more likely than
LTC
synthase to be rate-limiting.
None of the other
cytokines tested elicited any change in expression of the enzymes of
the 5-LO/LTC 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 [
H]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
synthase
pathway proteins suggests that the selectivity for IL-3 is independent
of a common
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, 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
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
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.