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Department of Biology, Virginia Commonwealth University, Richmond, VA 23284
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA
induction after stem cell factor stimulation. These data offer a role
for STAT6 and phosphotidylinositide-3'-kinase in IL-4-mediated Kit
down-regulation, coupled with the novel observation that IL-10 is a
potent inhibitor of Kit expression and function. Regulating Kit
expression and signaling may be essential to controlling mast
cell-mediated inflammatory responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mast cell development, proliferation, and function require proper expression and signaling of several cytokine receptors, among which the Kit tyrosine kinase is perhaps best studied. Mast cell development is drastically reduced in animals with mutations in genes encoding Kit or its ligand, stem cell factor (SCF).3 These animals also suffer from macrocytic anemia, loss of melanocyte migration, and sterility (4, 5, 6, 7). Thus Kit expression and signaling play an essential role in the development of multiple organ systems.
In addition to its role in mast cell ontogeny, Kit expression is regulated on mature mast cells. Binding by its ligand, SCF, triggers ubiquitin-mediated degradation of Kit expression (8). IL-4 is also known to inhibit Kit expression on mature mast cells (9, 10, 11, 12). However, neither the mechanism nor the functional significance of IL-4-mediated Kit down-regulation has been fully determined. Previous reports have argued for either loss of Kit mRNA expression or loss of Kit protein following IL-4 stimulation (11, 12).
IL-4 is produced by T lymphocytes (13), mast cells (14), and basophils (15) following receptor-mediated activation. It exerts a number of biological activities within the hemopoietic system. Previous studies have shown that IL-4 is a potent regulator of myeloid progenitor and mast cell growth, proliferation, and gene regulation (13, 16, 17, 18). It also directs Th2 cell development and induces Ig class switching and IgE production by B cells (reviewed in Ref. 19). IL-4 mediates its biological responses by binding to a high-affinity receptor complex, which has a wide distribution on hemopoietic and nonhemopoietic cells (reviewed in Ref. 20).
Signaling via the IL-4R occurs through distinct pathways, which predominantly promote growth or gene expression (21, 22, 23, 24). Many IL-4-mediated effects on gene expression require activation of the transcription factor STAT6. Animals genetically deficient in STAT6 fail to develop Th2 cells, synthesize IgE, or induce the expression of CD23 and MHC class II molecules in response to IL-4 (25, 26, 27). Thus, STAT6 is considered an essential positive gene regulator of IL-4-mediated responses (reviewed in 28, 29, 30). However, recent data also support a role for STAT6 as a negative regulator of gene expression (31, 32).
In addition to STAT6, IL-4R mediates its responses through activation
of other pathways including phosphotidylinositide-3'-kinase (PI3K)
(reviewed in Refs. 33 and 34). PI3Ks are
ubiquitously expressed enzymes that phosphorylate the D3 position
of inositol rings (reviewed in Ref. 35). Several classes
of PI3Ks have been identified based on their sequence similarities and
substrate selectivity. The best studied of PI3Ks consist of a 110-kDa
catalytic subunit and a tightly associated regulatory subunit of 85,
55, or 50 kDa. PI3K has been proven to be a critical signaling
intermediate in response to a wide variety of extracellular stimuli and
is activated by a number of proteins containing intrinsic or
associated tyrosine kinase activities. PI3K expression has been
demonstrated to be essential in many cellular responses, such as
rearrangement of cytoskeletal actin, movement of organelle membranes,
and chemotaxis (36). It has recently been shown that
disruptions in the p85 subunit, the most abundantly expressed
regulatory isoform of PI3K, severely affect B cell development and
proliferation while disruption of the entire p85 gene (which also
encodes p55 and p50) can be lethal (37, 38).
Like IL-4, IL-10 is also produced by Th2 cells, as well as activated
macrophages, B cells, activated mast cells, and keratinocytes (reviewed
in Ref. 39). IL-10 possesses potent negative regulatory
activities, inhibiting the production of IFN-
by lymphocytes,
TNF- and IL-6 by macrophages, monocytes, and peritoneal mast cells,
and TNF- and GM-CSF production by eosinophils (40, 41, 42, 43, 44, 45, 46).
However, IL-10 has also been reported to synergize with IL-3 and IL-4
in supporting mast cell development and proliferation (47, 48).
The current study demonstrates that IL-4-mediated murine bone
marrow-derived mast cell (BMMC) Kit down-regulation is STAT6 and PI3K
dependent. Additionally, we find that IL-10 has potent inhibitory
effects on BMMC Kit expression and that the effects of IL-10 on Kit
expression are enhanced by IL-4 in a STAT6-independent, PI3K-dependent
manner. Our data also demonstrate that IL-4- and IL-10-mediated Kit
down-regulation is transient and requires constant stimulation with
IL-4 and/or IL-10. Loss of Kit expression is not explained by a
reduction in steady-state c-kit mRNA levels, but rather by a
loss of Kit protein expression without changes in protein
stability. We also report that Kit stimulation induces IL-13
transcription and that Kit-mediated induction of both IL-13 and TNF-
mRNAs is greatly reduced by IL-4 and IL-10.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells were maintained in RPMI 1640 medium (Life Technologies,
Grand Island, NY), supplemented with 10% FBS, 2 mM
L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin,
and 1 mM sodium pyruvate (all from Biofluids, Rockville, MD) (cRPMI),
supplemented with 10–20% WEHI-3 conditioned medium (cRPMI/WEHI-3-CM).
BMMC were derived from femurs of adult C57BL/6, CB57BL/6 x 129
STAT6 wild-type (WT), or STAT6-deficient (26) mice by
culture in cRPMI with 20–25% WEHI-3-CM for 3 wk. BMMC were also
derived from BALB/c IL-4-deficient (IL-4-/-)
and IL-4R-deficient (IL-4R-/-) mice (kind gifts
of Drs. Nancy Noben-Trauth and William E. Paul, National Institutes of
Health, Bethesda, MD; Refs. 49 and 50). The
phenotype of mast cells was determined by flow cytometry analysis with
Abs specific for Kit, CD13, IgE, FcRII/RIII, and by histochemical
staining (data not shown). LY294002, wortmannin, PD98059, and
chelerythrine chloride were purchased from Sigma (St. Louis,
MO).
Cytokines and Abs
Murine SCF, IL-3, IL-4, and IL-10 were purchased from R&D
Systems (Minneapolis, MN) or BioSource International (Camarillo, CA).
2.4G2 rat anti-mouse FcRII/RIII ascites was the generous gift of
Jane Hu-Li and William Paul (National Institutes of Health) or was
purchased from PharMingen (San Diego, CA). Purified mouse IgE and Abs
specific for mouse CD13, CD4, CD117 (Kit), or IgE were purchased from
PharMingen. Monoclonal rat anti-mouse IL-4R was purchased from
Genzyme Diagnostics (Cambridge, MA). FITC-labeled goat anti-rat IgG
(H+L chains) F(ab')2 was purchased from Southern
Biotechnology Associates (Birmingham, AL).
Tissue culture conditions for inhibition of BMMC Kit expression
To assess the regulation of Kit expression, BMMC were washed to
remove WEHI-3-CM and were incubated for 4–6 h at 37°C in cRPMI. BMMC
were then plated at 
300,000 cells/ml in a 200-µl volume using
96-well flat-bottom plates (Costar, Cambridge, MA). Cells were
incubated for the indicated times in cRPMI containing 5 ng/ml mouse
IL-3 and the indicated concentrations of mouse IL-4 and/or IL-10. For
long-term incubations, cultures were fed every 4 days by replacement of
half the medium and cytokines. LY294002, wortmannin, PD98059,
chelerythrine chloride, or DMSO (also purchased from Sigma) were added
to cell cultures at indicated concentrations. Cells were then incubated
for 30–60 min at 25° or 37°C, after which time they were treated
with IL-3, IL-4, and/or IL-10. Fresh inhibitors were added to cultures
daily. Kit levels were determined by flow cytometry.
Flow cytometry analysis
To detect Kit expression on BMMC, cells were incubated with 0.3
µl 2.4G2 rat anti-mouse FcRII/RIII ascites per 100 µl for 10
min at 4°C, followed by 10 µg/ml FITC-conjugated rat anti-mouse
Kit for 30 min at 4°C in PBS/3% FBS/0.1% sodium azide (FACS
buffer). Cells were then washed twice and analyzed in the presence of
propidium iodide with a Becton Dickinson FACScan flow cytometer (Becton
Dickinson, San Jose, CA). Control samples were identically stained with
FITC-labeled rat anti-mouse CD4. Percent inhibition of Kit
expression was determined using mean fluorescence intensities (MFI),
with cells cultured in IL-3 alone as the baseline for all comparisons.
Fc
RI or CD13 expression was determined as described previously
(31).
RNase protection assay
For each sample, 5 x 106 BMMC
were stimulated with 5 ng/ml IL-3 with or without 10 ng/ml of IL-4
and/or IL-10, as described above for periods of 6 h to 7 days, and
cells were fed every 4 days. Cells were then washed and resuspended at
1 x 106 cells/ml in cRPMI with 1 ng/ml IL-3
(cRPMI-IL-3). SCF was added to a final concentration of 100 ng/ml, and
cells were incubated for 2 h at 37°C. RNA was harvested with
Trizol (Biotecx, Friendswood, TX), and an RNase protection assay was
performed using a custom-made probe set or the mCK-1 probe set from the
RiboQuant System (PharMingen) based on the manufacturer’s
instructions. Pixel intensity was determined using the Phosphorimaging
445SI System (Molecular Dynamics, Sunnyvale, CA).
EMSA
BMMC (1 x 107) were resuspended in cRPMI without WEHI-3-CM at 2 x 106/ml, incubated for 4 h at 37°C, washed, and resuspended in cRPMI at 2 x 106/ml for 1.5 h at 37°C. Cells were incubated with DMSO, LY294002, or wortmannin at indicated concentrations 30 min at 37°C. IL-3 and IL-4 were added to cells, at 100 ng/ml each, and cells were incubated for 10 min at room temperature. Total cell lysates were obtained as previously described (31), and 7.5 µg of lysate was subjected to EMSA analysis using a double-stranded oligonucleotide derived from the mouse IL-4 promoter (4GL3): gatc AAGACC-162TTCACAGGAA-152CTTTAATCT gatc as described previously (31). 4GL3 has been shown to specifically bind to STAT6 (31, 51).
Western blot analysis
BMMC were treated with IL-3 alone or combinations of IL-3, IL-4, and IL-10 for 3 days. Cells were then collected and washed. Pellets were lysed in lysis buffer (50 mM Tris, pH 7.2, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 0.5% Triton X-100, 10 mM EDTA plus protease inhibitors (Boehringer Mannheim, Indianapolis, IN)). Total protein lysates (25 µg) were subjected to SDS-PAGE on a 10% polyacrylamide gel and transferred to Nitran Membrane (Bio-Rad, Hercules, CA). Blots were blocked with 5% nonfat dry milk in HEPES-buffered saline (HBS: 50 mM HEPES, pH 7.2, 150 mM NaCl, and 10 mM EDTA) for 30 min at 37°C. Polyclonal Anti-Kit (Oncogene Science, Uniondale, NY) was added at 0.5 µg/ml in 5% nonfat dry milk in HBS and rocked overnight at 4°C. Blots were then washed three times, for 10 min each, with HBS. A secondary F(ab')2 HRP-conjugated donkey anti-rabbit IgG (Amersham, Arlington Heights, IL) was then added at a 1:1000 dilution in 5% nonfat dry milk in HBS for 1 h at room temperature. Blots were then washed three times, for 10 min each, in HBS. Kit expression was detected using enhanced chemiluminescence reagents (Pierce, Rockford, IL) as directed by the manufacturer. Quantitation of Kit band intensity was achieved using a personal densitometer SI (Molecular Dynamics, Sunnyvale, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the role of STAT6 in IL-4-mediated Kit regulation, we
derived BMMC populations from mice bearing a targeted disruption in the
STAT6 gene (STAT6-/-).
STAT6-/- BMMC had mast cell morphology and
expressed FcRI, Kit, CD13, Fc receptors, and T1/ST2 at levels
comparable to WT BMMC and proliferated normally in response to IL-4
(Ref. 31 and data not shown). While
STAT6-/- BMMC expressed Kit at normal levels,
IL-4-mediated down-regulation of Kit was greatly diminished in these
cells (Fig. 1
). The lack of IL-4
responsiveness in STAT6-/- BMMC did not appear
to be due to a deficiency in IL-4R expression. These cells have
previously been shown to express IL-4R at levels similar to WT BMMC
populations and to exhibit similar proliferative responses to IL-4 in
combination with suboptimal doses of IL-3 or SCF (31).
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We assessed the role of PI3K in the IL-4-mediated inhibition of
Kit activity using the PI3K inhibitor, LY294002. As shown in Fig. 2, IL-4-mediated inhibition of Kit
expression was diminished in cells treated with LY294002, but not in
cells treated with DMSO, the vehicle control. This effect was dose
dependent, with optimal inhibition of IL-4 effects observed when 10
µM LY294002 was used. Similar inhibitory effects were noted when
LY294002 was added daily or as a single dose at the initiation of
cultures (data not shown).
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PI3K is known to activate the serine/threonine kinase protein
kinase B (Akt), with subsequent downstream activation of other kinases
(reviewed in Ref. 52). Because serine phosphorylation has
been demonstrated to be necessary for full STAT functionality in some
systems (53, 54, 55), we wished to determine whether the PI3K
and STAT6 pathways intersect during IL-4-mediated Kit down-regulation.
As shown in Fig. 3, the PI3K inhibitors,
LY294002 or wortmannin, had no effect on STAT6 DNA binding
activity.
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IL-10 has been shown to regulate mast cell growth, mediator
release, and cytokine production, and synergizes with IL-4 in some of
these activities (46, 47, 48). In an effort to determine the
role of this Th2 cytokine on Kit expression, we treated BMMC with IL-10
and assessed Kit levels by flow cytometry. As shown in Fig. 4, cells cultured in the presence of IL-3
and IL-10 showed significantly reduced Kit levels compared with those
cultured in IL-3 alone. IL-10-mediated inhibition of Kit expression did
not appear to be due to intrinsic IL-4 production, as BMMC derived from
IL-4-/- or IL-4R-/-
mice (49, 50) also showed reduced Kit levels when cultured
in medium containing IL-3 and IL-10 (Table I). Furthermore, STAT6 was not required
for this effect, as STAT6-/- BMMC responded
normally to IL-10 (Fig. 5).
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Because IL-10 has been demonstrated to synergize with IL-3 and
IL-4 to enhance mast cell development (47, 48), we
examined the combined effects of IL-4 and IL-10 on Kit expression. As
shown in Fig. 5
A, stimulation with IL-4 or IL-10 decreased
Kit levels an average of 55%, while combined stimulation with IL-4
and IL-10 reduced Kit levels nearly 80% compared with cells cultured
in IL-3 alone (p < 0.05; Tukey test). Given
that STAT6 expression is required for full IL-4-mediated Kit
inhibition, we examined the role of STAT6 in Kit inhibition mediated by
costimulation with IL-4 and IL-10. Treatment of
STAT6-/- BMMC with IL-3 plus IL-4 and IL-10 led
to a greater reduction in Kit expression than did culture in IL-3 and
IL-10 (Fig. 5). Thus, the ability of IL-4 to enhance IL-10-mediated Kit
inhibition is not STAT6 dependent. This effect was also independent of
endogenously produced IL-4, but required expression of the IL-4R. As
demonstrated in Table I, IL-4-/-, but not
IL-4R-/- BMMC demonstrated IL-4 enhancement of
IL-10-mediated Kit inhibition.
Because PI3K activity is required for IL-4-mediated Kit
down-regulation, we determined the role of PI3K in the enhanced Kit
down-regulation observed when BMMC are stimulated with IL-4 and IL-10.
As shown in Fig. 5B, the ability of IL-4 to enhance
IL-10-mediated Kit down-regulation was diminished in both the WT and
STAT6-/- BMMC upon treatment with LY294002.
Thus, PI3K activation is essential for the IL-4-mediated enhancement of
IL-10 signaling.
The effect of IL-4 and IL-10, alone or combined, was sensitive and
transient. IL-4-mediated inhibition of Kit expression could be observed
at concentrations as low as 1.25 ng/ml IL-4. IL-10 gave comparable
results at 0.4 ng/ml (Fig. 5C). While inhibition increased
over time, peaking at days 2–3, Kit expression returned to normal
levels by day 7 of culture, even with continuous cytokine stimulation
(Fig. 5D). Cells stimulated with IL-10 alone or with IL-4
and IL-10 demonstrated an insignificant second peak of inhibition on
day 14 of culture (p 
0.05). It should be
noted that IL-4 and/or IL-10 stimulation of BMMC did not appear to
mediate a decrease in expression of all surface proteins, as CD13
expression was not decreased by IL-4 or IL-10 stimulation (data not
shown).
Inhibition of Kit expression requires constant stimulation with IL-4 and IL-10
To further investigate the kinetics of IL-4- and IL-10-mediated
inhibition of Kit expression, BMMC were cultured in the presence of
IL-3, with or without IL-4 and/or IL-10, for 2 days, washed
extensively, and recultured in IL-3 alone for 16 h (Fig. 6, bottom). Kit levels on BMMC
deprived of IL-4 and/or IL-10 for 16 h were comparable to cells
cultured in IL-3 alone. Control cultures stimulated with IL-3 plus IL-4
and/or IL-10 for 3 (Fig. 6, top) or 2 days (Fig. 6, middle) demonstrated Kit repression, as expected. Therefore,
IL-4- and IL-10-mediated Kit down-regulation was not only transient,
but also rapidly reversible.
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To determine whether IL-4- and/or IL-10-mediated Kit down-regulation correlated with a loss of c-kit mRNA production in murine BMMC, cells were cultured in the presence of IL-3, with or without IL-4 and/or IL-10 for periods of 6 h to 7 days, before assessing c-kit mRNA expression by RNase protection assay (RPA). Murine BMMC showed no significant reduction of c-kit mRNA expression following treatment with IL-3 plus IL-4 or IL-3 plus IL-10. However, stimulation with all three cytokines did reduce steady-state c-kit mRNA levels by an average of 30% (mean of four independent experiments) compared with cells cultured in IL-3 alone (Fig. 7A). These data indicate that loss of c-kit mRNA alone fails to explain IL-4- or IL-10-mediated Kit repression and that combined stimulation with IL-4 and IL-10 may alter Kit expression by a mechanism not employed by either cytokine alone.
We next assessed Kit protein expression and stability following
stimulation with IL-4 and/or IL-10. In keeping with a previous study
(12), we found that BMMC cultured with IL-3 plus IL-4 had
a 62% reduction in total Kit levels, compared with cells cultured in
IL-3 alone (Fig. 7B). Similarly, culturing BMMC in IL-3 plus
IL-10 yielded a 49% reduction in Kit protein levels, where stimulation
with IL-3 plus IL-4 plus IL-10 resulted in a drastic 84% loss of Kit
expression, relative to IL-3-treated control BMMC. These data correlate
well with our observed loss in surface Kit staining.
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B).
Western blot analysis, coupled with densitometry measurements,
indicated that treatment with IL-4 and/or IL-10 failed to decrease the
half life of Kit protein. BMMC cultured in IL-3 alone had a half life
of 3.5 h; those treated with IL-3 plus IL-4, IL-3 plus IL-10, or
IL-3 plus IL-4 plus IL-10 had half lives of 4.3 h, 5.0 h, and
5.8 h, respectively (all measurements are means of two experments,
comparing cycloheximide-treated to DMSO-treated cells). Thus IL-4 and
IL-10 reduce Kit protein expression without increasing degradation. IL-4- and IL-10-mediated inhibition of Kit expression affects production of SCF-induced cytokine mRNA
We wished to determine whether the IL-4- and IL-10-mediated
reduction in Kit expression levels translated to a loss of Kit
functionality. As SCF stimulation has been shown to induce production
of TNF- and IL-6 from BMMC (56), RPA analysis was used
to assess induction of multiple cytokine mRNAs following SCF
stimulation (Fig. 8). In addition to
TNF- production, we noted a significant induction of IL-13 mRNA
following SCF stimulation. IL-6 mRNA was constitutively expressed by
these BMMC populations and showed little increase after SCF stimulation
(data not shown). Culturing BMMC in the presence of IL-3 plus IL-4
and/or IL-10 greatly reduced their ability to induce TNF- or IL-13
mRNA following SCF stimulation, compared with BMMC cultured in IL-3
alone. In fact, TNF- and IL-13 mRNA levels were reduced by
nearly 70% in cells cultured in IL-3 plus IL-4 plus IL-10 before
SCF-stimulation. Interestingly, culturing BMMC in IL-3 plus IL-4 plus
IL-10 led to constitutive IL-13 mRNA transcription, a result observed
in two experiments (data not shown). Taken together, these data suggest
that costimulation with IL-4 and IL-10 not only leads to reduced
Kit expression, but that the remaining surface Kit has lost
functionality.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain and is thought to be due to the selective
interaction of IL-4R with secondary signaling proteins
(21, 23, 29). The IL-4R gene regulation domain is
known to bind and activate STAT6, while the growth domain binds the
adapter proteins insulin receptor substrate (IRS)-1 and IRS-2, which
allow for subsequent activation of PI3K, protein kinase C, and/or the
Ras/mitogen-activated protein kinase pathways (reviewed in Ref.
30).
STAT6 has been known to act as a positive regulator of transcription.
However, recent evidence shows that STAT6 is necessary for inhibiting
FcRI expression on mast cells, E-selection expression on endothelial
cells, and macrophage cytokine production (31, 32, 57, 58). Our data indicate that STAT6 expression is essential for
IL-4-mediated Kit down-regulation, as STAT6-/-
BMMC decrease Kit levels only slightly following IL-4 stimulation.
We also assessed the role of other signal transduction pathways known
to be activated by the IL-4R growth domain by using selective
inhibitors of PI3K, protein kinase C, mitogen-activated protein
kinase/extracellular signal-related kinase, or p70 S6 kinase. The PI3K
inhibitor LY294002 completely abrogated IL-4-mediated Kit
down-regulation, while all other inhibitors had no effect (Fig. 2, and data not shown). Therefore, rather than the selective activation of
either STAT6-related or IRS-related signaling pathways, our data
demonstrate that both pathways are required for IL-4-mediated Kit
down-regulation in murine BMMC. A molecular mechanism for the
cooperation between these two pathways is not due to PI3K-dependent
modifications in STAT6 DNA binding ability (Fig. 3). However, a
potential explanation could involve PI3K-mediated alterations in STAT6
transcriptional activation functions. Taken together, our data indicate
that both PI3K and STAT6 are required for IL-4-mediated Kit repression
and that these pathways may operate separately.
Because IL-10 is both a Th2 cytokine and a known inhibitor of gene
expression (reviewed in Ref. 59), we assessed its effect
on BMMC Kit expression. Indeed, IL-10 potently down-regulated Kit
expression in a sensitive, dose-responsive manner (Figs. 4 and 5).
Inhibitors of PI3K, protein kinase C, mitogen-activated protein
kinase/extracellular signal-related kinase, and p70 S6 kinase had no
effect on IL-10-mediated Kit repression (data not shown). We also found
that IL-4 enhanced this IL-10-mediated effect on Kit expression through
a mechanism that required PI3K function but was independent of
STAT6 expression. Both cytokines appeared to act in a
strain-independent manner, as we obtained similar results using BMMC
populations derived from C57BL/6, C57BL/6 x 129, and BALB/c mice
(Figs. 1 and 2, also Table I). A role for both IL-4 and IL-10 in Kit
regulation emphasizes the importance of Th2 function in allergic
diseases involving mast cell activation. Coupled with our earlier
observation of IL-4-mediated FcRI down-regulation, these data argue
for an ability of Th2 cytokines to negatively regulate mast cell
function. In fact, preliminary evidence indicates that IL-10 is also
capable of inhibiting FcRI expression (C.P.S. and J.J.R.,
unpublished observations).
Unlike data obtained using the human mast cell line HMC-1 (11), we found that IL-4- and IL-10-mediated Kit repression occurs largely at a posttranscriptional level. Although mast cells stimulated with IL-3 plus IL-4 or IL-3 plus IL-10 demonstrated a 50% reduction in Kit surface expression, there was no change in c-kit mRNA levels. Similarly, BMMC cultured in IL-3 plus IL-4 plus IL-10 exhibited a drastic (80%) reduction in Kit surface expression, with a modest (30%, mean of four experiments) c-kit mRNA. This latter result was interesting, as it argues for use of a distinct mechanism for Kit down-regulation in BMMC stimulated with IL-4 and IL-10. Determining this mechanism will require further study.
In agreement with an earlier study (12), we found that
IL-4 reduced total Kit protein levels. Similarly, treatment with IL-3
plus IL-10 or IL-3 plus IL-4 plus IL-10 also led to loss of Kit protein
expression. This reduction in protein expression was not coupled
with increased degradation, as shown by an overall increase, rather
than a decrease of Kit half-life (Fig. 7B). These data,
coupled with the minimal changes in Kit mRNA levels we have observed,
argue for cytokine-mediated control of Kit translation. This hypothesis
fits well with our observed transient and reversible nature of Kit
down-regulation. While the role of STAT6 and PI3K in this process
remains to be determined, each could be proposed to function in
expression or activation of translational cofactors.
Although IL-4- and IL-10-mediated Kit inhibition was transient, the
functional effects of this regulation were evidenced by assessment of
Kit-mediated cytokine production. SCF-stimulated BMMC induced
transcription of TNF-, as reported previously (56). We
also detected an increase in IL-13 mRNA following SCF stimulation, the
first report of such regulation (Fig. 8). However, BMMC cultured in the
presence of IL-3 plus IL-4 and/or IL-10 showed a remarkable loss of
mRNAs encoding TNF- and IL-13, compared with those cultured in IL-3
alone, following SCF activation (Fig. 8). IL-13 has recently been
demonstrated to play an important role in allergic asthma (60, 61). Therefore, regulation of IL-13 transcription by IL-4 and
IL-10 could be an important mechanism in the asthmatic response.
As Kit is known to be important for mast cell survival and cytokine
production, the decrease in both Kit expression and function argues for
key roles of IL-4 and IL-10 in mast cell-related allergic disease.
Together, with our previous data demonstrating IL-4-mediated FcRI
down-regulation, we postulate the use of IL-4 and IL-10 as homeostatic
factors to limit mast cell-mediated inflammatory responses.
Note added in proof. While this manuscript was in publication, Kanbe et al. reported that cord-blood-derived human cultured mast cells produce IL-13 in the presence of SCF (62).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John J. Ryan, Department of Biology, Box 842012, Virginia Commonwealth University, Richmond, VA 23284-2012. E-mail address:
3 Abbreviations used in this paper: SCF, stem cell factor; PI3K, phosphotidylinositide-3'-kinase; BMMC, bone marrow-derived mast cells; cRPMI, complete RPMI 1640 medium; CM, conditioned medium; WT, wild type; RPA, RNase protection assay; IRS, insulin receptor substrate; MFI, mean fluorescent intensity; HBS, HEPES-buffered saline.
Received for publication September 16, 1998. Accepted for publication June 16, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain. Immunity 4:113.[Medline]
RI expression through a STAT6-dependent mechanism. J. Immunol. 161:6915.-stimulated E-selection gene transcription is mediated by STAT6 antagonist of NF-
B. J. Biol. Chem. 272:10212.. Science 283:393. subunit of phosphoinositide 3-kinase. Science 283:390.- and interferon ß-stimulated gene expression through STAT proteins. Science 269:1721. and IL-12 by STAT6-dependent and -independent mechanisms. J. Immunol. 162:5224.This article has been cited by other articles:
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  S. Mithraprabhu and K. L Loveland Control of KIT signalling in male germ cells: what can we learn from other systems? Reproduction, November 1, 2009; 138(5): 743 - 757. [Abstract] [Full Text] [PDF]
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 K. Speiran, D. P. Bailey, J. Fernando, M. Macey, B. Barnstein, M. Kolawole, D. Curley, S. S. Watowich, P. J. Murray, C. Oskeritzian, et al. Endogenous suppression of mast cell development and survival by IL-4 and IL-10 J. Leukoc. Biol., May 1, 2009; 85(5): 826 - 836. [Abstract] [Full Text] [PDF]
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K. Ogawa, M. Funaba, and M. Tsujimoto A dual role of activin A in regulating immunoglobulin production of B cells J. Leukoc. Biol., June 1, 2008; 83(6): 1451 - 1458. [Abstract] [Full Text] [PDF]
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 S. Kennedy Norton, B. Barnstein, J. Brenzovich, D. P. Bailey, M. Kashyap, K. Speiran, J. Ford, D. Conrad, S. Watowich, M. R. Moralle, et al. IL-10 Suppresses Mast Cell IgE Receptor Expression and Signaling In Vitro and In Vivo J. Immunol., March 1, 2008; 180(5): 2848 - 2854. [Abstract] [Full Text] [PDF]
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M. Kashyap, A. M. Thornton, S. K. Norton, B. Barnstein, M. Macey, J. Brenzovich, E. Shevach, W. J. Leonard, and J. J. Ryan Cutting Edge: CD4 T Cell-Mast Cell Interactions Alter IgE Receptor Expression and Signaling J. Immunol., February 15, 2008; 180(4): 2039 - 2043. [Abstract] [Full Text] [PDF]
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 J. V. Spencer The Cytomegalovirus Homolog of Interleukin-10 Requires Phosphatidylinositol 3-Kinase Activity for Inhibition of Cytokine Synthesis in Monocytes J. Virol., February 15, 2007; 81(4): 2083 - 2086. [Abstract] [Full Text] [PDF]
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K. Ogawa, M. Funaba, Y. Chen, and M. Tsujimoto Activin A Functions as a Th2 Cytokine in the Promotion of the Alternative Activation of Macrophages J. Immunol., November 15, 2006; 177(10): 6787 - 6794. [Abstract] [Full Text] [PDF]
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D. P. Bailey, M. Kashyap, L. A. Bouton, P. J. Murray, and J. J. Ryan Interleukin-10 induces apoptosis in developing mast cells and macrophages J. Leukoc. Biol., September 1, 2006; 80(3): 581 - 589. [Abstract] [Full Text] [PDF]
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 Z. Orinska, E. Bulanova, V. Budagian, M. Metz, M. Maurer, and S. Bulfone-Paus TLR3-induced activation of mast cells modulates CD8+ T-cell recruitment Blood, August 1, 2005; 106(3): 978 - 987. [Abstract] [Full Text] [PDF]
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T. R. Kataoka, N. Komazawa, E. Morii, K. Oboki, and T. Nakano Involvement of connective tissue-type mast cells in Th1 immune responses via Stat4 expression Blood, February 1, 2005; 105(3): 1016 - 1020. [Abstract] [Full Text] [PDF]
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S. R. Gillespie, R. R. DeMartino, J. Zhu, H. J. Chong, C. Ramirez, C. P. Shelburne, L. A. Bouton, D. P. Bailey, A. Gharse, P. Mirmonsef, et al. IL-10 Inhibits Fc{epsilon}RI Expression in Mouse Mast Cells J. Immunol., March 1, 2004; 172(5): 3181 - 3188. [Abstract] [Full Text] [PDF]
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M. A. Sherman, D. R. Powell, and M. A. Brown IL-4 Induces the Proteolytic Processing of Mast Cell STAT6 J. Immunol., October 1, 2002; 169(7): 3811 - 3818. [Abstract] [Full Text] [PDF]
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E. D. White, R. P. Andrews, and G. K. K. Hershey Sulfhydryl-2 domain-containing protein tyrosine phosphatase-1 is not a negative regulator of interleukin-4 signaling in murine mast cells J. Leukoc. Biol., May 1, 2001; 69(5): 825 - 830. [Abstract] [Full Text]
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 C. F. Yeatman II, S. M. Jacobs-Helber, P. Mirmonsef, S. R. Gillespie, L. A. Bouton, H. A. Collins, S. T. Sawyer, C. P. Shelburne, and J. J. Ryan Combined Stimulation with the T Helper Cell Type 2 Cytokines Interleukin (Il)-4 and IL-10 Induces Mouse Mast Cell Apoptosis J. Exp. Med., October 16, 2000; 192(8): 1093 - 1104. [Abstract] [Full Text] [PDF]
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