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RI Expression Through a STAT6-Dependent Mechanism1
Departments of
*
Biology and
Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA 23284
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI, is poorly understood. We show that IL-4 can inhibit FcRI
expression on mouse bone marrow-derived mast cells and fetal
liver-derived mast cell progenitors. This effect could be observed at
2.5 ng/ml IL-4 and was dose dependent. IL-4-mediated inhibition of
cultured BMMC required 4 days of stimulation and was sustained at
maximum levels for at least 21 days. The inhibition of FcRI
expression resulted in decreased sensitivity to IgE-mediated
stimulation, as measured by serotonin release, and the induction of
mRNA for IL-4, IL-5, IL-6, and IL-13. Additionally, IL-4 could abrogate
the IgE-mediated increase in FcRI expression. Lastly, IL-4-mediated
inhibition was dependent upon expression of the STAT6 transcription
factor, as STAT6-deficient bone marrow-derived mast cells did not
decrease FcRI levels in response to IL-4. These data argue for a
homeostatic role of IL-4 in the regulation of FcRI expression, a
role that could be critical to understanding atopic
disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI) is a potent
physiologic stimulus for the release of the preformed mediators and of
the synthesis and secretion of cytokines by mast cells (for review, see
2 .
FcRI is expressed on mast cells and basophils (for review, see 3 , eosinophils (4), Langerhans cells (5, 6, 7), and some monocytes (8).
The receptor is a heterotetramer consisting of an 
-chain, a
ß-chain, and a dimer of two 
-chains. Disruption of the -chain
(9) or -chain (10) results in the failure of knockout mice to
express FcRI and to respond to challenge with IgE-mediated stimuli,
emphasizing the critical role played by FcRI in atopy.
IL-4 is a pleiotropic cytokine produced by T cells (11), mast cells (12), and basophils (13). A key cytokine in atopy, IL-4, directs Th2 development, induces Ig class switching and the production of IgE (reviewed in 14 , and is a cofactor for proliferation of mast cell lines (15). Recent reports have linked an IL-4R polymorphism to allergic disease (16), emphasizing the role of this cytokine in atopic phenomena.
The IL-4R signals via distinct growth-promoting and gene expression pathways (reviewed in 17 . Many effects of IL-4 on gene expression appear to require activation of the transcription factor STAT6. Animals genetically deficient in STAT6 fail to develop Th2 cells, synthesize IgE, or induce expression of CD23 and MHC class II molecules (18, 19, 20) in response to IL-4. Thus, STAT6 is regarded as a necessary element for IL-4-mediated positive gene control.
Regulation of FcRI expression is poorly understood, although
prospective DNA regulatory sequences have been proposed (21). IL-4 has
been shown to induce FcRI expression on developing human mast cells
(22, 23), but the mechanism for this expression is not known. Recent
reports have also demonstrated a dramatic increase in mast cell FcRI
expression in response to IgE stimulation (24, 25). Control of mast
cell FcRI expression may therefore be dependent upon IL-4 via direct
or indirect mechanisms due to its effects on mast cells and its ability
to induce IgE synthesis.
In the present study we investigated the role of IL-4 in regulating the
expression of mouse mast cell FcRI expression. In contrast to the
human system, mouse fetal liver-derived mast cell progenitors and more
mature bone marrow-derived mast cell
(BMMC)3 populations exhibited
a striking decrease in FcRI expression following IL-4 stimulation.
This unexpected response was time and dose dependent and was maintained
for long periods in culture. Importantly, the ability of IL-4 to
diminish FcRI levels was STAT6 dependent. BMMC derived from
STAT6-deficient animals failed to decrease FcRI levels, although
their heterozygous littermates responded normally. We conclude that in
the mouse, IL-4 acts as an inhibitory signal for FcRI expression,
possibly allowing this cytokine to act as a regulator of mast cell
activation by IgE-mediated stimuli. The possible implications for
atopic disease and the reasons for the disparity in data from the mouse
and human systems are discussed.
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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) or
in 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-deficient or heterozygous littermate mice by
culture in cRPMI/WEHI for 3 wk, after which time >99% express
FcRI, c-Kit, and CD13 and have mast cell morphology by histochemical
staining. STAT6-deficient and heterozygous littermate mice have been
described previously (19) and were provided by Dr. James Ihle (Memphis,
TN). The phenotype of BMMC was determined by flow cytometric analysis
using Abs specific for c-Kit, CD13, and IgE and by histochemical
staining (data not shown).
Cytokines and Abs
Murine SCF, IL-3, and IL-4 were purchased from R&D Systems
(Minneapolis, MN). 2.4G2 rat anti-mouse FcRII/II ascites was a
gift from Jane Hu-Li and William Paul (National Institutes of Health,
Bethesda, MD) or was purchased from PharMingen (San Diego, CA).
FITC-labeled rat anti-mouse CD13, FITC-labeled rat anti-mouse
CD4, FITC-labeled or unlabeled rat anti-mouse CD23 (clone B3B4),
and murine IgE were purchased from PharMingen (San Diego, CA). Rat
anti-mouse IL-4R Ab was purchased from Genzyme (Cambridge, MA).
Goat anti-rat IgG (H+L chains) F(ab')2 and FITC-labeled
rat anti-mouse IgE were purchased from Southern Biotechnology
Associates (Birmingham, AL). Rabbit-anti mouse STAT6 antisera was a
gift from Dr. James Ihle (Memphis, TN).
Tissue culture conditions for inhibition of BMMC FcRI expression
To examine the regulation of FcRI expression, BMMC were
washed to remove WEHI-3-CM and were incubated for 4–6 h at 37°C.
BMMC were then plated at 500,000 cells/ml in a 200-ml 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 mouse IgE.
Cultures were fed every 4 days by replacement of half the culture
medium and cytokines. FcRI levels were determined by flow cytometric
analysis.
Culture of fetal liver-derived progenitors
BALB/c mice were bred, and the day of vaginal plug observation was denoted day 0 of gestation. Embryos were harvested on day 14 of gestation, and livers were dissected and dispersed by manual disruption with glass slides. Cells were cultured in cDMEM supplemented with 15% embryonic stem cell FBS (Life Technologies), 100 ng/ml SCF, and 100 ng/ml IL-3 with and without 20 ng/ml IL-4 for 23 days at 37°C in 5% CO2 and 5% O2. Day 23 cultures contained >99% granulated metachromatic cells, as determined by histochemistry with acid toluidine blue.
Flow cytometric analysis
To detect expression of FcRI on BMMC, cells were incubated
with 0.3 ml of 2.4G2 rat anti-mouse FcRII/III ascites/100 ml of
solution for 10 min at 4°C, followed by 10 µg/ml IgE for 30 min at
4°C in PBS/3% FCS/0.1% sodium azide (FACS buffer). Cells were then
washed twice and stained with 10 µg/ml FITC-conjugated rat
anti-mouse IgE for 30 min at 4°C, washed twice, and analyzed in
the presence of propidium iodide with a Becton Dickinson FACScan
(Becton Dickinson, San Jose, CA). Control samples were identically
stained in the absence of IgE. These BMMC populations do not express
detectable levels of the low affinity IgE receptor, CD23, either
basally or following IL-4 stimulation, as determined by flow cytometric
analysis (data not shown). The percent inhibition of FcRI expression
was determined using mean fluorescence intensities, with cells cultured
in IL-3 alone as the baseline for all comparisons. To detect expression
of FcRI on fetal liver-derived mast cells, cells were stained as
described above, with the inclusion of 10 mg/ml rat anti-mouse CD23
in combination with 2.4G2 Ab. To detect expression of CD13 or Kit,
cells were first incubated with 2.4G2 as described above; then stained
with FITC-labeled anti-CD13, anti-Kit, or anti-CD4 (as a
control stain); and washed as described above.
Serotonin release assay
Cells were incubated for 11–13 days in cRPMI supplemented with 5 ng/ml IL-3 with or without 20 ng/ml IL-4, then incubated overnight at approximately 5 x 105 cells/ml with 2 µCi/ml 5-hydroxy-[G-3H]tryptamine creatinine sulfate (Amersham, Arlington Heights, IL). Cells were washed twice, resuspended at 1 x 106 cells/ml, and incubated for 45 min at 37°C in cRPMI supplemented with 1 ng/ml IL-3 and 2 mg/ml IgE in 96-well V-bottom plates (Costar). Excess IgE was removed by washing twice in cRPMI, after which cells were plated at 1 x 105 cells/well and incubated for 30 min at 37°C with the indicated concentration of rat anti-mouse IgE or with 2 mM ionomycin. To samples containing rat anti-mouse IgE, goat anti-rat IgG F(ab')2 was added at a 1 mg/ml final concentration for 30 min at 37°C. Control samples contained IgE alone, which resulted in higher background release of serotonin than anti-IgE alone (data not shown). Supernatants were harvested, and lysates were made using 100 µl of lysis buffer (50 mM HEPES (pH 7.5), 0.5% Triton X-100, and 5 mM EDTA). Radioactivity in supernatants and lysates was determined by scintillation counting, and the percent radioactivity in supernatants was calculated. All samples were studied in duplicate.
RNase protection assay (RPA)
For each sample, 5 x 106 BMMC were stimulated with 5 ng/ml IL-3 alone or IL-3 and 10 ng/ml IL-4 as described above for 6–13 days, with feeding of cells every 4 days. Cells were then washed and resuspended at 1 x 106/ml in cRPMI with 1 ng/ml IL-3 (cRPMI/IL-3). IgE (2 mg/ml) and 2.4G2 (5 mg/ml) were added for 45 min at 4°C. Cells were washed twice in cRPMI and resuspended at 1 x 106/ml in cRPMI/IL-3. Cells were then incubated with 1 mg/ml rat anti-mouse IgE for 30 min at 37°C, after which time goat anti-rat IgG F(ab')2 was added to a final concentration of 1 mg/ml for 5 h at 37°C. Control samples were incubated with goat anti-rat IgG F(ab')2 alone or with 2 mM ionomycin for 5 h. RNA was harvested with RNAzol (Tel-Test, Friendswood, TX). RPA was performed using the mCK-1 probe set from the RiboQuant System (PharMingen) to the manufacturer’s specifications. Pixel intensity was determined using a Phosphorimaging 445si System (Molecular Dynamics, Sunnyvale, CA).
Proliferation experiments
Cells were washed twice to remove WEHI-3-CM, incubated for 16 h in cRPMI followed by 2 h in RPMI, and plated at 2 x 104 cells/well in a 96-well flat-bottom plate for 48 h in cRPMI with the indicated cytokines. Cultures were pulsed with 1 mCi/well tritiated thymidine deoxyribonucleotide for 16 h before harvesting. The mean counts per minute of triplicate samples were determined.
Electrophoretic mobility shift assay (EMSA)
BMMC (3 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 RPMI at 2 x 106/ml for 2 h at 37°C. After a final wash, BMMC were resuspended at 6 x 107/ml in RPMI/50 mM sodium orthovanadate (vanadate) and stimulated with 100 ng/ml IL-3 or IL-4 for 10 min at room temperature. Reactions were stopped by addition of cold RPMI/100 mM vanadate. Cell pellets were resuspended in 10 ml of lysis buffer (0.5% Nonidet P-40, 50 mM Tris (pH 8.0), 10% glycerol, 100 µM EDTA (pH 8.0), 50 mM NaF, 150 mM NaCl, 100 µM Na3VO4, 1 mM DTT, and 1x complete protease inhibitor mixture (Boehringer Mannheim, Indianapolis, IN)) and incubated on ice for 60 min. Lysates were cleared by centrifugation at 15,000 rpm for 15 min at 4°C, and supernatants were harvested, resuspended to a final concentration of 7.6 µg/µl, and stored at -70°C. For assays, 38 µg of cell lysate was incubated with 100 ng of 32P-labeled oligonucleotide in reaction buffer (40 mM KCl, 1 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 20 mM HEPES (pH 7.9), 6% glycerol, 1 mg/ml BSA, and 0.1 mg/ml poly(dI-dC)) for 15 min room temperature. Reactants were loaded (without loading dye) onto a 6% polyacrylamide/0.22x TBE gel that had been pre-run at 200 V for 60 min and electrophoresed at 300 V for 2 h. Gels were dried and exposed directly to film. To detect STAT DNA binding activity, a double-stranded oligonucleotide corresponding to a GAS-like element found in the mouse IL-4 promoter element (4GL-3) was used: gatcAAGACC-162TTCACAGGAA-152CTTTAATCT gatc. To detect STAT3 and -5 (induced by IL-3), a GAS-like element found in the human CD23 promoter was used: 5'-gatcAAGACC-227TTTCTAAGAA-217CTTTAATCTgatc. Lysates were confirmed to contain STAT6 by supershift analysis with anti-STAT6 Abs. Oligonucleotides were synthesized with a 5'ACTG overhang on each end (denoted by lowercase letters) and were labeled using Klenow DNA polymerase and [32P]dCTP by standard techniques.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI expression on murine BMMC and developing
fetal liver progenitor cells
Given the role of IL-4 in IgE synthesis and the recent
demonstration that IgE can up-regulate mast cell FcRI expression
(22, 23), we assessed the effect of IL-4 stimulation on BMMC expression
of FcRI. As shown in Fig. 1
, BMMC
cultured in the presence of IL-3 and IL-4 exhibited diminished FcRI
expression compared with cells cultured in IL-3 alone, as assessed by
flow cytometry. This effect varied between different populations of
BMMC, with an average inhibition of nearly 60% based on mean
fluorescent intensities. IL-4-mediated inhibition of FcRI expression
could be observed at concentrations as low as 2.5 ng/ml IL-4. Maximal
activity was observed using 20 ng/ml IL-4, with no further inhibition
at higher concentrations (Fig. 1B).
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RI expression required several days of IL-4
stimulation and was prolonged with continuous exposure to IL-4. As
shown in Fig. 1
C, maximal inhibition of FcRI expression
was observed after 4 days of stimulation and was sustained for 21 days
of culture, the longest time point examined. Thus, the effects of IL-4
appeared to be both sensitive and sustainable. Further, IL-4
stimulation of BMMC did not appear to mediate a generalized decrease in
the expression of all surface proteins, as CD13 expression was not
diminished by IL-4 stimulation (data not shown).
After observing that IL-4 could inhibit FcRI expression on
differentiated mast cells, we wished to determine whether these effects
were similar on developing mast cell progenitors. Bone marrow cells
cultured in IL-3 and SCF yielded mast cells, as expected. However,
attempts to culture bone marrow-derived progenitors in the presence of
IL-3, SCF, and IL-4 led to a complete failure of mast cell development
(data not shown). The ability of IL-4 to enhance monocyte development
from bone marrow progenitors (26) may explain these results, as our
cultures appeared to produce monocytic cells.
Since bone marrow progenitors were unsuitable for these studies, we
assessed the effect of IL-4 stimulation on cultures of developing
progenitors from day 14 murine fetal liver. As shown in Fig. 2, the addition of IL-4 to cultures of
developing mast cells from fetal liver progenitors led to a
dose-dependent decrease in FcRI staining. The inhibitory effect of
IL-4 was similar to that observed with BMMC, with an approximately 50%
decrease in FcRI expression levels. Thus, IL-4 is able to inhibit
FcRI expression on both differentiated mast cells and developing
mast cell progenitor populations.
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To determine the effect of IL-4 stimulation on FcRI function,
we measured serotonin release in response to FcRI cross-linkage with
anti-IgE Abs in BMMC cultured with IL-3 alone or with 20 ng/ml
IL-4. As shown in Fig. 3, IL-4 treatment
of BMMC led to only a slight decrease in maximal serotonin release
following IgE cross-linkage. However, there was a more potent effect on
the concentration of anti-IgE necessary to induce half-maximal
serotonin release. Cells cultured in IL-3 and IL-4 required an average
11.5-fold more anti-IgE to reach half-maximal serotonin release
than cells cultured in IL-3 alone (0.75 vs 0.065 mg/ml, respectively).
Thus, IL-4-mediated inhibition of FcRI expression coincided with a
decrease in sensitivity to IgE-mediated stimulation.
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). These data indicate that IL-4
stimulation of BMMC reduces both immediate and delayed effects of
IgE-mediated stimulation, and that these effects coincide with the
IL-4-mediated inhibition of FcRI expression.
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RI expression
The time required for FcRI modulation by IL-4 is similar to
that reported for IgE-mediated enhancement of receptor expression (24).
This led us to examine the coordinated effects of IL-4 and IgE on
FcRI expression. BMMC were cultured in IL-3 alone, in IL-3 with
increasing concentrations of IgE, or in IL-3, IgE, and 10 ng/ml IL-4.
IL-4 repressed the IgE-mediated increase in FcRI expression (Fig. 5). This effect was clearly dependent
upon the concentration of IgE. BMMC cultured with 1 mg/ml IgE showed a
modest reduction in FcRI levels when costimulated with IL-4, while
cells cultured with 0.2 mg/ml IgE expressed FcRI at control (IL-3
alone) levels when costimulated with IL-4.
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). BMMC cultured
in IL-3 plus IL-4 for 4 days before the addition of IgE showed lower
FcRI levels than cells cultured in IL-3 alone before the addition of
IgE, an effect that closely mirrored the simultaneous addition of IL-4
and IgE. BMMC cultured with IL-3 and IgE for 4 days before the addition
of IL-4 showed less reduction in FcRI levels than cells precultured
with IL-3 and IL-4. Thus, we found that IL-4 and IgE have opposing
effects on FcRI expression, with the end effect on FcRI levels
contingent upon the concentration and timing of each stimulus.
STAT6 expression is required for IL-4-mediated inhibition of BMMC
FcRI expression
IL-4R signaling is known to proceed through at least two pathways
(reviewed in 17 . Of these, the STAT6 pathway has been shown
essential for IL-4-induced gene expression (18, 19, 20). While STAT6 is
generally thought to be a positive regulator of gene transcription,
there is some evidence for its requirement in gene repression
activities (27). Thus, it seemed possible that STAT6 functions in the
IL-4-mediated inhibition of FcRI expression.
To determine the role of STAT6 in IL-4 regulation of FcRI, we
derived BMMC populations from mice bearing a targeted disruption in the
STAT6 gene or their heterozygous littermates. STAT6-deficient BMMC
derived from WEHI-3CM cultures had mast cell morphology and expressed
normal levels of FcRI, c-Kit receptors, and CD13 (data not shown).
However, STAT6-deficient BMMC exhibited little or no inhibition of
FcRI expression in response to IL-4 (Fig. 6A), while wild-type and
heterozygous littermate control BMMC responded with a substantial
reduction in FcRI staining.
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RI regulation in STAT6-deficient BMMC
did not appear to represent a deficiency in IL-4R expression or
function. STAT6-deficient BMMC expressed IL-4R at similar levels to
wild-type and heterozygote littermate BMMC populations (Fig. 6B), and all populations exhibited similar proliferative
responses to IL-4 in combination with suboptimal doses of IL-3 or SCF
(Fig. 6C). The STAT6-deficient phenotype of these BMMC
populations was confirmed by EMSA analysis. IL-4, and to a lesser
extent IL-3, activated a DNA binding factor in wild-type, but not
STAT6-deficient, BMMC that bound an oligonucleotide containing a
canonical STAT6 site (Fig. 6D). This DNA:protein complex
could be supershifted with anti-STAT6 antisera (data not shown).
Using a probe that binds STAT3 and STAT5, wild-type and STAT6-deficient
BMMC were shown to activate similar DNA-binding factors in response to
IL-3 stimulation (data not shown). Thus, STAT6-deficient BMMC did not
appear to have a global loss of transcription factor activation. These
data indicate a requirement for STAT6 in IL-4-mediated inhibition of
FcRI staining. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI on human
mast cells (22, 23).
However, in these experiments using murine mast cells, we found that
IL-4 exhibits a dose- and time-dependent inhibition of FcRI levels.
These results were consistent in 30 experiments using 13 individual
BMMC populations derived from 10 animals, with an average IL-4-mediated
inhibition of nearly 60% compared with that of cells cultured in IL-3.
This effect was delayed but sustainable; maximum inhibition required 4
days of stimulation and was maintained for 21 days of culture.
IL-4-mediated inhibition of Fc[espilon]RI expression did not appear
to be strain dependent, as we have obtained similar results using BMMC
populations derived from C57BL/6, C57BL/6 x 129, and BALB/c mice.
We also find that IL-4 stimulation of developing mast cell progenitors
from day 14 fetal liver yields mast cells with decreased FcRI levels
compared with cells grown in medium lacking IL-4. Conversely, in human
fetal liver progenitor cells IL-4 induces, rather than represses,
FcRI expression (22). This discrepancy could be explained by
differential cytokine responsiveness across or within species. For
example, mouse mast cell progenitors develop in response to IL-3 and
SCF, while human progenitors do not respond to IL-3, but develop in
response to SCF alone (30). Further, our own findings show a disparity
in IL-4 responsiveness in mouse mast cell progenitors derived from
different organs. While bone marrow mast cell progenitor development is
nearly completely inhibited by IL-4 stimulation, fetal liver
progenitors are less dramatically affected in their development, but do
show reduced FcRI expression. The precise explanation for
differences in these experimental systems remains to be determined.
The functional consequence of inhibiting FcRI levels was evident by
measurements of serotonin release. We found an 11.5-fold decrease in
sensitivity to anti-IgE stimulation when measuring serotonin
release of BMMC cultured with IL-3 and IL-4 vs that of cells cultured
in IL-3 alone. While IL-4 has been previously reported to enhance
serotonin release (28), these studies examined the effects of IL-4
after a 2-day stimulation. Since our data indicate that IL-4 has little
or no effect on FcRI expression at this time point, the difference
between our data and those reported earlier is probably due to this
timing effect. A diminished response to activation stimuli could play a
role in modulating mast cell degranulation, a critical step in allergic
disease. What role, if any, this plays in vivo remains to be
determined.
Stimulation with IL-4 was also found to result in potent inhibition of
IgE-mediated cytokine mRNA induction. RPA studies indicated that BMMC
cultured in IL-3 and IL-4 before cross-linkage of surface-bound IgE
expressed mRNA for IL-4, IL-5, IL-6, and IL-13 at approximately 20% of
the levels elicited by IgE-mediated stimulation of cells cultured in
IL-3 alone. Coupled with the decrease in serotonin release, these data
argue for a potentially important role of IL-4 in regulating the early
and late effects of IgE-mediated mast cell stimulation. Whether these
effects are entirely due to a reduction in FcRI expression or to
other effects on cytokine transcription is as yet unknown.
In addition to the effects on FcRI signaling, IL-4 was also capable
of abrogating the dramatic increase in BMMC FcRI expression driven
by long-term incubation with IgE. This effect displayed crucial timing
and concentration elements, with maximal inhibitory effects observed
under conditions where IL-4 was given before or during IgE stimulation
of BMMC. These data may be important to understanding the role of IL-4
in allergic disease. The recently reported role of IgE in enhancing the
expression of its own receptor (24, 25) offers a mechanism for positive
feedback in atopy that could hypersensitize mast cells to allergens,
potentiating the allergic response. An ability to both direct IgE
synthesis and inhibit expression of FcRI would place IL-4 at a
pivotal junction in the regulation of atopy, offering a mechanism for
homeostasis in this potentially damaging immune response (Fig. 7).
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B
site (27). Our data indicate that STAT6 plays an essential role in the
IL-4-mediated inhibition of FcRI expression. It is interesting to
note that the murine FcRIa chain promoter possesses a canonical
STAT6 DNA binding sequence -285TTC TGTT
GAA-295 (31). We postulate that STAT6 binding to this
element may lead to decreased transcription from this promoter,
resulting in lowered FcRI levels. Our studies coupled with those of
Bennett and colleagues argue for a reassessment of STAT6 as both a
positive and a negative regulator of gene expression. In summary, these data indicate that IL-4 may function to both initiate and dampen atopic responses. Both positive and negative regulatory events appear to require STAT6 activation. While further studies are needed to fully elucidate the mechanism of these responses and understand their in vivo roles in normal and atopic individuals, IL-4 may function as a homeostatic factor in the atopic response.
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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: BMMC, bone marrow-derived mast cells; SCF, stem cell factor; cRPMI, RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 mM sodium pyruvate; WEHI-3-CM, WEHI-3-conditioned medium; RPA, ribonuclease protection assay; EMSA, electrophoretic mobility shift assay.
Received for publication April 21, 1998. Accepted for publication August 26, 1998.
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References |
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|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RI mediates IgE binding to human epidermal Langerhans cells. J. Invest. Dermatol. 99:30S.[Medline]
RI). J. Exp. Med. 175:1285.RI. J. Exp. Med. 175:1353.RI) on monocytes from atopic individuals. J. Exp. Med. 179:745. chain gene. Cell 75:969.[Medline]
chain deletion results in pleiotrophic effector cell defects. Cell 76:519.[Medline]
chain of Fc and T cell receptors. J. Biol. Chem. 268:1355. receptor type I expression in recombinant human stem cell factor-dependent fetal liver-derived human mast cells. J. Immunol. 159:2911.[Abstract]
RI) on human mast cells by IL-4. Int. Immunol. 8:1367.RI expression in vitro and in vivo: evidence for a novel amplification mechanism in IgE-dependent reactions. J. Exp. Med. 185:663.RI expression in the rat intestinal mucosa and tongue is enhanced during Nippostrongylus brasiliensis infection and can be up-regulated by the in vivo administration of IgE. J. Immunol. 158:3805.[Abstract]
-stimulated E-selectin gene transcription is mediated by STAT6 antagonism of NF-B. J. Biol. Chem. 272:10212.-chain of the mouse high affinity receptor for IgE (FcRI). J. Immunol. 149:897.[Abstract]
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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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|
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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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K. Suzuki, H. Nakajima, K. Ikeda, Y. Maezawa, A. Suto, H. Takatori, Y. Saito, and I. Iwamoto IL-4-Stat6 Signaling Induces Tristetraprolin Expression and Inhibits TNF-{alpha} Production in Mast Cells J. Exp. Med., December 1, 2003; 198(11): 1717 - 1727. [Abstract] [Full Text] [PDF]
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M. Hasegawa, C. Nishiyama, M. Nishiyama, Y. Akizawa, K. Takahashi, T. Ito, S. Furukawa, C. Ra, K. Okumura, and H. Ogawa Regulation of the Human Fc{epsilon}RI {alpha}-Chain Distal Promoter J. Immunol., April 1, 2003; 170(7): 3732 - 3738. [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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K. Suzuki, H. Nakajima, S.-i. Kagami, A. Suto, K. Ikeda, K. Hirose, T. Hiwasa, K. Takeda, Y. Saito, S. Akira, et al. Proteolytic Processing of Stat6 Signaling in Mast Cells as a Negative Regulatory Mechanism J. Exp. Med., July 1, 2002; 196(1): 27 - 38. [Abstract] [Full Text] [PDF]
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R. Malaviya and F. M. Uckun Role of STAT6 in IgE Receptor/Fc{varepsilon}RI-Mediated Late Phase Allergic Responses of Mast Cells J. Immunol., January 1, 2002; 168(1): 421 - 426. [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 9, 2000; 192(8): 1093 - 1104. [Abstract] [Full Text] [PDF]
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K. Suzuki, H. Nakajima, N. Watanabe, S.-i. Kagami, A. Suto, Y. Saito, T. Saito, and I. Iwamoto Role of common cytokine receptor gamma chain (gamma c)- and Jak3-dependent signaling in the proliferation and survival of murine mast cells Blood, September 15, 2000; 96(6): 2172 - 2180. [Abstract] [Full Text] [PDF]
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 H. Yoshidome, A. Kato, M. Miyazaki, M. J. Edwards, and A. B. Lentsch IL-13 Activates STAT6 and Inhibits Liver Injury Induced by Ischemia/Reperfusion Am. J. Pathol., October 1, 1999; 155(4): 1059 - 1064. [Abstract] |
